Tazemetostat

Current developer:  Epizyme, Inc., Cambridge, MA 02139.

EPZ-6438 (Tazemetostat)
CAS: 1403254-99-8

Chemical Formula: C34H44N4O4
Exact Mass: 572.33626

N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide
SIMLES: O=C(C1=CC(C2=CC=C(CN3CCOCC3)C=C2)=CC(N(CC)C4CCOCC4)=C1C)NCC5=C(C)C=C(C)NC5=O

(1,1′-Biphenyl)-3-carboxamide, N-((1,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(4-morpholinylmethyl)-

N-((4,6-Dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(oxan-4-yl)amino)-4-methyl-4′-((morpholin-4-yl)methyl)(1,1′-biphenyl)-3-carboxamide

UNII-Q40W93WPE1

WO 2012142504 PRODUCT PAT

SEE  Proceedings of the National Academy of Sciences of the United States of America (2013), 110(19), 7922-7927, S7922/1-S7922/5….http://www.pnas.org/content/110/19/7922.abstract

http://www.epizyme.com/wp-content/uploads/2014/11/Ribrag-ENA-FINAL.pdf

Tazemetostat, also known as EPZ-6438,  is a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity. EPZ-6438 induces apoptosis and differentiation specifically in SMARCB1-deleted MRT cells.

Treatment of xenograft-bearing mice with EPZ-6438 leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation.

These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers. EPZ-6438 is currently in clinical trials.

Epizyme, Inc., Eisai R&D Management Co.Ltd.

Epizyme is developing tazemetostat, a lead from several small molecule EZH2 inhibitors, for treating cancer (phase 1 clinical, as of April 2015). Japanese licensee Eisai was developing the program for the potential oral treatment of cancers, including non-Hodgkin’s lymphoma; however, in March 2015, Epizyme regained worldwide, ex-Japan, rights to the program.

It appeared that Eisai was planning to investigate the program in Japan .

WO-2015057859 From, Eisai Research Institute; Epizyme Inc, indicates Novel crystalline polymorphic form C of tazemetostat, useful for treating an EZH2-mediated cancer, including non-Hodgkin’s lymphoma and breast cancer.

see WO2013155317, claiming novel hydrobromide salt of tazemetostat.

PREDICT

………………………………….

PATENT

WO 2012142504

http://www.google.com/patents/WO2012142504A1?cl=en

Example 44: Synthesis of N-((4,6-dimethyl-2-oxo-l ,2-dihydropyridin-3- yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(moφholinomethyl)-[l , – biphenyl]-3-carboxamide

Compound 44

[Step 1 : Synthesis of 5-brom -2-methyl-3-nitrobenzoic acid

To stirred solution of 2-methyl-3-nitrobenzoic acid ( 100 g, 552 mmol) in cone. H₂S0₄ (400 mL), 1 ,3-dibromo-5,5-dimethyl-2,4-imidazolidinedione (88 g, 308 mmol) was added in a portion wise manner at room temperature and the reaction mixture was then stirred at room temperature for 5 h. The reaction mixture was poured onto ice cold water, the precipitated solid was filtered off, washed with water and dried under vacuum to afford the desired compound as a solid ( 140 g, 98%). The isolated compound was taken directly into the next step. Ή NMR (DMSO-4$, 400 MHz) δ 8.31 (s, 1 H), 8.17 (s, 1 H), 2.43 (s, 3H).

Step 2: Synthesis of methyl -bromo-2-methyl-3-nitrobenzoate

To a stirred solution of 5-bromo-2-methyl-3-nitrobenzoic acid (285 g, 1 105 mmol) in DMF (2.8L) at room temperature was added sodium carbonate (468 g, 4415 mmol) followed by addition of methyl iodide (626.6 g, 4415 mmol). The resulting reaction mixture was heated at 60 °C for 8 h. After completion (monitored by TLC), the reaction mixture was filtered (to remove sodium carbonate) and washed with ethyl acetate ( 1 L X 3). The combined filtrate was washed with water (3L X 5) and the aqueous phase was back extracted with ethyl acetate (1L X 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford the title compound as a solid (290g, 97% yield). The isolated compound was taken directly into the next step. Ή NMR (CDC1₃, 400 MHz) δ 8.17 (s, 1H), 7.91 (s, 1H), 3.96 (s, 3H), 2.59 (s, 3H).

Step 3: Synthesis of methyl 3-amino-5-bromo-2-methylbenzoate

To a stirred solution of methyl 5-bromo-2-methyl-3-nitrobenzoate (290 g,

1058 mmol) in ethanol (1 .5L) was added aqueous ammonium chloride (283 g, 5290 mmol dissolved in 1.5L water). The resulting mixture was stirred at 80°C to which iron powder (472 g, 8451 mmol) was added in a portion wise manner. The resulting reaction mixture was heated at 80 °C for 12 h. Upon completion as determined by TLC, the reaction mixture was hot filtered over celite® and the celite bed was washed with methanol (5L) followed by washing with 30% MeOH in DCM (5L). The combined filtrate was concentrated in-vacuo, the residue obtained was diluted with aqueous sodium bicarbonate solution (2L) and extracted with ethyl acetate (5L X 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford the title compound as a solid (220 g, 85%). The compound was taken directly into the next step. Ή NMR (CDC1₃, 400 MHz) δ 7.37 (s, 1 H), 6.92 (s, 1 H), 3.94 (s, 3H), 3.80 (bs, 2H), 2.31 (s, 3H).

Step 4: Synthesis of methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate

To a stirred solution of methyl 3-amino-5-bromo-2-methylbenzoate (15 g, 61 .5 mmol) and dihydro-2H-pyran-4(3)-one (9.2 g, 92 mmol) in dichloroethane (300 mL) was added acetic acid (22 g, 369 mmol) and the reaction mixture stirred at room temperature for 15 minutes, then the reaction mixture was cooled to 0°C and sodium triacetoxyborohydnde (39 g, 184 mmol) was added. The reaction mixture was stirred overnight at room temperature. Upon completion of the reaction as determined by TLC, aqueous sodium bicarbonate solution was added to the reaction mixture until a pH of 7-8 was obtained. The organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (100-200 mesh silica gel) eluting with ethyl acetate: hexane to afford the desired compound as a solid ( 14 g, 69%). ‘H NMR (DMSO-<fc, 400 MHz) δ 7.01 (s, 1 H), 6.98 (s, 1 H), 5.00 (d, 1 H, J=7.6 Hz), 3.84-3.87 (m, 2H), 3.79 (s, 31 1), 3.54-3.56 (m_f 1 H), 3.43 (L 21 1, J 12 Hz), 2.14 (s. 31 1). 1 . 1 – 1 .84 (m_: 211). 1 .47- 1 .55 (m, 2H).

Step 5: Synthesis of methyl 5-bromo-3-(ethyl (tetrahydro-2H-pyran-4-yl) amino)-2- methylbenzoate

To a stirred solution of methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (14 g, 42.7 mmol) in dichloroethane (150 mL) was added acetaldehyde (3.75 g, 85.2 mmol) and acetic acid ( 15.3 g, 256 mmol). The resulting reaction mixture was stirred at room temperature for 15 minutes. The mixture was cooled to 0 °C and sodium

triacetoxyborohydnde (27 g, 128 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. Upon completion of the reaction as determined by TLC, aqueous sodium bicarbonate solution was added to the reaction mixture until a pH 7-8 was obtained, the organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (100- 200 mesh silica gel) eluting with ethyl acetate: hexane to afford the desired compound as a viscous liquid (14 g, 93%). Ή NMR (DMSO-cfo 400 MHz) δ 7.62 (s, 1 H), 7.52 (s, 1 H), 3.80 (bs, 5H), 3.31 (t, 2H), 2.97-3.05 (m, 2H), 2.87-2.96 (m, 1 H), 2.38 (s, 3H), 1.52-1.61 (m, 2H), 1 .37-1.50 (m, 2H), 0.87 (t, 3H, J=6.8 Hz).

Step 6: Synthesis of 5-bromo-N-((4, 6-dimethyl-2-oxo-l , 2-dihydropyridin-3-yl) methyl)-3 -(ethyl (tetrahydro-2H-pyra -4-yl) amino)-2-methylbenzamide

To a stirred solution of 5-bromo-3-(ethyl (tetrahydro-2H-pyran-4-yl) amino)-2- methylbenzoate (14 g, 39.4 mmol) in ethanol ( 100 mL) was added aqueous NaOH (2.36 g, 59.2 mmol in 25mL water) and the resulting mixture was stirred at 60 °C for 1 h. Upon completion of the reaction as determined by TLC, the solvent was removed under reduced pressure and the residue obtained was acidified with IN HC1 until a pH 7 was obtained and then aqueous citric acid solution was added until a pH 5-6 was obtained. The aqueous layer was extracted with 10% MeOH in DCM (200mL X 3), the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the respective acid (14 g, 100%).

The above acid (14 g, 40.9 mmol) was then dissolved in DMSO (70 mL) and 3- (amino methyl)-4, 6-dimethylpyridin-2( l H)-one ( 12.4 g, 81 .9 mmol) was added to it. The reaction mixture was stirred at room temperature for 15 minutes, then PYBOP (31.9 g, 61.4 mmol) was added and stirring was continued for overnight at room temperature. Upon completion of the reaction as determined by TLC, the reaction mixture was poured onto ice- cold water (700 mL), stirred for 30 minutes and the precipitated solid was collected by filtration, washed with water (500 mL) and air dried. The solid obtained was stirred with acetonitrile (75mL X 2), filtered and air dried. The solid obtained was again stirred with 5% MeOH in DCM ( l OOmL), filtered and dried completely under vacuum to afford the title compound as a solid ( 14 g, 74 %). Ή NMR (DMSO- ₆, 400 MHz) δ 1 1.47 (s, 1 H), 8.23 (t, 1 H), 7.30 (s, 1 H), 7.08 (s, 1 H), 5.85 (s, 1 H), 4.23 (d, 2H, J=4.4 Hz), 3.81 (d, 2H, J=l 0.4 Hz), 3.20-3.26 (m, 2H), 3.00-3.07 (m, I H), 2.91 -2.96 (m, 2H), 2.18 (s, 3H), 2.14 (s, 3H), 2.10 (s, 3H), 1.58-1.60 (m, 2H), 1.45-1.50 (m, 2H), 0.78 (t, 3H, J=6.8 Hz).

Step 7: Synthesis of N-((4, 6-dimethyl-2-oxo-l , 2-dihydropyridin-3-yl) methyl)-5- (ethyl (tetrahydro-2H-pyran-4-yl) amino)-4-methyl-4′-(morpholinomethyl)-[l , l ‘-biphenyl]-3- carboxamide

TITLE COMPD

To a stirred solution of 5-bromo-N-((4, 6-dimethyl-2-oxo-l , 2-dihydropyridin-3-yl) methyl)-3-(ethyl (tetrahydro-2H-pyran-4-yl) amino)-2-methylbenzamide (14 g, 29.5 mmol) in dioxane/ water mixture (70 mL/ 14 mL) was added 4-(4-(4, 4, 5, 5-tetramethyl- l , 3, 2- dioxaborolan-2-yl) benzyl) morpholine (13.4 g, 44.2 mmol) followed by addition of Na₂C0₃ (1 1 .2 g, 106.1 mmol). The solution was purged with argon for 15 minutes and then Pd (PPh₃)₄ (3.40 g, 2.94 mmol) was added and the solution was again purged with argon for a further 10 min. The reaction mixture was heated at 100°C for 4 h. After completion (monitored by TLC), the reaction mixture was diluted with water and extracted with 10% MeOH/DCM.

The combined organic layers were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (100- 200 mesh silica gel) eluting with methanol: DCM to the title compound as a solid (12 g, 71 %).

Analytical Data: LCMS: 573.35 (M + 1 )⁺; HPLC: 99.5% (@ 254 nm) (R,;3.999; Method: Column: YMC ODS-A 1 50 mm x 4.6 mm x 5 μ; Mobile Phase: A; 0.05% TFA in water/ B; 0.05% TFA in acetonitrile; Inj. Vol : 10 μΐ, Col. Temp.: 30 °C; Flow rate: 1 .4 mL/min.;

Gradient: 5% B to 95% B in 8 min, Hold for 1 .5 min, 9.51 -12 min 5% B);

Ή NMR (DMSO-i ₆, 400 MHz) 5 1 1 .46 (s, I H), 8. 19 (t, 1 H), 7.57 (d, 2H, J=7.2 Hz), 7.36-7.39 (m, 3H), 7.21 (s, I H), 5.85 (s, I H), 4.28 (d, 2H, J=2.8 Hz), 3.82 (d, 2H, J=9.6 Hz), 3.57 (bs, 4H), 3.48 (s, 2H), 3.24 (t, 2H, J=10.8Hz), 3.07-3.09 (m, 2H), 3.01 (m, I H), 2.36 (m, 4H), 2.24 (s, 3H), 2.20 (s, 3H), 2.10 (s, 3H), 1 .64-1 .67 (m, 2H), 1 .51 – 1 .53 (m, 2H), 0.83 (t, 3H, J=6.4 Hz).

TRIHYDROCHLORIDE

Step 8: Synthesis of N-((4,6-dimethyl-2-oxo-l ,2-dihydropyridin-3-yl)methyl)-5- (ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[ 1 , 1 ‘-biphenyl]-3- carboxamide trihydrochloride

N-((4, 6-dimethyl-2-oxo-l , 2-dihydropyridin-3-yl) methyl)-5-(ethyl (tetrahydro- 21 l-pyran-4-yl) amino)-4-methyI-4′-(niorpholinornethyl)-[ 1 , 1 ‘-biphenyl]-3-carboxamide ( 12 g, 21.0 mmol) was dissolved in methanolic HC1 (200 mL) and stirred at room temperature for 3 h. After three hours of stirring, the reaction mixture was concentrated under reduced pressure. The solid obtained was stirred with ether ( l OOmL X 2) to afford the desired salt as a solid ( 1 1 g, 77 %).

Analytical Data of the tri-HCl salt: LCMS: 573.40 (M + 1 )⁺; HPLC: 99.1 % (@ 254 nm) (R,;3.961 ; Method: Column: YMC ODS-A 150 mm x 4.6 mm x 5 μ; Mobile Phase: A; 0.05% TFA in water/ B; 0.05% TFA in acetonitrile; Inj. Vol: 10 pL, Col. Temp.: 30 °C; Flow rate: 1.4 mL/min.; Gradient: 5% B to 95% B in 8 min, Hold for 1.5 min, 9.51 -12 min 5% B);

Ή NMR (D₂0 400 MHz) δ 7.92 (bs, I H,) 7.80 (s, I H), 7.77 (d, 2H, J=8 Hz), 7.63 (s, I H), 7.61 (s, I H), 6.30 (s, I H), 4.48 (s, 2H), 4.42 (s, 2H), 4.09-4.1 1 (m, 4H), 3.95-3.97 (m, 2H), 3.77 (t, 3H, J=10.4 Hz), 3.44-3.47 (m, 3H), 3.24-3.32 (m, 3H), 2.42 (s, 3H), 2.35 (s, 3H), 2.26 (s, 3H), 2.01 (m, 2H), 1 .76 (m, 2H), 1 .04 (t, 3H, J=6.8 Hz).

…………………………………………

PATENT

WO2013155317

 http://www.google.com/patents/WO2013155317A1?cl=en

N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’- biphenyl] -3-carboxamide hydrobromide:

N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-5-(ethyl

(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’-biphenyl]-3- carboxamide hydrobromide:

As used herein, “Compound I” refers to N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)-5-(ethyl (tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’- biphenyl]-3-carboxamide. The hydrobromide of Compound I can be used to inhibit the histone methyltransferase activity of EZH2, either in a subject or in vitro. The hydrobromide of Compound I can also be used to treat cancer in a subject in need thereof.

Scheme 1

……………………………………..Compound I                                       Compound I – HBr

HPLC

HPLC was conducted on an Agilent 1200 HPLC quaternary pump, low pressure mixing, with an in-line degasser. Analytical method conditions: 8 μΐ_^ sample (20 mg of ER-581982-06 diluted with 50 mL of a methanol to provide approximately 0.4 mg/mL solution) was injected onto a Agilent Zorbax Eclipse XDB-C18 (4.6 x 150 mm, 3.5 um), Chromatography conditions: mobile phase A, water with 5mM ammonium formate; mobile phase B, 5 mM ammonium formate in 50/45/5 acetonitrile/methanol/water; flow rate, 1.5 ml/min.; gradient: isocratic at 10% B from 0 to 3 min; linear increase to 70% B from 3 to 7 min; isocratic at 70% B from 7 to 12 min; linear increase to 100% B from 12 to 15 min isocratic at 100% B from 15 to 20 min;

column temperature, 35 °C; detection, UV 230 nm. Approximate retention time of Compound I = 10.7 min.

Synthesis of Polymorph A

5-bromo-2-methyl-3-nitrobenzoic acid stirred solution of 2-methyl-3-nitrobenzoic acid (100 g, 552 mmol) in cone. H₂S0₄ (400 mL), l,3-dibromo-5,5-dimethyl-2,4- imidazolidinedione (88 g, 308 mmol) was added in a portion wise manner at room temperature and the reaction mixture was then stirred at room temperature for 5 h. The reaction mixture was poured onto ice cold water, the precipitated solid was filtered off, washed with water and dried under vacuum to afford the desired compound as a solid (140 g, 98%). The isolated compound was taken directly into the next step. 1H NMR (DMSO-J₆, 400 MHz) δ 8.31 (s, 1H), 8.17 (s, 1H), 2.43 (s, 3H).

Methyl 5-bromo-2-methyl-3-nitrobenzoate To a stirred solution of 5-bromo-2- methyl-3-nitrobenzoic acid (285 g, 1105 mmol) in DMF (2.8L) at room temperature was added sodium carbonate (468 g, 4415 mmol) followed by addition of methyl iodide (626.6 g, 4415 mmol). The resulting reaction mixture was heated at 60 °C for 8 h. After completion (monitored by TLC), the reaction mixture was filtered (to remove sodium carbonate) and washed with ethyl acetate (1L X 3). The combined filtrate was washed with water (3L X 5) and the aqueous phase was back extracted with ethyl acetate (1L X 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford the title compound as a solid (290g, 97% yield). The isolated compound was taken directly into the next step. 1H NMR (CDC1₃, 400 MHz) δ 8.17 (s, 1H), 7.91 (s, 1H), 3.96 (s, 3H), 2.59 (s, 3H).

Methyl 3-amino-5-bromo-2-methylbenzoate (1) To a stirred solution of methyl 5- bromo-2-methyl-3-nitrobenzoate (290 g, 1058 mmol) in ethanol (1.5L) was added aqueous ammonium chloride (283 g, 5290 mmol dissolved in 1.5L water). The resulting mixture was stirred at 80°C to which iron powder (472 g, 8451 mmol) was added in a portion wise manner. The resulting reaction mixture was heated at 80 °C for 12 h. Upon completion as determined by TLC, the reaction mixture was hot filtered over celite® and the celite bed was washed with methanol (5L) followed by washing with 30% MeOH in DCM (5L). The combined filtrate was concentrated in- vacuo, the residue obtained was diluted with aqueous sodium bicarbonate solution (2L) and extracted with ethyl acetate (5L X 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford the title compound as a solid (220 g, 85%). The compound was taken directly into the next step. 1H

NMR (CDCI₃, 400 MHz) δ 7.37 (s, 1H), 6.92 (s, 1H), 3.94 (s, 3H), 3.80 (bs, 2H), 2.31 (s, 3H).

Methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (2) A reactor was charged with methyl 3-amino-5-bromo-2-methylbenzoate (455.8 g, 1.87 mol), 1,2- Dichloroethane (4.56 L), and acetic acid (535 ml, 9.34 mol). To the mixture were added dihydro-2H-pyran-4(3H)-one (280 g, 2.80 mol) and sodium triacetoxyborohydride (594 g, 2.80 mol) maintaining the internal temperature below 40 °C. The mixture was stirred at 25 °C for 2.5 h and then the reaction was quenched with a solution of sodium hydroxide (448 g, 11.20 mol) in water (5.61 L). After stirring for 20 minutes at ambient temperature, the organic layer was separated and the aqueous layer was extracted with ethyl acetate (3.65 L). The organic layers were combined, washed with brine (1.5 L), and concentrated under vacuum.

The residue was treated with ethyl acetate (1.8 L) and heated to 65-70 °C. The mixture was stirred at 65-70 °C for 15 minutes to give a clear solution and then treated with n-heptane (7.3 L) maintaining the temperature between 60-70 °C. Once the heptane was completely added to the solution, the mixture was held at 65-70 °C for 15 minutes and then allowed to cool to 18- 22 °C over 3 h. The resulting suspension was stirred at 18-22 °C for 4 h, cooled to 0-5 °C over 1 h, and held at 0-5 °C for 2 h. The precipitate was filtered, washed twice with n-heptane (1.4 L), and dried under vacuum to give the title compound (540 g, 88%). The XRPD pattern of this compound is shown in Figure 17.

Methyl 5-bromo-3-(ethyl (tetrahydro-2H-pyran-4-yl) amino)-2-methylbenzoate (3)

To a stirred solution of methyl 5-bromo-2-methyl-3-((tetrahydro-2H-pyran-4-yl) amino) benzoate (14 g, 42.7 mmol) in dichloroethane (150 mL) was added acetaldehyde (3.75 g, 85.2 mmol) and acetic acid (15.3 g, 256 mmol). The resulting reaction mixture was stirred at room temperature for 15 minutes. The mixture was cooled to 0 °C and sodium triacetoxyborohydride (27 g, 128 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. Upon completion of the reaction as determined by TLC, aqueous sodium bicarbonate solution was added to the reaction mixture until a pH 7-8 was obtained, the organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (100-200 mesh silica gel) eluting with ethyl acetate: hexane to afford the desired compound as a viscous liquid (14 g, 93%). 1H NMR DMSO-d₆, 400 MHz) δ 7.62 (s, 1H), 7.52 (s, 1H), 3.80 (bs, 5H), 3.31 (t, 2H), 2.97-3.05 (m, 2H), 2.87-2.96 (m, 1H), 2.38 (s, 3H), 1.52-1.61 (m, 2H), 1.37-1.50 (m, 2H), 0.87 (t, 3H, J=6.8 Hz).

Methyl 5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-

[l,l’-biphenyl]-3-carboxylate (4): A mixture of methyl 5-bromo-3-(ethyl(tetrahydro-2H-pyran- 4-yl)amino)-2-methylbenzoate (580 g, 1.63 mol), 4-(4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan- 2-yl)benzyl)morpholine (592 g, 1.95 mol), 1,4-dioxane (3.86 L), sodium carbonate (618 g, 5.83 mol), and water (771 ml) was degassed by bubbling nitrogen through the mixture at 20 °C for 20 minutes and treated with tetrakis(triphenylphosphine)palladium(0) (14.11 g, 12.21 mmol). The resulting mixture was degassed for an additional 20 minutes and then heated to 87-89 °C for 17 h. After cooling to 20 °C, the mixture was diluted with ethyl acetate (5.80 L) and a solution of (R)-2-Amino-3-mercaptopropionic acid (232 g) in water (2.320 L). After stirring for 1 h at 20 °C, the organic layer was separated and washed again with a solution of (R)-2-Amino-3- mercaptopropionic acid (232 g) in water (2.320 L). The aqueous layers were combined and extracted with ethyl acetate (5.80 L). The organic layers were combined, washed with a solution of sodium hydroxide (93 g) in water (2.32 L), and concentrated under vacuum at 35 °C to give the title compound as an orange oil (1.21 kg, 164% yield).

5-(Ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’- biphenyl]-3-carboxylic acid (5): Methyl 5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′- (morpholinomethyl)-[l,l’-biphenyl]-3-carboxylate (69.0 g, 152.5 mmol) (based on the theoretical yield from the previous step) was suspended in ethanol (380 mL) and treated with a solution of sodium hydroxide (24.84 g, 621.0 mmol) in water (207 mL). The mixture was stirred at 40°C for 18 h. After cooling to 0-5 °C, the mixture was neutralized to pH 6.5 with 1 N hydrochloric acid (580 mL) maintaining the temperature below 25 °C. Then, the mixture was extracted twice with a mixture of dichloromethane (690 mL) and methanol (69.0 mL). The organic layers were combined and concentrated under vacuum to give a crude product as a yellow solid (127g).

The crude product was dissolved in 2-methyltetrahydrofuran (656 mL) at 70 °C and then treated with IPA (828 mL). The mixture was allowed to cool to rt over 3-4 h and then stirred overnight at rt. The precipitate was filtered, washed twice with IPA (207 mL), and dried under vacuum to give the title compound as an off white solid (53.54 g, 80%). The XRPD pattern of this compound is shown in Figure 9.

N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H- pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[l,l’-biphenyl]-3-carboxamide

(Compound I): A mixture of 5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′- (morpholinomethyl)-[l,l’-biphenyl]-3-carboxylic acid (540 g, 1.23 mol) and 3-(aminomethyl)- 4,6-dimethyl-dihydro-pyridin-2(lH)-one hydrochloride (279 g, 1.48 mol) was suspended in DMSO (2.70 L) and treated with triethylamine (223 ml, 1.60 mol). The mixture was stirred at 25 °C for 30 min and treated with EDC-HC1 (354 g, 1.85 mol) and HOBT hydrate (283 g, 1.85 mol). The reaction mixture was stirred at rt for 16 h. After addition of triethylamine (292 ml, 2.09 mol), the mixture was cooled to 15 °C, diluted with water (10.1 L) maintaining the temperature below 30 °C, and stirred at 19-25 °C for 4 h. The resulting precipitate was filtered, washed twice with water (2.70 L), and dried under vacuum to give a crude product (695 g, wt-wt analysis = 78%).

For the further purification of the product, recrystallization was conducted. A crude product (20.00 g, 34.92 mmol) was suspended in a mixture of ethanol (190 ml) and water (10.00 ml) and heated to 75°C until a clear solution was obtained. The solution was allowed to cool to rt overnight. The precipitate was filtered, washed twice with a mixture of ethanol (30.0 ml) and water (30.0 ml), and dried under vacuum at 35 °C to give the title compound as an off white solid (14.0 g, 70% recovery from the crude and 90% yield based on wt-wt assay).

4-((3′-(((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′- (ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[l,l’-biphenyl]-4-yl)methyl)morpholin- 4-ium bromide (Polymorph A): A crude N-((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)am

biphenyl]-3-carboxamide (595 g, 464 g based on wt-wt assay, 810.3 mmol) was suspended in ethanol (3.33 L). After heating to 70 °C, the mixture was treated with 48% aqueous HBr (97 ml, 850.8 mmol) and stirred at 70 °C for 30 min. The resulting orange-red solution was treated with ethyl acetate (3.33 L) maintaining the temperature above 60 °C. The mixture was slowly cooled to rt over 18 h. The mixture was cooled to 0 °C over 1 h and stirred at that temperature for 5.5 h. The resulting precipitate was filtered, washed twice with ethyl acetate (1.39 L), and dried under vacuum to give the title compound as an off white solid (515 g, 97% yield).

Recrystallization of Polymorph A: 4-((3′-(((4,6-dimethyl-2-oxo-l,2-dihydropyridin-3- yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′-methyl-[l,l’-biphenyl]-4- yl)methyl)morpholin-4-ium bromide (0.50 g, 0.77 mmol; 95.6% pure by HPLC) was suspended in ethanol (3.0 mL) and heated to 80 °C until a clear solution was obtained. To the solution was added MTBE (5.0 mL) slowly. The resulting solution was allowed to cool to 18-22 °C over 3 h and stirred at 18-22 °C for 15 h. The precipitate was filtered, washed twice with MTBE (2 mL) and dried under vacuum to give 0.45 g of the title compound (89% recovery, 96.6% pure by HPLC).

Compound I is protonated at the nitrogen of the morpholino substituent, providing a monohydrobromide of Compound I having the following structure:

This particular monohydrobromide can be referred to as “4-((3′-(((4,6-dimethyl-2-oxo- l,2-dihydropyridin-3-yl)methyl)carbamoyl)-5′-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4′- methyl-[l, -biphenyl]-4-yl)methyl)morpholin-4-ium bromide.” Figure 11 depicts the X-ray crystal structure of this particular salt form.

…………………………………………………………..

see

WO-2015057859

Eisai Research Institute; Epizyme Inc

Novel crystalline polymorphic form C of tazemetostat, useful for treating an EZH2-mediated cancer, including non-Hodgkin’s lymphoma and breast cancer.

…………………

PAPER

RSC Advances (2015), 5(33), 25967-25978

http://pubs.rsc.org/en/content/articlelanding/2015/ra/c5ra02365c#!divAbstract

DOI: 10.1039/C5RA02365C

The histone lysine methyltransferase EZH2 has been implicated as a key component in cancer aggressiveness, metastasis and poor prognosis. This study discovered a new class of hexahydroisoquinolin derivatives as EZH2 inhibitors. A structure–activity relationship study showed that the steric hindrance was important to the activity for EZH2. A preliminary optimization study led to the discovery of several potent compounds with low nanomolar to sub-nanomolar potency for EZH2. Biological evaluation indicated that SKLB1049 was a highly potent with improved solubility compared to EPZ6438, SAM-competitive, and cell-active EZH2 inhibitor that decreased global H3K27me3 in SU-DHL-6 and Pfeiffer lymphoma cells in a concentration- and time-dependent manner. Further study indicated that SKLB1049 caused cell arrest in G₀/G₁ phase. These compounds would be useful as chemical tools to further explore the biology of EZH2 and provided us with a start point to develop new EZH2 inhibitors.

1: Knutson SK, Warholic NM, Johnston LD, Klaus CR, Wigle TJ, Iwanowicz D, Littlefield BA, Porter-Scott M, Smith JJ, Moyer MP, Copeland RA, Pollock RM, Kuntz KW, Raimondi A, Keilhack H. Synergistic Anti-Tumor Activity of EZH2 Inhibitors and Glucocorticoid Receptor Agonists in Models of Germinal Center Non-Hodgkin Lymphomas. PLoS One. 2014 Dec 10;9(12):e111840. doi: 10.1371/journal.pone.0111840. eCollection 2014. PubMed PMID: 25493630; PubMed  Central PMCID: PMC4262195.

2: Knutson SK, Kawano S, Minoshima Y, Warholic NM, Huang KC, Xiao Y, Kadowaki T,  Uesugi M, Kuznetsov G, Kumar N, Wigle TJ, Klaus CR, Allain CJ, Raimondi A, Waters NJ, Smith JJ, Porter-Scott M, Chesworth R, Moyer MP, Copeland RA, Richon VM, Uenaka T, Pollock RM, Kuntz KW, Yokoi A, Keilhack H. Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma. Mol Cancer Ther. 2014 Apr;13(4):842-54. doi: 10.1158/1535-7163.MCT-13-0773. Epub 2014 Feb 21. PubMed PMID: 24563539

3. Inhibitors of human histone methyltransferase EZH2, and methods of use thereof for treating cancer. By Kuntz, Kevin W.; Knutson, Sarah K.; Wigle, Timothy James Nelson . From U.S. Pat. Appl. Publ. (2013), US 20130040906 A1 20130214.

4. Aryl-or heteroaryl-substituted benzamide compounds as anticancer agents and their preparation By Kuntz, Kevin Wayne; Chesworth, Richard; Duncan, Kenneth William; Keilhack, Heike; Warholic, Natalie; Klaus, Christine; Zheng, Wanjun; Seki, Masashi; Shirotori, Syuji; Kawano, Satoshi From PCT Int. Appl. (2012), WO 2012142504 A1 20121018.

5: Knutson SK, Warholic NM, Wigle TJ, Klaus CR, Allain CJ, Raimondi A, Porter Scott M, Chesworth R, Moyer MP, Copeland RA, Richon VM, Pollock RM, Kuntz KW, Keilhack H. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2. Proc Natl Acad Sci U S A. 2013 May 7;110(19):7922-7. doi: 10.1073/pnas.1303800110. Epub 2013 Apr 25. PubMed PMID: 23620515; PubMed Central PMCID: PMC3651445.

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Jason Rhodes (left) has been appointed to president of Epizyme Inc.,

100 Technology Square

Evofosfamide, HAP-302 , TH-302

Evofosfamide

Names
IUPAC name (1-Methyl-2-nitro-1H-imidazol-5-yl)methyl N,N’-bis(2-bromoethyl)phosphorodiamidate
Other names TH-302; HAP-302
Identifiers
CAS Registry Number	918633-87-1
ChemSpider	10157061
InChI[show]
Jmol-3D images	Image
PubChem	11984561
SMILES[show]
Properties
Molecular formula	C9H16Br2N5O4P
Molar mass	449.04 g·mol−1
Solubility in water	6 to 7 g/l

TH-302 is a nitroimidazole-linked prodrug of a brominated derivative of an isophosphoramide mustard previously used in cancer drugs

evofosfamide (first disclosed in WO2007002931), useful for treating cancer.

Threshold Pharmaceuticals and licensee Merck Serono are codeveloping evofosfamide, the lead in a series of topoisomerase II-inhibiting hypoxia-activated prodrugs and a 2-nitroimidazole-triggered bromo analog of ifosfamide, for treating cancer, primarily soft tissue sarcoma and pancreatic cancer (phase 3 clinical, as of April 2015).

In November 2014, the FDA granted Fast Track designation to the drug for the treatment of previously untreated patients with metastatic or locally advanced unresectable soft tissue sarcoma.

Evofosfamide (INN,^[1] USAN;^[2] formerly known as TH-302) is an investigational hypoxia-activated prodrug that is in clinical development for cancer treatment. The prodrug is activated only at very low levels of oxygen (hypoxia). Such levels are common in human solid tumors, a phenomenon known as tumor hypoxia.^[3]

Evofosfamide is being evaluated in clinical trials for the treatment of multiple tumor types as a monotherapy and in combination with chemotherapeutic agents and other targeted cancer drugs
Discovered at Threshold, TH-302 is a hypoxia-activated prodrug (HAP) designed to exploit low oxygen levels in hypoxic tumor regions. Therapeutics that specifically target resistant hypoxic zones could provide significant additional antitumor activity and clinical benefit over current chemotherapeutic and radiation therapies.

Evofosfamide (TH-302) was developed by Threshold Pharmaceuticals Inc. (Threshold).^[4] The company is located in South San Francisco, CA, USA.

In 2012, Threshold signed a global license and co-development agreement for evofosfamide with Merck KGaA, Darmstadt, Germany, which includes an option for Threshold to co-commercialize eofosfamide in the United States. Threshold is responsible for the development of evofosfamide in the soft tissue sarcoma indication in the United States. In all other cancer indications, Threshold and Merck KGaA are developing evofosfamide together.^[5] From 2012 to 2013, Merck KGaA paid 110 million US$ for upfront payment and milestone payments to Threshold. Additionally, Merck KGaA covers 70% of all evofosfamide development expenses.^[6]
Discovered at Threshold, TH-302 is a hypoxia-activated prodrug (HAP) designed to exploit low oxygen levels in hypoxic tumor regions. Therapeutics that specifically target resistant hypoxic zones could provide significant additional antitumor activity and clinical benefit over current chemotherapeutic and radiation therapies.

History

SEE

WO2007002931

http://www.google.com/patents/WO2007002931A2?cl=en

Example 8

Synthesis of Compounds 25, 26 [0380] To a solution of 2-bromoethylammmonium bromide (19.4 g) in DCM (90 mL) at – 1O⁰C was added a solution OfPOCl₃ (2.3 mL) in DCM (4 mL) followed by addition of a solution of TEA (14.1 mL) in DCM (25 mL). The reaction mixture was filtered, the filtrate concentrated to ca. 30% of the original volume and filtered. The residue was washed with DCM (3×25 mL) and the combined DCM portions concentrated to yield a solid to which a mixture of THF (6 mL) and water (8 mL) was added. THF was removed in a rotary evaporator, the resulting solution chilled overnight in a fridge. The precipitate obtained was filtered, washed with water (10 mL) and ether (30 mL), and dryed in vacuo to yield 2.1 g of:

Isophosphoramide mustard

can be synthesized employing the method provided in Example 8, substituting 2- bromoethylammmonium bromide with 2-chloroethylammmonium chloride. Synthesis of Isophosphoramide mustard has been described (see for example Wiessler et al., supra).

The phosphoramidate alkylator toxin:

was transformed into compounds 24 and 25, employing the method provided in Example 6 and the appropriate Trigger-OH.

Example 25

Synthesis of l-N-methyl-2-nitroimidazole-5-carboxylis acid

A suspension of the nitro ester (39.2 g, 196.9 rnmol) in IN NaOH (600 mL) and water (200 mL) was stirred at rt for about 20 h to give a clear light brown solution. The pH of the reaction mixture was adjusted to about 1 by addition of cone. HCl and the reaction mixture extracted with EA (5 x 150 mL). The combined ethyl acetate layers were dried over MgS O4 and concentrated to yield l-N-methyl-2-nitroimidazole-5-carboxylis acid (“nitro acid”) as a light brown solid (32.2 g, 95%). Example 26

Synthesis of l-N-methyl-2-nitroimidazole-5-carboxylis acid

A mixture of the nitro acid (30.82 g, 180.23 mmol) and triethylamine (140 niL, 285 mmol) in anhydrous THF (360 mL) was stirred while the reaction mixture was cooled in a dry ice-acetonitrile bath (temperature < -20 ⁰C). Isobutyl chloroformate (37.8 mL, 288 mmol) was added drop wise to this cooled reaction mixture during a period of 10 min and stirred for 1 h followed by the addition of sodium borohydride (36 g, 947 mmol) and dropwise addition of water during a period of 1 h while maintaining a temperature around or less than O⁰C. The reaction mixture was warmed up to O⁰C. The solid was filtered off and washed with THF. The combined THF portions were evaporated to yield l-N-methyl-2- nitroimidazole-5-methanol as an orange solid (25 g) which was recrystallized from ethyl acetate.

……………………………………….

WO-2015051921

EXAMPLE 1

1

N-Formylsarcosine ethyl ester 1 (1 ,85 kg) was dissolved in toluene (3,9 kg) and ethyl formate (3,28 kg) and cooled to 10 °C. A 20 wt-% solution of potassium tert-butoxide (1 ,84 kg) in tetrahydrofuran (7,4 kg) was added and stirring was continued for 3h. The reaction mixture was extracted 2x with a solution of sodium chloride in water (10 wt-%) and the combined water extracts were washed lx with toluene.

Aqueous hydrogen chloride (25% wt-%; 5,62 kg) was added to the aqueous solution, followed by ethylene glycol (2,36 kg). The reaction mixture was heated to 55-60 °C for lh before only the organic solvent residues were distilled off under vacuum.

Aqueous Cyanamide (50 wt-%, 2,16 kg) was then added at 20 °C, followed by sodium acetate (3,04 kg). The resulting reaction mixture was heated to 85-90 °C for 2h and cooled to 0-5 °C before a pH of ~ 8-9 was adjusted via addition of aqueous sodium hydroxide (32% wt-%; 4,1 kg). Compound 3 (1,66 kg; 75%) was isolated after filtration and washing with water.

Ή-NMR (400 MHz, d6-DMSO): δ= 1,24 (3H, t, J= 7,1 Hz); 3,53 (3H, s); 4,16 (2H, q, J= 7,0 Hz) ; 6,15 (s, 2 H); 7,28 (s, 1H).

HPLC (R_t = 7,7 min): 97,9% (a/a).

REFERENCES

1

• WHO Drug Information; Recommended INN: List 73
• 2
• Adopted Names of the United States Adopted Names Council
• 3
• Duan J; Jiao, H; Kaizerman, J; Stanton, T; Evans, JW; Lan, L; Lorente, G; Banica, M et al. (2008). “Potent and Highly Selective Hypoxia-Activated Achiral Phosphoramidate Mustards as Anticancer Drugs”. J. Med. Chem. 51 (8): 2412–20. doi:10.1021/jm701028q. PMID 18257544.
• 4
• Website of Threshold Pharmaceuticals Inc.
• 5
• Threshold Pharmaceuticals and Merck KGaA Announce Global Agreement to Co-Develop and Commercialize Phase 3 Hypoxia-Targeted Drug TH-302 – Press release from 3 February 2012
• 6

Threshold Pharmaceuticals Form 8-K from 3 Nov 2014

DHAKA BANGLADESH

.

Steamers and ferries in Sadarghat Port

Kawran Bazar

.

Dry fish sellers at the Karwan Dry Fish Market (Bazar), Dhaka, Bangladesh.

4-[4-[2-(difluoromethyl)benzimidazol-1-yl]-6-morpholin-4-yl-1,3,5-triazin-2-yl]morpholine

ZSTK474; 475110-96-4; 4,4′-(6-(2-(Difluoromethyl)-1H-benzo[d]imidazol-1-yl)-1,3,5-triazine-2,4-diyl)dimorpholine; ZSTK-474; ZSTK 474; TCMDC-137004;

2-(2-Difluoromethylbenzimidazol-1-yl)-4,6-bis(morpholino)-1,3,5-triazine

2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-triazine

Zenyaku Kogyo (Innovator)

phase2………Treatment of Solid Tumors Therapy

ZSTK474 is a cell permeable and reversible P13K inhibitor with an IC₅₀ at 6nm. It was identified as part of a screening library, selected for its ability to block tumor cell growth. ZSTK474 has shown strong antitumor activities against human cancer xenographs when administered orally to mice without a significant toxic effect.

Phosphatidylinositol 3-kinase (PI3K) has been implicated in a variety of diseases including cancer. A number of PI3K inhibitors have recently been developed for use in cancer therapy. ZSTK474 is a highly promising antitumor agent targeting PI3K. We previously reported that ZSTK474 showed potent inhibition against four class I PI3K isoforms but not against 140 protein kinases.

However, whether ZSTK474 inhibits DNA-dependent protein kinase (DNA-PK), which is structurally similar to PI3K, remains unknown. To investigate the inhibition of DNA-PK, we developed a new DNA-PK assay method using Kinase-Glo. The inhibition activity of ZSTK474 against DNA-PK was determined, and shown to be far weaker compared with that observed against PI3K. The inhibition selectivity of ZSTK474 for PI3K over DNA-PK was significantly higher than other PI3K inhibitors, namely NVP-BEZ235, PI-103 and LY294002.

Other Names: ZSTK-474

Chemical Formula:  C₁₉H₂₁F₂N₇O₂

CAS Number: 475110-96-4

Molecular Weight: 417.41

WO 2002088112

http://www.google.co.in/patents/EP1389617A1?cl=en

The condensation of 2,4-dichloro-6-(4-morpholinyl)-1,3,5-triazine

with 2-(difluoromethyl)-1H-benzimidazole  by means of K2CO3 in DMF gives

2-chloro-4-[2-(difluoromethyl)-1H-benzimidazol-1-yl]-6-(4-morpholinyl)-1,3,5-triazine ,

which is then condensed with morpholine by means of K2CO3 in DMF to afford the target trisubstituted triazine.

^aReagents and conditions: (i) K₂CO₃, DMF, room temp; (ii) morpholine, DMF or THF, room temp; (iii) NaH or K₂CO₃, DMF or DMSO, 120 °C.

• 2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-triazine(compound 19)
  Melting point: 211-214°C
  NMR(CDCl₃) δ : 3.79(8H, t, J=4Hz), 3.88(8H, t, J=4Hz), 7.3-7.4(2H, m), 7.56(1H, t, J=53Hz), 7.88(1H, d, J=7Hz), 8.32(1H, d, J=7Hz)
  MS m/z: 417(M⁺

……………………

TRIAZINE, PYRIMIDINE AND PYRIDINE ANALOGS AND THEIR USE AS THERAPEUTIC AGENTS AND DIAGNOSTIC PROBES [US2011275762]2011-11-10

…………..

Zenyaku Kogyo

Otsuka

Bunkyo

BEZ235 (NVP-BEZ235)Dactolisib

4-​[2,​3-​dihydro-​3-​methyl-​2-​oxo-​8-​(3-​quinolinyl)-​1H-​imidazo[4, ​5-​c]quinolin-​1-​yl]-​α,​α-​dimethyl-​benzeneacetonitrile

2-methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-1H,2H,3H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile

2-Methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)- phenyl]-propionitrile

Chemical Formula:  C₃₀H₂₃N₅O

CAS Number: 915019-65-7

Molecular Weight: 469.54

PHASE 2, NOVARTIS

CANCER, BLADDER

NVP-BEZ235 is a dual inhibitor of phosphatidylinositol 3-kinase (P13K)and the downstream mammalian target of rapamycin (mTOR) by binding to the ATP-binding cleft of these enzymes. It specifically blocks the dysfunctional activation of the P13K pathway and induce G(1) arrest. NPV-BEZ235 has been shown to inhibit VEGF induced cell proliferation and survival in vitro and VEGF induced angiogenesis in vivo. It has also been shown to inhibit the growth of human cancer in animal models.

BEZ-235 is an orally active phosphatidylinositol 3-kinase (PI3K) inhibitor in early clinical trials at Novartis for the treatment of advanced breast cancer, renal cell carcinoma, solid tumors and castration-resistant prostate cancer. Phase I clinical trials were also under way at the company for the treatment of glioma, however, no developments in this indication has been reported. Phase II clinical trials are ongoing at Johann Wolfgang Goethe Universität for the treatment of relapsed or refractory acute leukemia.
PI3Ks perform various functions, promoting cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. Mutations leading to increased activity of PI3Ks, including faulty production or action of PI3K antagonists, have been found in many cancers.

……………………………..

WO 2006122806

http://www.google.com/patents/WO2006122806A2?cl=en

…………………………..

WO 2008064093

2-methyl-2-[4-(3-methyl- 2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrile of formula I (compound I),

Example 1

2-Methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)- phenyl]-propionitrile

In a suitable lab glass reactor are placed 45.0 g of starting 2[4-(8-bromo-3-methyl-2-oxo-2,3- dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]2-methyl-propionitrile together with 2.25 g of bistriphenylphosphine’palladium dichloride in 445 ml N,N-dimethylformamide. This mixture is heated to 95 ⁰C and then a solution of 22.2 g of 3-quinoline boronic acid in a mixture of 225 ml DMF, 300 ml H₂O and 60 g of KHCO₃ is added. This mixture is heated for 2 h at 95 ⁰C. Then 1080 ml H₂O are added. The product 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl- 2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]propionitrile precipitates. The mixture is cooled within 1.5 h to 0 – 5 °C. After stirring at that temperature for 2 h the crude product is filtered and washed with 300 ml H₂O. This product is dried in vacuo at 60 ⁰C for 18 h, to yield crude product.

40 g of this crude product is dissolved in 200 ml formic acid at 60 ⁰C. 8 g of active charcoal and Smopex 234 are added. The mixture is stirred at 60 ⁰C for 1 h, the charcoal is filtered, the residue washed with 80 ml formic acid and then 175 ml formic acid are distilled off in vacuo. Then 320 ml methanol are added and the mixture is heated at reflux for 3 h. The purified product precipitates from the reaction mixture. The mixture is cooled to 0 – 5 ⁰C within 1 h, then stirred 2 h at that temperature is finally filtered and washed with 80 ml cold methanol. This recrystallisation procedure is repeated again. Finally the twice recrystallised material is dried in vacuo at 60 ⁰C to yield purified 2-Methyl-2-[4-(3-methyl-2-oxo-8-quinolin- 3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]propionitrile.

Example 1a 5-Bromo-2-(2-nitro-vinylamino)-benzoic acid

A suspension of 25 g (16 mmol) of 2-amino-5-bromo-benzoic acid (Fluka, Buchs, Switzerland) in H₂O-HCI (37%) (10:1) is stirred for 8 h and then filtered (solution A). 8.17 g (255 mmol) of nitromethane (Fluka, Buchs, Switzerland) are added over 10 min to an ice- bath cooled mixture of 35 g of ice and 15.3 g (382 mmol) of NaOH. After stirring for 1 h at 0 ⁰C and 1 h at rt, the solution is added at 0 ⁰C to 28 g of ice and 42 ml of HCI (37%) (solution B). Solutions A and B are combined and the reaction mixture is stirred for 18 h at rt. The yellow precipitate is filtered off, washed with H₂O and dried in vacuo at 40⁰C to give the title compound. ES-MS: 287, 289 (M + H)⁺, Br pattern; ¹H NMR (DMSO-d₆): δ 13.7-14.6/br s (1 H), 12.94/d (1 H), 8.07/d (1 H), 8.03/dd (1 H), 7.83/dd (1 H), 7.71/d (1 H), 6.76/d (1 H).

Example 1b 6-Bromo-3-nitro-quinolin-4-ol

29 g (101 mmol) of 5-bromo-2-(2-nitro-vinylamino)-benzoic acid (Example 1a) and 11.9 g (121 mmol) of potassium acetate in 129 ml (152 mmol) of acetic anhydride are stirred for 1.5 h at 120 ⁰C. The precipitate is filtered off and washed with acetic acid until the filtrate is colorless, then is washed with H₂O and dried in vacuo to give the title compound. ES-MS: 269, 271 (M + H)⁺, Br pattern; analytical HPLC: W= 2.70 min (Grad 1).

Example 1c 6-Bromo-4-chloro-3-nitro-quinoline

20 g (74.3 mmol) of 6-bromo-3-nitro-quinolin-4-ol (Example 1b) in 150 ml (1.63 mol) of POCI₃ are stirred for 45 min at 120 °C. The mixture is cooled to rt and poured slowly into ice- water. The precipitate is filtered off, washed with ice-cold water, and dissolved in CH₂CI₂. The organic phase is washed with cold brine, and the aqueous phase is discarded. After drying over MgSO₄, the organic solvent is evaporated to dryness to provide the title compound. ¹H NMR (CDCI₃): J9.20/S (1H), 8.54/d (1H), 8.04/d (1H), 7.96/dd (1H); analytical HPLC: W= 4.32 min (Grad 1).

Example 1d 2-Methyl-2-(4-nitro-phenyl)-propionitrile

O .

To 15 g (92.5 mmol) of (4-nitro-phenyl)-acetonitrile (Fluka, Buchs, Switzerland), 1.64 mg (5.09 mmol) of tetrabutylammonium bromide (Fluka, Buchs, Switzerland) and 43.3 g (305 mmol) of iodomethane in 125 mL of CH₂CI₂ are added 1O g (250 mmol) of NaOH in 125 ml of water. The reaction mixture is stirred for 20 h at RT. After this time, the organic layer is separated, dried over MgSO₄, and evaporated to dryness. The residue is dissolved in diethylether and treated with black charcoal for 30 min, filtered over Celite and evaporated in vacuo to give the title compound as a pale yellow solid. Analytical HPLC: t_ret= 3.60 minutes (Grad 1).Example 1e (2-(4-Amino-phenyl)-2-methyl-propionitrile

16 g (84.1 mmol) of 2-methyl-2-(4-nitro-phenyl)-propionitrile (Example 1d) and 4.16 g of Raney-Ni are shacked in 160 ml of THF-MeOH (1:1) under 1.1 bar of H₂ for 12 h at rt. After completion of the reaction, the catalyst is filtered-off and the filtrate is evaporated to dryness. The residue is purified by flash chromatography on silica gel (hexane-EtOAc 3:1 to 1:2) to provide the title compound as an oil. ES-MS: 161 (M + H)⁺; analytical HPLC: t_ret= 2.13 minutes (Grad 1).

Example 1f 2-[4-(6-Bromo-3-nitro-quinolin-4-ylamino)-phenyl]-2-methyl-propionitrile

18 g (62.6 mmol) of 6-bromo-4-chloro-3-nitro-quinoline (Example 1c) and 11 g (68.9 mmol) of (2-(4-amino-phenyl)-2-methyl-propionitrile (Example 1e) are dissolved in 350 ml of acetic acid and stirred for 2 h. After this time, water is added and the yellow precipitate is filtered off and washed with H₂O. The solid is dissolved in EtOAc-THF (1 :1), washed with sat. aqueous NaHCO₃ and dried over MgSO₄. The organic phase is evaporated to dryness to give the title compound as a yellow solid. ES-MS: 411 , 413 (M + H)⁺, Br pattern; analytical HPLC: t_ret= 3.69 min (Grad 1).

Example 1q 2-[4-(3-Amino-6-bromo-quinolin-4-ylamino)-phenyl]-2-methyl-propionitrile

24 g (58.4 mmol) of 2-[4-(6-bromo-3-nitro-quinolin-4-ylamino)-phenyl]-2-methyl-propionitrile (Example 1e) is shacked in 300 ml of MeOH-THF (1:1) under 1.1 bar of H₂ in the presence of 8.35 g of Raney-Ni for 1 h. After completion of the reaction, the catalyst is filtered off and the filtrate is evaporated to dryness to give the title compound as a yellow foam. ES-MS: 381 , 383 (M + H)⁺, Br pattern; analytical HPLC: W= 3.21 min (Grad 1).

Example 1h

2-[4-(8-Bromo-2-oxo-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-2-methyl- propionitrile

A solution of 5 g (13.1 mmol) of 2-[4-(3-amino-6-bromo-quinolin-4-ylamino)-phenyl]-2- methyl-propionitrile (Example 1g) and 1.59 g (15.7 mmol) of triethylamine in 120 ml CH₂CI₂ is added over 40 min to a solution of 2.85 g (14.4 mmol) of trichloromethyl chloroformate (Fluka, Buchs, Switzerland) in 80 ml of CH₂CI₂ at 0⁰C with an ice-bath. The reaction mixture is stirred for 20 min at this temperature then is quenched with sat. aqueous NaHCO₃, stirred for 5 min and extracted with CH₂CI₂. The organic layer is dried over Na₂SO₄, filtered and evaporated in vacuo to give crude title compound as a brownish solid. ES-MS: 407, 409 (M + H)⁺, Br pattern; analytical HPLC: t_ret= 3.05 min (Grad 1). Example 1i

2-[4-(8-Bromo-3-methyl-2-oxo-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-2- methyl-propionitrile

To a solution of 3.45 g (8.47 mmol) of 2-[4-(8-bromo-2-oxo-2,3-dihydro-imidazo[4,5- c]quinolin-1-yl)-phenyl]-2-methyl-propionitrile (Example 1h), 1.8 g (12.7 mmol) of iodomethane (Fluka, Buchs, Switzerland) and 273 mg (0.847 mmol) of tetrabutylammonium bromide (Fluka, Buchs, Switzerland) in 170 ml of CH₂CI₂ is added a solution of 508 mg (12.7 mmol) of NaOH (Fluka, Buchs, Switzerland) in 85 ml of H₂O. The reaction mixture is stirred for 2 days and 900 mg (6.35 mmol) of iodomethane and 254 mg (6.35 mmol) of NaOH in 5 ml of H₂O are added. The reaction mixture is stirred for 1 day at rt . After this time, the reaction is quenched with H₂O and extracted with CH₂CI₂ (2*). The organic layer is washed with brine, dried over Na₂SO₄, filtered and evaporated in vacuo to give the title compound as a beige solid. ES-MS: 421 , 423 (M + H)⁺, Br pattern; analytical HPLC: t_ret= 3.15 min (Grad 1).

Example 2

2-Methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)- phenyl]propionitrile p-toluenesulfonate salt

26.5 g of 2-Methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1- yl)-phenyl]propionitrile are placed together with 55 ml formic acid into a glass reactor. This mixture is heated to 60 ⁰C to get a clear solution. This solution is clearfiltered and washed with 36 ml formic acid. Then formic acid is distilled off until the volume of the residual solution is 55 ml. Then a solution of 11.3 g of p-toluenesulfonic acid in 228 ml acetone is added at 50 ⁰C, followed by further addition of 822 ml acetone within 30 minutes. The salt precipitates from the reaction mixture. The mixture is cooled to 0 ⁰C within 2 h, stirred at that temperature for 3 h, is then filtered and washed with 84 ml acetone. The product^‘ is dried at 60 ⁰C in vacuo for 18 h to yield 29.8 g (82.4 %) of the 2-Methyl-2-[4-(3-methyl-2-oxo-8- quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]propionitrile p-toluenesulfonate salt (crystalline form A). The crystalline forms of the present invention are synthesized in accordance with the following examples which are illustrative without limiting the scope of the present invention.

Example 3:

Preparation of form A of 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro- imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrile

Form A of compound I can be manufactured in the following way: 241 g of free base are dissolved 2.4 I acetic acid at 50 ⁰C. The solution is clearfiltered, washed with 250 ml acetic acid and then at 50 ⁰C 7.2 I of water are added. The free base starts precipitating. The mixture is cooled within 1 h to 25 ⁰C, is then filtered and washed with 10 I H₂O. The free base is then dried in vacuo at 50 ⁰C over night to yield 204 g of free base.

References

Magnolia virginiana

Honokiol, from magnolia bark, shuts down cancer cells in lab

Compound in magnolia may combat head and neck cancers

Honokiol, from magnolia bark, shuts down cancer cells in lab

Magnolias are prized for their large, colorful, fragrant flowers. Does the attractive, showy tree also harbor a potent cancer fighter?

Yes, according to a growing number of studies, including one from VA and the University of Alabama at Birmingham that is now online in the journal Oncotarget.

The study focused on squamous cell head and neck cancers, a scourge among those who use tobacco and alcohol. According to the National Cancer Institute, at least 3 in 4 head and neck cancers are caused by the use of tobacco and alcohol. The cancers have only a 50 percent survival rate, killing some 20,000 Americans each year.

Enter honokiol–chemical formula C18H18O2. As one of the major active compounds in magnolia extract, the phytochemical has been used for centuries in traditional Chinese and Japanese medicine to treat anxiety and other conditions. More recently, scientists have been discovering that the compound, found in magnolia bark, is a wily and versatile adversary of cancer. It seems to exploit many biochemical pathways to shrink tumors of various types, or to keep them from growing in the first place.

The Alabama scientists have now shown how it works against head and neck cancers: It blocks a protein called epidermal growth factor receptor, or EGFR. Prior research has found that almost all head and neck cancer cells display an over-abundance of the protein, and it had been suggested in the literature as a potential target.

The VA-UAB team says, based on its lab studies, that honokiol binds more strongly with EGFR than does the drug gefitinib (sold as Iressa), which is commonly used to treat head and neck cancers.

The researchers tested honokiol on cell lines derived from human cancers of the oral cavity, larynx, tongue, and pharynx. In all cases, the botanical shut down the aberrant cells. The team also tested it against tumors implanted into mice, with similar results.

Senior author Dr. Santosh K. Katiyar and his colleagues wrote, “Conclusively, honokiol appears to be an attractive bioactive small molecule phytochemical for the management of head and neck cancer which can be used either alone or in combination with other available therapeutic drugs.”

Katiyar has published extensively in the past on other natural substances that work against tumors, especially skin cancer. Some of his recent work has focused on compounds in green tea, for example, and grape seed proanthocyanidins.

Purification

There are several methods for purifying and isolating honokiol. In nature, honokiol exists with its structural isomer magnolol, which differs from honokiol only by the position of onehydroxyl group. Because of the very similar properties of magnolol and honokiol, purification has often been limited to a HPLC or electromigration. However, methods developed in 2006 by workers in the lab of Jack L. Arbiser, took advantage of the proximity of the phenolic hydroxyl groups in magnolol, which form a protectable diol, to generate amagnolol acetonide (Figure 1), with a subsequent simple purification via flash chromatography over silica.^[4]

Figure 1

Magnolol and Honokiol are normally inseparable. Honokiol is easily separable from the protected magnolol acetonide

Additionally a rapid separation approach was published in the Journal of Chromatography A in 2007. The process uses high-capacity high-speed countercurrent chromatography(high-capacity HSCCC).^[5] Through this method honokiol can be separated and purified to above 98% purity with a high yield in under an hour.

Honokiol is a lignan isolated from the bark, seed cones, and leaves of trees belonging to the genus Magnolia. It has been identified as one the chemical compounds in some traditional eastern herbal medicines along with magnolol, 4-O-methylhonokiol, andobovatol.

Traditional medicine

Seed Cone

Extracts from the bark or seed cones of the Magnolia tree have been widely used in traditional medicine in China, Korea, and Japan.^[2]

Houpu has traditionally been used in Eastern medicine as analgesic and to treat anxiety and mood disorders.^[2][6] However, it has been shown to treat a number of other conditions. In China, magnolia bark is called Houpu and is most commonly taken from the Magnolia obovata and the Magnolia officinalis species.^[7] Some Chinese traditional formulas containing Houpu include Banxia Houpu Tang (半夏厚朴丸), Xiao Zhengai Tang, Ping Wei San(平胃散) and Shenmi Tang.^[2] Japanese Kampo formulas include, Hange-koboku-to (半夏厚朴湯) and Sai-boku-to (柴朴湯).^[2][6]

Seeds

Modern medicine

In the late 1990s, honokiol saw a revival in interest as a potent and highly tolerable antitumorigenic and neurotrophiccompound.

Alternative medicine

Currently there are a large number of supplements containing honokiol on the market, and its use has been widely well received among practitioners of new age, homeopathic, and holistic medicine

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Mature Magnolia fruit just starting to open, with a few seeds visible

Honokiol

Names
IUPAC name 2-(4-hydroxy-3-prop-2-enyl-phenyl)- 4-prop-2-enyl-phenol
Other names houpa, hnk
Identifiers
CAS Registry Number	35354-74-6
ChEMBL	ChEMBL16901
ChemSpider	65254
InChI[show]
Jmol-3D images	Image
KEGG	C10630
PubChem	72303
SMILES[show]
Properties
Chemical formula	C18H18O2
Molar mass	266.334 g/mol
Appearance	White solid
Solubility in water	sparingly (25 °C)
Related compounds
Related biphenols	diethylstilbestrol, dihydroxyeugenol
Related compounds	magnolol. 4-O-Methylhonokiol
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).

Magnolia seeds and fruit on a tree in northern Argentina

Magnolia grandiflora

http://www.google.com/patents/WO2006107451A2?cl=en

The root and stem bark of Magnolia has been used as a traditional Chinese medicine for the treatment of thrombotic stroke, gastrointestinal complaints, and anxiety. Honokiol (HNK), a substituted biphenyl and an active component isolated and purified from Magnolia, has anti-oxidant, antithrombosis, antibacterial, neurotrophic, xanthine oxidase inhibitory, and anxiolytic effects (Taira et al., Free Radic Res Commun. 1993;19 Suppl l:S71-77; Teng et al. Thromb Res. 1988;50:757-765; Clark et al., J. Pharm. Sci. 1981;70:951-952; Chang et al., Anticancer Res. 1994;14:501-506; Kuribara et al., J. Pharm Pharmacol. 1998;50:819-826; Esumi et al., Bioorg & Medicinal Chem Let 2004, 14: 2621-25).

In the early 1990s, reports of HNK’s anticancer effects were published. In 1994, Hirano et al (Life Sci. 1994;55(13): 1061-9) examined the anti leukemic-cell efficacy of 28 naturally occurring and synthetic flavonoids and 11 naturally occurring ligands on human promyelocytic leukemic cell line HL-60, and cytotoxicity of these compounds was compared with four clinical anti-cancer agents. HNK was identified as one of the most potent compounds in this screen, with an IC₅₀ value less than 100 ng/ml. In 1998, Hibasami et al. demonstrated that HNK induced apoptosis in human lymphoid leukemia Molt 4B cells (Hibasami et al., Int. J. MoI. Med. 1998).

HNK has also been found to induce apoptosis in human squamous cell lung cancer CH27 cells (Yang SE, et al Biochem Pharmacol. 2002;63:1641-1651) and in human colorectal RKO cells (Wang et al World J Gastroenterol. 2004; 10:2205-2208). In 2004, Chen et al. (World J Gastroenterol. 2004; 10: 3459-3463) reported that HNK was effective in an in vivo animal model of human colon cancer by inhibiting tumor growth and prolonging the lifespan of tumor bearing mice.

Honokiol is an inhibitor of angiogenesis and antitumor activity in vivo. HNK can cause apoptosis in tumor cells and inhibit angiogenesis through blocking phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), the major mitogenic and chemoattractant endothelial growth factor (Bai et al. (2003) J. Biol. Chem. 278, 35501- 35507). Honokiol also exhibits direct antitumor activity through induction of apoptosis through tumor necrosis factor apoptosis-inducing ligand (TRAIL/ Apo2L) signaling and has been found to be highly effective against angiosarcoma in nude mice in vivo (Bai et al. (2003) J. Biol. Chem. 278, 35501-35507).

Esumi et al. (Biorganic & Medicinal Chemistry Letters (2004) 14: 2621-2625) describe a synthesis method to produce HNK. This report also evaluates the structure activity relationship of O-methylated and/or its hydrogenated analogs of HNK in an in vitro neurotrophic assay. Esumi et al. conclude that the 5-allyl and 4′-hydroxyl groups are essential for the neurotrophic activity of HNK.

PCT Publication No. WO 02/076393 and U.S. Publication No. 2004/0105906 to Emory University describe pharmaceutical compositions and methods of treating conditions such angiogenic-, neoplastic-, and cancer-related conditions and skin conditions by administration of honokiol-type and/or magnolol-type compounds, as shown in Figures 1-4. For example, such compositions comprise at least one compound of formula Al :

AI wherein R₁, R₂, R₃, R₄, R₅, R₁, R₂, R₃, R₄, and R’₅ can be independently selected from groups that include, but are not limited to, hydrogen, hydroxyl groups, amides, amines, hydrocarbons, halogenated hydrocarbons, cyclic hydrocarbons, cyclic heterocarbons, halogenated cyclic heterocarbons, benzyl, halogenated benzyl, organo selenium compounds, sulfides, carbonyl, thiol, ether, dinitrogen ring compounds, thiophenes, pyridines, pyrroles, imidazoles, and pyrimidines. Honokiol-type and magnolol-type compounds are shown to inhibit SVR cell proliferation.

In November of 2004, Arbiser et al. reported that honokiol inhibited the growth of multuple myeloma cell lines via induction of Gl growth arrest, followed by apoptosis with IC50 values at 48h of 5 to 10 μg/mL. It was also reported that honokiol inhibited growth of doxorubin (Dox)-resistant (RPMI-Dox40), mephalan resistant (RPMI-LR5) and dexamethasone (Dex)-resistant (MM. IR) cell lines. It was suggested that the mechanism of honokiol triggered cytotoxicity is the honokiol induced increased expressin of Bax and Bad, down-regulated Mc-I protein expression, followed by caspase-8/9/3 cleavage, (Arbiser, J. et al. Poster at the American Society of Hematology Annual Meeting, 2004. Abstract published online November 4, 2004).

In July of 2005, Battle et al. reported that honokiol induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells (Blood. July 2005; 106:690- 697). Honokiol induced caspase-dependent cell death in all of the B-CLL cells examined, which were primary tumor cells derived from B-CLL patients, and was more toxic toward B- CLL cells than to normal mononuclear cells. The honokiol-induced apoptosis was characterized by the activation of caspase-3, -8, and -9 and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP). It was also reported that honokiol enhanced cytotoxicity induced by fludarabine, cladribine, or chlorambucil.

In September 2005, Ishitsuka et al reported that honokiol overcomes conventional drug resistance in human multiple myeloma by induction of caspase-dependent and – independent apoptosis (Blood, 1 September 2005, Vol. 106, No. 5, pp.1794-1800). HNK induced cytotoxicity in human multiple myeloma (MM) cell lines and tumor cells from patients with relapsed refractory MM through induction of apoptosis via both caspase- dependent and -independent pathways. HNK also enhanced MM cell cytotoxicity and apoptosis induced by bortezomib.

It is an object of the present invention to provide new compounds, compositions, methods and uses for the treatment of disorders associated with angiogenesis, cell proliferation, tumor growth, tumorogenesis, and myeloma.

the intermediates for the synthesis of honokiol are 3-allyl-4- hydroxybenzeneboronate 5 and 4-allyl-2-bromophenol 9. The boronate 5 can be prepared from 2-iodophenol 1 by bromination, followed by Suzuki coupling to introduce the allyl group, and boronation under Suzuki conditions. Compound 9 can be prepared from 4- iodophenol 6 by bromination and allylation (Suzuki coupling). The coupling of 5 and 9 under Suzuki conditions can yield honokiol from Suzuki coupling, not other allyl-oriented products from the Heck reaction, as it was shown that Suzuki coupling can succeed in the presence of C=C double bond (see Miyaura, N.; Suzuki, A. (1995), Chem. Rev. 95, 2457- 2483; and Suzuki, A. (1999), J. Organometal. Chem. 576, 147-168, and the references cited therein). Thus, honokiol and derivatives can be synthesized from commercially available starting materials in 6 steps (Scheme 1). Scheme 1

Treatment of honokiol with TMS-diazomethane in methanol results in mono- and di- methylated compounds I-III, and hydrogenation of honokiol with Wilkinson’s catalyst yields di- and tetrahydrohonokiols VI-VIII, as reported by Esumi, T. et al. (2004), Bioorg. Med. Chern. Lett. 14, 2621-2625. The amino and fluoro analogues (IV and V) can be constructed from iodoacetanilide under Suzuki coupling conditions. From 2-iodoacetanilide 10, after bromination, allylation, and boronation, the boronated intermediate 13 can be prepared. The other bromo intermediate 16 can be prepared from 4-iodoacetanilide 14 via bromination and allylation. The coupling of boronate 13 and bromide 16 under Suzuki conditions can afford, after deprotection, the compound IV. Diazotization followed by Schiemann reaction can convert the amino analogue TV to fluoro analogue V (Scheme 2).

The dimethoxy honokiol derivative, III, can also be prepared, for example, by the treatment of honokiol with potassium carbonate, iodomethane. (Scheme 2a). The hydrogenated honokiol analog can alternatively be prepared by the hydrogenation of honokiol with sodium borohydride and nickel(II) chloride to yields tetrahydrohonokiols VI- Vπi. (Scheme 2a). Scheme 2a

The preparation of the vinyl analogue IX is based on combining the Wittig reaction with Suzuki coupling. The intermediate aldehyde 18 can be prepared from 4-iodophenol 17 via the Reimer-Tiemann reaction, while 3-bromo-4-hydroxybenzenealdehyde 23 can be prepared from para-hydroxybenzoic ester 21 via bromination and reduction. The Wittig reaction of these two aldehydes can yield the corresponding vinyl substituted benzenes 19 and 24. Compound 19 can afford the boronate 20, which can be coupled with 24, to yield the compound IX (Scheme 3).

Reagents and conditions: (a) CHCl₃, aq. NaOH, 70 °C; (b) Ph₃PCH₃Br₁ n-BuLi, THF; (c) PdC!₂(dppf), dppf, KOAc, dioxane, bis(pinacoato)diboron, 80 °C; (d) DIBALH, -70 °C; (e) PdCI₂(dppf), dppf, K₃PO₄, dioxane, reflux.

For the synthesis of honokiol analogues with changed positions of the allyl or hydroxyl groups, the boronate 5, and the bromophenols 4 and 9 can be used as intermediates. Suzuki coupling of one of these intermediates with an appropriate halide or boronate can provide the compounds X-XVII. Compounds X-XII and XTV-XV can be prepared by Suzuki coupling of boronate 5 with an appropriate halide. Halide 25, needed for compound X, can be prepared from 2-bromo-6-iodophenol 2 via allylation, while the intermediate, 5-allyl-2- bromophenol 29 for compound XI, can be furnished from 3-iodophenol 26 via bromination and allylation. The preparation of halide 5-allyl-3-bromophenol 33, an intermediate for the synthesis of compound XIV, requires an organothallium reagent. The thallation of 3- bromophenol 30 followed by treatment with iodide can yield 3-bromo-5-iodophenol 32. After allylation, the allyl-substituted intermediate 33 can be prepared. The synthesis of compound XII can begin with 2-iodoacetanilide 10, via sulfonation, nitration, and reduction to obtain the intermediate 36. Aniline 36, after diazotization, followed by acid and base treatments, will afford 2-amino-3-iodophenol 37. Diazotization, Sandmeyer reaction, and allylation of compound 37 will yield halide 39. By a coupling reaction of these halides (25, 29, 33, and 39) with boronate 5, these compounds (X-XII, and XTV) can be prepared. Compound XV can be synthesized by Suzuki coupling of halide 4 with boronate 5 (Scheme 4).

Scheme 4

Alternatively, compounds X, XV, and XVII can be synthesized by an allylation- Claisen pathway. Biphenol compounds can be reacted first with potassium carbonate and allyl bromide, followed by reaction with BCl₃ to yield honokiol-like compounds, for example, X, XV, and XVII. (Scheme 4a). To a cooled solution (O⁰C with an ice bath) of diallyl starting material (1 eq.) in dry diehloromethane (Concentration of the solution : 0.1 mol.L^“1) was added dropwise a solution Of BCl₃ (IM in diehloromethane; 1.5 eq. = 0.75 eq for each allyl group). The reaction is then stirred at O⁰C until disappearance of the starting material on TLC (If after 15 minutes, the reaction is not complete, 1 more equivalent of BCl₃ can be added). After hydrolysis with water (about same volume than diehloromethane), the two layers are separated. The organic layer is washed again with water, dried under MgSO₄ then evaporated under vacuum. The residue is finally purified by column chromatography to give the di- hydroxy derivative . Scheme 4a

Bromide 9 is also a useful intermediate for coupling with some boronates. For example, Suzuki coupling of bromide 9 with boronate 42, which is prepared from 4-bromo-3- iodophenol 40 via allylation and boronation, can yield the compound XIII. Similarly, the coupling between bromide 9 and boronate 43 can afford the compound XVT. The compound XVII can be prepared from 4-allyl-2-bromophenol 9 via boronation followed by Suzuki coupling with 2-allyl-6-bromophenol 25 (Scheme 5).

The compounds XVIII and XIX can be synthesized from commercially available bisphenol 45 and the dihydroxynaphthalene-disulfuric acid salt 47. Thus, the bisphenol 45, through the Williamson reaction and Claisen rearrangement, can be converted to compound XVπi. Similarly, desulfonation of dihydroxynaphthalene-disulfuric acid salt .47, followed by the Williamson reaction and Claisen rearrangement, can produce the compound XIX (Scheme 6).Scheme 6

Dioxolane compounds can be prepared from magnoliol by reaction of magnoliol with 2,2′-dimethoxypropane and p-toluenesulfonic acid. (Scheme 7). This synthesis also provides a method of separating mixtures of honokiol and magnoliol. Scheme 7

The following examples are offered by way of illustration and not by way of limitation.

……..

http://www.google.com/patents/US20080300298

Axitinib (AG013736; trade name Inlyta) is a small molecule tyrosine kinase inhibitor developed by Pfizer. It has been shown to significantly inhibit growth of breast cancer in animal (xenograft) models^[2] and has shown partial responses in clinical trials with renal cell carcinoma (RCC)^[3] and several other tumour types.^[4] It was approved by the U.S. Food and Drug Administration after showing a modest increase in progression-free survival,^[5] though there have been reports of fatal adverse effects.^[6]

Axitinib, a small-molecule indazole derivative chemically known as (E)-N-methyl-2-(3-(2-(pyridin-2-yl)-vinyl)-1H-indazol-6-ylthio)benzamide developed by Pfizer, was approved in January 2012 by the U.S. FDA with the trade name Inlyta. It selectively inhibits vascular endothelial growth factor receptors for the treatment of renal cell carcinoma

On January 27, 2012, axitinib was approved with the trade name INLYTA for treatment of patients in the United States with advanced renal cell carcinoma after failure of one prior systemic therapy.

It has received FDA (27 January 2012), EMA (13 September 2012), MHRA (3 September 2012) and TGA (26 July 2012) approval for use as a treatment for renal cell carcinoma.^{[11][12][13][14]}

A study published in 2015^[15] showed that axitinib effectively inhibits a mutated gene (BCR-ABL1[T315I]) that is common in chronic myeloid leukemias and adult acute lymphoblastic leukemias which have become resistant to other tyrosine kinase inhibitors likeimatinib. This is one of the first examples of a new indication for an existing drug being discovered by screening known drugs using a patient’s own cells.

The discovery and development of an efficient synthesis route to axinitib is reported. The first-generation route researched by Pfizer implemented two Pd-catalyzed coupling reactions as key steps. In this work, the development of Heck-type and C–S coupling reactions catalyzed by CuI is briefly described, using an economial and practical protocol. Aspects of this route, such as selecting optimal ligands, solvent, and other conditions, are discussed in detail. The scale-up experiment was carried out to provide more than 300 g of active pharmaceutical ingredients of axitinib in Form XLI with 99.9% purity in 39% yield. In short, we provide a new choice of synthesis route to axitinib, through two copper-catalyzed coupling reactions with good yield.

(E)-N-Methyl-2-(3-(2-(pyridin-2-yl)vinyl)-1H-indazol-6-ylthiol)benzamide (Axitinib) Form XLI (326.4 g in 96% yield with purity 99.91%). Residual Cu content was determined to be 2.2 ppm by atomic absorption spectroscopy: mp 227.7 °C; 

1H NMR (300 MHz, DMSO-d6) δ 13.27 (s, 1H), 8.60 (d, J = 4.8 Hz, 1H), 8.29 (d, J = 5.4 Hz, 1H), 8.18 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 16.4 Hz, 1H), 7.81 (t, J = 7.5 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.63–7.44 (m, 3H), 7.29 (p, J = 7.4, 6.6 Hz, 3H), 7.19 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 7.4 Hz, 1H), 2.78 (d, J = 4.6 Hz, 3H); 

13C NMR (75 MHz, DMSO-d6) δ 167.89, 154.86, 149.54, 142.01, 141.86, 136.92, 136.88, 135.67, 132.52, 130.32, 129.99, 129.25, 127.80, 126.15, 125.59, 123.66, 122.68, 122.50, 121.79, 120.29, 114.76, 26.13.

………………………..

Axitinib (Axitinib, AG-013736, CAS: 319460-85-0) is a Pfizer research and development by the United States of new, mainly targeting VEGFR kinase GABA, inhibiting angiogenesis anticancer small molecule drug, trade name Inlyta, for other systems therapy for advanced renal cell carcinoma (Renal Cell Carcinoma, RCC), 2008 has been approved in the domestic clinical, and Pfizer’s cancer drug Sutent another similar imatinib (Sunitinib) , Axitinib also potent and selective multi-targeted tyrosine kinase inhibitor, can inhibit the vascular endothelial growth factor receptor (Vascular EndothelialGrowth Factor Rec India tor, VEGFR), including VEGFl receptor, VECF2 receptors and VECF3 receptor, can inhibit platelet-derived growth factor receptor (Platelet-derived growth factor receptor, PDGFR) and c_KIT. Axitinib is called sunitinib second generation, better than sunitinib adverse reactions.

Axitinib (II) chemical name 6- [2_ (methylcarbamoyl) phenylsulfanyl] -3-E- [2_ (Batch-2-yl) ethenyl] indazole structural formula as follows:

Axitinib (II)

Assi synthesis method for Nepal mainly in the following three ways:

(I) Patent US20060094881 (Agouron Pharmaceuticals), EP2163544 (Pfizer) reported the first synthesis method Axitinib to 3,6-diiodo-indazole as a starting material, first-iodo-6-position is substituted mercapto group, protection of the NH group, then the Heck reaction occurs (pyridine-2-yl) vinyl 3-position, after deprotection Axitinib whole synthesis route is as follows:

Axitinib Scheme I

This method although the synthesis route is shorter, but the catalyst and reagents used relatively expensive and require purified through the column, the total yield is low, is not conducive to industrial production.

[0004] (2) The second method of synthesis Axitinib e.g. W00102369 (Agouron Pharmaceuticals), US6531491 (Agouron Pharmaceuticals) reported in 6-nitro-indazole as a starting material, the 3-position first iodo, followed by the protecting group NH, Suzuki coupling reaction with boronic acid to give 3- styryl styryl-position, a nitro group reduced to an amino group, an amino diazotization reaction was iodo, the 3-position of the styrene-based ozone of the obtained aldehyde, followed by Wittig reaction to give the 3-position (pyridin-2-yl) ethenyl, 6-position is substituted mercapto iodine, alkaline hydrolysis then amidated, and finally deprotection Axitinib, the entire reaction formula as follows:

Axitinib Scheme 2

The method of synthesis route is long, harsh reaction conditions, complex process, the total yield is low, does not apply to industrial production.

[0005] (3) The third method is W02006048745 (Pfizer) discloses to 6-nitro-indazole as a starting material, the 3-position iodo first, followed by the protecting group NH, 3- bits Heck coupling reaction, a nitro group reduced to an amino group, an amino diazotization reaction was iodo, iodo-6-position is substituted mercapto group, and finally deprotected to give Axitinib, the entire reaction is as follows:

This method has an advantage over the first two methods, it is possible to enlarge the production, but the reaction was not complete in the reaction step, will generate new impurities through the column needs to be purified.

SYNTHESIS

aReagents and conditions: (a) I2, K2CO3, DMF; (b) CH2Cl2, CH3SO3H, dihydrofuran; (c) compound B, i-Pr2EtN, Pd(OAc)2, (o-Tol)3P, DMF; (d) iron, EtOH, NH4Cl; (e) AcOH, NaNO2, CH2Cl2, I2/KI; (f) compound C, Pd(dppf)Cl2, Cs2CO3, DMF; (h) 1, p-TsOH, MeOH; 2, NaHCO3; (i) AcOH, MeOH, Pd removal, recrystallization.

 http://www.google.com/patents/WO2006048745A1?cl=en

Example 15: Final deprotectioπ step to produce 6-r2-(methylcarbamoyl)phenylsulfanyll-3-E-f2- (pyridine-2-yl)ethenyllindazole

N-1 THP 6-[2-(methylcarbamoyl)phenylsulfanyl]-3-E-[2-(pyridine-2-yl)ethenyl]indazole (355 g) was suspended in 2,485 ml_ of methanol, after which p-toluenesulfonic acid monohydrate (718 g) was added. The mixture was then heated to 65 ⁰C (hard reflux) for 4 hours under argon while the reaction was monitored by HPLC (gluco method). Heating continued until less than 1% of the N-1 THP protected starting material persisted. The heating was then removed and the reaction was cooled to room temperature. The solid was filtered and the wet cake was washed with methanol (2 volumes, 710 mL) then the solids were rinsed with ethyl acetate (2 volumes, 710 mL). The wet cake was transferred to a reactor containing sodium bicarbonate (126.84 g), deionized water (1800 mL), and ethyl acetate (975 mL), which was then stirred for 2 hours at 2O⁰C. The solids were filtered and washed with 5 volumes of deionized water (1800 mL), then with 2 volumes of ethyl acetate (760 mL), and then dried in a vacuum oven at 40⁰C for 16 hours. The isolated yield for the reaction was 92.5% (274 g). The isolated material was identified as crystalline Form III free base (0.5 ethyl acetate solvate). ¹H NMR, 300 MHz, (DMSO-D6), ppm; 13.35 (1 H, s), 8.60 (1 H, d, J=3.8 Hz), 8.39 (1 H, m), 8.23 (1 H, d, J=8.5 Hz), 7.95 (1 H, d, J=16.4 Hz), 7.82 (1 H, ddd, J=7.7, 7.6, 1.8 Hz), 7.67 (1 H, d, J=7.8 Hz), 7.60 (a H, s), 7.57 (1 H, d, J=16.4 Hz), 7.49 (1 H, dd, J=7.1 , 1.6 Hz), 7.35-7.26 (3 H, m), 7.19 (1 H, d, J=8.4 Hz), 7.04 (1 H, d, J=7.8 Hz), 2.77 (3 H, d, J=4.6 Hz). ¹³C NMR, 75 MHz, (DMSO-D6) ppm: 168.23, 155.18, 149.81 , 142.35, 142.22, 137.31 , 136.00, 132.89, 130.64, 130.36, 129.51 , 128.14, 126.50, 125.93, 124.08, 123.01 , 122.85, 122.12, 120.642, 115.08, 26.45.

Example 21 : Preparation of 6-F2-(methylcarbamovDphenylsulfanyll-3-Z-r2-(pyridine-2- vDethenyllindazole

To a 100 ml_ 3-neck flask containing a solution of 0.95 g of 6-[2- (methylcarbamoyl)phenylsulfanyl]-3-[2-(pyridine-2-yl)ethynyl]indazole was added 2.5 g of phenyliodide diacetate followed by 1.0 mL of H₂NNH₂ H₂O. After the bubbling had settled, more phenyliodide diacetate and H₂NNH₂ H₂O were added in small portions, until LC/MS indicated the disappearance of 6-[2-(methylcarbamoyl)phenylsulfanyl]-3-[2-(pyridine-2-yl)ethynyl]indazole and the formation of 6-[2-(methylcarbamoyl)phenylsuIfanyl]-3-Z-[2-(pyridine-2-yl)ethenyl]indazole. Example 22: Palladium removal and polymorph control of 6-[2-(methylcarbamoyl)phenylsulfanvn- 3-E-r2-(pyridine-2-vDethenyllindazole

4) MeOH, reflux

Polymorph Form IV

5) HOAc/Xylenes

To a 12 L 3-neck flask, equipped with a mechanical stirrer, was added 160.20 g of 6-[2- (methylc’arbamoyl)phenylsulfanyl]-3-E-[2-(pyridine-2-yl)ethenyl]indazole and 1.6 L of DMA and 1.6 L of THF. After stirring for 20 minutes, the mixture became homogeneous. To the clear solution was added 800.99 g of 10% cysteine-silica and the resulting mixture was allowed to stir at room temperature overnight.

The mixture was filtered through a medium sintered glass fritted funnel, and the cake was washed with a solution of 500 mL of DMA and 500 mL of THF. The cake was further washed with 2.0 L of THF and the filtrate was collected into a separate flask. The volatile parts in the latter filtrate were removed in vacuo and the residue was combined with the main filtrate. The combined filtrate was recharged back into the 12 L flask, followed by 800 g of 10% cysteine-silica. The flask was equipped with a mechanical stirrer and stirred over the weekend at room temperature. The mixture was then filtered through a medium sintered glass fritted funnel and the silica was washed with a mixture of solvents of 500 ml. of DMA and 500 ml_ of THF, followed by 3.0 L of THF. The volatile parts in the filtrate were removed in vacuo and the remaining solution was transferred to a 22 L 3-neck flask and treated with 12 L of water (added over a 20 minute period of time), a thick precipitate formed at this stage. After stirring overnight, the mixture was filtered and the cake was washed with 2.0 L of water and sucked dry.

The cake was charged to a 5 L 3-neck flask, followed by 1.6 L of THF and 160 mL of DMF. The flask was equipped with a mechanical stirrer, a reflux condenser and the mixture was heated at reflux for 8 hours. After cooling overnight, the mixture was filtered through sharkskin filter paper and sucked dry. The cake was charged to a 5 L 3-neck flask and 1.6 L of MeOH was added. The flask was equipped with a mechanical stirrer, a water condenser and the contents were heated at reflux for 6 hours. After cooling overnight, the mixture was filtered through sharkskin filter paper and sucked dry.

The cake was dissolved into 1.6 L of HOAc with the assistance of gentle heating in the water bath of a rotary evaporator. The solution was filtered through #3 filter paper and the total volume of the filtrate was reduced to ~500 mL in volume on the rotary evaporator at 60 °C/60 mmHg. At this stage, the bulk of the mixture remained a yellow solution and a small amount of precipitate formed. To the flask was charged 500 mL of xylenes (precipitate formed) and the total volume was reduced to -500 mL in volume on the rotary evaporator at 60°C/60 mmHg. The process was repeated two more times. After cooling, the mixture was filtered, the cake was washed with 500 mL of xylenes and sucked dry. The cake was transferred to a glass dish and further dried at 80°C/27 inch vacuum overnight.

The cake was off-white in color and weighed 108.38g. X-ray powder diffraction analysis indicated that a crystalline form was present, which was characterized as Form IV by a powder X- ray diffraction pattern comprising peaks at the following approximate diffraction angles (20): 8.9, 12.0, 14.6, 15.2, 15.7, 17.8, 19.2, 20.5, 21.6, 23.2, 24.2, 24.8, 26.2, and 27.5.

While the invention has been illustrated by reference to specific and preferred embodiments, those skilled in the art will recognize that variations and modifications may be made through routine experimentation and practice of the invention. Thus, the invention is intended not to be limited by the foregoing description, but to be defined by the appended claims and their equivalents.

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Chekal, B. P.; Guinness, S. M.; Lillie, B. M.; McLaughlin, R. W.; Palmer, C. W.; Post, R. J.; Sieser, J. E.; Singer, R. A.; Sluggett, G. W.; Vaidyanathan, R.; Withbroe, G. Org. Process Res. Dev. 2014, 18, 266 http://pubs.acs.org/doi/abs/10.1021/op400088k

The manufacturing process of axitinib (1) involves two Pd-catalyzed coupling reactions, a Migita coupling and a Heck reaction. Optimization of both of these pivotal bond-formation steps is discussed as well as the approach to control impurities in axitinib. Essential to the control strategy was the optimization of the Heck reaction to minimize formation of impurities, in addition to the development of an efficient isolation of crude axitinib to purge impurities.

Babu, S.; Dagnino, R., Jr.; Ouellette, M. A.; Shi, B.; Tian, Q.; Zook, S. E. PCT Int. Appl. WO/2006/048745, 2006.

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 http://www.google.com/patents/CN103570696A?cl=en

formula:

A Axitinib intermediate (1) production method, based on 6-nitro-indazole as a starting material, in the first catalyst is reacted with 3,4-dihydro -2H- pyran, bits of NH the protecting group tetrahydro -2H- pyran-2-yl, then the three iodide, to give the key intermediate in high yield 3-iodo-6-nitro-1- (tetrahydro -2H- pyrazol pyran-2-yl) -1H- indazole (I), comprising the following synthetic steps:

(1) 6-nitro-indazole dissolved in an aprotic solvent, and 3,4-dihydro -2H- pyran catalyst, 6-nitro-indazole in the catalyst and the 3,4-dihydro -2H – pyran reaction, the protecting group NH-position, was prepared to give 6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, the reaction equation is:

Wherein the 3,4-dihydro -2H- pyran an amount of 3 equivalents wide;

Aprotic solvent is acetonitrile, ethyl acetate, toluene or xylene;

The catalyst is 2,3-dichloro-5,6-dicyano-p-benzoquinone, p-toluenesulfonic acid or methanesulfonic acid;

The reaction temperature is 7 (T90 ° C, the reaction time is 1 to 4 hours;

(2) 6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole dissolved in a polar aprotic solvent, iodine was added and the acid-binding agent, an inorganic base, to afford 3- iodo-6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (I), the reaction equation is:

Wherein the polar aprotic solvent is N, N- dimethylformamide (DMF), N, N- dimethylacetamide, N, N- diethylformamide, N, N- diethyl-acetamide ;

Inorganic base acid binding agent is potassium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, potassium bicarbonate, sodium bicarbonate, cesium carbonate, lithium hydroxide;

The reaction temperature is 2 (T40 ° C, the reaction time is 8 to 20 hours.

[0009] A Axitinib intermediate (1) in preparation for the Nepalese Asif application, based on intermediate (1) and 2-vinyl pyridine Heck coupling reaction, followed sequentially nitro reduction and the diazotization reaction of iodine, and finally with a 2-mercapto–N- methylbenzamide was prepared by deprotection docking axitinib, including the following synthetic steps:

(I) Intermediate (1) and be given 2_ vinylpyridine Jie Heck coupling reaction to give (E) _6_ nitro _3- [2_ (P than-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, the reaction equation is:

(2) (E) -6- nitro-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- nitro indazole group reduction reaction, to give (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, The reaction equation is:

(3) (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole diazo of the iodide to give (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole The reaction equation is:

(4) (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole with 2- mercapto-methylbenzamide reaction -N-, to give (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) -1- (tetrahydro -2H- pyrazol pyran-2-yl) -1H- indazol-6-yl] thio} benzamide, the reaction equation is:

(5) (E) -N- methyl-2- {[3- (2- (pyridin-2-yl) ethenyl) -1- (tetrahydro -2H- pyran-2-yl) -1H- indazol-6-yl] thio} benzamide deprotected Axitinib (II), the reaction equation is:

Example 1

A Assi intermediates for preparing Nigeria, comprising the steps of:

Synthesis of (I) 6- nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

A 5L reaction flask was added acetonitrile (2L), followed by addition of 6-nitro-indazole (163.1g, 1.0mol), 3, 4- dihydro -2H- pyran (168.2g, 2.0mol), 2,3- dichloro-5,6-dicyano-p-benzoquinone (22.7g, 0.1mol), was heated to 820C under reflux for 2 hours to complete the reaction, cooled to room temperature, rotary evaporated to dryness, added water and dichloromethane 2L 2L, stirring I hour, delamination, the organic phase washed with brine, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to dryness, and then dissolved in acetonitrile and 2L, stirring ice-salt bath chilled to _5 ° C for 2 hours, suction filtered, the filter cake washed with a small amount of cold acetonitrile, recrystallized from ethanol, 60 ° C and dried in vacuo 12 hours to give an off-white solid, 6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, 236.3 g, yield 95.6%, m.p. 110 ~ 120 ° C, 1Η NMR (CDCl3): δ 1.30-1.83 (m, 6Η, Η3, _Η5,), 3.82-3.93 (m, 2Η, Η6 ‘), 5.86 (m , 1Η, Η2 ‘), 8.10-8.12 (m, 2Η, Η3, Η5), 8.31 (m, 1Η; Η4), 8.55 (s, 1Η, Η7);

The reaction equation is as follows:

(2) 3-iodo-6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (I),

5L reaction flask in DMF 700mL, followed by addition of 6-nitro-_1_ (tetrahydro -2H- pyran-2-yl) -1H- indazole (225.0g, 0.91mol, l.0eq) and potassium carbonate ( 251.6g, 1.82mol, 2.0eq), ice-cooled (10 ° C or less), followed by stirring, iodine (415.8g, 1.64mol, 1.8eq) was dissolved in DMF 300mL, was added dropwise to the reaction system, addition time 2 hours , the reaction system was stirred at 25 ° C for 16 hours to complete the reaction, sodium thiosulfate was added (223.0g, 1.41mol, 1.55eq) and 1.50g of potassium carbonate aqueous solution (1.5L), while maintaining the internal temperature 30 ° C Hereinafter, stirred for 30 minutes at room temperature, water was added with stirring 2L, solid precipitated, stirred for 30 minutes at room temperature, suction filtered, the filter cake was washed with water, 60 ° C and dried in vacuo 12 hours to give a pale yellow solid (Ι), 326.5g, yield 96.2%, m.p. 135 ~ 137 ° C / H NMR (DMS0_d6): δ 1.60-1.61 (m, 2H, H4,, H5 ‘), 1.73-1.76 (m, 1H, H5’), 2.01-2.04 (m, 2H, H3 ‘, H4’), 2.35-2.38 (m, 1H, H3 ‘), 3.81-3.87 (m, 2H, H6’), 6.11-6.14 (dd, 1H, H2 ‘), 7.70-7.72 (d , 1H, H4),

8.05-8.07 (dd, 1H, H5), 8.79 (s, 1H, H7).

The reaction equation is as follows:

A Axitinib intermediate (1) in the preparation for the Nepalese Asif applications, including the following synthetic steps:

Synthesis of (I) (E) -6- nitro-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

A 5L reaction flask was added DMF (2L), followed by addition of the intermediate (1) (312.0g, 0.84mol), 2- vinylpyridine (127.5g, 1.21mol), N, N- diisopropylethylamine ( 205.3g, 1.59mol), tri-o-tolylphosphine (22.3g, 0.073mol) and palladium chloride (4.9g, 0.028mol), nitrogen, and heated to 100 ° C for 12 hours to complete the reaction, cooled to 45 ° C, isopropanol was added 1L, stirring at 45 ° C for 30 minutes, diluted with water and 5L, stirring at room temperature for I h, suction filtered, washed with water, isopropanol was added to the filter cake 1.2L, stirred at 55 ° C for 30 minutes, then stirred at room temperature for 30 minutes, suction filtered, the filter cake washed with cold isopropanol, 50 ° C and dried under vacuum for 12 hours to give (E) -6- nitro-3- [2- (pyridin-2 – yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, 275.3g, 94.0% yield, m.p. 175 ~ 176 ^, ¾ NMR (DMSO-Cl6): δ 1.63-1.64 (m, 2H, H4 ‘, H5’), 1.79-1.81 (m, 1H, H5 ‘), 2.05-2.07 (m, 2H, H3’, H4 ‘), 2.44-2.50 (m, 1H , H3 ‘), 3.86-3.90 (m, 2H, H6’), 6.15-6.18 (dd, 1H, H2 ‘), 7.30-7.33 (dd, 1H, pyridine H5), 7.65-7.69 (d, 1H, J = 16Hz, vinyl H2), 7.72-7.74 (d, 1H, pyridine H4), 7.82-7.86 (m, 1H, pyridine H3), 7.96-8.00 (d, 1H, J = 16Hz, vinyl HI), 8.07 -8.10 (dd, 1H, H4), 8.44-8.46 (d, 1H, H5), 8.63-8.64 (d, 1H, pyridine H6), 8.77-8.78 (d, 1H, H7);

The reaction equation is as follows:

Synthesis of (2) (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2Η-) -1H- indazole

5L reaction flask in ethanol HOOmLdjC 1000mL and ammonium chloride (300.0g, 5.61mol), was dissolved with stirring, followed by addition of (E) -6- nitro-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (255.0g, 0.73mol), was added iron powder (162.6g, 2.91mol), heated to 50 ° C the reaction was stirred for 2 hours to completion of the reaction, was cooled to 22 ° C, tetrahydrofuran 2L, stirred for I hour at room temperature, filtered through Celite, the filter cake washed with tetrahydrofuran and the filtrate was rotary evaporated to dryness, cooled to room temperature, water was added 2L, stirred for I hour at room temperature, pumping filtered, the filter cake washed with petroleum ether, 50 ° C and dried under vacuum for 12 hours to give a pale yellow solid 206.5g, (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole, yield 88.6%, m.p. 162 ~ 164 ° C / H NMR (CDCl3): δ 1.63-1.77 (m, 2H, H4 ‘, H5 ‘), 2.02-2.06 (m, 1H, H5’), 2.17-2.18 (m, 1H, H4 ‘), 2.55-2.60 (m, 1H, H3’) 3.70-3.72 (m, 2H, H3 ‘, H6 ‘), 3.91 (s, 2H, NH2), 4.04-4.07 (m, 1H, H6’), 5.57-5.60 (dd, 1H, H2 ‘), 6.64-6.66 (dd, 1H, H5), 6.74-6.75 (d, 1H, H7), 7.13-7.16 (dd, 1H, pyridine H5), 7.48-7.50 (d, 1H, pyridine H4), 7.49-7.53 (d, 1H, J = 16Hz, vinyl H2), 7.64 -7.68 (m, 1H, pyridine H3), 7.78-7.82 (d, 1H, J = 16Hz, vinyl Hl), 7.82-7.83 (d, 1H, H4), 8.60-8.61 (d, 1H, pyridine H6) ;

The reaction equation is as follows:

Synthesis of (3) (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole

A 5L reaction flask was added 600mL of water and sodium nitrite (70.2g, 1.02mol), stirred and dissolved, and cooled to (TC, (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl ] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (200.0g, 0.62mol) was dissolved in glacial acetic acid 1.3L, dropwise added to the system dropwise over I h, a solution process maintain an internal temperature of 0 ° C, the same temperature for I hour, dropping HCl solution (concentrated hydrochloric acid 112mL, water 200mL) at O ​​° C, the dropping time of 10 minutes, with the temperature for I h, TLC plate tracking point diazonium salt formation reaction (PE: EA = 1: 1). dropwise 800mL dichloromethane between 0 ° C, the dropping time of 5 minutes, potassium iodide (207.3g, l.25mol) and iodine (79.2g, 0.31mol) was dissolved water 600mL, in (TC dropwise added to the system at the same temperature for 2 hours to complete the reaction. The reaction mixture was poured into the system to 20% sodium thiosulfate solution (2L) and dichloromethane SOOmL and stirred, layered , the aqueous phase was extracted with dichloromethane frozen (2x800mL), dichloromethane phases were combined burning, 3M sodium hydroxide solution was added dropwise 3.5L, adjust the aqueous phase pH = 9 ~ 12, and water was added ammonia 200mL 400mL, stirred for 30 minutes , separated and the aqueous phase was extracted with dichloromethane (2×1.2L), the organic phases were combined, rotary evaporated to dryness, and purified through silica gel to give (E) -6- iodo-3- [2- (pyridin-2-yl ) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, 176.0g, 65.4% yield, m.p. 142 ~ 143 ° C, 1H NMR (DMS0_d6): δ 1.58- 1.61 (m, 2H, H4 ‘, H5,) 1.72-1.78 (m, 1H, H5,), 1.97-2.04 (m, 2H, H3,, H4,), 2.38-2.44 (m, 1H, H3,) , 3.79-3.81 (m, 1H, H6,), 3.88-3.90 (m, 1H, H6,), 5.91-5.94 (dd, 1H, H2,), 7.29-7.31 (m, 1H, pyridine H5), 7.56 -7.60 (d, 1H ,, J = 16Hz, vinyl H2), 7.57-7.59 (m, 1H, pyridine H4), 7.69-7.71 (d, 1H, pyridine H3), 7.80-7.84 (m, 1H, H4 ), 7.89-7.93 (d, 1H, J = 16Hz, vinyl HI), 8.01-8.03 (d, 1H, H5), 8.25 (s, 1H, H7), 8.61-8.62 (d, 1H, pyridine H6) ;

The reaction equation is as follows:

(4) (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) _1_ (tetrahydro -2H- pyran-2-yl) -1H- indazole 6-ylthio} benzamide]

A 5L reaction flask was added DMF (1750mL) and (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1H- indazole (175.0g, 0.41mol), nitrogen, was added [1, I, – bis (diphenylphosphino) ferrocene] dichloropalladium dichloromethane complex (14.9g, 0.018mmol ), cesium carbonate (198.3g, 0.61mol) and dichloromethane 20mL, was added 2-mercapto -N- methylbenzamide (84.9g, 0.5Imol), heated to 80 ° C for 16 hours to complete the reaction, spin distilled was removed DMF, cooled to room temperature, ethyl acetate was added 3L, water 4L, stirred for 40 minutes, the organic phase was separated, washed with brine, layered, dried over sodium sulfate, filtered, and rotary evaporated to dryness, to give (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) -1- (tetrahydro -2H- pyran-2-yl) -1H- indazol-6-yl] thio } benzamide, 165.6g, a yield of 86.7%, the melting point of 142 ~ 143 ° C;

The reaction equation is as follows:

(5) Synthesis of axitinib

In a 2L reaction flask was added (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) _1_ (tetrahydro -2H- pyran-2-yl) -1H – indazol-6-yl] thio} benzamide (150.0g, 0.32mol), p-toluenesulfonic acid monohydrate (303.2g, 1.59mol), methanol (800mL) and water (150mL), nitrogen, heated to 65 ° C for 4 hours, spin evaporated to dryness and ethanol (800mL), 65 ° C was stirred for I hour, the ethanol was removed by rotary evaporation, then repeated three times, TLC spot plate tracking reaction (petroleum ether: ethyl acetate = 1: 1). Completion of the reaction, cooled to room temperature, rotary evaporated to dryness, water was added 500mL, stirred for I h, filtered, and the filter cake was washed with methanol and ice, and then added to the reaction vessel, ethyl acetate was added 450mL, stirred at 65 ° C 30 minutes. cooled to room temperature, suction filtered, the filter cake washed with ethyl acetate and freeze paint, water paint, 50 ° C and dried under vacuum for 12 hours to give a white solid 117.5g, Axitinib (II), yield 95.4%, HPLC purity 98.8 % / H NMR (DMS0_d6): δ 2.78 (d, 3H, CH3), 7.05 (dd, 1H), 7.19 (dd, 1H), 7.36-7.23 (m, 3H), 7.50 (dd, 1H), 7.58 ( d, 1H), 7.61 (s, 1H), 7.66 (d, 1H), 7.85-7.76 (m, 1H), 7.96 (d, 1H, J = 16Hz), 8.21 (d, 1H), 8.39 (q, 1H), 8.61 (d, 1H), 13.35 (s, 1H).

The reaction equation is as follows:

Example 2

A Assi intermediates for preparing Nigeria, comprising the steps of:

Synthesis of (1) 6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

A 5L reaction flask was added ethyl acetate (2L), followed by addition of 6-nitro-indazole (163.14g, 1.0mol), 3, 4- dihydro -2H- pyran (210.3g, 2.5mol), toluene acid (20.7g, 0.12mol), heated to 78 ° C under reflux for 3 hours to complete the reaction, cooled to room temperature, rotary evaporated to dryness, added water and dichloromethane 2L 2L, stirred for I hour, stratification, the organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to dryness, and then dissolved in acetonitrile and 2L, stirring ice-salt bath chilled to _5 ° C for 2 hours, suction filtered, the filter cake washed with a small amount of cold acetonitrile, recrystallized from ethanol , 60 ° C and dried in vacuo 12 hours to give an off-white solid 223.3g, 6- nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, yield 90.3%, m.p. 110 ^ 11 TC;

The reaction equation is as follows:

(2) 3-iodo-6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (I),

5L reaction flask in DMF 700mL, followed by addition of 6-nitro-_1_ (tetrahydro -2H- pyran-2-yl) -1H- indazole (200.0g, 0.81mol, l.0eq) and sodium hydroxide (64.7g, 1.62mol, 2.0eq), ice-cooled (10 ° C or less), followed by stirring, iodine (369.6g, 1.46mol, 1.8eq) was dissolved in DMF 300mL, was added dropwise to the reaction system, addition time 2 hours, the reaction system was stirred at 25 ° C for 12 hours to complete the reaction, sodium thiosulfate was added (198.2g, 1.25mol, 1.55eq) and 1.50g of potassium carbonate aqueous solution (1.5L), while maintaining the temperature of 30 ° C or less, and stirred for 30 minutes at room temperature, water was added with stirring 2L, solid precipitated, stirred for 30 minutes at room temperature, suction filtered, the filter cake was washed with water, 60 ° C and dried in vacuo 12 hours to give a pale yellow solid

(1), 294.3g, 97.5% yield, m.p. 136 ~ 137. . .

[0014] The reaction equation is as follows:

A Axitinib intermediate (1) in the preparation for the Nepalese Asif applications, including the following synthetic steps:

Synthesis (1) (E) -6- nitro-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2Η-) -1H- indazole

A 5L reaction flask was added DMF (2L), followed by addition of the intermediate (1) (312.0g, 0.84mol), 2- vinylpyridine (127.5g, 1.21mol), N, N- diisopropylethylamine ( 205.3g, 1.59mol), tri-o-tolylphosphine (22.3g, 0.073mol) and palladium chloride (4.9g, 0.028mol), nitrogen, and heated to 100 ° C for 12 hours to complete the reaction, cooled to 45 ° C, isopropanol was added 1L, stirring at 45 ° C for 30 minutes, diluted with water and 5L, stirring at room temperature for I h, suction filtered, washed with water, isopropanol was added to the filter cake 1.2L, stirred at 55 ° C for 30 minutes, then stirred at room temperature for 30 minutes, suction filtered, the filter cake washed with cold isopropanol, 50 ° C and dried under vacuum for 12 hours to give (E) -6- nitro-3- [2- (pyridin _2 _-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, 275.3g, 94.0% yield, m.p. 175 ~ 176 ^, ¾ NMR (DMSO-Cl6): δ 1.63-1.64 (m, 2H, H4 ‘, H5’), 1.79-1.81 (m, 1H, H5 ‘), 2.05-2.07 (m, 2H, H3’, H4 ‘), 2.44-2.50 (m, 1H , H3 ‘), 3.86-3.90 (m, 2H, H6’), 6.15-6.18 (dd, 1H, H2 ‘), 7.30-7.33 (dd, 1H, pyridine H5), 7.65-7.69 (d, 1H, J = 16Hz, vinyl H2), 7.72-7.74 (d, 1H, pyridine H4), 7.82-7.86 (m, 1H, pyridine H3), 7.96-8.00 (d, 1H, J = 16Hz, vinyl HI), 8.07 -8.10 (dd, 1H, H4), 8.44-8.46 (d, 1H, H5), 8.63-8.64 (d, 1H, pyridine H6), 8.77-8.78 (d, 1H, H7);

The reaction equation is as follows:

Synthesis of (2) (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

5L reaction flask in ethanol HOOmLdjC 1000mL and ammonium chloride (300.0g, 5.61mol), was dissolved with stirring, followed by addition of (E) -6- nitro-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (255.0g, 0.73mol), was added iron powder (162.6g, 2.91mol), heated to 50 ° C the reaction was stirred for 2 hours to completion of the reaction, was cooled to 22 ° C, tetrahydrofuran 2L, stirred for I hour at room temperature, filtered through Celite, the filter cake washed with tetrahydrofuran and the filtrate was rotary evaporated to dryness, cooled to room temperature, water was added 2L, stirred for I hour at room temperature, pumping filtered, the filter cake washed with petroleum ether, 50 ° C and dried under vacuum for 12 hours to give a pale yellow solid 206.5g, (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole, yield 88.6%, m.p. 162 ~ 164 ° C / H NMR (CDCl3): δ 1.63-1.77 (m, 2H, H4 ‘, H5 ‘), 2.02-2.06 (m, 1H, H5’), 2.17-2.18 (m, 1H, H4 ‘), 2.55-2.60 (m, 1H, H3’) 3.70-3.72 (m, 2H, H3 ‘, H6 ‘), 3.91 (s, 2H, NH2), 4.04-4.07 (m, 1H, H6’), 5.57-5.60 (dd, 1H, H2 ‘), 6.64-6.66 (dd, 1H, H5), 6.74-6.75 (d, 1H, H7), 7.13-7.16 (dd, 1H, pyridine H5), 7.48-7.50 (d, 1H, pyridine H4), 7.49-7.53 (d, 1H, J = 16Hz, vinyl H2), 7.64 -7.68 (m, 1H, pyridine H3), 7.78-7.82 (d, 1H, J = 16Hz, vinyl Hl), 7.82-7.83 (d, 1H, H4), 8.60-8.61 (d, 1H, pyridine H6) ;

The reaction equation is as follows:

Synthesis of (3) (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole

A 5L reaction flask was added 600mL of water and sodium nitrite (70.2g, 1.02mol), stirred and dissolved, and cooled to (TC, (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl ] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (200.0g, 0.62mol) was dissolved in glacial acetic acid 1.3L, dropwise added to the system dropwise over I h, a solution process maintain an internal temperature of 0 ° C, the same temperature for I hour, dropping HCl solution (concentrated hydrochloric acid 112mL, water 200mL) at O ​​° C, the dropping time of 10 minutes, with the temperature for I h, TLC plate tracking point diazonium salt formation reaction (PE: EA = 1: 1). dropwise 800mL dichloromethane between 0 ° C, the dropping time of 5 minutes, potassium iodide (207.3g, l.25mol) and iodine (79.2g, 0.31mol) was dissolved water 600mL, in (TC dropwise added to the system at the same temperature for 2 hours to complete the reaction. The reaction mixture was poured into the system to 20% sodium thiosulfate solution (2L) and dichloromethane SOOmL and stirred, layered , the aqueous phase was extracted with dichloromethane frozen (2x800mL), dichloromethane phases were combined burning, 3M sodium hydroxide solution was added dropwise 3.5L, adjust the aqueous phase pH = 9 ~ 12, and water was added ammonia 200mL 400mL, stirred for 30 minutes , separated and the aqueous phase was extracted with dichloromethane (2×1.2L), the organic phases were combined, rotary evaporated to dryness, and purified through silica gel to give (E) -6- iodo-3- [2- (pyridin-2-yl ) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, 176.0g, 65.4% yield, m.p. 142 ~ 143 ° C, 1H NMR (DMS0_d6): δ 1.58- 1.61 (m, 2H, H4 ‘, H5,) 1.72-1.78 (m, 1H, H5,), 1.97-2.04 (m, 2H, H3,, H4,), 2.38-2.44 (m, 1H, H3,) , 3.79-3.81 (m, 1H, H6,), 3.88-3.90 (m, 1H, H6,), 5.91-5.94 (dd, 1H, H2,), 7.29-7.31 (m, 1H, pyridine H5), 7.56 -7.60 (d, 1H ,, J = 16Hz, vinyl H2), 7.57-7.59 (m, 1H, pyridine H4), 7.69-7.71 (d, 1H, pyridine H3), 7.80-7.84 (m, 1H, H4 ), 7.89-7.93 (d, 1H, J = 16Hz, vinyl HI), 8.01-8.03 (d, 1H, H5), 8.25 (s, 1H, H7), 8.61-8.62 (d, 1H, pyridine H6) ;

The reaction equation is as follows:

(4) (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) _1_ (tetrahydro -2H- pyran-2-yl) -1H- indazole 6-ylthio} benzamide]

A 5L reaction flask was added DMF (1750mL) and (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1H- indazole (175.0g, 0.41mol), nitrogen, was added [1, I, – bis (diphenylphosphino) ferrocene] dichloropalladium dichloromethane complex (14.9g, 0.018mmol ), cesium carbonate (198.3g, 0.61mol) and dichloromethane 20mL, was added 2-mercapto -N- methylbenzamide (84.9g, 0.5Imol), heated to 80 ° C for 16 hours to complete the reaction, spin distilled was removed DMF, cooled to room temperature, ethyl acetate was added 3L, water 4L, stirred for 40 minutes, the organic phase was separated, washed with brine, layered, dried over sodium sulfate, filtered, and rotary evaporated to dryness, to give (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) -1- (tetrahydro -2H- pyran-2-yl) -1H- indazol-6-yl] thio } benzamide, 165.6g, a yield of 86.7%, the melting point of 142 ~ 143 ° C;

The reaction equation is as follows:

(5) Synthesis of axitinib

In a 2L reaction flask was added (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) _1_ (tetrahydro -2H- pyran-2-yl) -1H – indazol-6-yl] thio} benzamide (150.0g, 0.32mol), p-toluenesulfonic acid monohydrate (303.2g, 1.59mol), methanol (800mL) and water (150mL), nitrogen, heated to 65 ° C for 4 hours, spin evaporated to dryness and ethanol (800mL), 65 ° C was stirred for I hour, the ethanol was removed by rotary evaporation, then repeated three times, TLC spot plate tracking reaction (petroleum ether: ethyl acetate = 1: 1). Completion of the reaction, cooled to room temperature, rotary evaporated to dryness, water was added 500mL, stirred for I h, filtered, and the filter cake was washed with methanol and ice, and then added to the reaction vessel, ethyl acetate was added 450mL, stirred at 65 ° C 30 minutes. cooled to room temperature, suction filtered, the filter cake washed with ethyl acetate and freeze paint, water paint, 50 ° C and dried under vacuum for 12 hours to give a white solid 117.5g, Axitinib (II), yield 95.4%, HPLC purity 98.8 % / H NMR (DMS0_d6): δ 2.78 (d, 3H, CH3), 7.05 (dd, 1H), 7.19 (dd, 1H), 7.36-7.23 (m, 3H), 7.50 (dd, 1H), 7.58 ( d, 1H), 7.61 (s, 1H), 7.66 (d, 1H), 7.85-7.76 (m, 1H), 7.96 (d, 1H, J = 16Hz), 8.21 (d, 1H), 8.39 (q, 1H), 8.61 (d, 1H), 13.35 (s, 1H).

The reaction equation is as follows:

Example 3

A Assi intermediates for preparing Nigeria, comprising the steps of:

Synthesis of (1) 6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

5L reaction flask in toluene (2L), followed by addition of 6-nitro-indazole (163.lg, 1.0mol), 3,4- dihydro -2H- pyran (193.5g, 2.3mol), methanesulfonic acid (14.4g, 0.15mol), heated to 85 ° C under reflux for 3.5 hours, to complete the reaction, cooled to room temperature, rotary evaporated to dryness, added water and dichloromethane 2L 2L, stirred for I hour, stratification, the organic phase was washed with brine wash, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to dryness, and then dissolved in acetonitrile and 2L, stirring ice-salt bath chilled to _5 ° C for 2 hours, suction filtered, the filter cake washed with a small amount of cold acetonitrile and paint, and recrystallized from ethanol , 60 ° C and dried in vacuo 12 hours to give an off-white solid, 6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, 234.4g, 94.8% yield, m.p. 111 ~ 112.. ;

The reaction equation is as follows:

(2) 3-iodo-6-nitro-1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (I),

5L reaction flask in DMF 700mL, followed by addition of 6-nitro-_1_ (tetrahydro -2H- pyran-2-yl) -1H- indazole (225.0g, 0.91mol, 1.0eq) and potassium hydroxide ( 102.lg, 1.82mol, 2.0eq), ice-cooled below 10 ° C, with stirring, iodine (415.8g, 1.64mol, 1.8eq) was dissolved in DMF 300mL, was added dropwise to the reaction system dropwise over 2 hours, The reaction system was stirred at 30 ° C for 10 hours to complete the reaction, sodium thiosulfate was added (223.0g, 1.41mol, 1.55eq) and 1.50g of potassium carbonate aqueous solution (1.5L), while maintaining the internal temperature below 30 ° C , stirred for 45 minutes at room temperature, water was added with stirring 2L, solid precipitated, stirred for 45 minutes at room temperature, suction filtered, the filter cake was washed with water, 60 ° C and dried in vacuo 12 hours to give a pale yellow solid

(1), 317.2g, 93.4% yield, m.p. 135 ~ 136 ° C.

The reaction equation is as follows:

A Axitinib intermediate (1) in the preparation for the Nepalese Asif applications, including the following synthetic steps:

Synthesis (1) (E) -6- nitro-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

A 5L reaction flask was added DMF (2L), followed by addition of the intermediate (1) (312.0g, 0.84mol), 2- vinylpyridine (127.5g, 1.21mol), N, N- diisopropylethylamine ( 205.3g, 1.59mol), tri-o-tolylphosphine (22.3g, 0.073mol) and palladium chloride (4.9g, 0.028mol), nitrogen, and heated to 100 ° C for 12 hours to complete the reaction, cooled to 45 ° C, isopropanol was added 1L, stirring at 45 ° C for 30 minutes, diluted with water and 5L, stirring at room temperature for I h, suction filtered, washed with water, isopropanol was added to the filter cake 1.2L, stirred at 55 ° C for 30 minutes, then stirred at room temperature for 30 minutes, suction filtered, the filter cake washed with cold isopropanol, 50 ° C and dried under vacuum for 12 hours to give (E) -6- nitro-3- [2- (pyridin _2 _-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole, 275.3g, 94.0% yield, m.p. 175 ~ 176 ^, ¾ NMR (DMSO-Cl6): δ 1.63-1.64 (m, 2H, H4 ‘, H5’), 1.79-1.81 (m, 1H, H5 ‘), 2.05-2.07 (m, 2H, H3’, H4 ‘), 2.44-2.50 (m, 1H , H3 ‘), 3.86-3.90 (m, 2H, H6’), 6.15-6.18 (dd, 1H, H2 ‘), 7.30-7.33 (dd, 1H, pyridine H5), 7.65-7.69 (d, 1H, J = 16Hz, vinyl H2), 7.72-7.74 (d, 1H, pyridine H4), 7.82-7.86 (m, 1H, pyridine H3), 7.96-8.00 (d, 1H, J = 16Hz, vinyl HI), 8.07 -8.10 (dd, 1H, H4), 8.44-8.46 (d, 1H, H5), 8.63-8.64 (d, 1H, pyridine H6), 8.77-8.78 (d, 1H, H7);

The reaction equation is as follows:

Synthesis of (2) (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole

5L reaction flask in ethanol HOOmLdjC 1000mL and ammonium chloride (300.0g, 5.61mol), was dissolved with stirring, followed by addition of (E) -6- nitro-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (255.0g, 0.73mol), was added iron powder (162.6g, 2.91mol), heated to 50 ° C the reaction was stirred for 2 hours to completion of the reaction, was cooled to 22 ° C, tetrahydrofuran 2L, stirred for I hour at room temperature, filtered through Celite, the filter cake washed with tetrahydrofuran and the filtrate was rotary evaporated to dryness, cooled to room temperature, water was added 2L, stirred for I hour at room temperature, pumping filtered, the filter cake washed with petroleum ether, 50 ° C and dried under vacuum for 12 hours to give a pale yellow solid 206.5g, (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole, yield 88.6%, m.p. 162 ~ 164 ° C / H NMR (CDCl3): δ 1.63-1.77 (m, 2H, H4 ‘, H5 ‘), 2.02-2.06 (m, 1H, H5’), 2.17-2.18 (m, 1H, H4 ‘), 2.55-2.60 (m, 1H, H3’) 3.70-3.72 (m, 2H, H3 ‘, H6 ‘), 3.91 (s, 2H, NH2), 4.04-4.07 (m, 1H, H6’), 5.57-5.60 (dd, 1H, H2 ‘), 6.64-6.66 (dd, 1H, H5), 6.74-6.75 (d, 1H, H7), 7.13-7.16 (dd, 1H, pyridine H5), 7.48-7.50 (d, 1H, pyridine H4), 7.49-7.53 (d, 1H, J = 16Hz, vinyl H2), 7.64 -7.68 (m, 1H, pyridine H3), 7.78-7.82 (d, 1H, J = 16Hz, vinyl Hl), 7.82-7.83 (d, 1H, H4), 8.60-8.61 (d, 1H, pyridine H6) ;

The reaction equation is as follows:

Synthesis of (3) (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1Η- indazole

A 5L reaction flask was added 600mL of water and sodium nitrite (70.2g, 1.02mol), stirred and dissolved, and cooled to (TC, (E) -6- amino-3- [2- (pyridin-2-yl) ethenyl ] -1- (tetrahydro -2H- pyran-2-yl) -1H- indazole (200.0g,

0.62mol) was dissolved in glacial acetic acid 1.3L, dropwise added to the system dropwise over I hour, added dropwise to maintain the internal temperature process 0 ° C, the same temperature for I h, HCl solution was added dropwise at O ​​° C (112mL of concentrated hydrochloric acid , water 200mL), was added dropwise for 10 minutes, with the temperature for I h, TLC plate tracking point diazonium salt formation reaction (PE: EA = 1: 1). Solution of methylene chloride at 0 ° C and 800mL, dropping time of 5 minutes, potassium iodide (207.3g, l.25mol) and iodine (79.2g, 0.31mol) dissolved in water 600mL, at (TC dropwise added to the system, same temperature for 2 hours to complete the reaction. The reaction system was poured into a mixture of 20% sodium thiosulfate solution (2L) and dichloromethane SOOmL and stirred, layers were separated, the aqueous phase was extracted with dichloromethane frozen (2x800mL ), methylene chloride phases were combined burning, 3M sodium hydroxide solution was added dropwise 3.5L, adjust the aqueous phase pH = 9 ~ 12, and water was added ammonia 200mL 400mL, stirred for 30 minutes, separated and the aqueous phase extracted with dichloromethane ( 2×1.2L), the organic phases were combined, rotary evaporated to dryness, and purified through silica gel to give (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro -2H – pyran-2-yl) -1H- indazole, 176.0g, 65.4% yield, m.p. 142 ~ 143 ° C, 1H NMR (DMS0_d6): δ 1.58-1.61 (m, 2H, H4 ‘, H5,) 1.72-1.78 (m, 1H, H5,), 1.97-2.04 (m, 2H, H3,, H4,), 2.38-2.44 (m, 1H, H3,), 3.79-3.81 (m, 1H, H6,) , 3.88-3.90 (m, 1H, H6,), 5.91-5.94 (dd, 1H, H2,),

7.29-7.31 (m, 1H, pyridine H5), 7.56-7.60 (d, 1H ,, J = 16Hz, vinyl H2), 7.57-7.59 (m, 1H, pyridine H4), 7.69-7.71 (d, 1H, pyridine H3), 7.80-7.84 (m, 1H, H4), 7.89-7.93 (d, 1H, J = 16Hz, vinyl HI), 8.01-8.03 (d, 1H, H5), 8.25 (s, 1H, H7 ), 8.61-8.62 (d, 1H, pyridine H6); reaction equation is as follows:

(4) (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) _1_ (tetrahydro -2H- pyran-2-yl) -1H- indazole 6-ylthio} benzamide]

A 5L reaction flask was added DMF (1750mL) and (E) -6- iodo-3- [2- (pyridin-2-yl) ethenyl] -1- (tetrahydro-pyran-2-yl -2H-) -1H- indazole (175.0g, 0.41mol), nitrogen, was added [1, I, – bis (diphenylphosphino) ferrocene] dichloropalladium dichloromethane complex (14.9g, 0.018mmol ), cesium carbonate (198.3g, 0.61mol) and dichloromethane 20mL, was added 2-mercapto -N- methylbenzamide (84.9g, 0.5Imol), heated to 80 ° C for 16 hours to complete the reaction, spin distilled was removed DMF, cooled to room temperature, ethyl acetate was added 3L, water 4L, stirred for 40 minutes, the organic phase was separated, washed with brine, layered, dried over sodium sulfate, filtered, and rotary evaporated to dryness, to give (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) -1- (tetrahydro -2H- pyran-2-yl) -1H- indazol-6-yl] thio } benzamide, 165.6g, a yield of 86.7%, the melting point of 142 ~ 143 ° C;

The reaction equation is as follows:

(5) Synthesis of axitinib

In a 2L reaction flask was added (E) -N- methyl-2 – {[3- (2- (pyridin-2-yl) ethenyl) _1_ (tetrahydro -2H- pyran-2-yl) -1H – indazol-6-yl] thio} benzamide (150.0g, 0.32mol), p-toluenesulfonic acid monohydrate (303.2g, 1.59mol), methanol (800mL) and water (150mL), nitrogen, heated to 65 ° C for 4 hours, spin evaporated to dryness and ethanol (800mL), 65 ° C was stirred for I hour, the ethanol was removed by rotary evaporation, then repeated three times, TLC spot plate tracking reaction (petroleum ether: ethyl acetate = 1: 1). Completion of the reaction, cooled to room temperature, rotary evaporated to dryness, water was added 500mL, stirred for I h, filtered, and the filter cake was washed with methanol and ice, and then added to the reaction vessel, ethyl acetate was added 450mL, stirred at 65 ° C 30 minutes. cooled to room temperature, suction filtered, the filter cake washed with ethyl acetate and freeze paint, water paint, 50 ° C and dried under vacuum for 12 hours to give a white solid 117.5g, Axitinib (II),

yield 95.4%, HPLC purity 98.8 % / H NMR (DMS0_d6): δ 2.78 (d, 3H, CH3), 7.05 (dd, 1H), 7.19 (dd, 1H), 7.36-7.23 (m, 3H), 7.50 (dd, 1H), 7.58 ( d, 1H), 7.61 (s, 1H), 7.66 (d, 1H), 7.85-7.76 (m, 1H), 7.96 (d, 1H, J = 16Hz), 8.21 (d, 1H), 8.39 (q, 1H), 8.61 (d, 1H), 13.35 (s, 1H).

The reaction equation is as follows:

SEE NMR……….

http://orgspectroscopyint.blogspot.in/2015/07/axitinib.html

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http://dmd.aspetjournals.org/content/suppl/2014/03/07/dmd.113.056531.DC1/Supplemental__Data_Figures_56531.pdf

MASS

References

(R)-N4-[3-Chloro-4-(thiazol-2-ylmethoxy)-phenyl]-N6-(4-methyl-4,5-dihydro-oxazol-2-yl)-quinazoline-4,6-diamine

ASLAN001 , Varlitinib

C22H19ClN6O2S

Molecular Weight: 466.94

Elemental Analysis: C, 56.59; H, 4.10; Cl, 7.59; N, 18.00; O, 6.85; S, 6.87

CAS: 845272-21-1 (Varlitinib); 1146629-86-8 (Varlitinib tosylate).

ASLAN001; ASLAN-001; ASLAN 001; AR 00334543; ARRY-334543; ARRY334543; ARRY-543; ARRY543; ARRY 543.

(R)-N4-(3-chloro-4-(thiazol-2-ylmethoxy)phenyl)-N6-(4-methyl-4,5-dihydrooxazol-2-yl)quinazoline-4,6-diamine.

(R)-4-[[3-Chloro-4-[(thiazol-2-yl)methoxy]phenyl]amino]-6-[(4-methyl-4,5-dihydrooxazol-2-yl)amino]quinazoline

4,​6-​Quinazolinediamine, N4-​[3-​chloro-​4-​(2-​thiazolylmethoxy)​phenyl]​-​N6-​[(4R)​-​4,​5-​dihydro-​4-​methyl-​2-​oxazolyl]​-

ASLAN Pharmaceuticals, a Singapore-based drugmaker, announced The Food and Drug Administration (FDA) gave an orphan drug designation on August 13 to its pan-HER inhibitor ASLAN001 (varlitinib), a drug candidate created to treat a destructive form of bile duct cancer called cholangiocarcinoma that has no known cure.  ………http://www.dddmag.com/news/2015/08/aslan-pharmaceuticals-gains-orphan-designation-rare-cancer-drug

Current developer: Array Biopharma Inc,

Varlitinib, also known as ARRY-543 and ASLAN001, is an orally bioavailable inhibitor of the epidermal growth factor receptor family with potential antineoplastic activity.

Varlitinib (ASLAN-001) is an oncolytic drug in phase II clinical trials at ASLAN Pharmaceuticals for the treatment of gastric cancer and for the treatment of metastatic breast cancer in combination with capecitabine. Clinical development is also ongoing for the treatment of solid tumors in combination with cisplatin/FU and cisplatin/capecitabine. The product had been in phase I/II clinical trials at Array BioPharma for the treatment of patients with advanced pancreatic cancer. Phase II clinical trials had also been ongoing for the treatment of solid tumors. No recent development has been reported for this research

Varlitinib selectively and reversibly binds to both EGFR (ErbB-1) and Her-2/neu (ErbB-2) and prevents their phosphorylation and activation, which may result in inhibition of the associated signal transduction pathways, inhibition of cellular proliferation and cell death. EGFR and Her-2 play important roles in cell proliferation and differentiation and are upregulated in various human tumor cell types. Due to the dual inhibition of both EGFR and Her-2, this agent may be therapeutically more effective than agents that inhibit EGFR or Her-2 alone.

The drug is a dual inhibitor of the ErB-2 and EGFR receptor kinases, both of which have been shown to stimulate aberrant growth, prolong survival and promote differentiation of many tumor types. The compound behaves as a reversible ATP-competitive inhibitor with nanomolar potency both in vitro and in cell-based proliferation assays.

In 2011, the compound was licensed to Aslan Pharmaceuticals by Array BioPharma worldwide for the treatment of solid tumors, initially targeting patients with gastric cancer through a development program conducted in Asia.

In 2015, orphan drug designation was assigned to the compound in the U.S. for the treatment of cholangiocarcinoma.

SEE NMR ………….http://www.medkoo.com/Product-Data/Varlitinib/Varlitinib-QC-KB20121128web.pdf

……………..

https://www.google.co.in/patents/US20050043334

Example 52

(R)-N4-[3-Chloro-4-(thiazol-2-ylmethoxy)-phenyl]-N6-(4-methyl-4,5-dihydro-oxazol-2-yl)-quinazoline-4,6-diamine

Prepared using (R)-2-aminopropan-1-o1. MS APCI (+) m/z 467, 469 (M+1, Cl pattern) detected; ¹H NMR (400 mHz, DMSO-D6) δ 9.53 (s, 1H), 8.47 (s, 1H), 8.09 (s, 1H), 7.86 (d, 1H), 7.81 (d, 1H), 7.77 (d, 1H), 7.69 (m, 3H), 7.32 (d, 1H), 7.02 (s, 1H), 5.54 (s, 2H), 4.47 (m, 1H), 3.99 (m, 1H), 3.90 (m, 1H), 1.18 (d, 3H).

Example 53

(S)-N4-[3-Chloro-4-(thiazol-2-ylmethoxy)-phenyl]-N6-(4-methyl-4,5-dihydro-oxazol-2-yl)-quinazoline-4,6-diamine

Prepared using (S)-2-amino-propan-1-o1. MS APCI (+) m/z 467, 469 (M+1, Cl pattern) detected; ¹H NMR (400 mHz, DMSO-D6) δ 9.53 (s, 1H), 8.47 (s, 1H), 8.09 (s, 1H), 7.86 (d, 1H), 7.81 (d, 1H), 7.77 (d, 1H), 7.69 (m, 3H), 7.32 (d, 1H), 7.02 (s, 1H), 5.54 (s, 2H), 4.47 (m, 1H), 3.99 (m, 1H), 3.90 (m, 1H), 1.18 (d, 3H).

………………

PATENT

http://www.google.co.in/patents/WO2005016346A1?cl=en

Example 52

R VN4-r3-Chloro-4-(^‘thiazol-2-v-metho-xy)-phenyll-N6-(4-methyl-4,5-dihvdro-oxazol- 2-yl)-quinazoUne-4,6-diamine

[00194] Prepared using (R)-2-aminopropan- 1 -ol. MS APCI (+) m/z 467, 469

(M+l, CI pattern) detected; 1H NMR (400 mHz, DMSO-D6) δ 9.53 (s, IH), 8.47 (s, IH), 8.09 (s, IH), 7.86 (d, IH), 7.81 (d, IH), 7.77 (d, IH), 7.69 (m, 3H), 7.32 (d, IH), 7.02 (s, IH), 5.54 (s, 2H), 4.47 (m, IH), 3.99 (m, IH), 3.90 (m, IH), 1.18 (d, 3H). Example 53

(S)-N4-|^“3-Chloro-4- thiazol-2-ylmethoxy)-phenyll-N6-(^‘4-methyl-4,5-dihvdro-oxazol- 2-yl)-quinazoline-4,6-diamine [00195] Prepared using (S)-2-amino-propan- 1 -ol. MS APCI (+) m z 467, 469

(M+l, CI pattern) detected; 1H NMR (400 mHz, DMSO-D6) δ 9.53 (s, IH), 8.47 (s, IH), 8.09 (s, IH), 7.86 (d, IH), 7.81 (d, IH), 7.77 (d, IH), 7.69 (m, 3H), 7.32 (d, IH), 7.02 (s, IH), 5.54 (s, 2H), 4.47 (m, IH), 3.99 (m, IH), 3.90 (m, IH), 1.18 (d, 3H).

………

CAUTION a very similar molecule but not same 

NOTE……..METHYL NEXT TO OXYGEN ATOM

A novel type of quinazoline derivatives, which were designed by the combination of quinazoline as the backbone and oxazole scaffold as the substituent, have been synthesized and their biological activities were evaluated for anti-proliferative activities and EGFR inhibitory potency. Compound 12b demonstrated the most potent inhibitory activity (IC₅₀=0.95 µmol/L for EGFR), which could be optimized as a potential EGFR inhibitor in the further study. The structures of the synthesized quinazoline analogs and all intermediates were comfirmed by ¹H and ¹³C NMR, 2D NMR spectra, IR spectra and MS spectra.

12c: Employing the same method as above, compound 12c was prepared and the amino alcohol was (S)-2-amino-propan-1-ol. Yellow solid, yield 52 %. m.p. 243-244 °C; [α] 20D =﹢22.5 ° (c 1.0, CH3CN); 1 H NMR (DMSO-D6): δ 9.54 (s, 1 H), 8.46 (s, 1 H), 8.06 (s, 2 H), 7.85 (d, 2 H, J=3.3 Hz), 7.79 (d, 2 H, J=3.3 Hz), 7.75 (d, 1 H, J=8.9 Hz), 7.64 (d, 1 H, J=8.3 Hz), 7.30 (d, 1 H, J=9.0 Hz), 5.54 (s, 2 H), 4.76 (m, 1 H), 3.72 (s, 1 H), 3.19 (s, 1 H), 1.34 (d, 3 H, J=6.15 Hz). 13C NMR (DMSO-D6) δ: 165.8, 156.9, 152.0, 148.8, 145.3, 142.6, 134.3, 128.7, 128.0, 123.5, 121.7, 121.3, 121.0, 115.6, 114.6, 72.5, 67.7, 63.0, 29.8, 29.0, 20.0, 13.9. IR (KBr) ν: 3439, 3278, 3101, 2925, 1660, 1631, 1601, 1557, 1500, 1428, 1404, 1384, 1329, 1291, 1257, 1225, 1052 cm-1. Anal. calcd for C22H19N6O2SCl: C 55.59, H 4.10, N 18.00, O 6.85; found C 55.55, H 4.13, N 18.02, O 6.78; MS (ESI) m/z: 467.2 (M+H).

12d: Employing the same method as above, compound 12d was prepared and the amino alcohol was (R)-2-amino-propan-1-ol. Yellow solid, yield 60%. m.p. 242-243 °C; [α] 20D = ﹣22.3 ° (c 1.0, CH3CN); 1 H NMR (DMSO-D6): δ 9.52 (s, 1 H), 8.80 (s, 1 H), 8.52 (dd, 1 H, J=2.7 Hz, J=8.9 Hz), 8.45 (s, 1 H), 8.30 (s, 1 H), 8.07 (s, 1 H), 7.85 (d, 1 H, J=3.2 Hz), 7.79 (d, 1 H, J=3.2 Hz), 7.75 (s, 1 H), 7.63 (d, 1 H, J=8.2 Hz), 7.31 (d, 1 H, J=9.0 Hz), 5.53 (s, 2 H), 4.76 (m, 1 H), 3.81 (s, 1 H), 3.19 (s, 1 H), 1.34 (d, 3 H, J=6.2 Hz). 13C NMR (DMSO-D6) δ: 165.8, 156.9, 152.0, 148.8, 145.3, 142.6, 134.3, 128.7, 128.0, 123.5, 121.7, 121.3, 121.0, 115.6, 114.6, 72.5, 67.7, 63.0, 29.8, 29.0, 20.0, 13.9. IR (KBr) ν: 3439, 3278, 3101, 2925, 1660, 1631, 1601, 1557, 1500, 1428, 1404, 1384, 1329, 1291, 1257, 1225, 1052 cm-1. Anal. calcd for C22H19N6O2SCl: C 55.59, H 4.10, N 18.00, O 6.85; found C 55.55, H 4.13, N 18.02, O 6.78; MS (ESI) m/z: 467.20 (M+H).

The above paper allows you to synthesize the key amino int 11 ………N4-(3-chloro-4-(thiazol-2-ylmethoxy)phenyl)quinazoline-4,6-diamine (11)

this can be applied to varlitinib till int  11

6-Nitro-4-hydroxyquinazoline (3)

2-amino-5-nitrobenzoic acid (5.46 g, 30 mmol) was added to a 250 mL flask equipped with a reflux condenser. Then 50 mL formamide was added. The mixture was heated with vigorous stirring at 160 °C for 3 h. After cooling the solution was poured in ice-water to give 3 in almost pure form (Yellow solid 4.70 g, yield 82.0%). m.p. 317-318 °C; 1 H NMR (DMSO-d6): δ 12.74 (1 H, s, OH, exchangeable), 8.78 (1 H, d, J=2.4 Hz), 8.53 (1 H, dd, J=2.6 Hz, 9.0 Hz), 8.30 (s, 1 H), 7.84 (1 H, d, J=9.0 Hz); 13C NMR (DMSO-d6) δ: 160.1, 152.9, 148.9, 145.0, 129.1, 128.3, 122.7, 121.9. IR (KBr) ν: 3172, 3046, 2879, 1674, 1615, 1577, 1514, 1491, 1469, 1343, 1289, 1242, 1167, 1112, 928, 920, 901, 803, 753, 630, 574, 531 cm-1. Anal. calcd for C8H5N3O3: C 50.27, H 2.64, N 21.98; found C 50.30, H 2.65, N 21.96; MS (ESI) m/z: 189.97 (M-H).

13C NMR OF 3 IN DMSOD6

IR

4-chloro-6-Nitroquinazoline (4)

In a 100 mL flask equipped with a reflux condenser, 6-nitroquinazolin-4-one (2.86 g, 15 mmol) and thionyl chloride (SOCl2) 25 mL were added. The mixture was heated under reflux with vigorous stirring for 2 h. After the solution was clear, the reaction mixture was heated for another 2 h. Then, 150 mL of ice MeOH was dropped into it carefully, the mixture was extracted with CH2Cl2. The organic layer was S3 dried under MgSO4, filtered and the solvent removed to give 4-chloro-6-nitroquinazoline (4). Yellow solid 2.45 g, yield 78%. m.p. 134-135 °C; 1 H NMR (DMSO-d6): δ 8.80 (1 H, d, J=3.0 Hz), 8.54(1 H, dd, J=2.7 Hz, 9.0 Hz), 8.35(s, 1 H), 7.87 (1 H, d, J= 9.0 Hz); 13C NMR (DMSO-d6) δ: 160.0, 152.5, 149.1, 145.1, 128.7, 128.4, 122.7, 122.0. IR (KBr) ν: 3431, 3082, 3038, 2664, 2613, 2567, 1724, 1685, 1676, 1646, 1617, 1578, 1526, 1468, 1359, 1346, 1269 cm-1. Anal. calcd for C8H4N3O2Cl: C 45.84, H 1.92, N 20.05, O 15.27; found C 45.81, H 1.97, N 20.02, O 15.21; MS (ESI) m/z: 207.96 (M-H).

4 nmr dmsod6

13C NMR OF4 IN DMSOD6

IR

Thiazol-2-yl-methano1 (6)

Sodium borohydride (16.0 g, 140 mmol) was added to a stirred solution of thiazole-2-carbaldehyde (24.2 g, 214 mmol) in MeOH (400 mL) at 0 °C . The reaction mixture was warmed to room temperature. After 1 hour, the reaction mixture was quenched by the addition of water and the organics were removed by concentration. The resulting aqueous mixture was extracted with EtOAc. The combined organic extracts were dried under Na2SO4 and concentrated to give thiazol-2-yl-methano1 (23.39 g, 95%). bp:75-76 °C (0.2 mmHg) [lit.[19] bp:70-80 °C (0.2 mmHg)]; m. p. 63-64 °C. 1 H NMR (CDCl3) δ 4.91 (s, 2 H), 5.1(br, l H), 7.28(d, 1 H, J=3.2 Hz), 7.68 (d, 1 H, J=2.9 Hz). IR (KBr) ν: 3135, 3099, 3082, 2814, 1509, 1446, 1351, 1189, 1149, 1073, 1050, 977, 775, 745, 613, 603 cm-1. Anal. calcd for C4H5NOS: C 41.72, H 4.38, N 12.16; found C 41.74, H 4.33, N 12.18; MS (ESI) m/z: 116.11 (M+H).

6 in dmsod6 1H NMR

2-((2-Chloro-4-nitrophenoxy)methyl)thiazole (8)

2-(2-chloro-4-nitro-phenoxymethy1)-thiazole was prepared by adding thiazol-2-yl-methanol (5.48 g, 47.65 mmol) to a slurry of sodium hydride (2.42 g of a 60% dispersion in oil, 60.5 mmol) in THF (50 ml) at 0 °C After several minutes, 2-chloro-1-fluoro- 4-nitro-benzene (7.58 g, 43.60 mmol) was added and the reaction mixture warmed to room temperature. The reaction mixture was stirred at room temperature for 3 h, and 60 °C for 16 h. After cooling to room temperature, the reaction mixture was poured into 300 mL water. The resulting precipitate was collected by filtration, washed with water, and dried in vacuo to give 2-(2- chloro-4-nitrophenoxymethy1)-thiazole (11.06 g, 86%) which was used in next step without further purification. m.p. 170-171 °C; 1 H NMR (DMSO-d6): δ 8.35 (1 H, d, J=2.8 Hz), 8.25 (1 H, dd, J=2.8 Hz, 9.15 Hz), 7.87 (1 H, d, J=3.3 Hz), 7.83(1 H, d, J=3.3 Hz), 7.54 (1 H, d, J=9.2 Hz), 5.73(s, 1 H); 13C NMR (DMSO-d6) δ: 164.2, 158.5, 143.2, 141.7, 125.9, 124.9, 122.4, 122.2, 114.6, 68.4; IR (KBr) ν: 3112, 3009, 1587, 1509, 1500, 1354, 1319, 1284, 1255, 1154, 1125, 1054, 1006, 894, 780, 746, 728 cm-1. Anal. calcd for C10H7N2O3SCl: C 44.37, H 2.61, N 10.35, O 17.73; found C 44.31, H 2.67, N 10.29; MS (ESI) m/z: 268.89 (M-H).

1H NMR 8 DMSOD6

13C NMR OF 8 IN DMSOD6

3-Chloro-4-(thiazol-2-ylmethoxy)aniline (9)

In a flask equipped with a reflux condenser, the compound 8 15.00 g (55.6 mmol), reduced zinc powder 14.44 g (222.0 mmo1, 4 eq), saturated ammonia chloride (5 mL) and methanol (100 mL) were mixed. The mixture was stirred at a temperature of 40 °C for 1.5 h. Then the zinc powder was filtered off, the filtrate was concentrated to obtain yellow solid 13.21 g, yield 99%. m.p. 60-61 °C; 1 H NMR (DMSO-d6): δ 7.80 (1 H, d, J=3.3 Hz), 7.75 (1 H, d, J=3.3 Hz), 6.96 (1 H, d, J=8.8 Hz), 6.64(1 H, d, J=2.7 Hz), 6.46 (1 H, dd, J=2.7 Hz, J=8.7 Hz), 5.30 (s, 2 H), 5.04 (s, 2 H, NH2, exchangeable); 13C NMR (DMSO-d6) δ: 166.8, 145.1, 144.1, 142.80, 123.1, 121.5, 117.7, 115.2, 113.6, 69.1. IR (KBr) ν: 3322, 3192, 3112, 1607, 1499, 1457, 1436, 1291, 1274, 1221, 1191, 1144, 1057, 1027, 857, 797, 767, 733, 584 cm-1. Anal. calcd for C10H9N2OSCl: C 49.90, H 3.77, N 11.64, O 6.65; found C 49.95, H 3.76, N 11.66, O 6.60; MS (ESI) m/z: 239.01 (M-H).

1H NMR DMSOD6 OF 9

13C NMR OF 9 IN DMSOD6

N-(3-chloro-4-(thiazol-2-ylmethoxy)phenyl)-6-nitro- quinazolin-4-amine（10）

In a flask equipped with a reflux condenser, 6-nitro-4-chloro- quinazoline 8.0 g (38.3 mmol) and 3-Chloro-4-(thiazol-2-ylmethoxy)aniline 8.9 g (37.0 mmol) were dissolved into 150 mL of THF, and the solution was refluxed for 3 h.Then a lot of yellow solid was deposited. Then it was filtered affording to yellow solid 12.8 g, yield 81%. m.p. 183-184 °C (decompose); 1 H NMR (DMSO-d6): δ 11.97(s, 1 H, exchangeable), 9.84 (s, 1 H), 9.00 (s, 1 H), 8.76 (1 H, d, J=9.1 Hz), 8.12-8.14 (m, 1 H), 7.94 (1 H, d, J=2.3 Hz), 7.87 (1 H, d, J=3.2 Hz), 7.81 (1 H, d, J=3.2 Hz), 7.44 (1 H, d, J=9.0 Hz), 7.69 (1 H, dd, J=2.5 Hz, J=8.9 Hz), 5.61 (s, 2 H); 13C NMR (DMSO-d6) δ: 166.8, 145.1, 144.1, 142.8, 123.1, 121.5, 117.7, 115.2, 113.7, 69.1. IR (KBr) ν: 3442, 3100, 1636, 1618, 1570, 1552, 1523, 1492, 1442, 1400, 1377, 1344, 1301, 1267, 1069, 805 cm-1. Anal. calcd for C18H12N5O3SCl: C 52.24, H 2.92, N 16.92, O 11.60; found C 52.26, H 2.93, N 16.96, O 11.58; MS (ESI) m/z: 412.84 (M-H).

1H NMR DMSOD6 OF 10

13C NMR OF 10 IN DMSOD6

N4-(3-chloro-4-(thiazol-2-ylmethoxy)phenyl)quinazoline-4,6-diamine (11)

In a flask equipped with a reflux condenser, the compound 10 5.00 g (12.1 mmol), reduced zinc powder 3.2 g (48.5 mmo1, 4 eq), saturated ammonia chloride (3 mL) and methanol (60 mL) were mixed. The mixture was stirred at room temperature for 30 min. Then the zinc powder was filtered off, the filtrate was concentrated to obtain yellow solid 4.58 g, yield 98%. m.p. 197-198 °C (decompose); 1 H S4 NMR (DMSO-d6): δ 9.33(s, 1 H, exchangeable), 8.31 (s, 1 H), 8.05 (d, 1 H, J=2.6 Hz), 7.85 (d, 1 H, J=3.3 Hz), 7.79 (1 H, d, J=3.3 Hz), 7.73 (1 H, dd, J=2.5 Hz, J=9.0 Hz), 7.51 (1 H, d, J=8.9 Hz), 7.30 (1 H, d, J=2.4 Hz), 7.29 (1 H, d, J=4.7 Hz), 7.23 (1 H, dd, J=2.3 Hz, J=8.9 Hz), 5.57 (s, 2 H, exchangeable), 5.52 (s, 2 H); 13C NMR (DMSO-d6) δ: 165.9, 155.8, 149.7, 148.5, 147.3, 142.6, 142.5, 134.6, 128.7, 123.6, 123.2, 121.4, 121.3, 121.1, 116.5, 114. 7, 100.9, 67.8. IR (KBr) ν: 3443, 3358, 3211, 3100, 1631, 1596, 1577, 1560, 1530, 1494, 1431, 1383, 1217, 910 cm-1. Anal. calcd for C18H14N5OSCl: C 56.32, H 3.68, N 18.24, O 4.17; found C 56.34, H 3.70, N 18.22, O 4.14; MS (ESI) m/z: 382.66 (M-H).

11 1HNMR DMSOD6

13C NMR OF 11 IN DMSOD6

Construction finally as per patent ……….US20050043334

Treatment of N4-[3-chloro-4-(thiazol-2-ylmethoxy)phenyl]quinazoline-4,6-diamine (11) with 1,1′-thiocarbonyldiimidazole , followed by condensation with 2(R)-amino-1-propanol  in THF/CH2Cl2 affords thiourea derivative , which finally undergoes cyclization in the presence of TsCl and NaOH in THF/H2O to furnish varlitinib .

1. ASLAN Pharmaceuticals
2. Address: 10 Bukit Pasoh Rd, Singapore 089824

   Phone:+65 6222 4235

carl fith

Mr Carl Firth, CEO, Aslan Pharmaceuticals, Singapore (left) and Mr Dan Devine, CEO, Patrys, Australia (right)

///////ASLAN001, varlitinib, ASLAN Pharmaceuticals,  Orphan Designation, ARRY-534, ARRY-334543 , PHASE 2, ORPHAN DRUG DESIGNATION, array

Millennium Pharmaceuticals, Inc. INNOVATOR

Millennium Pharmaceuticals, Inc., a subsidiary of Takeda Pharmaceutical Company Limited,

MLN4924, MLN 4924-003, TAK-924

905579-51-3 BASE

1160295-21-5 HcL

A potent and selective inhibitor of NAE. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. The ubiquitin-proteasome pathway mediates the destruction of unwanted proteins.

(((1S,2S,4R)-4-{4-[(S)-2,3-Dihydro-1H-inden-1-ylamino]-7H-pyrrolo[2,3-d]pyrimidin-7-yl}-2-hydroxycyclopentyl)methyl sulfamate hydrochloride) (pevonedistat), a novel NEDD8-activating enzyme (NAE) inhibitor, has demonstrated in vitro cytotoxic activity against a variety of human malignancies and is currently being developed by Takeda Pharmaceuticals Company Limited as a clinical candidate for the treatment of cancer

In 2011, orphan drug designation was assigned to MLN-4924 for the treatment of MDS and for the treatment of acute myelogenous leukemia.

PHASE 1…….CANCER SOLID TUMOR

………………….

PATENT

http://www.google.com/patents/US20120330013

preparing a compound represented by the following formula 1 by reacting the compound of formula 11 with TFA (step 9):

The retrosynthetic analysis of MLN4924 (1), as the final desired nucleoside, is shown in the following.

MLN 4924 (1) can be synthesized by condensing cyclic sulfate 3 as the glycosyl donor with a purine base. The glycosyl donor 3 can be produced from diol 4, which in turn can be obtained from cyclopentanone 5 via a stereoselective reduction and a regioselective cleavage of the isopropylidene moiety. The cyclopentanone 5 can be synthesized from cyclopentenone 6 by stereoselective reduction. The intermediate cyclopentenone 6 can be easily derived from D-ribose according to our previously published procedure (Jeong, L. S. et al., J. Org. Chem. 2004, 69, 2634-2636).

The synthetic route for the glycosyl donor 3 is shown in the following scheme 1.

Example 1 Preparation of MLN4924 Step 1: Preparation of 6-(tert-butyl-diphenyl-silanyloxymethyl)-2,2-dimethyl-tetrahydro-cyclopenta[1,3]dioxol-4-one (Compound 5)

To a suspension of the compound 6 (20.0 g, 47.1 mmol) in methanol (400 ml) was added 10% palladium on activated carbon (1.0 g), and the mixture was stirred at room temperature overnight under H₂ atmosphere. After filtration of the reaction mixture, the solvent was removed and the residue was dissolved in methylene chloride and then filtered through short pad silica gel. Then, the solvent was evaporated to give the compound 5 (20.1 g, 100%) as a colorless syrup.

[α]²⁰ _D −28.32 (c 1.49, MeOH); HR-MS (ESI): m/z calcd for C₂₅H₃₂NaO₄Si [M+Na]⁺ 447.1968, Found 447.1956; ¹H NMR (400 MHz, CDCl₃) δ 7.69 (m, 4H), 7.40 (m, 6H), 4.84 (t, J=4.4 Hz, 1H), 4.22 (dd, J=1.2, 4.8 Hz, 1H), 3.96 (dd, J=8.0, 10.0 Hz, 1H), 3.82 (dd, J=6.8, 10.0 Hz, 1H), 2.37 (m, 1H), 2.30 (ddd, J=1.2, 8.4, and 18.4 Hz, 1H), 2.20 (ddd, J=1.2, 12.0, and 18.4 Hz, 1H), 1.37 (s, 3H), 1.35 (s, 3H), 1.06 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 112.6, 80.5, 77.6, 77.2, 76.9, 63.6, 38.1, 36.9, 27.1, 27.02, 27.01, 25.3, 19.5; Anal. Calcd for C₂₅H₃₂O₄Si: C, 70.72; H, 7.60. Found: C, 70.79; H, 7.75.

Step 2: Preparation of 6-(tert-butyl-diphenyl-silanyloxymethyl)-2,2-dimethyl-tetrahydro-cyclopenta[1,3]dioxol-4-ol (Compound 7)

To a suspension of the compound 5 (20.1 g, 47.1 mmol) in methanol (500 ml) were added sodium borohydride (2.17 g, 57.4 mmol) and cerium (III) chloride heptahydrate (21.3 g, 57.2 mmol) at 0° C., and the mixture was stirred at room temperature for 30 min. After the solvent was removed, the residue was partitioned between ethyl acetate and water. The organic layer was then washed with brine, dried with anhydrous MgSO₄, filtered, and evaporated. The residue was purified by silica gel column chromatography (hexane/ethyl acetate=5/1) to give the compound 7 (20.86 g, 98%) as a colorless syrup.

[α]²⁰ _D +34.55 (c 0.55, MeOH); HR-MS (ESI): m/z calcd for C₂₅H₃₄NaO₄Si [M+Na]⁺: 449.2124; Found: 449.2110; ¹H NMR (400 MHz, CDCl₃) δ 7.69 (m, 4H), 7.39 (m, 6H), 4.62 (t, J=5.6 Hz, 1H), 4.44 (t, J=5.6 Hz, 1H), 3.89 (dd, J=6.0, 7.6 Hz, 1H), 3.84 (m, 1H), 3.68 (dd, J=6.4, 10.0 Hz, 1H), 1.91 (m, 2H), 1.26 (m, 1H), 1.42 (s, 3H), 1.33 (s, 3H), 1.05 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 135.9, 135.8, 134.2, 134.1, 129.8, 129.7, 127.8, 127.7, 110.6, 79.4, 78.9, 77.6, 77.2, 76.9, 72.5, 62.9, 41.6, 33.4, 27.0, 25.9, 27.0, 25.9, 24.4, 19.5; Anal. Calcd for C₂₅H₃₄O₄Si: C, 70.38; H, 8.03. Found: C, 70.41; H, 8.08.

Step 3: Preparation of 3-tert-butoxy-4-(tert-butyl-diphenyl-silanyloxymethyl)-cyclopentane-1,2-diol (Compound 4)

To a solution of the compound 7 (20.86 g, 47.12 mmol) in methylene chloride was added trimethylaluminum (2.0 M in toluene, 132.1 ml) at 0° C., and the mixture was stirred at room temperature for 2 days. The mixture was cooled to 0° C., slowly quenched with an aqueous saturated ammonium chloride solution, filtered, and evaporated. The residue was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried with anhydrous MgSO₄, filtered, and evaporated. The residue was purified by silica gel column chromatography (hexane/ethyl acetate=2/1) to give the compound 4 (13.42 g, 62%) as a colorless syrup.

[α]²⁰ _D +3.30 (c 0.55, MeOH); HR-MS (ESI): m/z calcd for C₂₆H₃₈NaO₄Si [M+Na]⁺: 465.2437; Found: 465.2423; ¹H NMR (400 MHz, CDCl₃) δ 7.70 (m, 4H), 7.41 (m, 6H), 4.05 (dd, J=4.4, 7.2 Hz, 1H), 3.93 (m, 1H), 3.72 (m, 2H), 3.59 (dd, J=3.6, 12.0 Hz, 2H), 2.70 (d, J=20.8 Hz, 1H), 2.10 (m, 2H), 1.60 (m, 1H), 1.20 (s, 9H), 1.06 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 135.9, 133.5, 130.0, 129.9, 127.9, 127.9, 77.6, 77.2, 76.9, 74.9, 73.8, 72.7, 72.1, 63.3, 42.1, 34.0, 28.5, 27.0, 19.4; Anal. Calcd for C₂₆H₃₈O₄Si: C, 70.55; H, 8.65. Found: C, 70.61; H, 8.70.

Step 4: Preparation of (4-tert-butoxy-2,2-dioxo-tetrahydro-2-yl-6-cyclopenta[1,3,2]-dioxathiol-5-ylmethoxy)-tert-butyl-diphenyl-silane (Compound 3)

To a solution of the compound 4 (13.42 g, 30.3 mmol) in methylene chloride were added triethyl amine (14.5 ml, 101.0 mmol) and thionyl chloride (3.7 ml, 47.4 mmol) at 0° C., and the reaction mixture was stirred at 0° C. for 10 minutes. The reaction mixture was partitioned between methylene chloride and water. The organic layer was washed with brine, dried with anhydrous MgSO₄, filtered, and evaporated. The residue was purified by silica gel column chromatography (hexane/ethyl acetate=6/1) to give the cyclic sulfite (14.37 g, 97%) as a white foam.

[α]²⁰ _D +20.00 (c 0.05, MeOH); HR-MS (ESI): m/z calcd for C₂₆H₃₆NaO₅SSi [M+Na]⁺: 511.1950; Found: 511.1929; ¹H NMR (400 MHz, CDCl₃) δ 7.64 (m, 4H), 7.40 (m, 6H), 5.23 (m, 1H), 5.04 (dd, J=4.4, 6.0 Hz, 1H), 4.01 (t, J=4.8 Hz, 1H), 3.68 (dd, J=3.6, 10.4 Hz, 1H), 3.56 (dd, J=8.0, 10.4 Hz, 1H), 2.07 (m, 2H), 1.96 (m, 1H), 1.14 (s, 9H), 1.05 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 135.8, 135.7, 133.9, 133.8, 129.9, 129.9, 127.9, 127.8, 85.7, 83.2, 77.6, 77.2, 76.9, 75.0, 71.1, 62.7, 44.7, 31.4, 28.5, 27.1, 19.4; Anal. Calcd for C₂₆H₃₆O₅SSi: C, 63.90; H, 7.42; S, 6.56. Found: C, 63.94; H, 7.45; S, 6.61.

To a solution of the cyclic sulfite obtained above (14.37 g, 29.4 mmol) in the mixture of carbon tetrachloride, acetonitrile and water (1:1:1.5, 210 ml) were added sodium metaperiodate (18.56 g, 56.4 mmol) and ruthenium chloride (1.72 g, 8.25 mmol), and the reaction mixture was stirred at room temperature for 10 minutes. The reaction mixture was partitioned between methylene chloride and water. The organic layer was washed with brine, dried with anhydrous MgSO₄, filtered, and evaporated. The residue was purified by silica gel column chromatography (hexane/ethyl acetate=4/1) to give the compound 3 (13.36 g, 90%) as a white solid.

mp 101-104° C.; [α]²⁰ _D −80.00 (c 0.05, MeOH); HR-MS (ESI): m/z calcd for C₂₆H₃₆NaO₆SSi [M+Na]⁺: 527.1900; Found: 527.1881; ¹H NMR (400 MHz, CDCl₃) δ 7.64 (m, 4H), 7.41 (m, 6H), 5.13 (m, 1H), 4.83 (dd, J=4.4, 6.8 Hz, 1H), 4.13 (t, J=4.0 Hz, 1H), 3.92 (dd, J=6.4, 10.4 Hz, 1H), 3.69 (dd, J=5.2, 10.4 Hz, 1H), 2.11 (m, 2H), 2.02 (m, 1H), 1.15 (s, 9H), 1.05 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 135.7, 135.0, 133.8, 133.7, 130.0, 128.0, 127.9, 83.5, 82.2, 77.6, 77.2, 76.9, 75.4, 70.4, 70.4, 62.2, 43.9, 31.3, 28.2, 27.1, 26.8, 19.4; Anal. Calcd for C₂₆H₃₆O₆SSi: C, 61.87; H, 7.19; S, 6.35. Found: C, 61.91; H, 7.14; S, 6.30.

Step 5: Preparation of 2-tert-butoxy-3-(tert-butyl-diphenyl-silanyloxymethyl)-5-[4-(indan-1-ylamino)-pyrrolo[2,3-d]pyrimidin-7-yl]-cyclopentanol (Compound 8)

A suspension of N⁶-indanyl-7-deazaadenine (8.80 g, 35.2 mmol), sodium hydride (1.38 g, 45.7 mmol) and 18-crown-6 (9.11 g, 45.7 mmol) in THF (200 ml) was stirred at 80° C. To the reaction mixture was added a solution for the compound 3 (13.36 g, 26.5 mmol) in THF (150 ml), and the stirring was continued at 80° C. overnight. The reaction mixture was cooled down to 0° C., and conc. HCl was added slowly until pH reaches 1-2. Then the reaction mixture was further stirred at 80° C. for 2 hours. After neutralized with saturated aqueous NaHCO₃ solution, the reaction mixture was partitioned between ethyl acetate and water. The organic layer was washed with brine, dried with anhydrous MgSO₄, filtered, and evaporated. The residue was purified by silica gel column chromatography (hexane/ethyl acetate=2/1) to give the compound 8 (11.62 g, 65%) as a white foam.

UV (CH₂Cl₂) λ_max 272.5 nm; [α]²⁰ _D −8.89 (c 0.45, MeOH); HR-MS (ESI): m/z calcd for C₄₁H₅₁N₄O₃Si [M+H]⁺: 675.3730; Found: 675.3717; ¹H NMR (400 MHz, CDCl₃) δ 8.38 (s, 1H), 7.70 (m, 4H), 7.41 (m, 6H), 6.92 (d, J=3.6 Hz, 1H), 6.29 (d, J=3.2 Hz, 1H), 5.91 (dd, J=7.6, 14.8 Hz, 1H), 5.14 (br d, J=6.8 Hz, 1H), 4.77 (m, 1H), 4.36 (t, J=6.0 Hz, 1H), 4.22 (dd, J=5.2, 10.8 Hz, 1H), 3.84 (dd, J=5.6, 10.4 Hz, 1H), 3.73 (dd, J=8.4, 10.4 Hz, 1H), 3.37 (d, J=5.6 Hz, 1H), 3.06 (m, 1H), 2.95 (m, 1H), 2.75 (m, 1H), 2.75 (m, 1H), 2.58 (m, 1H), 2.38 (m, 1H), 2.15 (m, 1H), 1.98 (m, 1H), 1.65 (s, 1H), 1.55 (s, 1H), 1.16 (s, 9H), 1.07 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 156.4, 151.8, 150.3, 144.1, 143.8, 135.9, 134.0, 129.9, 128.2, 127.9, 127.9, 127.0, 125.1, 124.4, 123.3, 103.8, 97.4, 77.8, 77.6, 77.2, 76.9, 74.9, 72.4, 63.5, 62.1, 56.3, 43.9, 34.9, 30.5, 30.5, 28.5, 27.2, 19.5; Anal. Calcd for C₄₁H₅₀N₄O₃Si: C, 72.96; H, 7.47; N, 8.30. Found: C, 73.01; H, 7.45; N, 8.36.

Step 6: Preparation of {7-[3-tert-butoxy-4-(tert-butyl-diphenyl-silanyloxymethyl)-cyclopentyl]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}-indan-1-yl-amine (Compound 9)

To a solution of the compound 8 (11.62 g, 17.2 mmol) in methylene chloride (300 ml) were added N,N-dimethylaminopyridine (5.64 g, 51.6 mmol) and phenyl chlorothionocarbonate (4.3 ml, 34.4 mmol), and the reaction mixture was stirred at room temperature overnight. After the solvent was removed, the residue was purified by silica gel column chromatography (hexane/ethyl acetate=6/1) to give the thiocarbonate (13.82 g, 99%) as a white foam.

UV (MeOH) λ_max 271.50 nm; [α]²⁰ _D +10.00 (c 0.15, MeOH); HR-MS (ESI): m/z calcd for C₄₈H₅₅N₄O₄SSi [M+H]⁺: 811.3713; Found: 811.3687; ¹H NMR (400 MHz, CDCl₃) δ 8.36 (s, 1H), 7.61 (dd, J=1.6, 7.6 Hz, 4H), 7.34 (m, 5H), 7.26 (m, 4H), 7.18 (m, 6H), 6.86 (s, 1H), 6.25 (d, J=3.2 Hz, 1H), 6.00 (dd, J=3.2, 8.4 Hz, 1H), 5.83 (d, J=6.8 Hz, 1H), 5.19 (m, 1H), 5.07 (br s, 1H), 4.48 (t, J=3.6 Hz, 1H), 3.82 (dd, J=7.2, 10.4 Hz, 1H), 3.52 (dd, J=7.2, 10.0 Hz, 1H), 2.99 (m, 1H), 2.88 (m, 2H), 2.69 (m, 2H), 2.18 (dd, J=11.2, 13.6 Hz, 1H), 1.94 (m, 2H), 1.12 (s, 9H), 0.98 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 194.9, 153.5, 152.1, 143.9, 135.9, 135.8, 134.1, 129.9, 129.6, 128.3, 127.9, 127.0, 126.7, 125.1, 124.6, 123.2, 122.0, 87.9, 77.6, 77.2, 76.9, 74.6, 70.4, 63.5, 57.3, 42.8, 35.0, 30.7, 30.5, 29.9, 28.7, 27.1, 19.4; Anal. Calcd for C₄₈H₅₄N₄O₄SSi: C, 71.08; H, 6.71; N, 6.91; S, 3.95. Found: C, 71.14; H, 6.75; N, 6.95; S, 4.01.

To a solution of the thiocarbonate obtained above (13.82 g, 17.0 mmol) in toluene (200 ml) were added tri-n-butyltinhydride (9.4 ml, 34.1 mmol) and 2,2′-azo-bis-isobutyronitrile (4.32 g, 26.3 mmol), and the reaction mixture was stirred at 110° C. for 1 hour. After the mixture was cooled down, the solvent was removed. The resulting residue was purified by silica gel column chromatography (hexane/ethyl acetate=3/1) to give the compound 9 (9.21 g, 82%) as a white foam.

UV (MeOH) λ_max 272.50 nm; [α]²⁰ _D −10.00 (c 0.20, MeOH); HR-MS (ESI): m/z calcd for C₄₁H₅₁N₄O₂Si [M+H]⁺: 659.3781; Found: 659.3757; ¹H NMR (400 MHz, CDCl₃) δ 8.41 (s, 1H), 7.69 (m, 4H), 7.41 (m, 6H), 7.29 (m, 2H), 7.23 (m, 2H), 6.92 (d, J=3.6 Hz, 1H), 6.31 (d, J=3.6 Hz, 1H), 5.90 (dd, J=7.2, 14.8 Hz, 1H), 5.38 (m, 1H), 5.15 (br s, 1H), 4.33 (dd, J=5.2, 8.4 Hz, 1H), 3.88 (dd, J=6.4, 10.0 Hz, 1H), 3.68 (dd, J=7.2, 10.4 Hz, 1H), 3.05 (m, 1H), 2.96 (dd, J=7.6, 15.6 Hz, 1H), 2.76 (m, 1H), 2.45 (d, J=5.2 Hz, 1H), 2.29 (m, 2H), 2.06 (m, 1H), 1.95 (m, 2H), 1.55 (s, 1H), 1.13 (s, 9H), 1.06 (s, 9H);¹³C NMR (100 MHz, CDCl₃) δ 156.3, 151.9, 144.1, 143.9, 135.9, 135.8, 134.3, 129.8, 128.2, 127.8, 127.0, 125.1, 124.6, 121.8, 77.6, 77.2, 76.7, 73.5, 72.2, 63.6, 56.4, 52.8, 46.8, 42.8, 34.9, 34.5, 30.5, 28.6, 27.2, 28.7, 19.4; Anal. Calcd for C₄₁H₅₀N₄O₂Si: C, 74.73; H, 7.65; N, 8.30. Found: C, 74.79; H, 7.61; N, 8.25.

Step 7: Preparation of 2-tert-butoxy-4-[4-(indan-1-ylamino)-pyrrolo[2,3-d]pyrimidin-7-yl]-cyclopentanol (Compound 10)

To a solution of the compound 9 (9.21 g, 13.97 mmol) in the mixture of THF and pyridine (1:1, 160 ml) was added dropwise pyridine hydrofluoride (18.42 ml, 190.0 mmol) at 0° C., and the reaction mixture was stirred at room temperature for 1 hour. The mixture was neutralized with saturated aqueous NaHCO₃ solution and partitioned between ethyl acetate and water. The organic layer was washed with brine, dried with anhydrous MgSO₄, filtered, and evaporated. Then, the residue was purified by silica gel column chromatography (hexane/ethyl acetate=1/3) to give the compound 10 (5.63 g, 99%) as a white foam.

UV (MeOH) λ_max 273.00 nm; [α]²⁰ _D −6.36 (c 1.10, MeOH); HR-MS (ESI): m/z calcd for C₂₅H₃₃N₄O₂ [M+H]⁺: 421.2604; Found: 421.2599; ¹H NMR (400 MHz, CDCl₃) δ 8.34 (s, 1H), 7.30 (d, J=7.6 Hz, 1H), 7.22 (d, J=7.2 Hz, 2H), 7.15 (t, J=6.8 Hz, 1H), 6.88 (d, J=3.2 Hz, 1H), 6.23 (d, J=3.6 Hz, 1H), 5.83 (dd, J=7.2, 15.2 Hz, 1H), 5.28 (m, 1H), 5.06 (m, 1H), 4.47 (dd, J=5.6, 10.4 Hz, 1H), 3.78 (m, 1H), 3.70 (m, 1H), 3.24 (t, J=5.2 Hz, 1H), 2.98 (m, 1H), 2.87 (m, 1H), 2.68 (m, 1H), 2.46 (m, 1H), 2.37 (m, 2H), 1.93 (m, 2H), 1.18 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 156.2, 151.8, 147.9, 143.9, 143.9, 128.3, 126.9, 125.1, 124.5, 121.9, 97.7, 77.6, 77.2, 76.9, 75.5, 74.9, 63.4, 56.4, 53.8, 44.2, 42.2, 34.9, 33.2, 30.5, 28.6; Anal. Calcd for C₂₅H₃₂N₄O₂: C, 71.40; H, 7.67; N, 13.32. Found: C, 71.46; H, 7.60; N, 13.35.

Step 8: Preparation of sulfamic acid 2-tert-butoxy-4-[4-(indan-1-ylamino)-pyrrolo[2,3-d]pyrimidin-7-yl]-cyclopentylmethyl ester (Compound 11)

Preparation of 2.0 M solution of chlorosulfonamide in acetonitrile: Formic acid (14.15 ml, 166.0 mmol) was added dropwise to chlorosulfonyl isocyanate (32.0 ml, 162.5 mmol) under nitrogen atmosphere at 0° C. When the addition was completed, the mixture was solidified. To the mixture was added acetonitrile (61.3 ml), and the resulting solution was left to stand under nitrogen source at room temperature overnight.

To a solution of the compound 10 (5.63 g, 13.83 mmol) and triethyl amine (9.7 ml, 0.74 mmol) in acetonitrile (278 ml) was added 2.0 M solution of chlorosulfonamide in acetonitrile (13.83 ml, 27.76 mmol) at 0° C., and the reaction mixture was stirred at room temperature for 45 minutes. Additional 2.0 M chlorosulfonamide solution in acetonitrile (13.83 ml, 27.76 mmol) was added and the mixture was stirred at room temperature for 15 minutes. The reaction was quenched with methanol, and the solvent was removed. The residue was purified by silica gel column chromatography (methylene chloride/methanol=20/1) to give the compound 11 (6.37 g, 92%) as a white foam.

UV (MeOH) λ_max 273.00 nm; [α]²⁰ _D −18.00 (c 0.50, MeOH); HR-MS (ESI): m/z calcd for C₂₅H₃₄N₅O₄S [M+H]⁺: 500.2332; Found: 500.2331; ¹H NMR (400 MHz, CDCl₃) δ 8.38 (s, 1H), 7.36 (d, J=7.2 Hz, 1H), 7.29 (d, J=7.2 Hz, 1H), 7.22 (m, 2H), 6.95 (d, J=3.6 Hz, 1H), 6.31 (d, J=3.2 Hz, 1H), 5.89 (d, J=6.4 Hz, 1H), 5.10 (s, 2H), 4.41 (m, 2H), 4.26 (m, 1H), 3.05 (m, 1H), 2.94 (m, 1H), 2.76 (m, 2H), 2.27 (m, 3H), 2.06 (m, 1H), 1.97 (m, 1H), 1.76 (br s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 156.4, 151.9, 149.9, 143.9, 143.8, 128.3, 126.9, 125.1, 124.5, 121.9, 121.9, 103.5, 97.9, 77.4, 77.2, 76.9, 74.3, 71.9, 71.3, 56.4, 53.1, 49.0, 42.3, 34.9, 34.3, 30.5, 28.6; Anal. Calcd for C₂₅H₃₃N₅O₄S: C, 60.10; H, 6.66; N, 14.02; S, 6.42. Found: C, 60.15; H, 6.71; N, 13.98; S, 6.39.

Step 9: Preparation of sulfamic acid 2-hydroxy-4-[4-(indan-1-ylamino)-pyrrolo[2,3-d]pyrimidin-7-yl]-cyclopentylmethyl ester (Compound 1)

A solution of the compound 11 (6.37 g, 12.72 mmol) in 70% trifluoroacetic acid (149.24 ml) was stirred at room temperature for 2 hours. The solvent was removed and the residue was purified by silica gel column chromatography (hexane/ethylene acetate=1/2) to give the compound 1 (5.08 g, 90%) as a white foam.  BASE

UV (MeOH) λ_max 279.50 nm; [α]²⁰ _D −6.41 (c 2.34, MeOH);

HR-MS (ESI): m/z calcd for C₂₁H₂₆N₅O₄S [M+H]⁺: 444.1705; Found: 444.1706;

¹H NMR (400 MHz, CD₃OD) δ 8.17 (d, J=1.6 Hz, 1H), 7.25 (m, 2H), 7.18 (m, 2H), 6.64 (d, J=3.6 Hz, 1H), 5.86 (t, J=7.6 Hz, 1H), 5.46 (m, 1H), 4.49 (d, J=2.8 Hz, 1H), 3.07 (m, 1H), 2.92 (m, 1H), 2.80 (m, 1H), 2.64 (m, 1H), 2.35 (m, 1H), 2.25 (m, 2H), 2.03 (m, 2H);

¹³C NMR (100 MHz, CD₃OD) δ 152.1, 145.3, 144.6, 128.8, 127.6, 125.7, 125.2, 122.6, 100.5, 73.1, 70.9, 56.9, 54.0, 44.8, 43.6, 34.9, 34.6, 31.1;

Anal. Calcd for C₂₁H₂₅N₅O₄S: C, 56.87; H, 5.68; N, 15.79; S, 7.23. Found: C, 56.91; H, 5.73; N, 15.82; S, 7.26.

…………………….

http://www.google.com/patents/WO2010132110A1?cl=en

((lS,2S,4R)-4-{4-[(lS)-2,3-dihydro-lH-inden-l-ylamino]-7H-pyrrolo[2,3-d]pyrimidin-7-yl }-2-hydroxycyclopentyl)methyl sulfamate (//) is described in Intl. App. Pub. No. WO 07/092213, U.S. App. Pub. No. 2007/0191293, and U.S. App. Pub. No. 2009/0036678. The potassium salt of ((lS,2S,4R)-4-{4-[( 1 S)-2,3-dihydro- 1 H-inden- 1 -ylamino]-7H-pyrrolo[2,3-d]pyrimidin-7-yl } -2-hydroxycyclopentyl)methyl sulfamate is disclosed in Intl. App. Pub. No. WO 07/092213 and U.S. App. Pub. No. 2007/0191293.

(H)

((lS,2S,4R)-4-{4-[(lS)-2,3-dihydro-lH- inden-l-ylamino]-7H-pyπOlo[2,3-d]pyrimidin-7-yl}-2-hydroxycyclopentyl)methyl sulfamate (/):

http://www.google.com/patents/WO2007092213A2?cl=en

Example 70: Diastereoisomeric mixture of (lS_/2R_/4R)-4-{4-[(lS)-2_/3-dihydro-lH-inden-l- ylaimnol-ZH-pyrrolop^-dlpyxirnidin-Z-ylJ^-hydroxycyclopentyl s ulf amate and (lR_f2S_/4S)-4-{4-[(lS)-2,3-dihydro-lH-inden-l-ylaminol-7H-pyrrolo[2,3d]- pyrimidin-7-yl}-2-hydroxycyclopentyl sulfamate (Compounds 1-77 and 1-78)

Step a: Cyclopent-3-en-l-yl methanesulfonate

[0335] 3-Cydopentene-l-ol (0.500 g, 5.94 mmol) was stirred in DCM (95 mL).

Pyridine (2.40 mL), N,N-dimethylaminopyridine (0.10 g, 1.00 mmol) and methanesulfonyl chloride (0.690 mL, 8.92 mmol) were added, and the reaction mixture was stirred at 35⁰C for 4 h. N,N-Dimethylarrιinopyridirιe (0.14 g, 1.2 mmol) and methanesulfonyl chloride (0.69 mL, 8.92 mmol) were added, and the reaction was stirred overnight. TLC indicated complete conversion. The reaction mixture was cooled and concentrated. The residue was purified by silica gel chromatography, eluting with DCM, to afford the title compound as a clear oil (0.660 g, 68%).

Step b: 7-Cyclopent-3-en-l-yl-N-r(lSV2,3-dihydro-lH-inden-l-yn-7H-pyrrolor2,3-rfl- pyrmτidin-4-arnine

[0336] N-[(lS)-2,3-DihydrcHlH-mden-l-yl]-7H-pyrrolo[2_/3-d]p3αimidin-4-amine (1.32 g, 5.29 mmol) was azeotroped with toluene and placed under high vacuum for 30 min. N,N-Dimethylformamide (17.7 mL) was added, followed by cesium carbonate (1.99 g, 6.10 mmol). The mixture was stirred at 70⁰C for 10 min. Cyclopent-3-en-l-yl methanesulfonate (0.660 g, 4.07 mmol) in N,N-dimethylformarnide (12.6 mL) was added dropwise. The reaction mixture was heated to 110⁰C for 1 h. The reaction mixture was cooled, quenched with brine and diluted with H₂O. The aqueous layer was extracted with EtOAc (3x), washed with H₂O and brine, dried (Na₂SO₄), filtered, and concentrated. The residue -was purified by via silica gel chromatography, eluting with a gradient of 0 to 5% MeOH in DCM followed by 25 to 50% EtOAc in hexanes, to afford the title compound as a pale brown solid (0.684 g, 53%). LC/MS: R₁ = 1.38 min, ES⁺ 317 (FA standard). Step c: (lR,2S,45)-4-{4-r(lS)-2,3-dihydro-lH-inden-l-ylaininol-7H-pyrrolof2.3- rf1pyrimidin-7-yl}cyclopentane-l,2-diol

[0337] 7-Cyclopent-3-en-l-yl-N-[(lS)-2^-dihyrdo-lH-inden-l-yl]-7H-pyrrolo[2,3- d]pyτimidin-4-amine (0.312 g, 0.986 mmol) was stirred in tert-butyl alcohol (4.9 mL) and H₂O (4.9 mL). AD-mix-α (Sigma- Aldrich, 1.4 g) was added, and the suspension was stirred at rt overnight. TLC indicated complete conversion. The reaction was quenched with sodium sulfite (1.48 g, 11.7 mmol), and the mixture was stirred for 5 h. The reaction mixture was diluted with EtOAc and H₂O, and the aqueous layer was extracted with EtOAc (2x). The organic layer was dried (Na₂SO₄), filtered, and concentrated. The residue was purified via silica gel chromatography, eluting with EtOAc, to afford the title compound as a white solid (0.190 g, 55%).

Step d: Diastereoisomeric mixture of (lS,2R,4R)-4-{4-r(15)-23-dihydro-lH-inden-l- ylarninoi^jH-pyrrolofΣ^dlpyrirnidin-y-yll-l-hydroxycyclopentyl sulfamate and (lR,2S,4S)-4-{4-iαSV2,3-dihydro-lH-inden-l-ylarninol-7H-pyrrolor2,3- rf1pyrimidm-7-yl)-2-hydroxycyclopenryl sulfamate (Compounds 1-77 and 1-78)

[0338] (lR,2S,4S)-4-{4-[(lS)-2,3-Dihydro-lH-inden-l-ylarnino]-7H-pyrrolo[2_/3- d]pyrimidin-7-ylJcyclopentane-l,2-diol (0.080 g, 0.23 mmol) was azeotroped with toluene and then was dissolved in anhydrous acetonitrile (2.3 mL). Pyridine (0.0369 mL, 0.458 mmol) was added. The reaction mixture was cooled to 0⁰C, and a 2N solution of chlorosulfonamide in acetonitrile (0.144 mL) was added dropwise. The reaction was stirred for 1 h, and then additional 2N chlorosulfonamide in acetonitrile (0.028 mL) was added. After 30 min, additional 2N chlorosulfonamide in acetonitrile (0.0342 mL) was added, and the reaction mixture was stirred for 2 h. The reaction was quenched with methanol, and the mixture was concentrated in vacuo. The residue was purified by preparative thin layer chromatography using DCM:AcCN:MeOH (50:45:5). The relevant band was cut, washed with acetone, filtered, and concentrated to give a mixture of diastereomers as a white solid. (11 mg, 11%). ¹H NMR (CDCl₃, 400 NMR, δ): 8.36-8.27 (m, IH); 7.38-7.09 (m, 5H); 6.90-6.80 (m, IH); 6.36- 6.20 (m, IH); 5.95-5.76 (m, IH); 5.51-5.22 (m, 2H); 4.83-4.68 (m, IH); 3.87-3.72 (m, IH); 3.12- 2.83 (m, 2H); 2.75-2.53 (m, IH); 2.50-2.14 (m, 2H); 2.08-1.79 (m, 2H) ppm. LC/MS: R, = 1.16 min, ES⁺ 430 (FA standard).

…………

WO 2012061551

http://www.google.im/patents/WO2012061551A1?cl=en

The compound ((lS,2S,4R)-4-(4-((lS)-2,3-dihydro-lH-inden-l-ylamino)-7H-pyrrolo[2,3-d]- pyrimidin-7-yl)-2-hydroxycyclopentyl)methyl sulfamate:

also known as MLN4924, is an inhibitor of NEDD8-activating enzyme (NAE). Inhibition of NAE has been shown to induce cancer cell death and inhibit the growth of tumors in xenograft models. See, e.g., T.A. Soucy et al., Nature, 2009, 458, 732-737; T.A. Soucy ei al., Clin. Cancer Res., 2009, 15 (12), 3912-3916; and J.E. Brownell et al., Mol. Cell., 2010, 37 (1), 102-111, each of which is hereby incorporated by reference herein in its entirety. MLN4924, pharmaceutical compositions of MLN4924, processes for its synthesis, and polymorphic forms have been described previously. See, e.g., US Patent Appl. Nos. 11/700,614 (Publ. No. 2007/0191293), 12/221,399 (Publ. No. 2009/0036678) and 12/779,331 (Publ. No. 2011/0021544),

……………

A practical synthesis of a novel NEDD8-activating enzyme (NAE) inhibitor pevonedistat (MLN4924) is described. Key steps include an enantioselective synthesis of an amino-diol cyclopentane intermediate containing three chiral centers and a novel, regioselective sulfamoylation using N-(tert-butoxycarbonyl)-N-[(triethylenediammonium)sulfonyl]azanide. The linear process, involving six solid isolations, has been carried out in multiple cGMP productions on 15–30 kg scale to produce pevonedistat in 98% (a/a) chemical purity and 25% overall yield.

((1S,2S,4R)-4-(4-(((S)-2,3-Dihydro-1H-inden-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methyl Sulfamate (1)

((1S,2S,4R)-4-(4-(((S)-2,3-Dihydro-1H-inden-1-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxycyclopentyl)methyl Sulfamate·Hydrochloride (Pevonedistat)

MLN4924 (1), which is in clinical trials as an anticancer agent, was stereoselectively synthesized from d-ribose via a route involving stereoselective reduction, regioselective cleavage of an isopropylidene moiety, and selective displacement of a cyclic sulfate moiety as key steps.

Sulfamic Acid 2-Hydroxy-4-[4-(indan-1-ylamino)pyrrolo[2,3-d]pyrimidin-7-yl]cyclopentylmethyl Ester (1)  BASE

purified by silica gel column chromatography (hexane/ethyl acetate = 1/2) to give 1 (5.08 g, 90%) as a white foam:

UV (MeOH) λ_max 279.50 nm;

[α]²⁰_D −6.41 (c 2.34, MeOH);

HR-MS (ESI) m/z calcd for C₂₁H₂₆N₅O₄S [M + H]⁺ 444.1705, found 444.1706;

¹H NMR (400 MHz, CD₃OD) δ 8.17 (d, J = 1.6 Hz, 1H), 7.25 (m, 2H), 7.18 (m, 2H), 6.64 (d, J = 3.6 Hz, 1H), 5.86 (t, J = 7.6 Hz, 1H), 5.46 (m, 1H), 4.49 (d, J = 2.8 Hz, 1H), 3.07 (m, 1H), 2.92 (m, 1H), 2.80 (m, 1H), 2.64 (m, 1H), 2.35 (m, 1H), 2.25 (m, 2H), 2.03 (m, 2H);

¹³C NMR (100 MHz, CD₃OD) δ 152.1, 145.3, 144.6, 128.8, 127.6, 125.7, 125.2, 122.6, 100.5, 73.1, 70.9, 56.9, 54.0, 44.8, 43.6, 34.9, 34.6, 31.1. Anal. Calcd for C₂₁H₂₅N₅O₄S: C, 56.87; H, 5.68; N, 15.79; S, 7.23. Found: C, 56.91; H, 5.73; N, 15.82; S, 7.26.

1H NMR PREDICT

13 C NMR

//////////Pevonedistat, MLN4924, Millennium Pharmaceuticals, TAKEDA, TAK-924 , PHASE 1, orphan drug designation

Vintafolide, EC-145  , MK-8109

mw 1917.041, cas 742092-03-1, mf C86 H109 N21 O26 S2

(2S)-2-[(4-{[(2-amino-4-oxo-3H-pteridin-6-yl)methyl]amino}phenyl)formamido]-4-{[(1S)-1-{[(1S)-4-carbamimidamido-1-{[(1S)-2-carboxy-1-{[(1S)-2-carboxy-1-{[(1R)-1-carboxy-2-({2-[({[(1R,9R,10S,11R,12R,19R)-12-ethyl-4-[(13S,15R,17S)-17-ethyl-17-hydroxy-13-(methoxycarbonyl)-1,11-diazatetracyclo[13.3.1.0⁴,¹².0⁵,¹⁰]nonadeca-4(12),5,7,9-tetraen-13-yl]-10,11-dihydroxy-5-methoxy-8-methyl-8,16-diazapentacyclo[10.6.1.0¹,⁹.0²,⁷.0¹⁶,¹⁹]nonadeca-2,4,6,13-tetraen-10-yl]formohydrazido}carbonyl)oxy]ethyl}disulfanyl)ethyl]carbamoyl}ethyl]carbamoyl}ethyl]carbamoyl}butyl]carbamoyl}-2-carboxyethyl]carbamoyl}butanoic acid

Vincaleukoblastin-23-oic acid, O⁴-deacetyl-, 2-[(2-mercaptoethoxy)carbonyl]hydrazide, disulfide with N-[4-[[(2-amino-1,4-dihydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-γ-glutamyl-L-α-aspartyl-L-arginyl-L-α-aspartyl-L-α-aspartyl-L-cysteine

Endocyte innovator

Vintafolide is an investigational targeted cancer therapeutic currently under development by Endocyte and Merck & Co.^[1] It is a small molecule drug conjugate consisting of a small molecule targeting the folate receptor, which is overexpressed on certain cancers, such as ovarian cancer, and a potent chemotherapy drug, vinblastine.^[2] It is being developed with a companion imaging agent, etarfolatide, that identifies patients that express the folate receptor and thus would likely respond to the treatment with vintafolide.^[3] A Phase 3 study evaluating vintafolide for the treatment of platinum-resistant ovarian cancer (PROCEED trial) and a Phase 2b study(TARGET trial) in non-small-cell lung carcinoma (NSCLC) are ongoing.^[4] Vintafolide is designed to deliver the toxic vinblastine drug selectively to cells expressing the folate receptor using folate targeting.^[5]

A Marketing Authorization Application (MAA) filing for vintafolide and etarfolatide for the treatment of patients withfolate receptor-positive platinum-resistant ovarian cancer in combination with doxorubicin, pegylated liposomal doxorubicin (PLD), has been accepted by the European Medicines Agency.^[6] The drug received an orphan drug status in Europe in March 2012.^[1] Merck & Co. acquired the development and marketing rights to this experimental cancer drug from Endocyte in April 2012.^[1] The drug received orphan drug status in Europe in March 2012.[3]Endocyte remains responsible for the development and commercialization of etarfolatide, a non-invasive companion imaging agent used to identify patients expressing the folate receptor that will likely respond to treatment with vintafolide.^[4] Vintafolide is designed to deliver the toxic vinblastine drug selectively to cells expressing the folate receptor using folate targeting.^[5]

In 2014 Merck and Endocyte stopped a late-stage study of vintafolide in treating ovarian cancer on the recommendation of a data safety monitoring board, saying that the drug failed to improve progression-free survival.^[7]

Vintafolide is folate-conjugated with DAVBLH, which is a derivative of the vinca alkaloid vinblastine.Vinblastine is a microtubule-destabilizing agent that binds tubulin and causes M phase-specific cell cycle arrest and apoptosis of mitotically active cells. Vinblastine is an extremely potent chemotherapeutic agent but has significant toxicities including bone marrow suppression, neurotoxicity, gastrointestinal toxicity and vesicant injury.

Endocyte’s desacetylvinblastinehydrazide/folate conjugate (EC-145) is a folate-targeted cytotoxic anticancer drug in early development for the treatment of non-small cell lung cancer (NSCLC) and breast cancer. The compound had been pre-registered in the E.U. by Merck for the treatment of ovarian cancer, but the application was withdrawn due to lack of efficacy.

In 2012, the product was licensed to Merck & Co. by Endocyte for worldwide exclusive development and commercialization. In 2014, however, this license agreement was terminated and Endocyte regained all rights.
Folates can serve as one-carbon donors in reactions that are critical in the de novo biosynthesis of purines and thymidylate, amino acid metabolism and methylation reactions. Folate can enter a cell by two routes: RFC or by membrane-bound FRs. RFC is a bidirectional anion transporter that is the normal entry method for reduced folates in most cells. By contrast, FRs are expressed in a limited distribution in normal tissues but are overexpressed in multiple cancers including ovarian, lung, breast and colorectal cancer. FRs bind folate derivatives with high affinity and mediate their internalization by endocytosis. Given that FRs are not typically expressed on the luminal surface of epithelial cells, making them inaccessible to normal circulation, they are attractive therapeutic targets with limited toxicity. In addition to the therapeutic agent vintafolide, a radiodiagnostic agent (^99mTc-etarfolatide [EC20]) has been developed to allow single-photon emission computed tomography (SPECT) imaging to identify FR-expressing tissues (tumors).

In 2012, orphan drug designations were assigned in the E.U. for the treatment of ovarian cancer and to be used with folic acid for the diagnosis of positive folate-receptor status in ovarian cancer. In 2013, orphan drug designation was assigned in the U.S. for the treatment of ovarian cancer.

Vintafolide is a water-soluble derivative of folic acid and the vinca alkaloid DAVLBH. The molecules are connected through a hydrophilic L-peptide spacer and a disulfide linker (Figure 1). The disulfide linker serves as a cleavable bond that is necessary for drug release following receptor mediated endocytosis. The disulfide bond is reduced in the acidic environment of the endosome, leading to efficient release of vinblastine.

Structure of vintafolide and mechanism of release of the payload in the endosome.

Mechanism of action

Folate is required for cell division, and rapidly dividing cancer cells often express folate receptors in order to capture enough folate to support rapid cell growth. Elevated expression of the folate receptor occurs in many diseases, including other aggressively growing cancers and inflammatory disorders.^[8] Vintafolide binds to the folate receptor and is subsequently taken up by the cell through a natural internalization process called endocytosis. Once inside the cell, vintafolide’s linker releases the chemotherapy drug which kills the cell.^[3]

……………

Bioorganic & Medicinal Chemistry Letters (2006), 16(19), 5093-5096

http://www.sciencedirect.com/science/article/pii/S0960894X06008079

An efficient synthesis of the folate receptor (FR) targeting conjugate EC145 is described. EC145 is a water soluble derivative of the vitamin folic acid and the potent cytotoxic agent, desacetylvinblastine monohydrazide. Both molecules are connected in regioselective manner via a hydrophilic peptide spacer and a reductively labile disulfide linker.

………approach for the design and regioselective synthesis of a FA-vinca alkaloid conjugate 1 (EC145,BELOW). As indicated in the retrosynthetic scheme, 1 can be assembled by tethering a FA-Spacer unit 2 to the highly potent cytotoxic molecule, desacetylvinblastine monohydrazide 3, via a linker containing a reducible disulfide bond. The latter is important for drug delivery applications since real-time imaging using a fluorescence resonance energy transfer technique has recently demonstrated that reduction-mediated release of the drug cargo from a disulfide linked FA-conjugate efficiently occurs within the endosomes of cancer cells.

Selected ¹H NMR data for 2 (D₂O, 300 MHz): δ 8.68 (s, 1H, FA H-7), 7.57 (d, 2H,J = 8.4 Hz, FA H-12 & 16), 6.67 (d, 2H, J = 9 Hz, FA H-13 & 15), 4.40–4.75 (series of m, 5H), 4.35 (m, 2H), 4.16 (m, 1H), 3.02 (m, 2H), 2.55–2.95 (series of m, 8H), 2.42 (m, 2H), 2.00–2.30 (m, 2H), 1.55–1.90 (m, 2H), 1.48 (m, 2H).

¹H NMR for compound 6 (DMSO-d₆, 300 MHz): δ 8.38 (m, 1H), 8.16 (dt, 1H, J = 8 Hz, 1 Hz), 8.02 (dt, 1H, J = 8 Hz, 1 Hz), 7.88 (ddd, 1H, J = 8 Hz, 7 Hz, 1 Hz), 7.7 (m, 2H), 7.63 (ddd, 1H, J = 8 Hz, 7 Hz, 1 Hz,), 7.4–7.2 (br, 1H), 7.2 (m, 1H), 4.72 (t, 2H,J = 6 Hz), 3.36 (t, 2H, J = 6 Hz).

Selected ¹H NMR data for

EC145 (D₂O, 300 MHz): δ 8.67 (s, 1H, FA H-7), 7.50 (br s, 1H, VLB H-11′), 7.30–7.40 (br s, 1H, VLB H-14′), 7.35 (d, 2H, J = 7.8 Hz, FA H-12 & 16), 7.25 (m, 1H, VLB H-13′), 7.05 (br s, 1H, VLB H-12′), 6.51 (d, 2H, J = 8.7 Hz, FA H-13 & 15), 6.4 (s, 2H, VLB H-14 & 17), 5.65 (m, 1H, VLB H-7), 5.5 (m, 1H, VLB H-6), 4.15 (m,1H, VLB H-8′), 3.82 (s, 3H, VLB C_18^′ –CO₂CH₃), 3.69 (s, 3H, VLB C₁₆ –OCH₃), 2.8 (s, 3H, VLB N–CH₃), 1.35 (br s, 1H, VLB H-3′), 1.15 (m, 1H, VLB H-2′), 0.9 (t, 3H, J = 7 Hz, VLB H-21′), 0.55 (t, 3H, J = 6.9 Hz, VLB H-21).

CLICK ON IMAGE FOR CLEAR VIEW

…………

WO 2004069159

http://www.google.com/patents/WO2004069159A2?cl=en

EXAMPLE 16b

The compounds of Examples 16a and 16b were prepared from the peptidyl fragment Pte-Glu-Asp-Arg-Asp-Asp-Cys-OH , prepared according to the general procedure described in Scheme 12. The Michael addition of this peptidyl fragment to the maleimido derivative of seco-CBI-bis-indole resulted in the folate conjugates Example 16a. The peptidyl fragment also reacted with either the thiosulfonate or pyridyldithio-activated vinblastine to form Example 16b. The maleimido derivative of seco-CBI-bis-indole, and the thiosulfonate and pyridyldithio- activated vinblastine intermediates were prepared using the procedures described herein for other examples.

……………..

https://www.google.com/patents/WO2012142281A1?cl=en

Folate-targeted drugs have been developed and are being tested in clinical trials as cancer therapeutics. EC145, also known as vintafolide, comprises a highly potent vinca alkaloid cytotoxic compound, desacetylvinblastine hydrazide (DAVLBH), conjugated to folate. The EC 145 molecule targets the folate receptor found at high levels on the surface of epithelial tumors, including non-small cell lung carcinomas (NSCLC), ovarian, endometrial and renal cancers, and others, including fallopian tube and primary peritoneal carcinomas. It is believed that EC 145 binds to tumors that express the folate receptor delivering the vinca moiety directly to cancer cells while avoiding normal tissue. Thus, upon binding, EC 145 enters the cancer cell via endocytosis, releases DAVLBH and causes cell death or inhibits cell function. EC 145 has the following formula

EC145

and has been accorded the Chemical Abstracts Registry Number 742092-03-1. As used herein, according to the context, the term EC 145 means the compound, or a pharmaceutically acceptable salt thereof; and the compound may be present in a solid, solution or suspension in an ionized form, including a protonated form. EC145 is disclosed in U.S. Patent No. 7,601,332; and particular uses and an aqueous liquid pH 7.4, phosphate-buffered formulation for intravenous administration are disclosed in WO 2011/014821. As described in WO 2011/014821, it is necessary to store the aqueous liquid formulation in the frozen state to ensure its stability. To avoid this necessity, a formulation is needed which has adequate stability at ambient temperature.

As one aspect of the invention described herein, there is provided a pharmaceutical composition of EC145 which is a lyophilized solid which has adequate stability for storage at ambient temperature and which is capable of redissolving in an aqueous diluent prior to administration.

In another aspect of the invention, there is provided a pharmaceutical composition of EC 145 which is an X-ray amorphous solid which has adequate stability for storage at ambient temperature and which is capable of redissolving in an aqueous diluent prior to administration.

Systematic (IUPAC) name
N-(4-{[(2-Amino-4-oxo-1,4-dihydropteridin-6-yl)methyl]amino}benzoyl)-L-γ-glutamyl-L-α-aspartyl-L-arginyl-L-α-aspartyl-L-α-aspartyl-L-cysteine disulfide with methyl (5S,7R,9S)-5-ethyl-9-[(3aR,4R,5S,5aR,10bR,13aR)-3a-ethyl-4,5-dihydroxy-8-methoxy-6-methyl-5-({2-[(2-sulfanylethoxy)carbonyl]hydrazinyl}carbonyl)-3a,4,5,5a,6,11,12,13a-octahydro-1H-indolizino[8,1-cd]carbazol-9-yl]-5-hydroxy-1,4,5,6,7,8,9,10-octahydro-2H-3,7-methanoazacycloundecino[5,4-b]indol-9-carboxylate
Clinical data
Legal status	IND
Identifiers
CAS Registry Number	742092-03-1
ATC code	L01CA06
ChemSpider	27444385
Synonyms	EC-145
Chemical data
Formula	C86H109N21O26S2
Molecular mass	1917 g/mol

References

////////Vintafolide, BMS-753493, DAVBLH,  Desacetylvinblastine hydrazide, EC-145 , MK-8109 , phase 2

Fruquintinib

Phase 3…cancer

Hutchison Medipharma Enterprises Limited

Hutchison MediPharma for the treatment of locally advanced or metastatic colorectal cancer

 C21H19N3O5
Exact Mass: 393.1325

cas 1194506-26-7, 6 ((6,7-dimethoxyquinazolin-4-yl) oxy) – N, 2-dimethylbenzofuran-3-carboxamide,

3-​Benzofurancarboxamid​e, 6-​[(6,​7-​dimethoxy-​4-​quinazolinyl)​oxy]​-​N,​2-​dimethyl-

Synonym: Fruquintinib; HMPL-013; HMPL 013; HMPL013.

HPLC.http://www.medkoo.com/Product-Data/Fruquintinib/QC-Fruquintinib-CRB50706web.pdf

Fruquintinib, also known as HMPL-013, is an orally available, small molecule inhibitor of vascular endothelial growth factor receptors (VEGFRs), with potential anti-angiogenic and antineoplastic activities.

HMPL-013, a novel small molecule compound that selectively inhibits vascular endothelial growth factor receptor (VEGFR), is in phase III clinical studies at Hutchison MediPharma for the treatment of locally advanced or metastatic colorectal cancer. Phase II clinical trials are also ongoing for the treatment of non-squamous non-small cell lung cancer.

Early clinical development is under way at the company for the treatment of gastric cancer in combination with paclitaxel.

Fruquintinib’s mechanism of action is the inhibition of all three forms of VEGF receptors (VEGFR-1, 2, 3). Competitive advantages over currently marketed therapies are the compound’s unique kinase profile, a highly potent efficacy and excellent kinase selectivity, large safety margin, a broad spectrum antitumor activity and a low cost of goods.
Upon oral administration, fruquintinib inhibits VEGF-induced phosphorylation of VEGFRs 1, 2, and 3 which may result in the inhibition of migration, proliferation and survival of endothelial cells, microvessel formation, the inhibition of tumor cell proliferation, and tumor cell death. Expression of VEGFRs may be upregulated in a variety of tumor cell types.

In 2013, the company entered into a licensing, co-development, and commercialization agreement in China with Eli Lilly.

Angiogenesis is a physiological process of growing new blood vessels from pre-existing vessels. It takes place in a healthy subject to heal wounds, i.e., restoring blood flow to tissues after injury or insult.

Excessive angiogenesis may be triggered by certain pathological conditions such as cancer, age-related macular degeneration, and chronic inflammatory disease. As a result, new blood vessels feed diseased tissues and destroy normal tissues. In cancer, new blood vessels also allow tumor cells to escape into the circulation and lodge in other organs.

Vascular endothelial growth factor (VEGF), a homodimeric glycoprotein, and its receptors, e.g., kinase insert domain receptor (KDR), constitute an important angiogenic pathway. Studies have shown that inhibition of KDR resulted in endothelial cell apoptosis and, thus, suppression of angiogenesis. See Rubin M. Tuder, Chest, 2000; 117: 281. KDR inhibitors are therefore potential candidates for treating an angiogenesis-related disorder.

Chi-Med Says Fruquintinib Successful in Lung Cancer Trial

Written by Richard Daverman, PhD, Executive Editor, Greg B. Scott.

Hutchison MediPharma, a division of Chi-Med reported that fruquintinib met its primary endpoint in a second proof-of-concept China trial, this time as a treatment for advanced non-squamous non-small cell lung cancer. The company said fruquintinib “clearly” met its primary endpoint of  progression-free survival, though specific data are being held for a scientific meeting. In 2013, Hutchison out-licensed China rights for the drug to Lilly. In May, the first proof-of-concept trial triggered two payments from Lilly to HMP totaling $18 million. More details…. http://www.chinabiotoday.com/articles/20150904

………….

Patent

US 20090281130

https://www.google.com.ar/patents/US20090281130

EXAMPLE 1 Synthesis of 6-(6,7-dimethoxyquinazolin-4-yloxy)-N,2-dimethylbenzofuran-3-carboxamide:

To a solution of 4-chloro-6,7-dimethoxyquinazoline (1 equiv.) in 2 ml CH₃CN were added 6-hydroxy-N,2-dimethylbenzofuran-3-carboxamide (1 equiv.) and K₂CO₃ (1.5 equiv.). The mixture was refluxed under stirring for 10 hr. After the solvent was evaporated, the residue was washed with water, dried over MgSO₄, filtered, concentrated, and purified by column chromatography to give the title compound in a yield of 85%.

¹H NMR (DMSO-d₆, 400 MHz) δ: 2.49 (s, 3H), 2.81 (d, J=8.4 Hz, 3H,10), 3.97 (s, 3H), 3.98 (s, 3H), 7.24 (dd, J=2.0, 8.4 Hz, 1H), 7.38 (s, 1H), 7.58 (s, 1H), 7.61 (d, J=2.0 Hz, 1H), 7.79 (d, J=8.4 Hz, 1H), 7.96 (m, 1H), 8.52 (s, 1H).

MS(m/e): 394.1 (M+1).

………………

WO 2009137797

https://www.google.com/patents/WO2009137797A2

……………….

CN 101575333

Example a: 6- (6,7-dimethoxy-quinazolin-4-oxo) -N, 2- dimethyl-benzofuran-3-carboxamide

[0048]

[0049] 4-Chloro-6,7-dimethoxy-quinazoline (1 mmol) was dissolved in 2 ml of acetonitrile, followed by addition of 6-hydroxy -N, 2- dimethyl-benzofuran-3- amide (1 mmol) and potassium carbonate (1.5 mmol). The reaction mixture was heated at reflux for 10 hours, concentrated to dryness, washed with water, and purified to give the desired product, yield 85%.

[0050] 1H NMR (DMS0-d6,400MHz) δ ppm:. 2 49 (s, 3H); 2.81 (d, J = 8. 4Hz; 3H, 10); 3.97 (s; 3H); 3.98 (s, 3H);. 7 24 (dd, J = 2. 0,8 4Hz;. 1H);. 7 38 (s, lH);. 7 58 (s, lH); 7.61 (d, J = 2. OHz; 1H);. 7 79 (d, J = 8. 4Hz; 1H);. 7 96 (m, 1H);. 8 52 (s, 1H).

[0051] MS (m / e)::. 394 1 (M + l).

………..

Simon To, M.B.A.
Chairman

Christian Hogg, M.B.A.
Chief Executive Officer, Hutchison China MediTech Limited and Director, Hutchison MediPharma Holdings Limited

Weiguo Su, Ph.D.
Executive Vice President and Chief Scientific Officer

Addresses in Chinese:

R & D Center ( A): Chinese Cai Lun Road, Zhangjiang Hi-Tech Park in Pudong New Area, Shanghai, Lane 720 (intermediate哈雷路爱迪way out), Building 4

Head Office (B): Harley Road, Zhangjiang Hi-Tech Park, Pudong New Area, China, Shanghai, Lane 917, Building 4

///////

Icaritin;  118525-40-9; AC1NSXIV; UNII-UFE666UELY;

3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-enyl)chromen-4-one

3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-enyl)chromen-4-one

The roots of Epimedium brevicornu Maxim

Beijing Shenogen Granted Fast Track Status for Novel Cancer Drug

Written by Richard Daverman, PhD, Executive Editor, Greg B. Scott.

Beijing Shenogen Biomedical announced that Icaritin, a China Class I cancer drug, was granted Fast Track Review status after the company filed its New Drug Approval submission to the Beijing Food & Drug Administration. Icaritin is an oral traditional Chinese medicine, derived from barrenwort, which targets the estrogen receptor α36. Shenogen has conducted clinical trials of Icaritin in patients with liver cancer, though it expects the drug will also prove effective in breast cancer and other estrogen-related cancers as well. More details…. http://www.chinabiotoday.com/articles/20150917

Antiproliferative agent (IC₅₀ values are 8,13 and 18 μM for K562, CML-CP and CML-BC cells respectively). Inhibits H/R-induced PTK activation. Induces G(2)/M cell cycle arrest and mitochondrial transmembrane potential drop. Modulates MAPK/ERK/JNK and JAK2/STAT3 /AKT signaling. Inhibits PPAR-g. Modulates differentiation. Inhibits cytochrome P450 in vivo. Orally active.

Cardiovascular function improvement, hormone regulation and antitumor activity.
2. The anti-MM activity of Icaritin was mainly mediated by inhibiting IL-6/JAK2/STAT3 signaling.
3. The inhibitory activity of Icariside II on pre-osteoclast RAW264.7 growth was synergized by Icaritin, which maybe contribute to the efficiency of Herba Epimedii extract on curing bone-related diseases, such as osteoporosis.
4. The Icaritin at low concentration (4 or 8 μmol/L) can promote rat chondrocyte proliferation and inhibit cell apoptosis, while the effect of Icaritin on rat chondrocyte at high concentration was reversed.
5. Icaritin might be a new potent inhibitor by inducing S phase arrest and apoptosis in human lung carcinoma A549 cells.
6. Icaritin dose-dependently inhibits ENKL cell proliferation and induces apoptosis and cell cycle arrest at G2/M phase. Additionally, Icaritin upregulates Bax, downregulates Bcl-2 and pBad, and activates caspase-3 and caspase-9.

What is Epimedium ?

Herba epimedii (Epimedium, also called bishop’s hat, horny goat weed or yin yang huo), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. It is a genus of about 60 flowering herbs, cultivated as a ground cover plant and an aphrodisiac. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Most of them bloom in the early spring, and the leaves of some species change colors in the fall, while other species retain their leaves year round.

Figure 1 Epimedium

Epimedium Raw Material

The herbs we used to extract icariin is one species of Epimedium, which name is Epimedium brevicornum Maxim. This kind of epimedium only can be abundantly found in Gansu province of China. And because of the growth habit of this kind of herb, which only grows under trees, it can’t to be planted, only can harvest the wild one.

This wild epimedium contains quite a bit of active components, depending on its long growth time and rich nutrient. Usually the content of the icariin is not lower than 1%.

Below photo is the herb specimen which we use. Picking in the epimedium full-bloom stage. And the medicinal value of the herb is the best at this time. The herb we select contains roots, stems, leaves and flowers. And we extract with the whole herb.

Figure 2 Epimedium for extract

Epimedium Extract

Epimedium extract is a herbal supplement claimed to be beneficial for the treatment of sexual problems such as impotence. It is believed to contain a number of active components, including plant compounds that may have antioxidant activity and estrogen-like compounds. The major components of Epimedium brevicornum are icariin, epimedium B and epimedium C. It is reported to have anti-inflammatory, anti-proliferative, and anti-tumor effects. It is also reported to have potential effects on the management of erectile dysfunction.

Figure 3 HPLC spectrum of icariin

Our specification available is Icariin HPLC 50%- 98%. Below please see the the information for reference:

      Figure 4 Epimedium Extract(Icariin)

Derivatives

The plant extracts of epimedium traditionally used for male impotence, and the individual compounds is icariin, were screened against phosphodiesterase-5A1 (PDE5A1) activity. Human recombinant PDE5A1 was used as the enzyme source. The E. brevicornum extract and its active principle icariin were active. To improve its inhibitory activity, some derivatives ware subjected to various structural modifications, which include icaritin, icariside II and 3,7-bis(2-hydroxyethyl) icaritin. There have some scientific papers report that the improved pharmacodynamic profile and lack of cytotoxicity on human fibroblasts make such compounds a promising candidate for further development. We hope that our new products can help you to find more commercial opportunity.

In this way, we can introduce those products as below, and we can also provide more details about the products according to your demand. The 1H-NMR of icaritin and 3,7-bis(2-hydroxyethyl) icaritin is as below.

Figure 4 1H-NMR of icaritin and 3,7-bis(2-hydroxyethyl) icaritin

Main Function of Epimedium Extract 

horny goat weed; epimedium; Icariin; penis medicine;epimedium p.e;epimedium brevicornum; shorthorned epimedium herb; Icariins; Icaritin; 3,7-Bis(2-Hydroxyethyl)Icaritin; icariin 60%; icariin 98%; epimedium graepimedium; icarisides II;epimedium sagittatum;epimedium leaf; barrenwort.powder extract

Epimedium has been used to treat male erectile dysfunction in Traditional Chinese Medicine for many centuries. The main functions of Epimedium brevicornum in ancient Chinese books focused on the nourishment of kidney viscera and reinforcement of ‘yang’, resulting in the restoration of erectile function in males.

Epimedium contains chemicals which might help increase blood flow and improve sexual function. It also contains phytoestrogens, chemicals that act somewhat like the female hormone estrogen that might reduce bone loss in postmenopausal women.

Figure 5 some products from epimedium extract

………..

PAPER

The novel total synthesis of icaritin (1), naturally occurring with important bioactive 8-prenylflavonoid, was performed via a reaction sequence of 8 steps including Baker-Venkataraman reaction, chemoselective benzyl or methoxymethyl protection, dimethyldioxirane (DMDO) oxidation, O-prenylation, Claisen rearrangement and deprotection, starting from 2,4,6-trihydroxyacetophenone and 4-hydroxybenzoic acid in overall yields of 23%. The key step was Claisen rearrangement under microwave irradiation. MS, ¹H and ¹³C NMR techniques have been used to confirm the structures of all synthetic compounds. – See more at: http://www.eurekaselect.com/124334/article

…….

PAPER

Synthesis of icariin from kaempferol through regioselective methylation and para-Claisen–Cope rearrangement

Qinggang Mei^1,2, Chun Wang¹, Zhigang Zhao³, Weicheng Yuan² and Guolin Zhang¹

¹Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
²Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, China
³College of Chemistry and Environmental Protection Engineering, Southwest University for Nationalities, Chengdu 610041, China…http://www.beilstein-journals.org/bjoc/single/articleFullText.htm?publicId=1860-5397-11-135

http://www.beilstein-journals.org/bjoc/content/supplementary/1860-5397-11-135-S1.pdf

Scheme 1: Reagents and conditions: (a) Ac₂O, pyridine, 94%; (b) BnBr, KI, K₂CO₃, acetone, 85%; (c) Me₂SO₄, K₂CO₃, acetone, MeOH, 82%; (d) MOMCl, N,N-diisopropylethylamine (DIPEA), CH₂Cl₂, 93%; (e) 3,3-dimethylallyl bromide, 18-crown-6, K₂CO₃, acetone, 86%; (f) Eu(fod)₃, NaHCO₃, PhCl, 85 °C, 61%; (g) MeOH, 3 M HCl (aq), reflux, 95%; (h) Pd/C, 1,4-cyclohexadiene, MeOH, 84%.

Scheme 2: Decomposition of 8.

Scheme 3: Claisen rearrangement of flavonol 8.

Scheme 4: Reagents and conditions: (a) 15, DMF/CHCl₃, Ag₂CO₃, molecular sieves (4 Å, powder); (b) 16, CH₂Cl₂, Ag₂O, molecular sieves (4 Å powder), 31% for 2 steps; (c) NH₃ (g), MeOH, 94%; (d) NH₃ (g), MeOH, 63% for 2 steps.

3 Nguyen, V.-S.; Shi, L.; Li, Y.; Wang, Q.-A. Lett. Org. Chem. 2014, 11, 677–681.

4. Dell’Agli, M.; Galli, G. V.; Dal Cero, E.; Belluti, F.; Matera, R.; Zironi, E.; Pagliuca, G.; Bosisio, E. J. Nat. Prod. 2008, 71, 1513–1517.

 1H NMR

13C NMR

HMBC

NOESY

………….

The present invention relates to compositions comprising icariside I, and to a novel, one step method of preparing such compositions, comprising converting specific prenylated flavonol glycosides such as epimedium A, epimedium B, epimedium C, icariin, and their corresponding acetate derivatives contained in an Epimedium plant extract to a single compound, namely icariside I shown below as compound I, which was surprisingly discovered to be a strong PDE-5 inhibitor.

This invention further comprises compositions enriched for anhydroicaritin, and to methods of preparing such compositions. One method of this invention for preparing compositions enriched for anhydroicaritin comprises a one-step method of converting prenylated flavonol glycosides, specifically the sagittatoside compounds A, B, and C, and the corresponding acetate derivatives, present in Epimedium plant extracts to a single compound, namely anhydroicaritin shown below as compound II, which was also discovered to be a strong PDE-5 inhibitor.

http://www.google.com/patents/US6399579

EXAMPLES Example 1 Acid Hydrolysis of a 50% EtOH Extract and Purification by Reversed Phase ChromatographyWhole Epimedium grandiflorum leaves were extracted with a 1:1 mixture of ethanol and water at 55° C. The resulting extract (referred to as a “50% EtOH extract”) was filtered and the filtrate concentrated at 40-50° C. under vacuum and then dried under vacuum at 60° C. to a dry solid. The dried extract (131 g) containing approximately 5.8 g of total PFG’s was placed in a 2 liter round bottom flask and 1 L of 90% ethanol was added. The mixture was heated to reflux to help dissolve the solids. Concentrated sulfuric acid (28 mL) was added. The mixture refluxed for 2 hr, cooled to room temperature, and 900 mL of water added with stirring. Next the mixture was filtered using vacuum to remove insoluble sulfate salts and other solids and loaded on a 2.5×56 cm (275 mL) column packed with 250-600 micron divinylbenzene cross-linked polystyrene resin (Mitsubishi Chemical). The column was washed with 2 column volumes (CVs) of 60% ethanol and the icariside I was eluted with 2 CVs of 95% ethanol. The product pool was air-dried producing 11.3 g of brown solids. HPLC analysis (FIG. 5) showed that the solids contained 18% icariside I (peak 15.27 min) and 12% anhydroicaritin (peak 25.15 min). The recovery of the icariside I in the product pool was 87% of the amount present in the hydrolyzate.

Example 2 Purification of a Hydrolyzate by Liquid/liquid ExtractionThe ethanolic hydrolyzate (25 mL) prepared in Example 1 was mixed with 62.5 mL of de-ionized water and the pH was adjusted to 7.0 using 50% (w/w) sodium hydroxide solution. The resulting mixture was extracted with three 25 mL portions of ethyl acetate and the combined ethyl acetate extracts were back extracted with 150 mL of water. The ethyl acetate layers were combined, dried, and assayed for icariside I. HPLC analysis (FIG. 6) showed that the dried EtOAc fractions contained 22% icariside I (peak 15.29 min) and 11% anhydroicaritin (peak 25.27 min), and icariside I recovery into the ethyl acetate was 97% of the amount present in the hydrolyzate. The partition coefficient for icariside I between ethyl acetate and water was found to be 16, indicating that the icariside I has a high affinity for ethyl acetate over water.

Example 3 Acid Hydrolysis of a 50% EtOH Extract and Purification by PrecipitationThe dried extract (204 g) described in Example 1 was mixed with 1 L of 90% EtOH and then heated to reflux to help dissolve the solids. Sulfuric acid (25 ML) was added slowly with swirling. The mixture was refluxed 90 minutes and immediately chilled to stop the reaction. After cooling to room temperature, the mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials, and the cake was washed with about 350 mL of 90% ethanol. The resulting ethanolic hydrolyzate (1.34 L) contained 4.1 g of icariside I.

The ethanolic hydrolyzate prepared above (1.32 L) was placed in a 10 L container and 40 g of 50% (w/w) sodium hydroxide solution was added followed by 20 mL of phosphoric acid. Next 3.3 L of deionized water was added with stirring. The pH of this mixture was 2.4. Sodium hydroxide solution (50% w/w ) was added until the pH was 8.25. The mixture was heated to 65° C. to assist with the coagulation of the precipitate. The mixture was cooled to room temperature and stirred for 0.5 hr at room temperature before filtering through a cellulose filter using vacuum. The resulting brown solids were washed with 715 mL of 10% ethanol and dried either under vacuum at room temperature or in air at 55° C. to yield brown solids. HPLC analysis (FIG. 7) showed the solids contained 20% icariside I (peak 15.27 min) and 10% anhydroicaritin. Recovery of icariside I using this precipitation procedure was 94% of the amount present in the hydrolyzate.

Example 4 Acid Hydrolysis of a Water Extract and Purification by PrecipitationGround Epimedium grandiflorum leaves (0.40 kg) were mixed with 5 L water in a 10 L round bottom flask. The flask was placed on a rotary evaporator for two hours at a rotation speed of 120 rpm and a water bath temperature of 90° C. The extract was filtered under reduced pressure through cellulose paper. The resulting filtrate (3.2 L) was evaporated using the rotary evaporator to a volume of 100 mL and dried under vacuum at 50° C.

The dark brown solids prepared above (40.4 g) were mixed with 200 mL of 90% ethanol and 6.0 mL of sulfuric acid in a 500 mL round bottom flask. The mixture was refluxed for 90 minutes and immediately chilled to stop the reaction. This mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials. The cake was washed with 15 mL of 90% ethanol. The resulting ethanolic hydrolyzate (215 mL) contained 0.53 g of icariside I.

The hydrolyzate prepared above (50 mL) was transferred to a 250 mL beaker and 2.5 mL of 50% (w/w) sodium hydroxide solution was added with stirring to adjust the pH of the solution to pH 9, followed by 1.5 mL of concentrated phosphoric acid. Deionized water (125 mL) was added, and the mixture was adjusted to pH 8.2 using 1.5 mL of 50% sodium hydroxide solution. The mixture was heated to 65° C. to assist with coagulation of the precipitate and cooled to room temperature. The mixture was allowed to sit undisturbed at room temperature for 30 minutes prior to filtration under reduced pressure through cellulose paper. The resulting olive-green solids were washed with 25 mL of de-ionized water and dried under vacuum at room temperature or in air at 80° C. to produce olive-green solids. HPLC analysis (FIG. 8) showed the solids contained 60% icariside I (peak 15.33 min) and 2.4% anhydroicaritin (peak 25.40 min). Recovery of icariside I using this precipitation procedure was 92% of the amount present in the hydrolyzate.

Example 5 Enzymatic Hydrolysis of Icariside Ia) The substrate was a partially purified icariside I product with 20% icariside I and 11% anhydroicaritin. About 50 mg was dissolved in 10 mL of ethanol, and water or buffer was added until the mixture became cloudy (about 20% ethanol). The following dry enzymes were added to separate samples: α-amylase, α-glucosidase, β-amylase, β-glucosidase, hesperidinase, lactase, and pectinase. The samples were incubated overnight at 40 ° C. and analyzed by HPLC. The results were only semi-quantitative due to the difficulty in dissolving the anhydroicaritin that precipitated from the samples. However, several of the chromatograms did show a definite reduction in icariside I and increase in the ratio of anhydroicaritin to icariside I. The best results were obtained using hesperidinase, lactase, β-glucosidase and pectinase.

A larger scale experiment was done using hesperidinase in order to isolate pure anhydroicaritin for characterization. Pure icariside I (20 mg )was dissolved in 10 mL of ethanol and 50 mL of water and 200 mg of hesperidinase enzyme was added and the mixture was incubated for 24 hr at 40 ° C. Crude anhydroicaritin was collected via filtration and purified on a 2.5×30 cm semi-prep C-18 HPLC column using a gradient of 50:50 (MeCN/H₂O) to 80:20 (MeCN/H₂O) in 20 min. The pure anhydroicaritin was analyzed by LC/MS and proton NMR.

b) Enzymatic Hydrolysis of PFG’s: The purified PFG solids (55.3%, purified by reversed-phase chromatography of a 50% EtOH extract) were subjected to enzymatic hydrolysis with the same enzymes and conditions described in part (a). Hesperidinase, lactase, β-glucosidase and pectinase appeared to convert the mixture of PFG’s to a mixture of sagittatosides, but no icariside I or anhydroicaritin were observed. This indicated that these enzymes were specific for the 7-β-glucosyl group and did not hydrolyze the 3-position sugar(s).

Example 6 Preparation of a High Anhydroicaritin-containing ProductA high sagittatosides Epimedium sagittatum extract containing 24.7% total sagittatosides (assayed as icariin) and 8.1% icariin and other expected prenylated flavonol glycosides was obtained from China. A 50 g portion of this extract was mixed with 250 mL of 90% ethanol and 7.5 mL of concentrated sulfuric acid in a 500 mL round bottom flask. The mixture was refluxed for 90 minutes, then allowed to cool to room temperature. The hydrolyzed mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials. The cake was washed with approximately 20 mL of 90% ethanol. The resulting filtered ethanolic hydrolyzate (305 mL) contained 3.75 g of anhydroicaritin and 2.50 g of icariside I.

The filtered hydrolyzate prepared above (200 mL) was transferred to a 1000 mL container and 8.0 mL of 50% (w/w) sodium hydroxide solution was added with stirring, followed by 4.0 mL of phosphoric acid. De-ionized water (500 mL) was then added. This mixture was adjusted to pH 4.9 using 50% sodium hydroxide solution. The mixture was allowed to sit undisturbed at room temperature for 24 hours prior to decanting off the liquid. The resulting solids were macerated using de-ionized water and filtered under reduced pressure through cellulose paper. The resulting dark brown solids (11.9 g) were washed with de-ionized water and dried in air overnight. The dark brown solids contained 20% anhydroicaritin and 12% icariside I and an anhydroicaritin/icariside I ratio of 1.66. The recovery of anhydroicaritin in the precipitation procedure was 94% from the hydrolyzate.

Example 7 Recrystallization of Icariside IIcariside 1 (30 mg) obtained by a method described in Example 1 was dissolved in a minimum of hot tetrahydrofuran (THF). Hot methanol (approximately 10 mL) was then added. The hot THF/MeOH solution was filtered through a PTFE filter into a vial and allowed to evaporate at room temperature to about 5 mL, whereupon crystals began to form, and then placed in a 4° C. refrigerator for 24 hours. The crystals were filtered and washed with cold methanol and dried in a vacuum. Icariside I (21 mg) was isolated as yellow crystals and had a chromatographic purity of 97.4%.

Example 8 Large Scale Acid Hydrolysis of an Epimedium extractAn 800 g portion of an Epimedium sagittatum powder extract obtained from China containing about 13% total prenylflavonol glycosides as icariin was mixed with 4.0 L of 90% ethanol and 120 mL of sulfuric acid in a 10 L round bottom flask. The mixture was refluxed for 90 minutes and immediately chilled to stop the reaction. This mixture was filtered under reduced pressure through cellulose paper to remove insoluble sulfates and other materials. The cake was washed with approximately 200 mL of 90% ethanol. The resulting ethanolic hydrolyzate (4.0 L) contained 33.7 g of icariside I.

The ethanolic hydrolyzate prepared above was transferred to a 34 L container and 200 mL of 50% (w/w) sodium hydroxide solution was added with stirring, followed by 120 mL of phosphoric acid. De-ionized water (10 L) was then added. This mixture was adjusted to pH 8.2 using 120 mL of 50% sodium hydroxide solution. The mixture was stirred for 10 minutes and allowed to sit undisturbed at room temperature for 60 minutes prior to filtration under reduced pressure through cellulose paper. The resulting olive-green solids were washed with 750 mL of de-ionized water and dried under vacuum at 50° C. or in air at 80° C. The olive-green solids contained 44.6% icariside I. Recovery of icariside I in the precipitation procedure was 96% from the hydrolyzate.

Example 9 Large Scale Purification of an Epimedium Extract Containing Prenylflavonoid GlycosidesA 3.7 kg portion of an Epimedium sagittatum powdered extract obtained from China containing approximately 10% total prenylflavonol glycosides (PFG’s) assayed as icariin was stirred with 35 L of 85/15 acetone/water (v/v) in a 50 L mixing tank. The mixture was stirred vigorously for 30 minutes and allowed to sit for 5 minutes. The acetone extract layer (36 L) was decanted from the tank and contained 362 g of PFG’s. Recovery of the PFG’s in this extraction procedure was 96%.

A portion (about 500 mL) of the acetone extract was dried under reduced pressure at 50° C. or less, providing 16.1 g of brown solids which were analyzed to contain 28.6% total PFG’s when assayed as icariin.

TABLE 1
			PDE-5
			IC50
Entry	Sample description	% PFG’s	(μg/mL)
1	Vat extraction of Epimedium leaves,	8.0	5.78
	refluxing for 17 hours with methanol
2	Extract prepared by extracting Epimedium	7.2	4.24
	leaves with 50% ethanol
3	Extract prepared by extracting Epimedium	10.2	12.50
	leaves with 90% ethanol
4	Extract prepared by extracting Epimedium	16.30	5.27
	leaves with 50% EtOH and then purifying
	the extract (after removal of EtOH) by
	liq/liq extraction with butanol. Sample
	tested was the butanol fraction.
5	Extract prepared by extracting Epimedium	19.3	3.97
	leaves with 50% EtOH and purifying by
	liquid/liquid extraction. Sample tested was
	the aqueous fraction of the liq/liq extraction.
6	Purification of a 90% ethanol extract on	65.60	1.87
	a HP-20 reversed phase column

TABLE 2
			PDE-5
		%	IC50
Entry	Sample description	icarside I	(μg/mL)
7	Crude hydrolyzate composition obtained	2.1	24.30
	from a 50% EtOH extract of Epimedium
	leaves
8	Crude hydrolyzate composition obtained	5.3	9.39
	from a 90% EtOH extract of Epimedium
	leaves
9	Icariside I fraction obtained from	21.4	1.50
	purifying hydrolyzate Sample No. 7 on a
	SP-70 reversed-phase column and
	eluting icariside I with alcohol
10	Pure (recrystallized) icariside I	100	0.33
11	Pure anhydroicaritin	0	1.50
12	icariside I hydrate	0	21.50
13	sildenafil	0	0.031

• Liang DL & Zheng SL Effects of icaritin on cytochrome P450 enzymes in rats. Pharmazie 69:301-5 (2014).Read more (PubMed: 24791596) »
• Guo Y  et al. An anticancer agent icaritin induces sustained activation of the extracellular signal-regulated kinase (ERK) pathway and inhibits growth of breast cancer cells. Eur J Pharmacol 658:114-22 (2011). Read more (PubMed: 21376032) »
• Zhu Jf  et al. Icaritin shows potent anti-leukemia activity on chronic myeloid leukemia in vitro and in vivo by regulating MAPK/ERK/JNK and JAK2/STAT3 /AKT signalings. PLoS One 6:e23720 (2011). Read more (PubMed: 21887305) »
• The roots of Epimedium brevicornu Maxim

/////////Beijing Shenogen,  Granted Fast Track Status,  Novel Cancer Drug, Icaritin, New Drug Approval submission,  Beijing Food & Drug Administration, oral traditional Chinese medicine, barrenwort

CEP-18770, Delanzomib

cas 847499-27-8

Chemical Formula: C21H28BN3O5

Exact Mass: 413.21220, UNII-6IF28942WO;

CT-47098
NPH 007098
NPH007098

[(1R)-1-[[(2S,3R)-3-Hydroxy-2-[[(6-phenylpyridin-2-yl)carbonyl]amino]-1-oxobutyl]amino]-3-methylbutyl]boronic acid

[(lR)-l-[[(2S,3R)-3-hydroxy-2- [6-phenyl-pyridine-2-carbonyl)amino]-l-oxobutyl]amino]-3-methylbutylboronic acid,

Boronic acid, ((1R)-1-(((2S,3R)-3-hydroxy-1-oxo-2-(((6-phenyl-2-pyridinyl)carbonyl)amino)butyl)amino)-3-methylbutyl)-

Cephalon, Inc.

In phase 2, multiple mylenoma, Ethical Oncology Science (EOS), licensee

CEP-18770 was discovered through collaboration between Cephalon and Novuspharma/CTI.

Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380, and Cell Therapeutics Europe S.r.l., Via L. Ariosto, 23, I-20091 Bresso, Italy

Cephalon was acquired by Teva in October 2011. In 2013, EOS was acquired by Clovis Oncology.

Chemical Process Research and Development, Teva Branded Pharmaceutical Products R&D Inc., 383 Phoenixville Pike, Malvern, Pennsylvania 19355, United States

CEP-18770 is a reversible P2 threonine boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Displays anti-multimyeloma (MM) effect.

HPLC………http://www.apexbt.com/downloader/document/A4009/HPLC.pdf

NMR………http://www.apexbt.com/downloader/document/A4009/NMR.pdf

CLICK ON IMAGE FOR CLEAR VIEW

Delanzomib, also known as CEP-18770,  is An orally bioavailable synthetic P2 threonine boronic acid inhibitor of the chymotrypsin-like activity of the proteasome, with potential antineoplastic activity. Proteasome inhibitor CEP 18770 represses the proteasomal degradation of a variety of proteins, including inhibitory kappaBalpha (IkappaBalpha), resulting in the cytoplasmic sequestration of the transcription factor NF-kappaB; inhibition of NF-kappaB nuclear translocation and transcriptional up-regulation of a variety of cell growth-promoting factors; and apoptotic cell death in susceptible tumor cell populations. In vitro studies indicate that this agent exhibits a favorable cytotoxicity profile toward normal human epithelial cells, bone marrow progenitors, and bone marrow-derived stromal cells relative to the proteasome inhibitor bortezomib. The intracellular protein IkappaBalpha functions as a primary inhibitor of the proinflammatory transcription factor NF-kappaB

New series of dipeptidyl boronate inhibitors of 20S proteasome were identified to be highly potent drug-like candidates with IC₅₀ values of 1.2 and 1.6 nM, respectively, which showed better activities than the drug bortezomib on the market

ref

The potent, selective, and orally bioavailable threonine-derived 20S human proteasome inhibitor that has been advanced to preclinical development, [(1R)-1-[ [ (2S,3R)- 3-hydroxy-2-[ (6-phenylpyridine- 2-carbonyl) amino]-1 -oxobutyl] amino]- 3-methylbutyl] boronic acid (CEP-18770, has been reported

ref .

Dorsey BD, Iqbal M, Chatterjee S, Menta E, Bernardini R, Bernareggi A, et al. Discovery of a potent, selective, and orally active proteasome inhibitor for the treatment of cancer. J Med Chem. 2008;51:1068–1072. [PubMed]

Further, the anti-multiple myeloma protea-some inhibitor CEP-18770 enhanced the anti-myeloma activity of bortezomib and melphalan. The combination of anti-multiple myeloma proteasome inhibitor CEP-18770 intravenously and bortezomib exhibited complete regression of bortezomib-sensitive tumours. Moreover, this combination markedly delayed progression of bortezomib-resistant tumours compared to treatment with either agent alone

Paper

Development and scale-up of an optimized route to the peptide boronic acid, CEP-18770
Org Process Res Dev 2013, 17(3): 422

http://pubs.acs.org/doi/abs/10.1021/op400010u

 USED AS PRODRUG

CEP-18770 is an unstable peptide boronic acid and an amorphous solid, making it a challenging synthetic target. Process R&D led to a new process that avoided chromatography through crystalline intermediates, increased atom and volume efficiency, provided a chromophore, and gave higher yields and purity. A stable, crystalline diethanolamine adduct was discovered that has the potential to be used as a prodrug.

Compound 8 proved to be a direct substitute for delanzomib in the formulation process. In the first step of the IV formulation process, delanzomib is dissolved in water along with several excipients. Predictably, the delanzomib degrades during this process. It was found that upon dissolution in the lyophilization medium, 8 hydrolyzes to delanzomib,

N-[(1S,2R)-1-[[[(1R)-1–1[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-2-hydroxypropyl]-6-phenyl-2-pyridinecarboxamide (5)

PAPER

Discovery of a Potent, Selective, and Orally Active Proteasome Inhibitor for the Treatment of Cancer

http://pubs.acs.org/doi/abs/10.1021/jm7010589

The ubiquitin−proteasome pathway plays a central role in regulation of the production and destruction of cellular proteins. These pathways mediate proliferation and cell survival, particularly in malignant cells. The successful development of the 20S human proteasome inhibitor bortezomib for the treatment of relapsed and refractory multiple myeloma has established this targeted intervention as an effective therapeutic strategy. Herein, the potent, selective, and orally bioavailable threonine-derived 20S human proteasome inhibitor that has been advanced to preclinical development, [(1R)-1-[[(2S,3R)-3-hydroxy-2-[(6-phenylpyridine-2-carbonyl)amino]-1-oxobutyl]amino]-3-methylbutyl]boronic acid 20 (CEP-18770), is disclosed.

 [(1R)-1-[[(2S,3R)-3-Hydroxy-2-[(6-phenylpyridine-2-carbonyl)amino]-1-oxobutyl]amino]-3-methylbutyl]boronic Acid (20)

¹H NMR (CD₃OD, 400 MHz) δ 8.17 (m, 2H), 8.13 (m, 1H), 8.05 (m, 2H), 7.5 (m, 3H), 4.75 (d, J = 3.04 Hz, 1H), 4.42 (dq, J = 6.4, 2.92 Hz, 1H), 2.77 (t, b, 1H), 1.61 (m, 1H), 1.35 (t, J = 7.48 Hz, 2H), 1.29 (d, J = 6.36 Hz, 3H), 0.89 (d, J = 6.52 Hz, 6H);

¹³C NMR (CD₃OD) δ 20.76, 22.64, 23.78, 27.17, 41.14, 57.19, 68.13, 121.93, 124.95, 128.16, 130.04, 131.18, 139.48, 140.24, 150.05, 157.79, 167.23, 177.43;

MS m/z 452 (M + K), 436 (M + Na), 396 (M − OH), 378, 352, 264.

HRMS (M + Na) Calcd: 435.2056. Found: 435.2057.

Anal. Calcd for C₂₁H₂₈BN₃O₅: C, 61.03; H, 6.83; N, 10.17%. Found: C, 63.22; H, 6.52; N, 10.17%.

Patent

http://www.google.com/patents/WO2010056733A1?cl=en

Preferred among these compounds is [(lR)-l-[[(2S,3R)-3-hydroxy-2- [6-phenyl-pyridine-2-carbonyl)amino]-l-oxobutyl]amino]-3-methylbutylboronic acid, also known as CEP- 18770, which has the following structure:

PATENT

http://www.google.co.in/patents/WO2005021558A2

NOT SAME BUT SIMILAR

Example E.4 Boronic acid, [(lR)-l-[[(2S,3R)-3-hydroxy-2-[[4-(3-pyridyl)benzoyl]amino]-l- oxobutyI]amino]-3-methyIbutyl].

[00275] A mixture of 4-(pyridin-3-yl)benzamide, N-[(1S,2R)-1-[[[(1R)-1-

[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-methano-l,3,2-benzodioxaborol-2- yl]-3-methylbutyl]amino]carbonyl]-2-hydroxypropyl]- of Example D.8.3 (155 mg, 0.283 mmol), 2-methylpropylboronic acid (81 mg, 0.793 mmol) and 2N aqueous hydrochloric acid (0.3 ml) in a heterogeneous mixture of methanol (3 ml) and hexane (3 ml) was stirred at room temperature for 24 hours. The hexane layer was removed and the methanolic layer was washed with fresh hexane (about 5 ml). Ethyl acetate (10 ml) was added to the methanol layer which was then concentrated. The residue was taken up with ethyl acetate and the mixture was concentrated. This step was repeated (2-3 times) until an amorphous white solid was obtained. The solid was then triturated with diethyl ether (5 ml) and the surnatant was removed by decantation. This step was repeated. The residue (126 mg) was combined with the product of a similar preparation (140 mg) and dissolved in ethyl acetate (about 40 ml) and a small amount of methanol (2-3 ml). The solution was washed with a mixture of NaCl saturated solution (7 ml) and 10% NaHCO₃ (2 ml). The layers were separated and the aqueous phase was further washed with ethyl acetate (2 x 20 ml). The combined organic phases were dried over sodium sulfate and concentrated. The residue was taken up with ethyl acetate (about 20 ml) and the minimum amount of methanol, and then concentrated to small volume (about 5 ml). The resulting white was collected by filtration and dried under vacuum at 50°C (160 mg, 65% overall yield).

1H NMR (MeOH-d4): 8.90 (IH, s); 8.49 (IH, d, J=4.0); 8.20 (IH, d, J=8.1); 8.06 (2H, d, J=8.1); 7.85 (2H, d, J=8.1); 7.58 (IH, t br., J=6.0); 4.80 (IH, d, J=3.9); 4.40-4.29 (IH, m); 2.78 (IH, t, J=7.5); 1.73-1.61 (IH, m); 1.38 (2H, t, J=6.9); 1.31 (3H, d, J=6.3); 0.94 (6H, d, J=6.31). [00276] Further compounds prepared according to the above procedure for

Example E.4 are reported in Table E-4. Table E-4

E.4.3 IS THE COMPD

D.8.12 Chemical Name: 6-Phenyl-2-pyridinecarboxamide,N-[(lS,2R)-l-[[[(lR)- l-[(3aS,4S,6S,7aR)-hexahydro-3a,5,5-trimethyl-4,6-

 THIS IS PRECURSOR OF FINAL PDT

methano-l,3,2-benzodioxaborol-2-yl]-3- methylbutyl]amino]carbonyl]-2-hydroxypropyl]. Analytical Data: Η -NMR (DMSO-d6): 9.20-8.95 (IH, m); 8.76 (IH, d, J=8.55 Hz); 8.26-8.16 (4H, m); 8.12 (IH, t, J= 7.77 Hz); 8.02 (IH, d, J= 7.56 Hz); 7.60-7.47 (4H, m); 5.27 (IH, d, J= 4.97 Hz); 4.50 (IH, dd, J= 4.22 Hz, J= 8.50 Hz); 4.16-4.07 (2H, m); 2.65-2.56 (IH, m); 2.25-2.15 (IH, m); 2.09-1.98 (IH, m); 1.84 (IH, t, J= 5.62 Hz); 1.79- 1.73 (IH, m); 1.73-1.66 (IH, m); 1.66-1.59 (IH, m); 1.40-1.26 (4H, m); 1.23 (7H, d, J= 10.89 Hz); 1.15-1.10 (4H, m); 0.85 (7H, d, J= 6.56 Hz); 0.79 (IH, bs).

1. Fuchs, Ota. Proteasome inhibition as a therapeutic strategy in patients with multiple myeloma. Multiple Myeloma (2009), 101-125. CODEN: 69MVM2 AN 2010:737549

2. Genin, E.; Reboud-Ravaux, M.; Vidal, J. Proteasome inhibitors: recent advances and new perspectives in medicinal chemistry. Current Topics in Medicinal Chemistry (Sharjah, United Arab Emirates) (2010), 10(3), 232-256. CODEN: CTMCCL ISSN:1568-0266. CAN 152:516315 AN 2010:423458

3. Sanchez, Eric; Li, Mingjie; Steinberg, Jeffrey A.; Wang, Cathy; Shen, Jing; Bonavida, Benjamin; Li, Zhi-Wei; Chen, Haiming; Berenson, James R. The proteasome inhibitor CEP-18770 enhances the anti-myeloma activity of bortezomib and melphalan. British Journal of Haematology (2010), 148(4), 569-581. CODEN: BJHEAL ISSN:0007-1048. AN 2010:353952

4. Dick, Lawrence R.; Fleming, Paul E. \Building on bortezomib: second-generation proteasome inhibitors as anti-cancer therapy. Drug Discovery Today (2010), 15(5/6), 243-249. CODEN: DDTOFS ISSN:1359-6446. AN 2010:318415

5. Ruggeri, Bruce; Miknyoczki, Sheila; Dorsey, Bruce; Hui, Ai-Min. The development and pharmacology of proteasome inhibitors for the management and treatment of cancer. Advances in Pharmacology (San Diego, CA, United States) (2009), 57(Contemporary Aspects of Biomedical Research: Drug Discovery), 91-135. CODEN: ADPHEL ISSN:1054-3589. AN 2010:62762

6. Chen-Kiang, Selina; Di Liberto, Maurizio; Huang, Xiangao. Targeting CDK4 and CDK6 kinases or genes thereof in cancer therapy for sensitizing drug-resistant tumors. PCT Int. Appl. (2009), 149pp. CODEN: PIXXD2 WO 2009061345 A2 20090514 CAN 150:531264 AN 2009:586623

7. Rickles, Richard; Lee, Margaret S. Use of adenosine A2A receptor agonists and phosphodiesterase (PDE) inhibitors for the treatment of B-cell proliferative disorders, and combinations with other agents. PCT Int. Appl. (2009), 70 pp. CODEN: PIXXD2 WO 2009011893 A2 20090122 CAN 150:160095 AN 2009:86451

8. Rickles, Richard; Pierce, Laura; Lee, Margaret S. Combinations for the treatment of B-cell proliferative disorders. PCT Int. Appl. (2009), 79pp. CODEN: PIXXD2 WO 2009011897 A1 20090122 CAN 150:160094 AN 2009:83374

9. Hoveyda, Hamid; Fraser, Graeme L.; Benakli, Kamel; Beauchemin, Sophie; Brassard, Martin; Drutz, David; Marsault, Eric; Ouellet, Luc; Peterson, Mark L.; Wang, Zhigang. Preparation and methods of using macrocyclic modulators of the ghrelin receptor. U.S. Pat. Appl. Publ. (2008), 178pp. CODEN: USXXCO US 2008194672 A1 20080814 CAN 149:288945 AN 2008:975261

10. Piva, Roberto; Ruggeri, Bruce; Williams, Michael; Costa, Giulia; Tamagno, Ilaria; Ferrero, Dario; Giai, Valentina; Coscia, Marta; Peola, Silvia; Massaia, Massimo; Pezzoni, Gabriella; Allievi, Cecilia; Pescalli, Nicoletta; Cassin, Mara; di Giovine, Stefano; Nicoli, Paola; de Feudis, Paola; Strepponi, Ivan; Roato, Ilaria; Ferracini, Riccardo; Bussolati, Benedetta; Camussi, Giovanni; Jones-Bolin, Susan; Hunter, Kathryn; Zhao, Hugh; Neri, Antonino; Palumbo, Antonio; Berkers, Celia; Ovaa, Huib; Bernareggi, Alberto; Inghirami, Giorgio. CEP-18770: a novel, orally active proteasome inhibitor with a tumor-selective pharmacologic profile competitive with bortezomib. Blood (2008), 111(5), 2765-2775. CODEN: BLOOAW ISSN:0006-4971. CAN 149:486154 AN 2008:292777

11. Dorsey, Bruce D.; Iqbal, Mohamed; Chatterjee, Sankar; Menta, Ernesto; Bernardini, Raffaella; Bernareggi, Alberto; Cassara, Paolo G.; D’Arasmo, Germano; Ferretti, Edmondo; De Munari, Sergio; Oliva, Ambrogio; Pezzoni, Gabriella; Allievi, Cecilia; Strepponi, Ivan; Ruggeri, Bruce; Ator, Mark A.; Williams, Michael; Mallamo, John P. Discovery of a Potent, Selective, and Orally Active Proteasome Inhibitor for the Treatment of Cancer. Journal of Medicinal Chemistry (2008), 51(4), 1068-1072. CODEN: JMCMAR ISSN:0022-2623. CAN 148:345774 AN 2008:146611

12. Dorsey, Bruce D.; Menta, Ernesto; Bernardini, Raffaella; Bernareggi, Alberto; Casara, Paolo G.; D’Arasmo, Germano; Ferretti, Edmondo; De Munari, Sergi; Oliva, Ambrogio; Iqbal, Mohamed; Chatterjee, Sankar; Ruggeri, Bruce; Ator, Mark A.; Williams, Michael; Mallamo, John P. CEP-18770: Discovery of a Potent, Selective and Orally Active Proteasome Inhibitor for the Treatment of Cancer. Frontiers in CNS and Oncology Medicinal Chemistry, ACS-EFMC, Siena, Italy, October 7-9 (2007), COMC-027. CODEN: 69KAR2 AN 2007:1171000

13. Marblestone Jeffrey G Ubiquitin Drug Discovery & Diagnostics 2009 – First Annual Conference. IDrugs : the investigational drugs journal (2009), 12(12), 750-3.

/////CEP-18770, delanzomib

B(C(CC(C)C)NC(=O)C(C(C)O)NC(=O)C1=CC=CC(=N1)C2=CC=CC=C2)(O)O

AMG-319

N-((1S)-1-(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine, WO2008118468

(S)-N-(1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine

 CAS 1608125-21-8

Chemical Formula: C21H16FN7
Exact Mass: 385.14512

Phosphoinositide-3 kinase delta inhibitor

AMGEN, PHASE 2

PI3K delta isoform selective inhibitor that is being investigated in human clinical trials for the treatment of PI3K-mediated conditions or disorders, such as cancers and/or proliferative diseases

Useful for treating PI3K-mediated disorders such as acute myeloid leukemia, myelo-dysplastic syndrome, myelo-proliferative diseases, chronic myeloid leukemia, T-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, non-Hodgkins lymphoma, B-cell lymphoma, or breast cancer.

Amgen is developing AMG-319, a small molecule PI3K-δ inhibitor, for treating lymphoid malignancies and solid tumors including, head and neck squamous cell carcinoma.

AMG-319 is a highly selective, potent, and orally bioavailable small molecule inhibitor of the delta isoform of the 110 kDa catalytic subunit of class IA phosphoinositide-3 kinases (PI3K) with potential immunomodulating and antineoplastic activities. PI3K-delta inhibitor AMG 319 prevents the activation of the PI3K signaling pathway through inhibition of the production of the second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), thus decreasing proliferation and inducing cell death. Unlike other isoforms of PI3K, PI3K-delta is expressed primarily in hematopoietic lineages. The targeted inhibition of PI3K-delta is designed to preserve PI3K signaling in normal, non-neoplastic cells.

PATENT

http://www.google.com/patents/WO2008118468A1?cl=en

PATENT

WO2013152150

http://www.google.com/patents/WO2013152150A1?cl=en

PATENT

WO-2015171725

Example 4: Method of making N-((lSM-(7-fluoro-2-(2-pyridinyl)- 3-quinolinyl)ethyl)-9H-purin-6-amine

N-((l S)- 1 -(7-Fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine (4) is synthesized in four steps beginning with (S)-l-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethanamine hydrochloride (1). A nucleophilic aromatic substitution between coupling partners 1 and purine 5 affords the penultimate intermediate 2. Cleavage of the p-methoxybenzyl (PMB) group leads to the isolation of the desired butyl acetate solvate 3. A crystalline form change is induced through an aqueous-acetone recrystallization to afford the target hydrate 4.

Synthetic Scheme

Step 1. Preparation of PMB protected pyridylpurinamine tosylate (2)

(S)- 1 -(7-Fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethanamine is prepared similar to that described in US20130267524. The (S)-l-(7-fluoro-2-(pyridin-2-

yl)quinolin-3-yl)ethanamine hydrochloride (1) is coupled to PMB-chloropurine (5, prepared similar to that described in J. Med. Chem. 1988,31, 606-612) in the presence of K₂CO₃ in IPA. Upon reaction completion the K₂CO₃ is removed via filtration and the product is crystallized by the addition of /?-toluenesulfonic acid (pTSA). Isolation of the PMB-protected pyridylpurinamine tosylate (2) is conducted via filtration.

Dry 100 L reactor under nitrogen. Set the temperature to 20 ± 5 °C. Charge (l S)-N-chloro-l-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethanamine HCl salt (1) to the reactor. Then 9-(4-methoxybenzyl)-6-chloro-9H-purine (5) is added. Potassium carbonate is added to the reactor. Isopropyl alcohol is added to the reactor and the mixture is heated to 80 °C and stirred for 24 hours. Additional isopropyl alcohol is added to the reactor and the mixture is cooled to 20 °C. The mixture is filtered through Celite and the solid is washed with isopropyl alcohol and the isopropyl alcohol solutions containing 9-(4-methoxybenzyl)-N-((S)- 1 -(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine are collected.

The 9-(4-methoxybenzyl)-N-((S)- 1 -(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine isopropyl alcohol solution is heated to 50 °C. /^-Toluene sulfonic acid monohydrate is dissolved in isopropyl alcohol and added to the 9-(4-methoxybenzyl)-N-((S)-l-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine in portions. The mixture is slowly cooled to 20 ± 5 °C over 6 ± 2 hrs. The crystalline 9-(4-methoxybenzyl)-N-((S)- 1 -(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)- 9H-purin-6-amine toluene sulfonic acid salt is collected, rinsed with isopropyl alcohol and dried with vacuum.

Example 5: Method of Making the Crystalline Hydrate Form of N-((1S)-1- (7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine Step 1: Isolation of a Butyl Acetate (BuOAc) Solvate of N-((lS)-l-(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine (3)

To a 2 L jacketed reactor equipped with a condenser, a mechanical stirrer, and a bubbler, under an atmosphere of N₂, was added N-((l S)-l-(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9-(4-methoxybenzyl)-9H-purin-6-amine (2, 100.0 g, 0.148 mol), followed by acetic acid (AcOH; 240 mL) and 1 -dodecanethiol (71.1 mL, 0.295 mol). The vessel was evacuated and back-filled with nitrogen three times. Methanesulfonic acid (MSA; 28.7 mL, 0.443 mol) was added to the vessel over 10 minutes. Then, the reaction was heated to 80 °C and stirred for 20 hrs. The reaction was then cooled to ambient temperature, after which toluene (1000 mL) and water (700 mL) were sequentially added. The solution was then stirred for 30 minutes. The phases were separated by removing the organic phase, adding another charge of toluene (1000 mL) to the aqueous phase, and the mixture was stirred for another 30 minutes. After removing the organic phase again, the aqueous phase was charged to a jacketed 5 L reactor equipped with a mechanical stirrer followed by n-butyl acetate (1500 mL,) and heated to 50 °C. The aqueous phase was neutralized to pH 6.3 with 10 N NaOH (350 mL). The organic (BuOAc) phase was azeotropically dried to 600 ppm water, while keeping a constant volume. The dried organic phase was polish filtered at 50 °C to remove salts, which were subsequently washed with hot BuOAc (285 mL). The BuOAc was charged back into the 2 L jacketed reactor equipped with a mechanical stirred and distillation apparatus, and then concentrated to 54 mg/g of N-((l S)-l-(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine in solution. The solution was then seeded with 1 wt% seed of the BuOAc solvate of N-((l S)- 1 -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine. The slurry was further concentrated to 300 mL total volume and cooled to ambient temperature over 1 hour. Heptane (460 mL) was added dropwise to the solution, and the solution was aged overnight. The supernatant concentration was checked, and determined to be 5.3 mg/g of N-((l S)-l-(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine. The supernatant was filtered and the resulting solid cake was washed with 1 : 1 BuOAc:heptane (280 mL), followed by heptane (280 mL). The washed cake was then

allowed to dry on the filter. The BuOAc solvate of N-((l S)- l -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine was obtained as a white solid (59.5 g, 99.6 LCAP, 86.3 wt%, 90 % corrected yield). ^!H NMR (400 MHz, CDC1₃) δ 13.72 (s, 1H), 8.80 (s, 1H), 8.37 (s, 1H), 8.31 (s, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.92 (d, J = 18.8 Hz, 2H), 7.76 (t, J = 1 1.6 Hz, 2H), 7.39 (s, 1H), 7.31 (td, J = 8.7, 2.5 Hz, 1H), 6.15 (s, 1H), 4.06 (t, J = 6.7 Hz, 1H), 2.04 (s, 1H), 1.65 – 1.44 (m, 3H), 1.39 (dt, J = 14.9, 7.4 Hz, 1H), 1.33 – 1.20 (m, 2H), 0.93 (t, J = 7.4 Hz, 1H), 0.88 (t, J = 6.8 Hz, 1H); ¹³C NMR (101 MHz, CDC1₃) δ 152.28 (s), 148.46 (s), 138.10 (s), 137.22 (s), 135.58 (s), 129.47 (s), 124.80 (s), 123.53 (s), 1 13.24 – 1 13.09 (m), 1 12.89 (d, J = 20.3 Hz), 64.40 (s), 48.60 (s), 31.91 (s), 30.67 (s), 29.05 (s), 22.72 (s), 19.15 (s), 14.15 (s); IR: 3193, 3087, 2967, 2848, 1738, 1609, 1493, 1267, 1242, 1 143, 933, 874, 763, 677, 646, 627, 606, 581 , 559, 474 cm^“1; exact mass m/z calcd for C₂iH₁₆FN₇, (M + H)⁺386.1451 , found 386.1529; MP = 144 °C.

Step 2: Isolation of the Crystalline Hydrate of N-((lS)-l-(7-fluoro-2-(2-pyridinvn-3-quinolinyl)ethyl)-9H-purin-6-amine 4

To a 100 L reactor with its jacket set to 20 °C, 1.206 kg butyl acetate solvate of N-((l S)- l -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine 3 was charged, followed by 6.8 L of acetone and 6.8 L of water. The resulting mixture was stirred at 90 rpm under nitrogen for 13 minutes to ensure complete dissolution of all solids. During these charges, the reactor contents increased in temperature that maximized at 26 °C. The solution was then transferred to another clean 100 L reactor through a 5 μιη filter, and stirred at 85 rpm under nitrogen. The solution was heated to 45 °C, and water (14.8 L) was added to reach a water content (by Karl Fischer, KF) of 75 wt%. The reactor solution was assayed by HPLC and shown to contain 42 mg/g N-((l S)- l -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine. The solution was seeded with a slurry of 1 13 g of the crystalline hydrate of N-((l S)- l -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine in 1 L water, and the seed slurry was rinsed into the reactor with an additional 1 L water. The reactor contents were cooled to 0 °C over 16 h and held at that temperature for 1 h. The supernatant was then assayed, and found to contain 7.6 mg/g of N-((l S)- l -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine. Next, 10 L of water was added to the reactor over 38 min and aged for 1 h. The supernatant was assayed at 4.9 mg/g, and the solids were isolated by filtration. The solids were washed with an acetone/water solution (140 mL acetone in 2.7 L water), then 4 L water, and dried under nitrogen on the filter for 68 h. The crystalline hydrate of N-((l S)-l -(7-fluoro-2-(2-pyridinyl)-3-quinolinyl)ethyl)-9H-purin-6-amine was isolated as an off-

white solid (1.12 kg, 616 ppm acetone, 3.73 wt% water, 99.56 LCAP, 95.88 wt%). This material was co-milled at 3900 rpm using a 0.024″ screen to yield an off-white powder (1.09 kg, 99.7 LCAP, 95.4 wt%, 75% yield). Calculated losses were 212 g (18%) to liquors, 5.5g (0.5%) to washes, and 23 g (2%) to fouling. ¾ NMR (400 MHz, DMSO) δ 12.86 (s, 1H), 8.69 (s, 1H), 8.64 (s, 1H), 8.27 (s, 1H), 8.10 (s, 1H), 8.06 – 7.91 (m, 4H), 7.76 (dd, J = 10.4, 2.4 Hz, 1H), 7.50 (ddd, J = 19.2, 9.5, 3.6 Hz, 2H), 6.03 (s, 1H), 3.38 (s, 2H), 1.63 (d, J = 6.6 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 163.58, 161.12, 158.36, 157.94, 151.99, 147.98, 146.49, 146.36, 136.82, 134.07, 130.24, 130.14, 124.69, 124.65, 123.30, 1 17.36, 1 17.1 1, 112.10, 1 1 1.90, 46.02, 22.01. HRMS m/z Calcd. for C₂iH₁₇FN₇ (M + H): 386.15295. Found: 386.15161.

PAPER

1: Cushing TD, Hao X, Shin Y, Andrews K, Brown M, Cardozo M, Chen Y, Duquette J, Fisher B, Gonzalez-Lopez de Turiso F, He X, Henne KR, Hu YL, Hungate R, Johnson MG, Kelly RC, Lucas B, McCarter JD, McGee LR, Medina JC, San Miguel T, Mohn D, Pattaropong V, Pettus LH, Reichelt A, Rzasa RM, Seganish J, Tasker AS, Wahl RC, Wannberg S, Whittington DA, Whoriskey J, Yu G, Zalameda L, Zhang D, Metz DP. Discovery and in vivo evaluation of (S)-N-(1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine (AMG319) and related PI3Kδ inhibitors for inflammation and autoimmune disease. J Med Chem. 2015 Jan 8;58(1):480-511. doi: 10.1021/jm501624r. Epub 2014 Dec 3. PubMed PMID: 25469863.

http://pubs.acs.org/doi/abs/10.1021/jm501624r

The development and optimization of a series of quinolinylpurines as potent and selective PI3Kδ kinase inhibitors with excellent physicochemical properties are described. This medicinal chemistry effort led to the identification of 1 (AMG319), a compound with an IC₅₀ of 16 nM in a human whole blood assay (HWB), excellent selectivity over a large panel of protein kinases, and a high level of in vivo efficacy as measured by two rodent disease models of inflammation.

(S)-N-(1-(7-Fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)-9H-purin-6-amine (1)

//////////

C[C@H](NC1=C2N=CNC2=NC=N1)C3=CC4=CC=C(F)C=C4N=C3C5=NC=CC=C5

Synthesis

Read also

http://newdrugapprovals.org/2014/07/08/japan-first-to-approve-alectinib-%E3%82%A2%E3%83%AC%E3%82%AF%E3%83%81%E3%83%8B%E3%83%96-%E5%A1%A9%E9%85%B8%E5%A1%A9-af-802-for-alk-nsclc/

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VAL-083

(1R,2S)-1-((R)-oxiran-2-yl)-2-((S)-oxiran-2-yl)ethane-1,2-diol

Galactitol, 1,​2:5,​6-​dianhydro-

• 1,2:5,6-Dianhydrodulcitol
• 1,2:5,6-Dianhydrogalactitol
• 1,2:5,6-Diepoxydulcitol

Dianhydrodulcitol; Dianhydrogalactitol; VAL083; VAL 083, Dulcitol diepoxide, NSC 132313

CAS 23261-20-3

MF C6H10O4, MW 146.14

VAL-083 is a bi-functional alkylating agent; inhibit U251 and SF188 cell growth in monolayer better than TMZ and caused apoptosis

VAL-083 is a bi-functional alkylating agent, with potential antineoplastic activity. Upon administration, VAL-083 crosses the blood brain barrier (BBB) and appears to be selective for tumor cells. This agent alkylates and crosslinks DNA which ultimately leads to a reduction in cancer cell proliferation. In addition, VAL-083 does not show cross-resistance to other conventional chemotherapeutic agents and has a long half-life in the brain. Check for active clinical trials or closed clinical trials using this agent

Currently, VAL-083 is approved in China to treat chronic myelogenous leukemia and lung cancer, while the drug has also secured orphan drug designation in Europe and the US to treat malignant gliomas.

LAUNCHED CHINA FOR Cancer, lung

Del Mar Pharmaceuticals Inc……..Glioblastoma…………..PHASE2

DelMar and MD Anderson to accelerate development of anti-cancer drug VAL-083
DelMar Pharmaceuticals has collaborated with the University of Texas MD Anderson Cancer Center (MD Anderson) to speed up the clinical development of its VAL-083 anti-cancer drug.

VAL-083 is a BI-Functional alkylating agent; INHIBIT U251 and SF188 Cell Growth in monolayer Better than TMZ and Caused apoptosis. IC50 Value : 5 uM (INHIBIT U251, SF188, T98G Cell Growth in monolayer after 72h) [1]. in vitro :.. VAL-083 INHIBITED U251 and SF188 Cell Growth in monolayer and as neurospheres Better than TMZ and Caused apoptosis after 72 hr Formation Assay In the colony, VAL-083 (5 uM) SF188 Growth suppressed by about 95% are T98G cells classically TMZ-resistant and express MGMT, but VAL-083 inhibited their growth in monolayer after 72 hr in a dose-dependent manner (IC50, 5 uM). VAL-083 also inhibited the growth of CSCs (BT74, GBM4, and GBM8) . by 80-100% in neurosphere self-Renewal assays Conversely, there was minimal normal Effect on Human Neural stem cells [1]. in Vivo : Clinical Trial : Safety Study of VAL-083 in Patients With Recurrent Malignant glioma or Secondary Progressive Brain Tumor. Phase 1 / Phase 2

VAL-083 has demonstrated activity in cyclophosphamide, BCNU and phenylanine mustard resistant cell lines and no evidence of cross-resistance has been encountered in published clinical studies. Based on the presumed alkylating functionality of VAL-083, published literature suggests that DNA repair mechanisms associated with Temodar and nitrosourea resistance, such as 06-methylguanine methyltransferace (MGMT), may not confer resistance to VAL-083.  VAL-083 readily crosses the blood brain barrier where it maintains a long half-life in comparison to the plasma. Published preclinical and clinical research demonstrates that VAL-083 is selective for brain tumor tissue.  VAL-083 has been assessed in multiple studies as chemotherapy in the treatment of newly diagnosed and recurrent brain tumors. In published clinical studies, VAL-083 has previously been shown to have a statistically significant impact on median survival in high grade gliomas when combined with radiation vs. radiation alone. The main dose-limiting toxicity related to the administration of VAL-083 in previous clinical studies was myelosuppression

Glioblastoma is the most common form of primary brain cancer

DelMar Pharmaceuticals has collaborated with the University of Texas MD Anderson Cancer Center (MD Anderson) to speed up the clinical development of its VAL-083 anti-cancer drug.

VAL-083 is a small-molecule chemotherapeutic designed to treat glioblastoma multiforme (GBM), the most common and deadly cancer that starts within the brain.

Under the deal, MD Anderson will begin a new Phase II clinical trial with VAL-083 in patients with GBM at first recurrence / progression, prior to Avastin (bevacizumab) exposure.

During the trial, eligible patients will have recurrent GBM characterised by a high expression of MGMT, the DNA repair enzyme implicated in drug-resistance, and poor patient outcomes following current front-line chemotherapy.

The company noted that MGMT promoter methylation status will be used as a validated biomarker for enrollment and tumours must exhibit an unmethylated MGMT promoter for patients to be eligible for the trial.

DelMar chairman and CEO Jeffrey Bacha said: “The progress we continue to make with our research shows that VAL-083 may offer advantages over currently available chemotherapies in a number of tumour types.

“This collaboration will allow us to leverage world-class clinical and research expertise and a large patient population from MD Anderson as we extend and accelerate our clinical focus to include GBM patients, following first recurrence of their disease.

“We believe that VAL-083’s unique cytotoxic mechanism offers promise for GBM patients across the continuum of care as a potential superior alternative to currently available cytotoxic chemotherapies, especially for patients whose tumours exhibit a high-expression of MGMT.”

The deal will see DelMar work with the scientists and clinicians at MD Anderson to accelerate its research in order to transform the treatment of patients whose cancers fail or are unlikely to respond to existing treatments.

In more than 40 clinical trials, VAL-083 showed clinical activity against several cancers including lung, brain, cervical, ovarian tumours and leukemia both as a single-agent and in combination with other treatments.

PATENT

WO 2012024368

https://www.google.com/patents/WO2012024368A3?cl=en

Dianhydrogalactitol (DAG or dianhydrodulcitol) can be synthesized from dulcitol which can be produced from natural sources (such as Maytenus confertiflora) or commercial sources.The structure of DAG is given below as Formula (I).

One method for the preparation of dulcitol from Maytenus confertiflora is as follows: (1) The Maytenus confertiflora plant is soaked in diluted ethanol (50-80%) for about 24 hours, and the soaking solution is collected. (2) The soaking step is repeated, and all soaking solutions are combined. (3) The solvent is removed by heating under reduced pressure. (4) The concentrated solution is allowed to settle overnight and the clear supernatant is collected. (5) Chloroform is used to extract the supernatant. The chloroform is then removed under heat and reduced pressure. (6) The residue is then dissolved in hot methanol and cooled to allow crystallization. (7) The collected crystals of dulcitol are filtered and dried under reduced pressure. The purified material is dulcitol, contained in the original Maytenus confertiflora plant at a concentration of about 0.1% (1/1000).

DAG can be prepared by two general synthetic routes as described below:

Route 1 :

Dulcitol DAG

Route 2. Dulcitol

In Route 1 , “Ts” represents the tosyl group, or p-toluenesulfonyl group. PATENT

However, the intermediate of Route 1, 1,6-ditosy)dulcitol, was prepared with low yield (~36%), and the synthesis of 1,6-ditosyldulcitol was poorly reproducible. Therefore, the second route process was developed, involving two major steps: (1) preparation of dibromodulcitol from dulcitol; and (2) preparation of dianhydrodulcitol from dibromodulcitol.

Dibromodulcitol is prepared from dulcitol as follows: (1) With an aqueous HBr solution of approximately 45% HBr concentration, increase the HBr concentration to about 70% by reacting phosphorus with bromine in concentrated HBr in an autoclave. Cool the solution to 0° C. The reaction is:

2P+3Br₂→2PBr₃+H₂0→HBr†+H₃P0₄. (2) Add the dulcitol to the concentrated HBr solution and reflux at 80° C to complete the reaction. (3) Cool the solution and pour the mixture onto ice water. Dibromodulcitol is purified through recrystallization.

The results for the preparation of dibromodulcitol (DBD) are shown in Table 1, below.

TABLE 1

For the preparation of DAG from DBD, DBD was poorly dissolved in methanol and ethanol at 40° C (different from what was described in United States PATENT

Patent No. 3,993,781 to Horvath nee Lengyel et al., incorporated herein by this reference). At refluxing, DBD was dissolved but TLC showed that new impurities formed that were difficult to remove from DBD.

The DBD was reacted with potassium carbonate to convert the DBD to dianhydrogalactitol.

The results are shown in Table 2, below.

TABLE 2

In the scale-up development, it was found the crude yield dropped significantly. It is unclear if DAG could be azeotropic with BuOH. It was confirmed that t-BuOH is essential to the reaction. Using MeOH as solvent would result in many impurities as shown spots on TLC. However, an improved purification method was developed by using a slurry with ethyl ether, which could provide DAG with good purity. This was developed after a number of failed attempts at recrystallization of DAG.

Bromination of dulcitol with HBr at 80°C gives dibromodulcitol , which upon epoxidation in the presence of K2CO3 in t-BuOH or NaOH in H2O  or in the presence of ion exchange resin Varion AD (OH) (4) affords the target dianhydrogalactitol .

PATENT

US 20140155638

SCHEME 5

PATENT

CN 103923039

http://www.google.com/patents/CN103923039A?cl=en

The resulting Dulcitol 9g and 18ml mass percent concentration of 65% hydrobromic acid at 78 ° C under reflux for 8 hours to give 1,6-dibromo dulcitol, and the product is poured into ice crystals washed anhydrous tert-butyl alcohol, and dried to give 1,6-dibromo dulcitol crystal, then 10.0gl, 6- dibromo dulcitol sample is dissolved in t-butanol, adding solid to liquid 2 % obtained through refining process 1,6_ dibromo dulcitol seed stirred and cooled to 0 ° C, allowed to stand for seven days to give 1,6_ dibromo dulcitol crystal, anhydrous t-butanol, dried to give 1,6-dibromo dulcitol. 5g of the resulting 1,6_ dibromo Euonymus dissolved in 50ml tert-butanol containing 5g of potassium carbonate, the elimination reaction, at 80 ° C under reflux time was 2 hours, the resulting product was dissolved in t-butanol, Join I% stock solution to the water quality of 1,2,4,5_ two Dulcitol including through a purification step to get less than 1% of 1,2,5,6_ two to water Dulcitol seeded stirring, cooling to 0 ° C, allowed to stand for I-day, two to go get 1,2,5,6_ water Dulcitol crystals washed anhydrous tert-butyl alcohol, and dried to give 1,2,5,6 two to crystalline water Dulcitol and lyophilized to give two to water Dulcitol lyophilized powder, containing I, 2,4,5- two to water Dulcitol less than 0.3%.

PATENT

WO 2005030121

PATENT

US 20140066642

• DAG can be prepared by two general synthetic routes as described below:
• In Route 1, “Ts” represents the tosyl group, or p-toluenesulfonyl group.
• However, the intermediate of Route 1, 1,6-ditosyldulcitol, was prepared with low yield (˜36%), and the synthesis of 1,6-ditosyldulcitol was poorly reproducible. Therefore, the second route process was developed, involving two major steps: (1) preparation of dibromodulcitol from dulcitol; and (2) preparation of dianhydrodulcitol from dibromodulcitol.
• Dibromodulcitol is prepared from dulcitol as follows: (1) With an aqueous HBr solution of approximately 45% HBr concentration, increase the HBr concentration to about 70% by reacting phosphorus with bromine in concentrated HBr in an autoclave. Cool the solution to 0° C. The reaction is: 2P+3Br₂→2PBr₃+H₂O→HBr↑+H₃PO₄. (2) Add the dulcitol to the concentrated HBr solution and reflux at 80° C. to complete the reaction. (3) Cool the solution and pour the mixture onto ice water. Dibromodulcitol is purified through recrystallization.

PATENT

US 20150329511

 PAPER

Antibacterial and Anti-Quorum Sensing Molecular Composition Derived from Quercus cortex (Oak bark) Extract

REFERENCES

Currently, VAL-083 is approved in China to treat chronic myelogenous leukemia and lung cancer, while the drug has also secured orphan drug designation in Europe and the US to treat malignant gliomas.

[1]. Fotovati A, Hu KJ, Wakimoto H, VAL-083, A NOVEL N7 ALKYLATING AGENT, SURPASSES TEMOZOLOMIDE ACTIVITY AND INHIBITS CANCER STEM CELLS, PROVIDING A NEW POTENTIAL TREATMENT OPTION FOR GLIOBLASTOMA MULTIFORME. Neuro-oncology, 2012, 14, AbsET-37, Suppl. 6

[2]. Fotovati A, Hu KJ, Wakimoto H, VAL-083, A NOVEL AGENT N7 alkylating, SURPASSES temozolomide Inhibits TREATMENT ACTIVITY AND STEM CELLS, PROVIDING A NEW TREATMENT OPTION FOR POTENTIAL glioblastoma multiforme. Neuro-oncology, 2012, 14, AbsET-37, Suppl. 6

1: Szende B, Jeney A, Institoris L. The diverse modification of N-butyl-N-(4-hydroxybutyl) nitrosamine induced carcinogenesis in urinary bladder by dibromodulcitol and dianhydrodulcitol. Acta Morphol Hung. 1992;40(1-4):187-93. PubMed PMID: 1365762.

2: Anderlik P, Szeri I, Bános Z. Bacterial translocation in dianhydrodulcitol-treated mice. Acta Microbiol Hung. 1988;35(1):49-54. PubMed PMID: 3293340.

3: Huang ZG. [Clinical observation of 15 cases of chronic myelogenous leukemia treated with 1,2,5,6-dianhydrodulcitol]. Zhonghua Nei Ke Za Zhi. 1982 Jun;21(6):356-8. Chinese. PubMed PMID: 6957285.

4: Anderlik P, Szeri I, Bános Z, Wessely M, Radnai B. Higher resistance of germfree mice to dianhydrodulcitol, a lymphotropic cytostatic agent. Acta Microbiol Acad Sci Hung. 1982;29(1):33-40. PubMed PMID: 6211912.

5: Bános Z, Szeri I, Anderlik P. Effect of Bordetella pertussis vaccine on the course of lymphocytic choriomeningitis (LCM) virus infection in suckling mice pretreated with dianhydrodulcitol (DAD). Acta Microbiol Acad Sci Hung. 1979;26(2):121-5. PubMed PMID: 539467.

6: Bános Z, Szeri I, Anderlik P. Dianhydrodulcitol treatment of lymphocytic choriomeningitis virus infection in suckling mice. Acta Microbiol Acad Sci Hung. 1979;26(1):29-34. PubMed PMID: 484266.

7: Gerö-Ferencz E, Tóth K, Somfai-Relle S, Gál F. Effect of dianhydrodulcitol (DAD) on the primary immune response of normal and tumor bearing rats. Oncology. 1977;34(4):150-2. PubMed PMID: 335301.

8: Kopper L, Lapis K, Institóris L. Incorporation of 3H-dibromodulcitol and 3H-dianhydrodulcitol into ascites tumor cells. Autoradiographic study. Neoplasma. 1976;23(1):47-52. PubMed PMID: 1272473.

9: Bános S, Szeri I, Anderlik P. Combined phytohaemagglutinin and dianhydrodulcitol treatment of lymphocytic choriomeningitis virus infection in mice. Acta Microbiol Acad Sci Hung. 1975;22(3):237-40. PubMed PMID: 1155228.

Carbohydrate Research, 1982 ,  vol. 108, p. 173 – 180

Deryabin, Dmitry G.; Tolmacheva, Anna A.
Molecules, 2015 ,  vol. 20,  9  pg. 17093 – 17108

Gati; Somfai-Relle
Arzneimittel-Forschung/Drug Research, 1982 ,  vol. 32,   2  pg. 149 – 151

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O[C@H]([C@H]1OC1)[C@@H](O)[C@H]2CO2

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Talazoparib, BMN-673, MDV-3800

(2S,3S)-methyl-7-fluoro-2-(4-fluorophenyl)-3-(1-methyl-1H-1,2,4-triazol-5-yl)-4-oxo-1,2,3,4-tetrahydroquinoline-5-carboxylate

(8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-8,9-dihydro-2H-pyrido[4,3,2-de]phthalazin-3(7H)-one

(8S,9R)-5-Fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one

CAS 1207456-01-6
Chemical Formula: C19H14F2N6O
Exact Mass: 380.11972

BMN673, BMN673, BMN-673, LT673, LT 673, LT-673,  Talazoparib

BioMarin Pharmaceutical Inc

phase 3

Poly ADP ribose polymerase 2 inhibitor; Poly ADP ribose polymerase 1 inhibitor

cancer

(85,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-8,9-dihydro-2H-pyrido[4,3,2-de]phthalazin-3(7H)-one toluenesulfonate salt

CAS 1373431-65-2(Talazoparib Tosylate)

1H NMR DMSOD6

13C NMR DMSOD6

HMBC NMR

HSQC NMR

Talazoparib (BMN-673) is an investigational drug that acts as a PARP inhibitor. It is in clinical trials for various cancers.

Medivation, under license from BioMarin Pharmaceuticals, following its acquisition of LEAD Therapeutics, is developing a PARP-1/2 inhibitor, talazoparib, for treating cancer, particularly BRCA-mutated breast cancer. In February 2016, talazoparib was reported to be in phase 3 clinical development

Talazoparib, also known as BMN-673, is an orally bioavailable inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) with potential antineoplastic activity (PARP1 IC50 = 0.57 nmol/L). BMN-673 selectively binds to PARP and prevents PARP-mediated DNA repair of single strand DNA breaks via the base-excision repair pathway. This enhances the accumulation of DNA strand breaks, promotes genomic instability and eventually leads to apoptosis. PARP catalyzes post-translational ADP-ribosylation of nuclear proteins that signal and recruit other proteins to repair damaged DNA and is activated by single-strand DNA breaks. BMN-673 has been proven to be highly active in mouse models of human cancer and also appears to be more selectively cytotoxic with a longer half-life and better bioavailability as compared to other compounds in development. Check for active clinical trials or closed clinical trials using this agent.

Talazoparib is C₁₉H₁₄F₂N₆O.

Talazoparib tosylate is C₂₆H₂₂F₂N₆O₄S.^[1]

Approvals and indications

None yet.

Mechanism of action

Clinical trials

After trials for advanced hematological malignancies and for advanced or recurrent solid tumors.^[2] it is now in phase 3 for metastatic germline BRCA mutated breast cancer.^[3] Trial estimated to complete in June 2016.^[4]

As of January 2016 it in 14 active clinical trials.^[5]

WO2010017055,  WO2015069851, WO 2012054698, WO 2011130661, WO 2013028495, US 2014323725, WO 2011097602

PAPER

Discovery and Characterization of (8S,9R)-5-Fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one (BMN 673, Talazoparib), a Novel, Highly Potent, and Orally Efficacious Poly(ADP-ribose) Polymerase-1/2 Inhibitor, as an Anticancer Agent

Abstract

We discovered and developed a novel series of tetrahydropyridophthlazinones as poly(ADP-ribose) polymerase (PARP) 1 and 2 inhibitors. Lead optimization led to the identification of (8S,9R)-47 (talazoparib; BMN 673; (8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one). The novel stereospecific dual chiral-center-embedded structure of this compound has enabled extensive and unique binding interactions with PARP1/2 proteins. (8S,9R)-47 demonstrates excellent potency, inhibiting PARP1 and PARP2 enzyme activity with K_i = 1.2 and 0.87 nM, respectively. It inhibits PARP-mediated PARylation in a whole-cell assay with an EC₅₀ of 2.51 nM and prevents proliferation of cancer cells carrying mutant BRCA1/2, with EC₅₀ = 0.3 nM (MX-1) and 5 nM (Capan-1), respectively. (8S,9R)-47 is orally available, displaying favorable pharmacokinetic (PK) properties and remarkable antitumor efficacy in the BRCA1 mutant MX-1 breast cancer xenograft model following oral administration as a single-agent or in combination with chemotherapy agents such as temozolomide and cisplatin. (8S,9R)-47 has completed phase 1 clinical trial and is currently being studied in phase 2 and 3 clinical trials for the treatment of locally advanced and/or metastatic breast cancer with germline BRCA1/2 deleterious mutations.

http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.5b01498

http://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.5b01498/suppl_file/jm5b01498_si_001.pdf

Preparation of (8S,9R)-5-Fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one Tosylate Salt ((8S,9R)-47 Tosylate Salt)

A suspension of (8S,9R)-47 (BMN 673) (400 mg, 1.05 mmol) in a mixture of acetone (27 mL) and THF (13 mL) was heated to reflux until the suspension became clear. TsOH (220 mg, 1.16 mmol) was then added to the solution. White solids started to precipitate out from the solution shortly after the addition of TsOH. After stirring at 25 °C for 30 min, the mixture was filtered to collect the white crystal solids, which were washed with a mixture of acetone (10 mL) and 1,4-dioxane (4 mL) and then dried under vacuum at 45 °C for 3 days. This afforded the product as a white crystalline solid (540 mg, yield 93%). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 2.29 (s, 3H), 3.67 (s, 3H), 4.97–5.06 (m, 2H), 6.91–6.94 (dd, J₁ = 2.0 Hz, J₂ = 10.8 Hz, 1H), 7.06–7.19 (m, 5H), 7.19–7.51 (m, 4H), 7.74 (s, 1H), 7.87 (s, 1H), 10.32 (brs, 1H), 12.36 (s, 1H). LC-MS (ESI)m/z: 381 (M + H)⁺. Anal. Calcd for C₁₉H₁₄F₂N₆O·toluene sulfonic acid: C, 56.52; H, 4.01; N, 15.21. Found: C, 56.49; H, 3.94; N, 15.39.

(8S,9R)-5-Fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one (8S,9R)-47 or BMN 673 and (8R,9S)-5-Fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one (8R,9S)-47

Compound 47 was dissolved in DMF, and chiral resolution was performed using supercritical-fluid chromatography (SFC) with a CHIRALPAK IA chiral column and methanol (20% with 0.1% DEA) and CO₂ (80%) as the eluents. Yield 90%. For (8S,9R)-47 (BMN 673): retention time 8.8 min and ee 99.3%. For (8R,9S)-47: retention time 10.2 min and ee 99.2%.

Alternatively, compound (8S,9R)-47 could also be made using (2S,3R)-60a as a starting material and employing the same procedure described for the conversion of 60a to 47.

The optical rotation for both (8S,9R)-47 and (8R,9S)-47 was measured using a RUDOLPH (AUTOPOL V) automatic polarimeter at a concentration of 6.67 mg/mL in MeOH/MeCN/DMF = 0.5:0.5:1 at 20 °C. The specific rotation for (8S,9R)-47 was +92.2°, whereas it was −93.4° for (8R,9S)-47.

PATENT

WO-2016019125

WO2016019125

The compound (85,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-8,9-dihydro-2H-pyrido[4,3,2-de]phthalazin-3(7H)-one toluenesulfonate salt (Compound (A))

Compound (A)

is an inhibitor of poly(ADP-ribose)polymerase (PARP). Methods of making it are described in WO2010017055, WO2011097602, and WO2012054698. However, the disclosed synthetic routes require chiral chromatography of one of the synthetic intermediates in the route to make Compound (A), methyl 7-fluoro-2-(4-fluorophenyl)-3-(l -methyl- lH-1, 2,4-triazol-5-yl)-4-oxo- 1 ,2,3,4-tetrahydroquinoline-5-carboxylate (Intermediate (A)),

Intermediate (A)

to yield the chirally pure (2S,35)-methyl 7-fluoro-2-(4-fluorophenyl)-3-(l-methyl-lH- 1,2,4-triazol-5-yl)-4-oxo-l,2,3,4-tetrahydroquinoline-5-carboxylate (Compound (1))

Compound (1).

Using conventional chiral chromatography is often solvent and time intensive.

Use of more efficient chromatography methods, such as simulated moving bed (SMB) chromatography still requires the use of expensive chiral chromatography resins, and is not practical on a large scale to purify pharmaceutical compounds. Also, maintaining

Compound (1) in solution for an extended time period during chromatography can lead to epimerization at the 9-position and cleavage of the methyl ester group in Compound (1). Replacing the chromatography step with crystallization step(s) to purify Compound (1) is desirable and overcomes these issues. Therefore, it is desirable to find an alternative to the use of chiral chromatography separations to obtain enantiomeric Compound (1).

Scheme 1 below describes use of Ac49 as a coformer acid for the preparation of Compound (la) and for the chiral resolution of Compound (1).

Scheme 1

Compound (1 )

Example 2 – Preparation of Compound (1) Using Scheme 1

Step la

Intermediate (A) (5 g, 12.5 mmol) was dissolved in 9: 1 v/v MIBK/ethanol (70 mL, 14 vol.) at 50 °C with stirring and dissolution was observed in less than about 5 minutes. [(lS)-en<io]-(+)-3-bromo-10-camphor sulfonic acid monohydrate (4.1 g, 12.5 mmol) was added and dissolution was observed in about 10-20 minutes. Seeding was then performed with Compound (la) (95% e.e., 5 mg, 0.1% w.) and the system was allowed to equilibrate for about 1 hour at 50 ^°C, was cooled to about 20 °C at 0.15 °C/min, and then equilibrated at 20 °C for 2 hours. The solid phase was isolated by filtration, washed with ethanol, and dried at about 50 °C and 3 mbar for about 2 to 3 hours to yield Compound (la) as a 0.6 molar equiv. EtOH solvate and 0.6 molar equiv. hydrate (93.4% e.e.).

Step lb

Compound (la) was then suspended in MIBK/ethanol 95/5% by volume (38 mL, 10 vol.) at 50 °C with stirring. After about 2 hours at 50 °C, the suspension was cooled to about 5 °C for 10 to 15 hours. The solid phase was recovered by filtration and dried at about 50 °C and 3 mbar for about 3 hours. Compound (la) (97.4% e.e.) was recovered. Step 2

000138] Compound (1) was released by suspending Compound (la) (3.9 g, 5.5 mmoi), without performing the optional reslurrying in Step 1, in 20 mL of water at room temperature and treating with 5M sodium hydroxide in water (1.3 mL, 1.2 mol). The mixture was kept at room temperature for about 15 hours and the solid was isolated by filtration and dried at 50 °C and 3 mbar for about 3 hours. Compound (1) was recovered (94.4% e.e.).

Example 3 – Large Scale Preparation of Compound (1) Using Scheme 1

The procedure of Example 1 was followed using 3.3 kg of Intermediate (A) and the respective solvent ratios to provide 95.7% e.e. in Step la; 99.2% e.e. in Step lb; and 99.2% e.e. in Step 2.

Example 4 – Alternative Preparation of Compound (1) Using Scheme 1

Step la

Intermediate (A) (751 mg, 1.86 mmol)) was dissolved in 9: 1 v/v

MIBK/ethanol (7.5 mL, 10 vol.) at 50 °C with stirring. [(15)-eni o]-(+)-3-bromo-10-camphor sulfonic acid monohydrate (620 mg, 1.88 mmol, 1 equiv.) was added. Formation of a precipitate was observed at about 1 hour at 50 °C. The system was then cooled to about 5 °C at 0.1 °C/min, and then equilibrated at 5 °C for about 60 hours. The solid phase was isolated by filtration and dried at about 50 °C and 3 mbar for about 2 hours to yield

Compound (la)(92% e.e.). See Figures 1-4 for XRPD (Figure 1), chiral HPLC (Figure 2), Ή NMR (Figure 3), and TGA/DSC analyses (Figure 4). The XRPD pattern from the material in Example 3 is similar to that in Example 1 with some slight shifts in the positions of specific diffraction peaks (highlighted by black arrows in Figure l). The ‘H NIVIR was consistent with a mono-salt of Compound (la) containing 0.5 molar equivalent of EtOH and 0.6% by weight residual MIBK. The TGA analysis showed a stepwise mass loss of 3.5% between 25 and 90 °C (potentially representing loss of the 0.5 molar equivalent of EtOH) and a gradual mass loss of 1.2% between 90 and 160 °C (potentially representing the loss of adsorbed water). The DSC analysis had a broad endotherm between 25 and 90 °C

representing desolvation and an endotherm at 135 °C representing melt/degradation.

Step lb

Compound (la) (100.3 mg, 0.141 mmol) was re-suspended in 95:5 v/v MIBK EtOH (1 mL, 10 vol.) at 50 °C and stirred for 1 hour before cooling to 5 °C at

0.1 °C/min. The solid (99.4% e.e.) was recovered by filtration after 1 night at 5 °C. Shifts in the XRPD diffraction peaks were no longer detected (Figure 5; compare Figure 1). Figure 6 shows the chiral HPLC for Compound (la).

Step 2

Compound (la) (100.2 mg, 0.141 mmol) from Step la was suspended in water (2 mL, 20 vol.) at 50 ^°C and 5 M NaOH in water (34 μL·, 1.2 molar equiv) was added. The resulting suspension was kept at 50 °C for one night, cooled to room temperature

(uncontrolled cooling) and filtered to yield Compound (1) (92% e.e.). The chiral purity was not impacted by this step and no [(15)-enJo]-(+)-3-bromo-10-camphor sulfonic acid was detected by NMR. Figure 7 compares the XRPD of Compound (1) in Step 2 with

Intermediate (A), the starting material of Step 1. Figure 8 shows the NMR of Compound (1) in Step 2 with Intermediate (A), the starting material of Step 1.

Example 5 – Alternative Preparation of Compound (1) Using Scheme 1 Step la

000144] Intermediate (A) (1 equiv.) was added with stirring to a solution of MIBK (12-13 vol), ethanol (1-1.5 vol), and water (0.05-0.10 vol) and the reaction was heated within 15 minutes to an internal temperature of about 48 °C to about 52 °C . [(lS)-endo]-(+)-3-bromo- 10-camphor sulfonic acid (1 equiv) was added and the reaction was stirred for about 5-10 mins at an internal temperature of about 48 °C to about 52 °C until dissolution occurred. Seed crystals of Compound (la) were added and the reaction was allowed to proceed for 1 hour at an internal temperature of about 48 °C to about 52 °C. The reaction was cooled at a rate of 0.15 °C /min to about 19-21 °C. The suspension was stirred for 2 hours at an internal temperature of about 19 °C to 21 °C and then was collected by filtration and washed twice with ethanol. The product was characterized by 1H NMR and ¹³C NMR (Figures 13a and 13b), IR Spectrum (Figure 14), DSC (Figure 15), and chiral HPLC (Figure 16).

Step 2a

To Compound (la) (1 equiv.) was added acetone (1.1 vol), IPA (0.55 vol), and methanol (0.55 vol) and the reaction was heated to an internal temperature of about 38 °C to 42 °C. Aqueous ammonia (25%) (1.3 equiv) was added and the reaction was stirred for about 10 minutes. The pH of the reaction was confirmed and the next step performed if > 7. Water was added (0.55 vol), the reaction was cooled to an internal temperature of about 35 °C, seed crystals of Compound (1) were added, and the reaction was stirred for about 10 mins. Water was added (3.3 vol) dropwise within about 30 minutes, the suspension was cooled within 30 minutes to an internal temperature of about 0 °C to 5 °C, and the reaction was stirred for 15 minutes. The solid was collected by filtration and washed three times with water.

Step 2b

To the product of Step 2a) was added acetone (4 vol), ΓΡΑ (1 vol), and methanol (1 vol) and the reaction was heated to an internal temperature of about 38 °C to 42 °C resulting in a clear solution. Water (2 vol) and seed crystals of Compound (1) were added and the system was stirred for about 15 minutes at an internal temperature of about 35 °C. Water (342 mL) was added dropwise in about 30 minutes. The suspension was then cooled in 30 min to an internal temperature of about 0 °C to 5 °C and was stirred for an additional 15 minutes. The solid was collected by filtration, washed twice with water, and chiral purity was determined. If > 99% e.e., then the solid was dried at an internal temperature of about 60 °C under reduced pressure to yield Compound (1). The product was characterized by Ή NMR (Figure 19), ¹³C NMR (Figure 20), IR (Figure 21), DSC (Figure 22), chiral HPLC (Figure 23).

Scheme 2 below describes use of Acl 10 as a coformer acid for the preparation of Compound (lb) and the chiral resolution of Compound (1).

Intermediate (A)

Compound (1 b)

Compound (1 )

Example 6 – Preparation of Compound (1) Using Scheme 2

Step la

Intermediate (A) (102 mg, 0.256 mmol) was dissolved in MIBK (1 mL, 10 vol.) at 65 ^°C with stirring. (lS)-phenylethanesulfonic acid, prepared using procedures known to one of skill in the art, in MIBK (3.8 M, 80 μί, 1 molar equiv.) was added and a suspension was observed after 30 minutes at 65 °C. The system was kept at 65 °C for another 30 minutes before cooling to 5 ^°C at 0.1 C/min. After one night at 5 °C, the solid was filtered, dried at 50 °C, 3 mbar pressure for about 2 hours to yield Compound (lb). See Figures 9-12 for XRPD (Figure 9), chiral HPLC (Figure 10), Ή NMR (Figure 11), and TGA/DSC analyses (Figures 12a and 12b). The XRPD diffraction pattern of the solid obtained in Example 5 differed from the XRPD pattern obtained with the solid from in the salt screen of Example 1 and was consistent with the production of different solids in Examples 1 and 5. The Ή NMR was consistent with the mono-salt with a 0.3% by weight residue of dioxane. In Figure 12a, the thermal behavior was consistent with a non-solvated form exhibiting a melt/degradation at 201 °C. Figure 12b compares the melt pattern of Compound (lb) in Example 5 with Compound (lb) in Example 1.

Steps lb and 2 can be carried out using procedures similar to those used in Examples 2-5.

Example 7 – Polymorphism of Compound (la)

Compound (1) (92% e.e., 10 mg, mmol) was placed in 1.5 mL vials and the solvents (1 mL or less) of Table 3 were added at 50 °C until dissolution was achieved. [(1S)-eni o]-(+)-3-bromo-10-camphorsulfonic acid was added as a solid at 50 °C. The samples were kept at 50 °C for about 1 hour prior to being cooled to room temperature overnight

(uncontrolled cooling rate). Clear solutions were successively cooled to 4 °C, -20 °C and evaporated at room temperature. Any gum obtained after evaporation was re-suspended in diethyl ether. The solid phases generated were characterized by XRPD and if relevant, by Ή NMR and TGA/DSC.

Table 3. Compound (la) Polymorphism Conditions

C.S. means clear solution and Susp. means suspension. “A” means the XRPD diffraction pattern was new but similar to that for Ac49 in

Example 1. “B” means the XRPD diffraction pattern was the same as that for Ac49 in Example 1. “M.E.” means molar equiv.

Page 38 of 64

NAI- 1500460480V I

Each of the seven solvents in which solvates were observed (heterosolvates not included) were mixed with MIBK (90% vol). Solutions of Intermediate (A) were prepared in the solvent mixtures (10 vol) at 50 C and [(15)-en<io]-(+)-3-bromo-10-camphor sulfonic acid (1 molar equivalent) was added. The resulting clear solutions were cooled to 5 °C at 0.2 C/min. Surprisingly, no crystallization was reported in any sample. Seeding was performed with a few crystals of each solvate at about 25 °C. The solid phases were analyzed by XRPD and the liquid phases were analyzed by chiral HPLC. See Table 4 for a summary of the results (where “Dias 2” is the (2R, 3R) diastereomer of Compound (la)) .

Table 4. Compound (la) Solvate Analysis

As seen in Table 4 above, the ethanol/MIBK system yielded 93% pure Compound (la) which demonstrates that Compound (la) does crystallize in a very pure form as an ethanolate solvate.

Other objects, features and advantages of the compounds, methods and compositions described herein will become apparent from the following description. It should be understood, however, that the description and the specific examples, while indicating specific embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present description will become apparent from this detailed description.

All publications including patents, patent applications and published patent applications cited herein are hereby incorporated by reference for all purposes.

PATENT

US 2011196153

http://www.google.co.ve/patents/US20110237581

Patent

US 2011237581

PATENT

PATENT

http://www.google.com/patents/WO2015069851A1?cl=en

SYNTHETIC EXAMPLES

Example 1

\ ,

(1 a) (2) (3) (la) (5)

To a flask was added N-methyl-l,2,4-triazole (la)(249.3 g, 3.0 mol, 1 equiv.),

2-methyl-THF (1020 mL, about 1 :4 m/v), and DMF (2)(230.2 g, 3.15 mol, 1.05 equiv.), in any order. The solution was cooled to an internal temperature of about -5 to 0 °C. To the flask was added LiHMDS (3) as a 20% solution in 2-methyl-THF (3012 g, 3.6 mol, 1.2 equiv.) dropwise within about 60 minutes. During the addition of the LiHMDS (3), the desired Compound (la) was precipitated as the 2-methyl-THF solvate, and the flask was cooled to about -30 °C. The reaction was stirred for about 30 minutes at an internal temperature of about -5 to 0 °C.

The precipitated crystals were removed from the reaction mixture by filtration and washed with 2-methyl-THF. The product, Compound (la) as the 2-methyl-THF solvate, was dried under vacuum at an internal temperature of about 60 °C (about 72.5% as measured by NMR) to yield Compound (la).

Example 2

As shown in Example 2, the Compounds of Formula I are useful in the synthesis of more complex compounds. See General Scheme 1 for a description of how the first step can be accomplished. Compounds of Formula I can be reacted with compound (6) to yield Compounds of Formula II. In Example 2, Compound (la) can be reacted with

Compound (6) to yield Compound (7). The remaining steps are accomplished using procedures known to one of ordinary skill in the art, for example, as disclosed in

WO2010017055 and WO2011097602 to yield Compound (12).

PATENT

US 2014323725/http://www.google.com/patents/WO2011097602A1

5-fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-8,9- dihydro-2H-pyrido[4,3,2-Je]phthalazin-3(7H)-one, as shown in formula (1), and its enantiomer compounds, as shown in formulas (la) and (lb):

Example 1

(Z)-6-Fluoro-3-(( 1 -methyl- IH- 1 ,2,4-triazol-5 -yl)methylene)-4-nitroisobenzofuran- 1 (3H)-one (3)

[0053] To a 80 L jacketed glass reactor equipped with a chiller, mechanical stirrer, thermocouple, and nitrogen inlet/outlet, at 15 – 25 °C, anhydrous 2-methyl-tetrahydrofuran (22.7 kg), 6-fluoro-4- nitroisobenzofuran-l(3H)-one (2) (2.4 kg, 12.2 mol, 1.00 eq.), and 2-methyl-2H-l,2,4-triazole-3- carbaldehyde (49.6 – 52.6 % concentration in dichloromethane by GC, 3.59 – 3.38 kg, 16.0 mol, 1.31 eq.) were charged consecutively. Triethylamine (1.50 kg, 14.8 mol, 1.21 eq.) was then charged into the above reaction mixture. The reaction mixture was stirred for another 10 minutes. Acetic anhydride (9.09 – 9.10 kg, 89.0 – 89.1 mol, 7.30 eq.) was charged into the above reaction mixture at room temperature for 20 – 30 minutes. The reaction mixture was heated from ambient to reflux temperatures (85 – 95 °C) for 80 – 90 minutes, and the mixture was refluxed for another 70 – 90 minutes. The reaction mixture was monitored by HPLC, indicating compound (2) was reduced to < 5 %. The resulting slurry was cooled down to 5 – 15 °C for 150 – 250 minutes. The slurry was aged at 5 – 15 °C for another 80 – 90 minutes. The slurry was filtered, and the wet cake was washed with ethyl acetate (2L x 3). The wet cake was dried under vacuum at 40 – 50 °C for 8 hours to give 2.65 – 2.76 kg of (Z)-6-fluoro-3-((l -methyl-lH-l ,2,4-triazol-3- yl)methylene)-4-nitroisobenzofuran-l(3H)-one (3) as a yellow solid (2.66 kg, yield: 75.3 %, purity: 98.6 – 98.8 % by HPLC). LC-MS (ESI) m/z: 291 (M+l)⁺. Ή-ΝΜΡ (400 MHz, DMSO-d₆) δ (ppm): 3.94 (s, 3H), 7.15 (s, 1H), 8.10 (s, 1H), 8.40-8.42 (dd, J_x = 6.4 Hz, J₂ = 2.4 Hz, 1H), 8.58-8.61 (dd, J_x = 8.8 Hz, J₂ = 2.4 Hz, 1H).

Example 2

Methyl 5- enzoate (4)

Example 2A

[0054] (¾-6-Fluoro-3-((l-methyl-lH-l,2,4-taazol-3-yl)m (3) (177 g, 0.6 mol, 1.0 eq.), and HC1 (2 N in methanol, 3 L, 6 mol, 10 eq.) were charged into a 5 L 3-neck flask equipped with mechanical stirrer, thermometer, and nitrogen inlet/outlet. The reaction mixture was stirred at room temperature for 25 hours. The reaction mixture was monitored by HPLC, indicating 0.8 % compound (3) remained. The reaction mixture was concentrated under vacuum at 40 °C to dryness, and methyl 5-fluoro-2-(2-(l -methyl- lH-l,2,4-triazole-3-yl)acetyl)-3-nitrobenzoate hydrochloride (4) was obtained as a yellow solid (201 g, yield: 93.4 %). It was used for the next step without further purification. LC-MS (ESI) m/z: 323 (M+l)⁺ ¾-NMR (400 MHz, DMSO-J₆) δ (ppm): 3.89 (s, 3H), 3.92 (s, 3H), 4.60 (s, 2H), 7.85 (s, 1H), 8.25-8.28 (dd, J_x = 8.4 Hz, J₂ = 2.8 Hz, 2H), 8.52-8.54 (dd, J_x = 8.4 Hz, J₂ = 2.8 Hz, 2H).

Example 2B

An alternative workup procedure to that illustrated in Example 2A follows. Instead of evaporating the reaction mixture to dryness, it was condensed to 2 volumes, followed by solvent exchange with 12 volumes of THF, and then 12 volumes of heptane. The slurry mixture was concentrated to 2 volumes and filtered to give the product. As such, 1.8 kilograms of (Z)-6-fluoro-3-((l-methyl-lH-l,2,4-triazol-3- yl)methylene)-4-nitroisobenzofuran-l(3H)-one (3) gave 2.15 kilograms (yield 96.4 %) of the product methyl 5-fluoro-2-(2-(l -methyl- lH-l,2,4-triazole-3-yl)acetyl)-3-nitrobenzoate hydrochloride (4).

Example 3

Methyl 7-fluoro-2-(4-fluorophenyl)-3-(l-methyl-lH-l,2,4-triazol-5-yl)-4-oxo-l,2,3,4- tetrahydroquinoline-5 -carboxylate (5)

Example 3A

To a suspension of methyl 5-fluoro-2-(2-(l-methyl-lH-l,2,4-triazol-5-yl)acetyl)-3-nitrobenzoate (4) (5 g, 15.5 mmol, leq.) and 4-fluorobenzaldehyde (3.6 g, 29 mmol, 1.87 eq.) in a mixture of solvents tetrahydrofuran (30 mL) and MeOH (5 mL) was added titanium(III) chloride (20 % w/w solution in 2N Hydrochloric acid) (80 mL, 6 eq.) dropwise with stirring at room temperature. The reaction mixture was allowed to stir at 30~50°C for 2 hours. The mixture was then diluted with water (160 mL), and the resulting solution was extracted with ethyl acetate (100 mL x 4). The combined organic layers were washed with saturated NaHC0₃ (50 mL x 3) and aqueous NaHS0₃ (100 mL x 3), dried by Na₂S0₄, and concentrated to dryness. This afforded a crude solid, which was washed with petroleum ether (120 mL) to obtain the title compound as a yellow solid (5.9 g, yield: 95 %, purity: 97 %). LC-MS (ESI) m/z: 399 (M+l)⁺. ^-NMR (400 MHz, CDCl_a) δ (ppm): 3.58 (s, 3H), 3.87 (s, 3H), 4.16-4.19 (d, J2=13.2 Hz, 1H), 4.88 (s, 1H), 5.37-5.40 (d, J2=13.2 Hz, 1H), 6.47-6.53 (m, 2H) , 6.97-7.01 (m, 2H), 7.37-7.41 (m, 2H), 7.80 (s, 1H).

Example 3B

An alternative workup procedure to that illustrated in Example 3A follows. After the completion of the reaction, the mixture was extracted with isopropyl acetate (20 volumes x 4) without water dilution. The product was isolated by solvent exchange of isopropyl acetate with heptanes followed by re-slurry with MTBE and filtration. As such, 3 kilograms of methyl 5-fluoro-2-(2-(l-methyl-lH-l,2,4-triazol-5- yl)acetyl)-3-nitrobenzoate (4) afforded 2.822 kilograms of the title compound (5) (yield 81 %).

Example 3C

To a stirred solution of methyl 5-fluoro-2-(2-(l-methyl-lH-l,2,4-triazol-5-yl)acetyl)-3- nitrobenzoate (4) (580 mg, 2 mmol) and 4-fluorobenzaldehyde (488 mg, 4 mmol) in methanol (0.75 mL) and tetrahydrofuran (4.5 mL) was added concentrated HC1 solution (w/w 37 %, 6 mL), then reductive powdered Fe (672 mg, 12 mmol) was added slowly to the reaction system. After the addition was complete, the resulting mixture was heated to 60 °C and kept at this temperature for 3 hours. After the disappearance of the starting material (4) as monitored by LC-MS, the reaction mixture was partitioned between ethyl acetate (30 mL) and water (30 mL) and the aqueous phase was extracted with ethyl acetate (20 mL x 3). The combined organic phase was dried with Na₂S0₄, concentrated in vacuo and purified by column chromatography (ethyl acetate: petroleum ether = 1 : 1) to give the title compound (5) as a pale yellow solid (300 mg, yield 40 %). LC-MS (ESI) m/z: 399 (M+l)⁺. ^LH-NMR (400 MHz, CDC1₃) δ (ppm): 3.58 (s, 3H), 3.87 (s, 3H), 4.17 (d, 1H), 4.87 (s, 1H), 5.38 (d, 1H), 6.50 (dd, 2H), 6.99 (dd, 2H), 7.38 (dd, 2H), 7.80 (s, 1H).

Example 3D

To a stirred solution of methyl 5-fluoro-2-(2-(l-methyl-lH-l,2,4-triazol-5-yl)acetyl)-3- nitrobenzoate (4) (580 mg, 2 mmol) and 4-fluorobenzaldehyde (488 mg, 4 mmol) in methanol (0.75 mL) and tetrahydrofuran (4.5 mL) was added SnCl₂ (2.28 g, 12 mmol) and concentrated HC1 (w/w 37 %, 6 mL), the resulting mixture was reacted at 45 °C for 3 hours, until LC-MS indicating the disappearance of the starting material (4) and about 50 % formation of the product. The mixture was then partitioned between ethyl acetate (30 mL) and water (30 mL) and the aqueous phase was extracted with ethyl acetate (20 mL x 3). The combined organic phase was dried with Na₂S0₄, concentrated in vacuo and purified by column chromatography (ethyl acetate: petroleum ether = 1 : 1) to give the title compound (5) as a pale yellow solid (10 mg, yield 1.3 %). LC-MS (ESI) m/z: 399 (M+l)⁺. ^LH-NMR (400 MHz, CDC1₃) δ (ppm): 3.58 (s, 3H), 3.87 (s, 3H), 4.17 (d, 1H), 4.87 (s, 1H), 5.38 (d, 1H), 6.50 (dd, 2H), 6.99 (dd, 2H), 7.38 (dd, 2H), 7.80 (s, 1H).

Example 3E

A solution of methyl 5-fluoro-2-(2-(l-methyl-lH-l,2,4-triazol-5-yl)acetyl)-3-nitrobenzoate (4) (580 mg, 2 mmol) and 4-fluorobenzaldehyde (488 mg, 4 mmol) in methanol (20 mL) and acetic acid (1 mL) was stirred at room temperature for 24 hours under hydrogen (1 barr) in the presence of a catalytic amount of 10 % Pd/C (212 mg, 0.2 mmol). After the reaction was complete, the catalyst was removed by filtration through a pad of Celite, the solvent was removed in vacuo, and the residue was purified by column chromatography (ethyl acetate: petroleum ether = 1 : 1) to give the title compound (5) as a pale yellow solid (63 mg, yield 8 %). LC-MS (ESI) m/z: 399 (M+l)⁺ . ¹HNMR (400 MHz, DMSO-d₆) δ (ppm): 3.56 (s, 3H), 3.86 (s, 3H), 7.02 (dd, 2H), 7.21 (dd, 2H), 7.90 (s, 1H), 8.08 (s, 1H), 8.26 (dd, 1H), 8.56 (dd, 1H).

Example 4

5-Fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-8,9-dihydro-2H-pyrido[4,3,2-

 Methyl 7-fluoro-2-(4-fluorophenyl)-3-(l -methyl-lH-l ,2,4-triazol-5-yl)-4-oxo-l,2,3,4- tetrahydroquinoline-5-carboxylate (5) (150 g, 0.38 mol, 1.0 eq.) and methanol (1.7 L) were charged into a 3 L 3-neck flask equipped with a mechanical stirrer, thermometer, and nitrogen inlet/outlet. The resulted suspension was stirred at room temperature for 15 minutes. Hydrazine hydrate (85 % of purity, 78.1 g, 1.33 mol, 3.5 eq.) was charged dropwise into the above reaction mixture within 30 minutes at ambient temperature. The reaction mixture was stirred at room temperature overnight. The reaction was monitored by HPLC, showing about 2 % of compound (5) left. The obtained slurry was filtered. The wet cake was suspended in methanol (2 L) and stirred at room temperature for 3 hours. The above slurry was filtered, and the wet cake was washed with methanol (0.5 L). The wet cake was then dried in vacuum at 45 – 55 °C for 12 hours. This afforded the title compound as a pale yellow solid (112 g, yield: 78.1 %, purity: 95.98 % by HPLC). LC-MS (ESI) m/z: 381 (M+l)⁺. ^-NMR (400 MHz, DMSO-J₆) δ (ppm): 3.66 (s, 3H), 4.97-5.04 (m, 2H), 6.91-6.94 (dd, J_x = 2.4, J₂ = 11.2 Hz, 1H), 7.06-7.09 (dd, J_x = 2.4, J₂ = 8.8 Hz, 1H), 7.14-7.18 (m, 3H), 7.47-7.51 (m, 2H), 7.72 (s, 1H), 7.80 (s, 1H), 12.35 (s, 1H).

Example 5

5 -Amino-7-flu in- 1 (2H)-one

To a solution of 6-fluoro-3-((l-methyl-lH-l,2,4-triazol-3-yl)methylene)-4-nitroiso-benzofuran- l(3H)-one (3) (4.0 g, 135 mmol) in THF (100 mL) was added hydrazine monohydrate (85 %) (6 mL) at room temperature under nitrogen atmosphere. The mixture was stirred for 2 hours, then acetic acid (6 mL) was added and the mixture was heated to and kept at 60 °C for 18 hours. The resulting mixture was diluted with water (100 mL) and extracted with ethyl acetate (100 mL x 3). The organic layer was dried over anhydrous Na₂S0₄ and evaporated to dryness to afford the title compound as a yellow solid (1.6 g, yield 42 %). LC-MS (ESI) m/z: 275(M+1)⁺.

Example 6

(£’)-7-fluoro-5-(4-fluorobenzylideneamino)-4-((l -methyl- IH- 1 ,2,4-triazol-5-yl)methyl)phthalazin- 1 (2H)- one

(7)

To a suspended of 5-amino-7-fluoro-4-((l-methyl-lH-l,2,4-triazol-3-yl)methyl) phthalazin- l(2H)-one (7) (1.6 g, 5.8 mmol) in acetonitrile (50 mL) was added 4-fluorobenzaldehyde (2.2 g, 17.5 mmol). The mixture was stirred under reflux under nitrogen for 48 hours. The precipitate was filtered and washed with a mixture of solvents (ethyl acetate/hexane, 1 :1, 10 mL). After drying in vacuum, it afforded the title compound as a yellow solid (1.2 g, yield 52 %). LC-MS (ESI) m/z: 381(M+1)⁺.

Example 7

5-Fluoro-8 4-fluorophenyl)-9 l-methyl H-l,2,4-triazol-5-yl)-8,9-dihydro-2H^yrido[4,3,2-

(8) (1 )

To a suspension of (£’)-7-fluoro-5-(4-fluorobenzylideneamino)-4-((l-methyl-lH-l,2,4-triazol-5- yl)methyl)phthalazin-l(2H)-one (8) (2.0 g, 5.3 mmol) in THF (80 mL) was added cesium carbonate (3.4 g, 10.6 mmol). The reaction mixture was stirred at 55 °C for 4 hours and cooled down to room temperature. The mixture was diluted with water (50 ml) and extracted with ethyl acetate (50 mL x 3). The combined organic layers were dried over anhydrous Na₂S0₄ and evaporated to dryness to afford the title compound as a white solid (1.6 g, yield 80 %). LC-MS (ESI) m/z: 381(M+1)⁺. ^-NMR (400 MHz, DMSO- ) δ (ppm): 3.66 (s, 3H), 4.97-5.04 (m, 2H), 6.91-6.94 (dd, J_x = 2.4, J₂ = 11.2 Hz, 1H), 7.06-7.09 (dd, Ji = 2.4, J₂ = 8.8 Hz, 1H), 7.14-7.18 (m, 3H), 7.47-7.51 (m, 2H), 7.72 (s, 1H), 7.80 (s, 1H), 12.35 (s, 1H).

Example 8

(£)-Methyl 5-fluoro-2-(3-(4-fluorophenyl)-2-(l-methyl-lH-l,2,4-triazol-5-yl)acryloyl)-3-nitrobenzoate

(9)

To a stirred solution of methyl 5-fluoro-2-(2-(l-methyl-lH-l,2,4-triazol-5-yl)acetyl)-3- nitrobenzoate (4) (580mg, 2 mmol) and 4-fluorobenzaldehyde (488 mg, 4 mmol) in dimethylsulfoxide (2 mL) was added L-proline (230 mg, 2 mmol). The resulting mixture was kept with stirring at 45 °C for 48 hours. The reaction system was then partitioned between ethyl acetate (50 mL) and water (30 mL), and the organic phase was washed with water (20 mL x 3), dried with Na₂S0₄, concentrated in vacuo, and purified by column chromatography (ethyl acetate: petroleum ether = 1 :3) to give the title compound (9) as a pale yellow foam (340 mg, yield 40 %). LC-MS (ESI) m/z: 429 (M+l)⁺. ^-NMR (400 MHz, DMSO-dg); δ (ppm): 3.56 (s, 3H), 3.86 (s, 3H), 7.02 (dd, 2H), 7.21 (dd, 2H), 7.90 (s, IH), 8.08 (s, IH), 8.26 (dd, IH), 8.56 (dd, IH).

Example 9

Methyl 7-fluoro-2-(4-fluorophenyl)- 1 -hydroxy-3-( 1 -methyl- IH- 1 ,2,4-triazol-5-yl)-4-oxo- 1 ,2,3,4- tetrahydroquinoline-5 -carboxylate (10)

To a solution of (£)-Methyl 5-fluoro-2-(3-(4-fluorophenyl)-2-(l-methyl-lH-l,2,4-triazol-5- yl)acryloyl)-3-nitrobenzoate (9) (200 mg, 0.467 mmol) in methanol (20 mL) was added 10 % Pd/C (24 mg). After the addition, the mixture was stirred under H₂ (1 atm) at room temperature for 0.5 h. The reaction system was then filtered and evaporated under reduced pressure. The residue was purified by chromatography (ethyl acetate: petroleum ether = 1 :1) to give the title compound (10) (110 mg, yield 57 %) as an off-white foam. LC-MS (ESI) m/z: 415 (M+H)⁺. ¾-NMR (400 MHz, DMSO-d₆) δ (ppm): 3.53 (s, 3H), 3.73 (s, 3H), 5.08 (d, 2H), 5.27 (d, 2H), 6.95 (dd, IH), 7.08 (dd, 2H), 7.15 (dd, IH), 7.42 (dd, 2H), 7.77 (s, IH), 9.92 (s, IH). Example 10

Methyl 7-fluoro-2-(4-fluorophenyl)-3-(l-methyl-lH-l,2,4-triazol-5-yl)-4-oxo-l,2,3,4-

(10) (5)

To a stirred solution of methyl 7-fluoro-2-(4-fluorophenyl)-l-hydroxy-3-(l-methyl-lH-l,2,4- triazol-5-yl)-4-oxo-l, 2,3, 4-tetrahydroquinoline-5 -carboxylate (10) (41.4 mg, 0.1 mmol) in methanol (5 mL) was added concentrated HCl solution (w/w 37 %, 1 mL) and reductive powdered Fe (56 mg, 1 mmol). The reaction mixture was refluxed for 3 hours. After the disappearance of compound (10) as monitored by LC-MS, the reaction system was partitioned between ethyl acetate (20 mL) and water (20 mL) and then the aqueous phase was extracted with ethyl acetate (10 mL x 3). The combined organic phase was dried with Na₂S0₄, concentrated in vacuo and purified by column chromatography (ethyl acetate: petroleum ether = 1 :1) to give the title compound (5) as a pale yellow solid (12 mg, yield 30 %). LC-MS (ESI) m/z: 399 (M+l)⁺. ¾-NMR (400 MHz, CDC1₃) δ (ppm): 3.58 (s, 3H), 3.87 (s, 3H), 4.17 (d, 1H), 4.87 (s, 1H), 5.38 (d, 1H), 6.50 (dd, 2H), 6.99 (dd, 2H), 7.38 (dd, 2H), 7.80 (s, 1H).

Example 11

Methyl 7-fluoro-2-(4-fluorophenyl)-3-(l-methyl-lH-l,2,4-triazol-5-yl)-4-oxo-l,2,3,4-

To a solution of (£)-Methyl 5-fluoro-2-(3-(4-fluorophenyl)-2-(l-methyl-lH-l,2,4-triazol-5- yl)acryloyl)-3-nitrobenzoate (9) (214 mg, 0.5 mmol) in methanol (5 mL) was added concentrated HCl solution (w/w 37 %, 1 mL), then reductive Fe powder (140 mg, 2.5 mmol) was added slowly to the reaction system. After the addition was complete the resulting mixture was refluxed for 24 hours. The reaction mixture was then filtered, concentrated, neutralized with saturated NaHC0₃ (20 mL), and extracted with ethyl acetate (10 mL x 3). The residue was purified by chromatography (ethyl acetate: petroleum ether = 1 : 1) to give the title compound (5) (30 mg, yield 15 %) as an off-white foam. LC-MS (ESI) m/z: 399 (M+H)⁺. ^-NMR (400 MHz, DMSO-d₆) δ (ppm): 3.56 (s, 3H), 3.86 (s, 3H), 7.02 (dd, 2H), 7.21 (dd, 2H), 7.90 (s, 1H), 8.08 (s, 1H), 8.26 (dd, 1H), 8.56 (dd, 1H).

Example 12

(8R,9S)-5-fluoro-8-(4-fluorophenyl)-9-(l-me

Je]phthalazin-3(7H)-one (la) and (8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-

(1) (la) (lb)

A chiral resolution of 5-fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5-yl)-8,9- dihydro-2H-pyrido[4,3,2-Je]phthalazin-3(7H)-one (1) (52.5 g) was carried out on a super-fluid chromatography (SFC) unit using a CHIRALPAK IA column and C0₂/methanol/diethylamine

(80/30/0.1) as a mobile phase. This afforded two enantiomers with retention times of 7.9 minute (23.6 g, recovery 90 %, > 98 % ee) and 9.5 minute (20.4 g, recovery 78 %, > 98 % ee) as analyzed with a CHIRALPAK IA 0.46 cm x 15 cm column and C0₂/methanol/diethylamine (80/30/0.1) as a mobile phase at a flow rate of 2 g/minute.

Example 13

(2R,3R)-methyl 7-fluoro-2-(4-fluorophenyl)-3-(l-methyl-lH-l,2,4-triazol-5-yl)-4-oxo-l,2,3,4- tetrahydroquinoline-5-carboxylate (6a) and (2S,3S)-methyl 7-fluoro-2-(4-fluorophenyl)-3-(l-methyl-lH-

(5) (6a) (6b)

Example 13A

The chiral resolution of compound (5) was carried out on a SFC unit with a CHIRALPAK®IC 3 cm (I.D.) x 25 cm, 5 μηι column, using C0₂/MeOH (80/20) as a mobile phase at a flow rate of 65 g/ minute while maintaining the column temperature at 35 °C and with a detection UV wavelength of 254 nm. As such, a racemate of compound (5) (5 g) in methanol solution was resolved, which resulted in two enantiomers with a retention times of 2.35 minute (2.2 g, 88 % recovery, >98 % ee) and 4.25 minute (2.3 g, 92 % recovery, >98 % ee), respectively when analyzed using CHIRALPAK®IC 0.46 cm x 15 cm column and CO₂/MeOH(80/20) as a mobile phase at a flow rate of 2 mL/ minute.

Example 13B

The chiral resolution of compound (5) was carried out on a SFC unit with a CHIRALPAK®IC 5cm (I.D.) x 25 cm, 5 μηι column, using C0₂/MeOH (75/25) as a mobile phase at a flow rate of 200 mL/ minute while maintaining the column temperature at 40 °C and with a detection UV wavelength of 255 nm. As such, a racemate of compound (5) (1.25 kg) in methanol solution was resolved, which resulted in two enantiomers in about 83 % yield and 97.4 % purity.

Example 13C

Alternatively, the separation can also be achieved on a Simulated Moving Bed (SMB) unit with a CHIRALPAK®IC column and acetonitrile as a mobile phase. The retention times for the two enantiomers are 3.3 and 4.1 minutes, respectively. In certain embodiments, the productivity can be greater than 6 kg Feed/day/kg CSP.

Example 14

(8R,9S)-5-fluoro-8 4-fluorophenyl)-9<l-me

Je]phthalazin-3(7H)-one (la) and (8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(l-methyl-lH-l,2,4-triazol-5- (lb)

Example 14A

To a solution of (2R,3R)-methyl 7-fluoro-2-(4-fluorophenyl)-3-(l-methyl-lH-l,2,4-triazol-5-yl)- 4-oxo-l,2,3,4-tetrahydroquinoline-5-carboxylate (6a) or (2S,3S)-methyl 7-fluoro-2-(4-fluorophenyl)-3-(l- methyl-lH-l,2,4-triazol-5-yl)-4-oxo-l,2,3,4-tetrahydroquinoline-5-carboxylate (6b) (400 mg, 1.0 mmol) in ethanol (8.0 mL) was added hydrazine monohydrate (85 %, 2.0 mL), and the solution stirred at room temperature for 2 hours. The resulting solution was then concentrated to a volume of 2 mL and filtered, and the resultant cake washed with ethanol (1 mL). After drying in vacuum at 50°C, this afforded the title compound as a white solid (209 mg, yield 55 %). LC-MS (ESI) m/z: 381(M+1)⁺. ^-NMR (400 MHz, DMSO-dg): δ (ppm): 3.681 (s, 3H), 4.99-5.06 (m, 2H), 6.92-6.96 (m, 1H), 7.08-7.11 (m, 1H), 7.16-7.21 (t, J= 8.8 Hz, 2H), 7.49-7.53 (m, 2H), 7.75 (s, 1H), 7.83 (s, 1H), 12.35 (s, 1H).

Example 14B

To a solution of (2R,3R)-methyl 7-fluoro-2-(4-fluorophenyl)-3-(l-methyl-lH-l,2,4-triazol-5-yl)- 4-oxo-l,2,3,4-tetrahydroquinoline-5-carboxylate (6a) or (2S,3S)-methyl 7-fluoro-2-(4-fluorophenyl)-3-(l- methyl-lH-l,2,4-triazol-5-yl)-4-oxo-l,2,3,4-tetrahydroquinoline-5-carboxylate (6b) (446 g) in acetonitrile (10 volume) was added hydrazine monohydrate (2.9 eq.), and the solution stirred at room temperature for 2 hours. The resulting solution was then concentrated to a volume of 2 mL and filtered. The crude product was re-slurried with water (3~5 volumes) at 15-16 °C. After drying in vacuum at 50 °C, this affords the title compound as a white solid (329 g, yield 77%, 99.93% purity). LC-MS (ESI) m/z:

381(M+1)⁺; ¾-NMR (400 MHz, DMSO-d₆) δ (ppm): 3.681 (s, 3H), 4.99-5.06 (m, 2H), 6.92-6.96 (m, 1H), 7.08-7.11 (m, 1H), 7.16-7.21 (t, J= 8.8 Hz, 2H), 7.49-7.53 (m, 2H), 7.75 (s, 1H), 7.83 (s, 1H), 12.35 (s, 1H).

References

External links

• Talazoparib. Chemspider.com structure etc

nmr……http://www.medkoo.com/uploads/product/Talazoparib__BMN-673_/qc/BMN673-QC-BBC20130523-Web.pdf

Patent                       Submitted                        Granted

PROCESSES OF SYNTHESIZING DIHYDROPYRIDOPHTHALAZINONE DERIVATIVES [US2014323725]2014-06-022014-10-30

CRYSTALLINE (8S,9R)-5-FLUORO-8-(4-FLUOROPHENYL)-9-(1-METHYL-1H-1,2,4-TRIAZOL-5-YL)-8,9-DIHYDRO-2H-PYRIDO[4,3,2-DE]PHTHALAZIN-3(7H)-ONE TOSYLATE SALT [US2014228369]2014-04-142014-08-14

Crystalline (8S,9R)-5-fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-8,9-dihydro-2H-pyrido[4,3,2-de]phthalazin-3(7H)-one tosylate salt [US8735392]2011-10-202014-05-27

DIHYDROPYRIDOPHTHALAZINONE INHIBITORS OF POLY(ADP-RIBOSE)POLYMERASE (PARP) [US8012976]2010-02-112011-09-06

DIHYDROPYRIDOPHTHALAZINONE INHIBITORS OF POLY(ADP-RIBOSE)POLYMERASE (PARP) FOR USE IN TREATMENT OF DISEASES ASSOCIATED WITH A PTEN DEFICIENCY [US2014066429]2013-08-212014-03-06

METHODS AND COMPOSITIONS FOR TREATMENT OF CANCER AND AUTOIMMUNE DISEASE [US2013184342]2013-03-132013-07-18

SEE………..http://orgspectroscopyint.blogspot.in/2016/02/talazoparib.html

http://apisynthesisint.blogspot.in/2016/02/talazoparib.html

Talazoparib

Systematic (IUPAC) name
(8S,9R)-5-Fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one
Clinical data
Legal status	Investigational
Chemical data
Formula	C19H14F2N6O
Molar mass	380.35 g/mol

/////////////BMN 673, talazoparib, phase 3, BMN673, BMN673, BMN-673, LT673, LT 673, LT-673, Poly ADP ribose polymerase 2 inhibitor, Poly ADP ribose polymerase 1 inhibitor, cancer, MDV-3800 , MDV 3800

Cn1c(ncn1)[C@H]2c3c4c(cc(cc4N[C@@H]2c5ccc(cc5)F)F)c(=O)[nH]n3

O=C1NN=C2C3=C1C=C(F)C=C3N[C@H](C4=CC=C(F)C=C4)[C@H]2C5=NC=NN5C

ODM 201, BAY 1841788; ODM-201

N-((S)-1-(3-(3-chloro-4-cyanophenyl)-1H-pyrazol-1-yl)propan-2-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide

CAS 1297538-32-9
Chemical Formula: C19H19ClN6O2
Exact Mass: 398.1258

SYNTHESIS SEE BELOW

Phase III Prostate cancer

• 12 Feb 2016 Bayer plans a phase I trial in healthy volunteers in Germany (NCT02671097)
• 01 Nov 2015 Orion Corporation completes a phase II trial in Prostate cancer (late-stage disease, second-line or greater) in USA, Czech Republic, Estonia, France, Finland and United Kingdom (NCT01429064)
• 16 Oct 2015 Phase-III clinical trials in Prostate cancer (Second-line therapy or greater) in Australia, Belarus, Canada, South Africa, South Korea, Russia, Spain, Taiwan and Ukraine (PO)

• Originator Orion

• Developer Bayer HealthCare; Orion

• Class Antineoplastics
• Mechanism of Action Androgen receptor antagonists

ODM-201 (also known as BAY-1841788) is a non-steroidal antiandrogen, specifically, a full and high-affinity antagonist of the androgen receptor (AR), that is under development by Orion and Bayer HealthCare[1] for the treatment of advanced, castration-resistant prostate cancer (CRPC).^[2][3]

Relative to enzalutamide (MDV3100 or Xtandi) and apalutamide (ARN-509), two other recent non-steroidal antiandrogens, ODM-201 shows some advantages.^[3] ODM-201 appears to negligibly cross the blood-brain-barrier.^[3] This is beneficial due to the reduced risk of seizures and other central side effects from off-target GABA_A receptor inhibition that tends to occur in non-steroidal antiandrogens that are structurally similar to enzalutamide.^[3] Moreover, in accordance with its lack of central penetration, ODM-201 does not seem to increase testosterone levels in mice or humans, unlike other non-steroidal antiandrogens.^[3] Another advantage is that ODM-201 has been found to block the activity of all tested/well-known mutant ARs in prostate cancer, including the recently-identified clinically-relevant F876L mutation that produces resistance to enzalutamide and ARN-509.^[3] Finally, ODM-201 shows higher affinity and inhibitory efficacy at the AR (K_i = 11 nM relative to 86 nM for enzalutamide and 93 nM for ARN-509; IC₅₀ = 26 nM relative to 219 nM for enzalutamide and 200 nM for ARN-509) and greater potency/efficaciousness in non-clinical models of prostate cancer.^[3]

ODM-201 has been studied in phase I and phase II clinical trials and has thus far been found to be effective and well-tolerated,^[4] with the most commonly reported side effects including fatigue, nausea, and diarrhea.^[5][6] No seizures have been observed.^[6][7] As of July 2015, ODM-201 is in phase III trials for CRPC.^[3]

ORM-15341 is the main active metabolite of ODM-201.^[3] It, similarly, is a full antagonist of the AR, with an affinity (K_i) of 8 nM and an IC₅₀ of 38 nM.^[3]

ODM-201 is a new-generation, potent and selective androgen receptor (AR) inhibitor which is potential useful for treatment of castration-resistant prostate cancer (CRPC). ODM-201 is a full and high-affinity AR antagonist that, similar to second-generation antiandrogens enzalutamide and ARN-509, inhibits testosterone-induced nuclear translocation of AR. Importantly, ODM-201 also blocks the activity of the tested mutant ARs arising in response to antiandrogen therapies, including the F876L mutation that confers resistance to enzalutamide and ARN-509. In addition, ODM-201 reduces the growth of AR-overexpressing VCaP prostate cancer cells both in vitro and in a castration-resistant VCaP xenograft model. ODM-201 overcomes resistance to AR-targeted therapies by antagonizing both overexpressed and mutated ARs. ODM-201 is currently in a phase 3 trial in CRPC

Figure 1: The structures of ODM-201 (A) and its main metabolite ORM-15341 (B).

WHIPPANY, N.J., Sept. 16, 2014 /PRNewswire/ — Bayer HealthCare and Orion Corporation, a pharmaceutical company based in Espoo, Finland, have begun to enroll patients in a Phase III trial with ODM-201, an investigational oral androgen receptor inhibitor in clinical development. The study, called ARAMIS, evaluates ODM-201 in men with castration-resistant prostate cancer who have rising Prostate Specific Antigen (PSA) levels and no detectable metastases. The trial is designed to determine the effects of the treatment on metastasis-free survival (MFS).

“The field of treatment options for prostate cancer patients is evolving rapidly.  However, once prostate cancer becomes resistant to conventional anti-hormonal therapy, many patients will eventually develop metastatic disease,” said Dr. Joerg Moeller, Member of the Bayer HealthCare Executive Committee and Head of Global Development. “The initiation of a Phase III clinical trial for ODM-201 marks the starting point for a potential new treatment option for patients whose cancer has not yet spread.  This is an important milestone for Bayer in our ongoing effort to meet the unmet needs of men affected by prostate cancer.”

Earlier this year, Bayer and Orion entered into a global agreement under which the companies will jointly develop ODM-201, with Bayer contributing a major share of the costs of future development. Bayer will commercialize ODM-201 globally, and Orion has the option to co-promote ODM-201 in Europe. Orion will be responsible for the manufacturing of the product.

About the ARAMIS Study
The ARAMIS trial is a randomized, Phase III, multicenter, double-blind, placebo-controlled trial evaluating the safety and efficacy of oral ODM-201 in patients with non-metastatic CRPC who are at high risk for developing metastatic disease. About 1,500 patients are planned to be randomized in a 2:1 ratio to receive 600 mg of ODM-201 twice a day or matching placebo. Randomisation will be stratified by PSA doubling time (PSADT less than or equal to 6 months vs. > 6 months) and use of osteoclast-targeted therapy (yes vs. no).

The primary endpoint of this study is metastasis-free survival (MFS), defined as time between randomization and evidence of metastasis or death from any cause. The secondary objectives of this study are overall survival (OS), time to first symptomatic skeletal event (SSE), time to initiation of first cytotoxic chemotherapy, time to pain progression, and characterization of the safety and tolerability of ODM-201.

About ODM-201
ODM-201 is an investigational androgen receptor (AR) inhibitor that is thought to block the growth of prostate cancer cells. ODM-201 binds to the AR and inhibits receptor function by blocking its cellular function.

About Oncology at Bayer
Bayer is committed to science for a better life by advancing a portfolio of innovative treatments. The oncology franchise at Bayer now includes three oncology products and several other compounds in various stages of clinical development. Together, these products reflect the company’s approach to research, which prioritizes targets and pathways with the potential to impact the way that cancer is treated.

About Bayer HealthCare Pharmaceuticals Inc.
Bayer HealthCare Pharmaceuticals Inc. is the U.S.-based pharmaceuticals business of Bayer HealthCare LLC, a subsidiary of Bayer AG. Bayer HealthCare is one of the world’s leading, innovative companies in the healthcare and medical products industry, and combines the activities of the Animal Health, Consumer Care, Medical Care, and Pharmaceuticals divisions. As a specialty pharmaceutical company, Bayer HealthCare provides products for General Medicine, Hematology, Neurology, Oncology and Women’s Healthcare. The company’s aim is to discover and manufacture products that will improve human health worldwide by diagnosing, preventing and treating diseases.

Bayer® and the Bayer Cross® are registered trademarks of Bayer.

SYNTHESIS

PATENT

US 2015203479

http://www.google.com/patents/WO2011051540A1?cl=en

PATENT

WO 2012143599

http://www.google.com/patents/US20140094474?cl=de

References

• 1 “Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms to androgen signaling-directed prostate cancer therapies.”. Sci Rep 5: 12007. 2015. doi:10.1038/srep12007. PMC 4490394. PMID 26137992.
• 2Fizazi K, Albiges L, Loriot Y, Massard C (2015). “ODM-201: a new-generation androgen receptor inhibitor in castration-resistant prostate cancer”. Expert Rev Anticancer Ther 15 (9): 1007–17. doi:10.1586/14737140.2015.1081566. PMID 26313416.
• 3Moilanen AM, Riikonen R, Oksala R, Ravanti L, Aho E, Wohlfahrt G, Nykänen PS, Törmäkangas OP, Palvimo JJ, Kallio PJ (2015). “Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms to androgen signaling-directed prostate cancer therapies”. Sci Rep 5: 12007. doi:10.1038/srep12007. PMC 4490394. PMID 26137992.
• 4“ODM-201 is safe and active in metastatic castration-resistant prostate cancer”. Cancer Discov 4 (9): OF10. 2014. doi:10.1158/2159-8290.CD-RW2014-150. PMID 25185192.
• 5Pinto Á (2014). “Beyond abiraterone: new hormonal therapies for metastatic castration-resistant prostate cancer”. Cancer Biol. Ther. 15 (2): 149–55. doi:10.4161/cbt.26724. PMC 3928129. PMID 24100689.
• 6Fizazi K, Massard C, Bono P, Jones R, Kataja V, James N, Garcia JA, Protheroe A, Tammela TL, Elliott T, Mattila L, Aspegren J, Vuorela A, Langmuir P, Mustonen M (2014). “Activity and safety of ODM-201 in patients with progressive metastatic castration-resistant prostate cancer (ARADES): an open-label phase 1 dose-escalation and randomised phase 2 dose expansion trial”. Lancet Oncol. 15 (9): 975–85. doi:10.1016/S1470-2045(14)70240-2. PMID 24974051.
• 7Agarwal N, Di Lorenzo G, Sonpavde G, Bellmunt J (2014). “New agents for prostate cancer”. Ann. Oncol. 25 (9): 1700–9. doi:10.1093/annonc/mdu038. PMID 24658665.

Fenner A. Prostate cancer: ODM-201 tablets complete phase I. Nat Rev Urol. 2015 Dec;12(12):654. doi: 10.1038/nrurol.2015.268. Epub 2015 Nov 3. PubMed PMID: 26526759.

2: Massard C, Penttinen HM, Vjaters E, Bono P, Lietuvietis V, Tammela TL, Vuorela A, Nykänen P, Pohjanjousi P, Snapir A, Fizazi K. Pharmacokinetics, Antitumor Activity, and Safety of ODM-201 in Patients with Chemotherapy-naive Metastatic Castration-resistant Prostate Cancer: An Open-label Phase 1 Study. Eur Urol. 2015 Oct 10. pii: S0302-2838(15)00964-1. doi: 10.1016/j.eururo.2015.09.046. [Epub ahead of print] PubMed PMID: 26463318.

3: Fizazi K, Albiges L, Loriot Y, Massard C. ODM-201: a new-generation androgen receptor inhibitor in castration-resistant prostate cancer. Expert Rev Anticancer Ther. 2015;15(9):1007-17. doi: 10.1586/14737140.2015.1081566. PubMed PMID: 26313416; PubMed Central PMCID: PMC4673554.

4: Bambury RM, Rathkopf DE. Novel and next-generation androgen receptor-directed therapies for prostate cancer: Beyond abiraterone and enzalutamide. Urol Oncol. 2015 Jul 7. pii: S1078-1439(15)00269-0. doi: 10.1016/j.urolonc.2015.05.025. [Epub ahead of print] Review. PubMed PMID: 26162486.

5: Moilanen AM, Riikonen R, Oksala R, Ravanti L, Aho E, Wohlfahrt G, Nykänen PS, Törmäkangas OP, Palvimo JJ, Kallio PJ. Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms to androgen signaling-directed prostate cancer therapies. Sci Rep. 2015 Jul 3;5:12007. doi: 10.1038/srep12007. PubMed PMID: 26137992; PubMed Central PMCID: PMC4490394.

6: Thibault C, Massard C. [New therapies in metastatic castration resistant prostate cancer]. Bull Cancer. 2015 Jun;102(6):501-8. doi: 10.1016/j.bulcan.2015.04.016. Epub 2015 May 26. Review. French. PubMed PMID: 26022286.

7: Bjartell A. Re: activity and safety of ODM-201 in patients with progressive metastatic castration-resistant prostate cancer (ARADES): an open-label phase 1 dose-escalation and randomised phase 2 dose expansion trial. Eur Urol. 2015 Feb;67(2):348-9. doi: 10.1016/j.eururo.2014.11.019. PubMed PMID: 25760250.

8: De Maeseneer DJ, Van Praet C, Lumen N, Rottey S. Battling resistance mechanisms in antihormonal prostate cancer treatment: Novel agents and combinations. Urol Oncol. 2015 Jul;33(7):310-21. doi: 10.1016/j.urolonc.2015.01.008. Epub 2015 Feb 21. Review. PubMed PMID: 25708954.

9: Boegemann M, Schrader AJ, Krabbe LM, Herrmann E. Present, Emerging and Possible Future Biomarkers in Castration Resistant Prostate Cancer (CRPC). Curr Cancer Drug Targets. 2015;15(3):243-55. PubMed PMID: 25654638.

10: ODM-201 is safe and active in metastatic castration-resistant prostate cancer. Cancer Discov. 2014 Sep;4(9):OF10. doi: 10.1158/2159-8290.CD-RW2014-150. Epub 2014 Jul 9. PubMed PMID: 25185192.

11: Fizazi K, Massard C, Bono P, Jones R, Kataja V, James N, Garcia JA, Protheroe A, Tammela TL, Elliott T, Mattila L, Aspegren J, Vuorela A, Langmuir P, Mustonen M; ARADES study group. Activity and safety of ODM-201 in patients with progressive metastatic castration-resistant prostate cancer (ARADES): an open-label phase 1 dose-escalation and randomised phase 2 dose expansion trial. Lancet Oncol. 2014 Aug;15(9):975-85. doi: 10.1016/S1470-2045(14)70240-2. Epub 2014 Jun 25. PubMed PMID: 24974051.

12: Agarwal N, Di Lorenzo G, Sonpavde G, Bellmunt J. New agents for prostate cancer. Ann Oncol. 2014 Sep;25(9):1700-9. doi: 10.1093/annonc/mdu038. Epub 2014 Mar 20. Review. PubMed PMID: 24658665.

13: Pinto Á. Beyond abiraterone: new hormonal therapies for metastatic castration-resistant prostate cancer. Cancer Biol Ther. 2014 Feb;15(2):149-55. doi: 10.4161/cbt.26724. Epub 2013 Nov 1. Review. PubMed PMID: 24100689; PubMed Central PMCID: PMC3928129.

14: Yin L, Hu Q, Hartmann RW. Recent progress in pharmaceutical therapies for castration-resistant prostate cancer. Int J Mol Sci. 2013 Jul 4;14(7):13958-78. doi: 10.3390/ijms140713958. Review. PubMed PMID: 23880851; PubMed Central PMCID: PMC3742227.

15: Leibowitz-Amit R, Joshua AM. Targeting the androgen receptor in the management of castration-resistant prostate cancer: rationale, progress, and future directions. Curr Oncol. 2012 Dec;19(Suppl 3):S22-31. doi: 10.3747/co.19.1281. PubMed PMID: 23355790; PubMed Central PMCID: PMC3553559.

ODM-201

Systematic (IUPAC) name
N((R)-1-(3-(4-Cyano-3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)propan-2-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide[1]
Identifiers
ChemSpider	38772320
Chemical data
Formula	C19H19ClN6O2
Molar mass	398.85 g·mol−1

/////

O=C(C1=NNC(C(O)C)=C1)N[C@@H](C)CN2N=C(C3=CC=C(C#N)C(Cl)=C3)C=C2

Trioxacarcin A, DC-45A

CAS No. 81552-36-5

• Molecular FormulaC₄₂H₅₂O₂₀
• Average mass876.850 Da
• 17′-[(4-C-Acetyl-2,6-dideoxyhexopyranosyl)oxy]-19′-(dimethoxymethyl)-10′,13′-dihydroxy-6′-methoxy-3′-methyl-11′-oxospiro[oxirane-2,18′-[16,20,22]trioxahexacyclo[17.2.1.0^2,15.0^5,14.0^7,12.0^17,21 ]docosa[2(15),3,5(14),6,12]pentaen]-8′-yl 4-O-acetyl-2,6-dideoxy-3-C-methylhexopyranoside
   (1S,2R,3aS,4S,8S,10S,13aS)-13a-(4-C-Acetyl-2,6-dideoxy-alpha-L-xylo-hexopyranosyloxy)-2-(dimethoxymethyl)-10,12-dihydroxy-7-methoxy-5-methyl-11-oxo-4,8,9,10,11,13a-hexahydro-3aH-spiro[2,4-epoxyfuro[3,2-b]naphtho[2,3-h]-1-benzopyran-1,2′-oxiran]-8-yl 4-O-acetyl-2,6-dideoxy-3-C-methyl-alpha-L-xylo-hexopyranoside
• Kyowa Hakko Kirin   INNOVATOR

Trioxacarcin B

Trioxacarcin B; Antibiotic DC 45B1; DC-45-B1; Trioxacarcin A, 14,17-deepoxy-14,17-dihydroxy-; AC1MJ5N1; 81534-36-3;

Trioxacarcin C

(CAS NO.81781-28-4):C₄₂H₅₄O₂₀
Molecular Weight: 878.8662 g/mol
Structure of Trioxacarcin C :

The trioxacarcins are polyoxygenated, structurally complex natural products that potently inhibit the growth of cultured human cancer cells

Natural products that bind and often covalently modify duplex DNA figure prominently in chemotherapy for human cancers. The trioxacarcins are a new class of DNA- modifying natural products with antiproliferative effects. The trioxacarcins were first described in 1981 by Tomita and coworkers (Tomita et al. , J. Antibiotics, 34( 12): 1520- 1524, 1981 ; Tamaoki et al., J. Antibiotics 34( 12): 1525- 1530, 1981 ; Fujimoto et al. , J. Antibiotics 36(9): 1216- 1221 , 1983). Trioxacarcin A, B, and C were isolated by Tomita and coworkers from the culture broth of Streptomyces bottropensis DO-45 and shown to possess anti-tumor activity in murine models as well as gram-positive antibiotic activity. Subsequent work led to the discovery of other members of this family. Trioxacarcin A is a powerful anticancer agent with subnanmolar IC70 values against lung (LXFL 529L, H-460), mammary (MCF-7), and CNS (SF-268) cancer cell lines. The trioxacarcins have also been shown to have antimicrobial activity {e.g., anti-bacterial and anti-malarial activity) (see, e.g. , Maskey et al., J. Antibiotics (2004) 57:771 -779).

trioxacarcin A

An X-ray crystal structure of trioxacarcin A bound to N-7 of a guanidylate residue in a duplex DNA oligonucleotide substrate has provided compelling evidence for a proposed pathyway of DNA modification that proceeds by duplex intercalation and alkylation (Pfoh et al, Nucleic Acids Research 36( 10):3508-3514, 2008).

All trioxacarcins appear to be derivatives of the aglycone, which is itself a bacterial isolate referred to in the patent literature as DC-45-A2. U.S. Patent 4,459,291 , issued July 10, 1984, describes the preparation of DC-45-A₂ by fermentation. DC-45-A2 is the algycone of trioxacarcins A, B, and C and is prepared by the acid hydrolysis of the fermentation products trioxacarcins A and C or the direct isolation from the fermentation broth of Streptomyces bottropensis.

Based on the biological activity of the trioxacarcins, a fully synthetic route to these compounds would be useful in exploring the biological and chemical activity of known trioxacarcin compounds and intermediates thereto, as well as aid in the development of new trioxacarcin compounds with improved biological and/or chemical properties.

PAPER

Component-Based Syntheses of Trioxacarcin A, DC-45-A1, and Structural Analogs
T. Magauer, D. Smaltz, A. G. Myers, Nat. Chem. 2013, 5, 886–893. (Link)

An 11-step synthesis of trioxacarcin A was achieved by the convergent assembly of five modular components of similar structural complexity, each denoted with a coloured circle. This strategy provides a general route to analogues with deep-seated structural variations. MOM, methoxymethyl;BrBcat, B-bromocatecholborane.

PAPER

A schematic shows a trioxacarcin C molecule, whose structure was revealed for the first time through a new process developed by the Rice lab of synthetic organic chemist K.C. Nicolaou. Trioxacarcins are found in bacteria but synthetic versions are needed to study them for their potential as medications. Trioxacarcins have anti-cancer properties. Source: Nicolaou Group/Rice University

A team led by Rice University synthetic organic chemist K.C. Nicolaou has developed a new process for the synthesis of a series of potent anti-cancer agents originally found in bacteria.

The Nicolaou lab finds ways to replicate rare, naturally occurring compounds in larger amounts so they can be studied by biologists and clinicians as potential new medications. It also seeks to fine-tune the molecular structures of these compounds through analog design and synthesis to improve their disease-fighting properties and lessen their side effects.

Such is the case with their synthesis of trioxacarcins, reported this month in the Journal of the American Chemical Society.

PAPER

PATENT

http://www.google.com/patents/EP2550285A1?cl=en

(S)-9-Hvdrox v- 10-methoxy-5-(4-methoxybenzylox v)- 1 -(methoxymethox y)-3- methyl-8-oxo-5,6.7.8-tetrahvdroanthracene-2-carbaldehvde. Potassium osmate dihydrate (29 mg, 0.079 mmol, 0.05 equiv) was added to an ice -cooled mixture of (S,£)-9-hydroxy- 10- methoxy-4-(4-methoxybenzyloxy)-8-(methoxymethoxy)-6-methyl-7-(prop- l -enyl)-3,4- dihydroanthracen-l -one (780 mg, 1.58 mmol, 1 equiv), 2,6-lutidine (369 μί, 3.17 mmol, 2.0 equiv), and sodium periodate ( 1.36 g, 6.33 mmol, 4.0 equiv) in a mixture of tetrahydrofuran (20 mL) and water ( 10 mL). After 10 min, the cooling bath was removed and the reaction flask was allowed to warm to 23 °C. After 1.5 h, the reaction mixture was partitioned between water ( 100 mL) and ethyl acetate (150 mL). The layers were separated. The organic layer was washed with aqueous sodium chloride solution (50 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (20% ethyl acetate- hexanes) to provide 498 mg of the product, (5)-9-hydroxy- 10-methoxy-5-(4- methoxybenzyloxy)- l -(methoxymethoxy)-3-methyl-8-oxo-5,6,7,8-tetrahydroanthracene-2- carbaldehyde, as an orange foam (65%). Ή NMR (500 MHz, CDC1₃): 15.17 (s, 1 H), 10.74 (s, 1 H), 7.66 (s, 1 H), 7.27 (d, 2H, 7 = 8.5 Hz), 6.86 (d, 2H, 7 = 8.6 Hz), 5.30-5.18 (m, 3H), 4.63 (d, 1H,7= 11.1 Hz), 4.52 (d, 1H,7 = 12.0 Hz), 3.86 (s, 3H), 3.79 (s, 3H), 3.62 (s, 3H), 3.22 (m, 1H), 2.75 (s, 3H), 2.63 (m, 1H), 2.54 (m, 1H), 2.08 (m, 1H). ^I3C NMR (125 MHz, CDC1₃): 204.9, 193.2, 163.2, 161.7, 159.2, 144.4, 141.7, 137.0, 130.1, 129.4, 120.7, 117.9, 113.8, 110.0, 102.8, 70.4, 67.2, 62.9, 58.3, 55.2, 32.3, 26.3, 22.2. FTIR, cm^-1 (thin film): 2936 (m), 2907 (m), 1684 (s), 1611 (s), 1377 (s), 1246 (s). HRMS (ESI): Calcd for

(C₂₇H2808+K)⁺: 519.1416; Found 519.1368. TLC (20% ethyl acetate-hexanes): R,= 0.17 (CAM).

86% yield

[00457] (S)-l,9-Dihvdroxy-10-methoxy-5-(4-methoxybenzyloxy)-3-methyl-8-oxo-5,6,7,8- tetrahydroanthracene-2-carbaldehyde. A solution of B-bromocatecholborane (418 mg, 2.10 mmol, 2.0 equiv) in dichloromethane (15 mL) was added to a solution of (S)-9-hydroxy-10- methoxy-5-(4-methoxybenzyloxy)-l-(methoxymethoxy)-3-methyl-8-oxo-5,6,7,8- tetrahydroanthracene-2-carbaldehyde (490 mg, 1.05 mmol, 1 equiv) in dichloromethane (15 mL) at -78 °C. After 50 min, the reaction mixture was diluted with saturated aqueous sodium bicarbonate solution (25 mL) and dichloromethane (100 mL). The cooling bath was removed, and the partially frozen mixture was allowed to warm to 23 °C. The biphasic mixture was diluted with 0.2 M aqueous sodium hydroxide solution (100 mL). The layers were separated. The aqueous layer was extracted with dichloromethane (100 mL). The organic layers were combined. The combined solution was washed sequentially with 0.1 M aqueous hydrochloric acid solution (100 mL), water (2 x 100 mL), then saturated aqueous sodium chloride solution (100 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to provide 380 mg of the product, (S)-\ ,9- dihydroxy-10-methoxy-5-(4-methoxybenzyloxy)-3-methyl-8-oxo-5,6,7,8- tetrahydroanthracene-2-carbaldehyde, as a yellow foam (86%). Ή NMR (500 MHz, CDCI3):

15.89 (brs, 1H), 12.81 (br s, 1H), 10.51 (s, 1H), 7.27-7.26 (m, 3H), 6.86 (d, 2H, J = 9.2 Hz), 5.14 (app s, 1H),4.62 (d, \H,J= 11.0 Hz), 4.51 (d, 1H,7= 11.0 Hz), 3.85 (s, 3H), 3.80 (s, 3H), 3.21 (m, 1H), 2.73 (s, 3H), 2.62 (m, 1H), 2.54 (m, 1H), 2.07 (m, 1H). ^I3C NMR (125 MHz, CDCI3): 204.4, 192.7, 166.6, 164.3, 159.3, 144.4, 142.7, 137.9, 130.4, 130.2, 129.4, 114.9, 114.2, 113.9, 113.8, 109.4, 70.4, 67.1,62.8, 55.3, 31.8, 26.5. FTIR, cm^-1 (thin film): 3316 (brw), 2938 (m), 1678 (m), 1610 (s), 1514 (m), 1393 (m), 1246 (s). HRMS (ESI): Calcd for (C₂₅H₂₄0₇+Na)⁺ 459.1414; Found 459.1354. TLC (50% ethyl acetate-hexanes): R = 0.30 (CAM).

[00458] (5)-2,2-Di-/erf-butyl-7-methoxy-8-(4-methoxybenzyloxy)-5-methyl- 1 1 -oxo- 8,9, 10, 1 1 -tetrahydroanthra[9, 1 -de \ 1 ,3,21dioxasiline-4-carbaldehyde. Όι^‘-tert- butyldichlorosilane (342 μL·, 1.62 mmol, 1.8 equiv) was added to a solution of (5)-l ,9- dihydroxy- 10-methoxy-5-(4-methoxybenzyloxy)-3-methyl-8-oxo-5,6,7,8- tetrahydroanthracene-2-carbaldehyde (380 mg, 0.90 mmol, 1 equiv), hydroxybenzotriazole (60.8 mg, 0.45 mmol, 0.50 equiv) and diisopropylethylamine (786 μί, 4.50 mmol, 5.0 equiv) in dimethylformamide (30 mL). The reaction flask was heated in an oil bath at 55 °C. After 2 h, the reaction flask was allowed to cool to 23 °C. The reaction mixture was partitioned between saturated aqueous sodium bicarbonate solution (100 mL) and ethyl acetate (150 mL). The layers were separated. The organic layer was washed sequentially with water (2 x 100 mL) then saturated aqueous sodium chloride solution (100 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (10% ethyl acetate- hexanes) to provide 285 mg of the product, (S)-2,2-di-/<?ri-butyl-7-methoxy-8-(4- methoxybenzyloxy)-5-methyl- 1 1 -oxo-8,9, 10, 1 1 -tetrahydroanthra[9, 1 -de] [ 1 ,3,2]dioxasiline-4- carbaldehyde, as a yellow foam (56%). The enantiomeric compound (/?)-2,2-di-½ri-butyl-7- methoxy-8-(4-methoxybenzyloxy)-5-methyl- l 1 -oxo-8,9, 10, 1 1 -tetrahydroanthra[9, 1 – i/e][ l ,3,2]dioxasiline-4-carbaldehyde has been prepared using the same route by utilizing R- (4-methoxybenzyloxy)cyclohex-2-enone as starting material. Ή NMR (500 MHz, CDCI3): 10.84 (s, 1 H), 7.37 (s, 1 H), 7.25 (d, 2H, J = 8.8 Hz), 6.85 (d, 2H, = 8.7 Hz), 5.20 (app s, 1 H), 4.62 (d, 1 H, 7 = 10.0 Hz), 4.51 (d, 1H, J = 1 1.4 Hz), 3.88 (s, 3H), 3.78 (s, 3H), 3.03 (m, 1H), 2.73 (s, 3H), 2.57-2.53 (m, 2H), 2.07 (m, 1H), 1.16 (s, 9H), 1.14 (s, 9H). ¹³C NMR (125 MHz, CDCl₃): 195.6, 190.9, 160.5, 159.2, 150.4, 145.7, 140.4, 134.0, 133.9, 130.3, 129.4, 1 19.5, 1 16.6, 1 15.8, 1 15.3, 1 13.8, 70.4, 67.8, 62.9, 55.2, 34.0, 26.0, 26.0, 22.5, 21.3, 21.1. FTIR, cm^“1 (thin film): 2936 (m), 2862 (m), 1682 (s), 1607 (s), 1371 (s), 1244 (s) 1057 (s). HRMS (ESI): Calcd for (C₃3H₄o0₇Si+H)⁺ 577.2616; Found 577.2584. TLC (10% ethyl acetate-hexanes): R/ = 0.19 (CAM). Alternative Routes to (4S,6S)-6-(½rt-Butyldimethylsilyloxy)-4-(4-methoxybenzyloxy) cyclohex-2-enone.

Alternative Route 1.

[00459] (25,45,55)-2,4-Bis(ferf-butyldimethylsilyloxy)-5-hvdroxycvclohexanone. Dess- Martin periodinane (6.1 1 g, 14.4 mmol, 1.1 equiv) was added to a solution of diol (5.00 g, 13.3 mmol, 1 equiv) in tetrahydrofuran (120 mL) at 23 °C (Lim, S. M.; Hill, N.; Myers, A. G. J. Am. Chem. Soc. 2009, 131, 5763-5765). After 40 min, the reaction mixture was diluted with ether (300 mL). The diluted solution was filtered through a short plug of silica gel (-5 cm) and eluted with ether (300 mL). The filtrate was concentrated. The bulk of the product was transformed as outlined in the following paragraph, without purification. Independently,

s

an analytically pure sample of the product was obtained by flash-column chromatography (20% ethyl acetate-hexanes) and was characterized by Ή NMR, ^{l 3}C NMR, IR, and HRMS. TLC: (17% ethyl acetate-hexanes) R = 0.14 (CAM); Ή NMR (500 MHz, CDCI3) δ: 4.41 (dd, 1 H, 7 = 9.8, 5.5 Hz), 4.05 (m, l H), 4.00 (m, 1H), 2.81 (ddd, 1 H, 7 = 14.0, 3.7, 0.9 Hz), 2.52 (ddd, 1 H, 7 = 14.0, 5.3, 0.9 Hz), 2.29 (br s, 1 H), 2.18 (m, 1H), 1.98 (m, 1 H), 0.91 (s, 9H), 0.89 (s, 9H), 0.13 (s, 3H), 0.1 1 (s, 3H), 0.09 (s, 3H), 0.04 (s, 3H); ^{l 3}C NMR (125 MHz, CDCI3) δ: 207.9, 73.9, 73.3, 70.5, 43.3, 39.0, 25.7, 25.6, 18.3, 17.9, -4.7, -4.8, -4.9, -5.4; FTIR (neat), cm^“‘ : 3356 (br), 2954 (m), 2930 (m), 2857 (m), 1723 (m), 1472 (m). 1253 (s), 1 162 (m), 1 105 (s), 1090 (s), 1059 (s), 908 (s), 834 (s), 776 (s), 731 (s); HRMS (ESI): Calcd for (C|₈H380₄Si2+H)⁺ 375. 2381 , found 375.2381.

[00460] (4 ,6 )-4.6-Bis(fcr/-butyldimethylsilyloxy)cvclohex-2-enone. Trifluoroacetic anhydride (6.06 mL, 43.6 mmol, 3.3 equiv) was added to an ice-cooled solution of the alcohol ( 1 equiv, see paragraph above) and triethylamine ( 18.2 mL, 131 mmol, 9.9 equiv) in dichloromethane (250 mL) at 0 °C. After 20 min, the cooling bath was removed and the reaction flask was allowed to warm to 23 °C. After 18 h, the reaction flask was cooled in an ice bath at 0 °C, and the product solution was diluted with water ( 100 mL). The cooling bath was removed and the reaction flask was allowed to warm to 23 °C. The layers were separated. The aqueous layer was extracted with dichloromethane (2 x 200 mL). The organic layers were combined. The combined solution was washed with saturated aqueous sodium chloride solution ( 100 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash- column chromatography (6% ethyl acetate-hexanes) to provide 3.02 g of the product, (4S,65)-4,6-bis(/eri-butyldimethylsilyloxy)cyclohex-2-enone, as a colorless oil (64% over two steps). TLC: (20% ethyl acetate-hexanes) R = 0.56 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 6.76 (dd, 1 Η, / = 10.1 , 3.6 Hz), 5.88 (d, 1 H, 7 = 10.1 Hz), 4.66 (ddd, 1 H, 7 = 5.6, 4.1 , 3.6 Hz), 4.40 (dd, 1 H, 7 = 8.1 , 3.7 Hz), 2.26 (ddd, 1 H, / = 13.3, 8.0, 4.1 Hz), 2.1 1 (ddd, 1 H, J = 13.2, 5.6, 3.8 Hz), 0.91 (s, 9H), 0.89 (s, 9H), 0.12 (s, 3H), 0. 1 1 (s, 3H), 0. 10 (s, 3H), 0.10 (s, 3H); ¹³C NMR ( 125 MHz, CDC1₃) δ: 197.5, 150.3, 127.0, 71 .0, 64.8, 41.6, 25.7, 25.7, 18.3, 18.1 , -4.7, -4.8, -4.8, -5.4; FTIR (neat), cm^-1 : 3038 (w), 2955 (m), 2930 (m), 1705 (m), 1472 (m), 1254 (m), 1084 (m), 835 (s), 777 (s), 675 (s); HRMS (ESI): Calcd for (C,8H360₂Si2+Na)⁺ 379. 2095, found 379. 2080.

[00461] (4S,6S)-6-(/er/-Butyldimethylsilyloxy)-4-hydroxycvclohex-2-enone. Tetra- j- butylammonium fluoride ( 1 .0 M solution in tetrahydrofuran, 8.00 mL, 8.00 mmol, 1 .0 equiv) was added to an ice-cooled solution of the enone (2.85 g, 8.00 mmol, 1 equiv) and acetic acid (485 ί, 8.00 mmol, 1 .0 equiv) in tetrahydrofuran (80 mL) at 0 °C. After 2 h, the cooling bath was removed and the reaction flask was allowed to warm to 23 °C. After 22 h, the reaction mixture was partitioned between water ( 100 mL) and ethyl acetate (300 mL). The layers were separated. The aqueous layer was extracted with ethyl acetate (2 x 300 mL). The organic layers were combined. The combined solution was washed sequentially with saturated aqueous sodium bicarbonate solution ( 100 mL) then saturated aqueous sodium chloride solution ( 100 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash- column chromatography (25% ethyl acetate-hexanes) to provide 760 mg of the product, (4S,6S)-6-(ferNbutyldimethylsilyloxy)-4-hydroxycyclohex-2-enone, as a white solid (39%). TLC: (20% ethyl acetate-hexanes) R/ = 0.20 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 6.87 (dd, 1 Η, 7 = 10.2, 3.2 Hz), 5.95 (dd, 1H, J = 10.3, 0.9 Hz), 4.73 (m, 1 H), 4.35 (dd, 1 H, 7 = 7.6, 3.7 Hz), 2.39 (m, 1 H), 2. 13 (ddd, 1 H, J = 13.3, 6.2, 3.4 Hz), 1.83 (d, 1 H, J = 6.2), 0.89 (s, 9H), 0.10 (s, 3H), 0. 10 (s, 3H); ¹³C NMR ( 125 MHz, CDCb) δ: 197.3, 150.0, 127.5, 70.9, 64.2, 41 .0, 25.7, 18.2, -4.8, -5.4; FTIR (neat), cm^“1 : 2956 (w), 293 1 (w), 2858 (w), 1694 (m); HRMS (ESI): Calcd for (C |₂H220₃Si+H)⁺ 243.141 1 , found 243. 1412.

82″:.

[00462] (45.6S)-6-(fgrf-Butyldimethylsilyloxy)-4-(4-methoxybenzyloxy)cvclohex-2- enone. Triphenylmethyl tetrafluoroborate ( 16 mg, 50 μπιοΐ, 0.050 equiv) was added to a solution of 4-methoxybenzyl-2,2,2-trichloroacetimidate (445 μΙ_, 2.5 mmol, 2.5 equiv) and alcohol (242 mg, 1 .0 mmol, 1 equiv) in ether ( 10 mL) at 23 °C. After 4 h, the reaction mixture was partitioned between saturated aqueous sodium bicarbonate solution ( 15 mL) and ethyl acetate (50 mL). The layers were separated. The aqueous layer was extracted with ethyl acetate (50 mL). The organic layers were combined. The combined solution was washed with water (2 x 20 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash column chromatography (5% ethyl acetate-hexanes initially, grading to 10% ethyl acetate-hexanes) to provide 297 mg of the product, (4S,6S)-6-(im-butyldimethylsilyloxy)-4-(4- methoxybenzyloxy)cyclohex-2-enone, as a colorless oil (82%).

Alternative Route 2.

[00463] (5)-?erf-Butyl(4-(4-methoxybenzyloxy)cvclohexa- 1.5-dienyloxy)dimethylsilane. rerr-Butyldimethylsilyl trifluoromethanesulfonate (202 iL, 0.94 mmol, 2.0 equiv) was added to an ice-cooled solution of triethylamine (262 μί, 1.88 mmol, 4.0 equiv) and enone ( 109 mg, 0.47 mmol, 1 equiv) in dichloromethane (5.0 mL). After 30 min, the reaction mixture was partitioned between saturated aqueous sodium bicarbonate solution ( 10 mL), water (30 mL), and dichloromethane (40 mL). The layers were separated. The organic layer was washed sequentially with saturated aqueous ammonium chloride solution (20 mL) then saturated aqueous sodium chloride solution (20 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography with triethylamine-treated silica gel (5% ethyl acetate-hexanes), to provide 130 mg of the product, (5)-ierr-butyl(4-(4- methoxybenzyloxy)cyclohexa- l ,5-dienyloxy)dimethylsilane, as a colorless oil (80%). Ή

NMR (500 MHz, CDC1₃): 7.27 (d, 2H, J = 8.7 Hz), 6.88 (d, 2H, J = 8.6 Hz), 5.96 (dd, 1 H, J = 9.9, 3.5 Hz), 5.87 (d, 1 H, 7 = 9.6 Hz), 4.94 (m, l H), 4.46 (s, 2H), 4.14 (m, 1 H), 3.81 (s, 3H), 2.49 (m, 2H), 0.93 (s, 9H), 0. 16 (s, 3H), 0.15 (s, 3H). ^{, 3}C NMR ( 125 MHz, CDC1₃): 159.1 , 147.5, 130.9, 129.2, 128.6, 128.1 , 1 13.8, 101.4, 70.2, 69.0, 55.3, 28.5, 25.7, 18.0, ^1.5, -4.5. FTIR, cm^-1 (thin film): 2957 (m), 2931 (m), 2859 (m), 1655 (w), 1613 (w), 1515 (s), 1248 (s), 1229 (s), 1037 (m), 910 (s). HRMS (ESI): Calcd for (C₂oH3o0₃Si+H)⁺ 347.2037; Found 347.1912. TLC (20% ethyl acetate-hexanes): R = 0.74 (CAM).

OP B OPMB DM 00 ,,Α,,

c Ύ’^“ -ietone ii ·η- ) ‘”OH

OTBS 82 ^Q

[00464] (4S,6S)-6-Hvdroxy-4-(4-methoxybenzyloxy)cvclohex-2-enone. A solution of dimethyldioxirane (0.06 M solution in acetone, 2.89 mL, 0.17 mmol, 1.2 equiv) was added to an ice-cooled solution of (S)-ieri-butyl(4-(4-methoxybenzyloxy)cyclohexa- l ,5- dienyloxy)dimethylsilane (50 mg, 0.14 mmol, 1 equiv). After 10 min, the reaction mixture was partitioned between dichloromethane ( 15 mL) and 0.5 M aqueous hydrochloric acid ( 10 mL). The layers were separated. The organic layer was washed sequentially with saturated aqueous sodium bicarbonate solution ( 10 mL) then water ( 10 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography to provide 30 mg of the product, (4S,6S)-6-hydroxy-4-(4-methoxybenzyloxy)cyclohex-2-enone, as a colorless oil (82%). Ή NMR (500 MHz, CDC1₃): 7.28 (d, 2H, J = 8.2 Hz), 6.89 (m, 3H), 6.09 (d, 1 H, J = 10.1 Hz), 4.64 (m, 2H), 4.53 (d, 1 H, 7 = 1 1 .4 Hz), 4.24 (m, 1 H), 3.81 (s, 3H), 3.39 (d, 1 H, 7 = 1.4 Hz), 2.67 (m, 1 H), 1 .95 (ddd, 1 H, 7 = 12.8, 12.8, 3.6 Hz). ^{I 3}C NMR ( 125 MHz, CDC1₃): 200.4, 159.5, 146.6, 129.7, 129.4, 127.8, 1 14.0, 71.6, 69.8, 68.9, 55.3, 35.1 . FTIR, cm^-1 (thin film): 3474 (br), 2934 (m), 2864 (m), 1692 (s), 1613 (m), 1512 (s), 1246 (s), 1059 (s), 1032 (s). HRMS (ESI): Calcd for (C,₄H_l6O₄+Na)⁺ 271.0941 ; Found 271.0834. TLC (50% ethyl acetate-hexanes): R/ = 0.57 (CAM).

[00465] (45,65)-6-(½rt-Butyldimethylsilyloxy)-4-(4-methoxybenzyloxy)cvclohex-2- enone. rerr-Butyldimethychlorosilane (26 mg, 0.18 mmol, 1.5 equiv) was added to an ice- cooled solution of (45,65)-6-hydroxy-4-(4-methoxybenzyloxy)cyclohex-2-enone (29 mg, 0.12 mmol, 1 equiv) and imidazole (24 mg, 0.35 mmol, 3 equiv) in dimethylformamide (0.5 mL). After 45 min, the reaction mixture was partitioned between water (15 mL), saturated aqueous sodium chloride solution (15 mL), and ethyl acetate (20 mL). The layers were separated. The organic layer was washed with water (2 x 20 mL) and the washed solution was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography to provide 29 mg of the product, (4S,6S)-6-(rm-butyldimethylsilyloxy)-4-(4-methoxybenzyloxy)cyclohex-2- enone, as a colorless oil (87%).

Glycosylation experiments

[00466] Glycosylation experiments demonstrate that the chemical process developed allows for the preparation of synthetic, glycosylated trioxacarcins. Specifically, the C4 or CI 3 hydroxyl group may be selectively glycosylated with a glycosyl donor (for example, a glycosyl acetate) and an activating agent (for example, TMSOTf), which enables preparation of a wide array of trioxacarcin analogues.

Selective Glycosylation of the C4 Hydroxyl Group

[00467] 2,3-Dichloro-5,6-dicyanobenzoquinone ( 19.9 mg, 88 μιτιοΐ, 1.1 equiv) was added to a vigorously stirring, biphasic solution of differentially protected trioxacarcin precursor (60 mg, 80 μιτιοΐ, 1 equiv) in dichloromethane ( 1.1 mL) and pH 7 phosphate buffer (220 μί) at 23 °C. The reaction flask was covered with aluminum foil to exclude light. Over the course of 3 h, the reaction mixture was observed to change from myrtle green to lemon yellow. The product solution was partitioned between water (5 mL) and dichloromethane (50 mL). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C 18 column, 10 μιτι, 30 x 150 mm, UV detection at 270 nm, gradient elution with 40→90% acetonitrile in water, flow rate: 15 mL/min) to provide 33 mg of the product as a yellow-green powder (65%).

[00468] Trimethylsilyl triflate ( 10% in dichloromethane, 28.3 μί, 16 μπιοΐ, 0.3 equiv) was added to a suspension of deprotected trioxacarcin precursor (33 mg, 52 μπιοΐ, 1 equiv), 1 -0- acetyltrioxacarcinose A ( 14.1 mg, 57 μιτιοΐ, 1.1 equiv), and powdered 4- A molecular sieves (-50 mg) in dichloromethane (1 .0 mL) at -78 °C. After 5 min, the mixture was diluted with dichloromethane containing 10% triethylamine and 10% methanol (3 mL). The reaction flask was allowed to warm to 23 °C. The mixture was filtered and partitioned between

dichloromethane (40 mL) and saturated aqueous sodium chloride solution (5 mL). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C 18 column, 10 μπι, 30 x 150 mm, UV detection at 270 nm, gradient elution with 40→90% acetonitrile in water, flow rate: 15 mL/min) to provide 20 mg of the product as a yellow-green powder (47%). TLC: (5% methanol-dichloromethane) R = 0.40 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 7.47 (s, 1H), 5.38 (d, 1H, J = 3.6 Hz), 5.35 (app s, 1 H), 5.26 ppm (d, 1 H, 7 = 4.0 Hz), 4.84 (d, 1 H, J = 4.0 Hz), 4.78 (dd, 1 H, 7 = 12.3, 5.2 Hz), 4.75 (s, 1H), 4.71 (s, 1 H), 4.52 (q, 1H, J = 6.6 Hz), 3.86 (s, 1 H), 3.83 (s, 3H), 3.62 (s, 3H), 3.47 (s, 3H), 3.15 (d, l H, y = 5.3 Hz), 3.05 (d, 1 H, 7 = 5.3 Hz), 2.60 (s, 3H), 2.58 (m, 1H), 2.35 (m, 1 H), 2.14 (s, 3H), 1.96 (dd, 1 H, 7 = 14.6, 4.1 Hz), 1.62 (d, 1 H, 7 = 14.6 Hz), 1.26 (s, 1 H), 1.23 (d, 3H, J = 6.6 Hz), 1.08 (s, 3H), 0.95 (s, 9H), 0.24 (s, 3H), 0.16 (s, 3H); ‘³C NMR ( 125 MHz, CDC1₃) 6: 202.8, 170.5, 163.2, 151.8, 144.4, 142.4, 135.2, 126.6, 1 16.8, 1 15.2, 1 15.1 , 108.3, 104.0, 100.3, 98.6, 98.3, 74.6, 73.4, 69.8, 69.5, 69.5, 68.9, 69.5, 69.5, 68.9, 68.4, 62.9, 62.7, 57.2, 56.8, 50.7, 38.8, 36.8, 26.0, 25.9, 21.1 , 20.6, 18.6, 17.0, -4.2, -5.3; FTIR (neat), cm^“‘ : 2953 (w), 2934 (w), 2857 (w), 1749 (w), 1622 (m), 1570 (w), 1447 (w), 1391 (m), 1321 (w), 1294 (w), 1229 (m), 1 159 (m), 1 121 (s), 1084 (s), 1071 (m), 1020 (m), 995 (s), 943 (s), 868 (m), 837 (m), 779 (m); HRMS (ESI): Calcd for (C₄oH₅₄0i₆Si+Na)⁺ 841.3073, found

841.3064.

Glycosylation of a Cycloaddition Coupling Partner

[00469] 2,3-Dichloro-5,6-dicyanobenzoquinone ( 14.3 mg, 63 μπιοΐ, 1.2 equiv) was added to a vigorously stirring, biphasic solution of differentially protected aldehyde (37 mg, 52 μιτιοΐ, 1 equiv) in dichloromethane (870 μί) and water (175 μί) at 23 °C. The reaction flask was covered with aluminum foil to exclude light. Over the course of 2 h, the reaction mixture was observed to change from myrtle green to lemon yellow. The product solution was partitioned between water (5 mL) and dichloromethane (40 mL). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography (5% ethyl acetate-hexanes initially, grading to 10% ethyl acetate-hexanes) to provide 28 mg of the product as a yellow powder (91 %). TLC: (20% ethyl acetate-hexanes) R/ = 0.37 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 10.83 (s, 1H), 7.30 (s, 1 H), 5.45 (m, 1H), 4.68 (dd, 1H, / = 10.3, 4.2 Hz), 3.97 (s, 3H), 3.31 (brs, 1H), 2.72 (s, 3H), 2.51-2.45 (m, 1H), 2.41-2.37 (m, 1H), 1.15 (s, 9H), 1 , 13 (s, 9H), 0.88 (s, 9H), 0.15 (s, 3H), 0.1 1 (s, 3H); ^{l 3}C NMR (125 MHz, CDCI3) δ: 194.6, 191 , 160.5, 150.2, 146, 140.8, 135.8, 134, 1 19.6, 1 16.2, 1 15.4, 1 14.7, 72.7, 63.7, 62.4, 38.8, 29.9, 62.4, 38.8, 63.7, 62.4, 38.8, 63.7, 62.4, 38.8, 29.9, 26.2, 26.1 , 26, 22.7, 21.4; FTIR (neat), cm^“1 : 3470 (br, w), 2934 (w), 2888 (w), 1684 (s), 1607 (s), 1560 (w), 1472 (m), 1445 (w), 1392 (m), 1373 (s), 1242 (s), 1 153 (s), 1 1 19 (w), 1074 (m), 1044 (s), 1013 (s), 982 (w), 934 (m), 907 (w), 870 (m), 827 (s), 795 (s), 779 (s), 733 (s), 664 (s); HRMS (ESI): Calcd for (C₃iH₄₆0₇Si₂+H)⁺ 587.2855, found 587.2867.

[00470] Trimethylsilyl triflate (10% in dichloromethane, 25.9 μί, 14 μπιοΐ, 0.3 equiv) was added to a suspension of deprotected aldehyde (28 mg, 48 μηιοΐ, 1 equiv), 1-0- acetyltrioxacarcinose A (12.9 mg, 52 μπιοΐ, 1.1 equiv), and powdered 4-A molecular sieves (-50 mg) in dichloromethane ( 1.0 mL) at -78 °C. After 5 min, the mixture was diluted with dichloromethane containing 10% triethylamine and 10% methanol (3 mL). The reaction flask was allowed to warm to 23 °C. The mixture was filtered and partitioned between dichloromethane (40 mL) and saturated aqueous sodium chloride solution (5 mL). The layers were separated. The organic layer was dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by preparatory HPLC (Agilent Prep-C 18 column, 10 μπι, 30 x 150 mm, UV detection at 270 nm, gradient elution with 80→98% acetonitrile in water, flow rate: 15 mL/min) to provide 15 mg of the product as a yellow powder (41 %). TLC: (20% ethyl acetate-hexanes) R/ = 0.29 (CAM); Ή NMR (500 MHz, CDC1₃) δ: 10.83 (s, 1 H), 7.32 (s, 1 H), 5.43 (d, 1 H, J = 3.9 Hz), 5.32 (m, 1H), 4.74 (s, 1 H), 4.67 (dd, 1 H, J = 12.3, 5.0 Hz), 4.54 (q, 1H, J = 6.6 Hz), 3.91 (s, 1H), 3.88 (s, 3H), 2.72 (s, 3H), 2.59 (ddd, 1 H, J = 13.8, 5.0, 3.2 Hz), 2.34 (m, 1H), 2.14 (s, 3H), 1.97 (dd, 1H, J = 14.2, 4.2 Hz), 1.71 (d, 1 Η, / = 14.6 Hz), 1.22 (d, 3H, J = 6.3 Hz), 1.15 (s, 9H), 1.15 (s, 9H), 1.08 (s, 3H), 0.93 (s, 9H), 0.23 (s, 3H), 0.13 (s, 3H); ¹³C NMR (125 MHz, CDC1₃) δ: 193.9, 191.0, 170.5, 146.4, 140.9, 134.0, 132.4, 1 19.8, 1 16.8, 1 15.8, 1 15.0, 1 10.8, 99.6, 74.6, 71.5, 70.4, 68.9, 62.9, 62.7, 39.1 , 36.9, 26.2, 26.1 , 26.1 , 25.9, 24.1 , 22.7, 21.5, 21.3, 21.1 , 18.7, 16.9, -4.1 , -5.3; FTIR (neat), cm^-1 : 3524 (br, w), 2934 (m), 2861 (m), 1749 (m), 1686 (s), 1607 (s), 1560 (m), 1474 (m), 1447 (m), 1424 (w), 1375 (s), 1233 (s), 1 159 (s), 1 1 17 (m), 1080 (m), 1049 (s), 1015 (s), 997 (s), 937 (m), 883 (m), 872 (m), 827 (s), 797 (m), 781 (m), 737 (w), 677 (w), 667 (m); HRMS (ESI): Calcd for (C₄₀H₆₀O, ,Si₂+H)⁺773.3747, found 773.3741.

General Glycosylation Procedure of the C13 Hydroxyl Group

[00471] Crushed 4-A molecular sieves (-570 mg / 1 mmol sugar donor) was added to a stirring solution of the sugar acceptor (1 equiv.) and the sugar donor (30.0 equiv.) in dichloromethane ( 1.6 mL / 1 mmol sugar donor) and diethylether (0.228 mL / 1 mmol sugar donor) at 23 °C. The bright yellow mixture was stirred for 90 min at 23 °C and finally cooled to -78 °C. TMSOTf (10.0 equiv.) was added over the course of 10 min at -78 °C. After 4 h, a second portion of TMSOTf (5.0 equiv.) was added at -78 °C and stirring was continued for 1 h. The last portion of TMSOTf (5 equiv.) was added. After 1 h, triethylamine (20 equiv.) was added and the reaction the product mixture was filtered through a short column of silica gel deactivated with triethylamine (30% ethyl acetate-hexanes initially, grading to 50% ethyl acetate-hexanes). H NMR analysis of the residue showed minor sugar donor remainings and that the sugar acceptor had been glycosylated. The residue was purified by preparatory HPLC (Agilent Prep-C 18 column, 10 μπι, 30 x 150 mm, UV detection at 270 nm, gradient elution with 40→100% acetonitrile in water, flow rate: 15 mL/min) to provide the glycosylation product as a bright yellow oil

Three Specific Compounds Prepared by the General Glycosylation Procedure for the CI 3 Hydroxyl Group:

[00472] 10% yield; TLC: (50% ethyl acetate-hexane) R = 0.58 (UV, CAM); Ή NMR (600 MHz, CDC1₃) δ: 7.43 (s, 1 H), 5.84 (t, J = 3.6 Hz, 1 H), 5.29 (d, J = 4.2 Hz, 1 H), 5.19 (d, J = 4.2 Hz, 1 H), 5.01 (q, J = 6.6 Hz, 1 H), 4.75 (t, J = 3.6 Hz, 1 H), 4.73 (s, 1 H), 3.88 (s, OH), 3.77 (s, 3H), 3.63 (s, 3H), 3.47 (s, 3H), 3.03 (app q, J = 5.4 Hz, 2H), 2.84 (d, J = 6.0 Hz, 1 H), 2.77 (d, J = 6.0 Hz, 1 H), 2.72 (t, J = 6.6 Hz, 2H), 2.58 (s, 3H), 2.36 (s, 3H), 2.33 (t, J = 3.0 Hz, 2H), 2.23 (s, 3H), 2.1 1 -2.06 (m, 2H), 1.08 (d, J = 6.0 Hz, 3H). ^‘

[00473] 81 % yield, TLC: (50% ethyl acetate-hexane) R = 0.30 (UV, CAM); Ή NMR (600 MHz, CDCI3) δ: 7.46 (s, 1 H), 7.28 (d, J = 9 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2 H), 5.83 (dd, J = 3.6, 1.8 Hz, 1 H), 5.30 (d, J = 4.2 Hz, 1 H), 5.19 (d, J = 4.2 Hz, 1 H), 5.19 (m, 1 H), 5.00 (q, J = 6.0 Hz, 1 H), 4.96 (dd, J = 12.0, 4.8 Hz, 1 H), 4.75 (t, J = 3.6 Hz, 1 H), 4.74 (s, l H), 4.70 (d, y = 10.8 Hz, 1 H), 4.59 (d, J = 10.8 Hz, 1 H), 3.86 (s, OH), 3.83 (s, 3H), 3.80 (s, 3H), 3.63 (s, 3H), 3.47 (s, 3H), 2.81 (d, J = 6.0 Hz, 1 H), 2.73-2.68 (m, 1 H), 2.70 (d, J = 6.0 Hz, 1 H), 2.59 (s, 3H), 2.35 (s, 3H), 2.33-2.28 (m, 2H), 2.22 (s, 3H), 2.19- 2.1 3 (m, 1 H), 1 .08 (d, J = 6.0 Hz, 3H), 0.97 (s, 9H), 0.25 (s, 3H), 0.17 (s, 3H); HRMS (ESI): Calcd for (C₄9H₆₂0₁₈Si+H)⁺ 967.3778, found 967.3795; HRMS (ESI): Calcd for (C ¾₂0,₈Si+Na)⁺ 989.3598, found 989.3585.

[00474] Compound Detected by ESI Mass Spectrometry: Calculated Mass for

[C52H₇| N₃02i Si-Hr^l = 1 100.4277, Measured Mass = 1 100.4253.

PATENT

US 4511560

https://www.google.com/patents/US4511560

The physico-chemical characteristics of DC-45-A and DC-4-5-B₂ according to this invention are as follows:

(1) DC-45-A

(1) Elemental analysis: H:5.74%, C:55.11%

(2) Molecular weight: 877

(3) Molecular formula: C₄₂ H₅₂ O₂₀

(4) Melting point: 180° C.±3° C. (decomposed)

(5) Ultraviolet absorption spectrum: As shown in FIG. 1 (in 50% methanol)

(6) Infrared absorption spectrum: As shown in FIG. 2 (KBr tablet method)

(7) Specific rotation: [α]_D ²⁵ =-15.3° (c=1.0, ethanol)

(8) PMR spectrum (in CDC]₃ ; ppm): 1.07 (3H,s); 1.10 (3H, d, J=6.8); 1.24 (3H,d, J=6.5); many peaks between 1.40-2.30; 2.14 (3H,s); 2.49 (3H,s); 2.63 (3H,s); many peaks between 2.30-2.80; 2.91 (1H,d, J=5.6); 3.00 (1H,d, J=5.6); 3.49 (3H,s); 3.63 (3H,s); 3.85 (3H, s); many peaks between 3.60-4.00; 4.18 (1H,s); 4.55 (1H,q, J=6.8); many peaks between 4.70-4.90; 5.03 (1H, q, J=6.5); 5.25 (1H,d, J=4.0); 5.39 (1H, d, J=4.0); 5.87 (1H, m); 7.52 (1H,s); 14.1 (1H,s)

(9) CMR spectrum (in CDCl₃ ; ppm): 210.9; 203.8; 170.3; 162.1; 152.5; 145.2; 142.3; 135.3; 126.7; 117.0; 114.2; 108.3; 105.3; 99.7; 97.2; 93.7; 85.1; 79.0; 74.6; 71.1; 69.6; 69.3; 68.8; 67.9; 66.3; 64.0; 62.8; 57.3; 55.9; 36.5; 32.2; 28.0; 25.7; 20.9; 20.2; 17.0; 14.7

(10) Solubility: Soluble in methanol, ethanol, water and chloroform; slightly soluble in acetone and ethyl acetate, and insoluble in ether and n-hexane

(2) DC-45-B₂

(1) Elemental analysis: H: 6.03%, C: 54.34%

(2) Molecular weight: 879

(3) Molecular formula: C₄₂ H₅₄ O₂₀

(4) Melting point: 181°-182° C. (decomposed)

(5) Ultraviolet absorption spectrum: As shown in FIG. 5 (in 95% ethanol)

(6) Infrared absorption spectrum: As shown in FIG. 6 (KBr tablet method)

(7) Specific rotation: [α]_D ²⁵ =-10° (c=0.2, ethanol)

(8) PMR spectrum (in CDCl₃ ; ppm): 1.07 (3H,s); many peaks between 1.07-1.5; many peaks between 1.50-2.80; 2.14 (3H,s); 2.61 (3H, broad s); 2.86 (1H, d, J=5.7); 2.96 (1H, d, J=5.7); 3.46 (3H,s); 3.63 (3H, s); 3.84 (3H, s); many peaks between 3.65-4.20; many peaks between 4.40-5.00; many peaks between 5.10-5.50; 5.80 (1H, broad s); 7.49 (1H, d, J=1.0); 14.1 (1H, s)

(9) CMR spectrum (in CDCl₃ ; ppm): 202.8; 170.2; 163.1; 151.8; 144.8; 142.9; 135.4; 126.5; 116.8; 114.9; 107.3; 104.6; 101.5; 99.6; 98.0; 94.4; 74.4; 72.5; 71.4; 70.4; 69.1; 68.8; 68.3; 67.9; 67.5; 66.4; 62.9; 62.7; 56.8; 56.5; 48.0; 36.7; 32.3; 25.7; 20.8; 20.3; 18.2; 16.9; 15.5

(10) Solubility: Soluble in methanol, ethanol, acetone, ethyl acetate and chloroform; slightly soluble in benzene, ether and water; and insoluble in n-hexane.

//////

CC1C(C(CC(O1)OC2CC(C(=O)C3=C(C4=C5C(=C(C=C4C(=C23)OC)C)C6C7C(O5)(C8(CO8)C(O6)(O7)C(OC)OC)OC9CC(C(C(O9)C)(C(=O)C)O)O)O)O)(C)O)OC(=O)C

EGF 816, Nazartinib

EGF-816; EGFRmut-TKI EGF816

Novartis Ag innovator

(R,E)-N-(7-chloro-1-(1-(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-1H-benzo[d]imidazol-2-yl)-2-methylisonicotinamide

(R,E)-N-(7-chloro-l-(l-(4-(dimethylamino)but-2-enoyl)azepan-3-yl)-lH-benzo[d]imidazol-2 -yl)-2-methylisonicotinamide

NCI-H1975 (L858R/T790M): 25 nM
H3255 (L858R): 9 nM
HCC827 (Del ex19): 11 nM

EGF816 is a novel covalent inhibitor of mutant-selective EGFR; overcomes T790M-mediated resistance in NSCLC.

Epidermal growth factor receptor antagonists; Protein tyrosine kinase inhibitors

• Phase IINon-small cell lung cancer
• Phase I/IISolid tumours
• • 01 Feb 2015Phase-II clinical trials in Non-small cell lung cancer (Late-stage disease, Combination therapy) in Singapore (PO) (NCT02323126)
  • 24 Nov 2014Phase-I/II clinical trials in Non-small cell lung cancer (Combination therapy, Late-stage disease) in Spain (PO) after November 2014 (EudraCT2014-000726-37)
  • 24 Nov 2014Phase-I/II clinical trials in Non-small cell lung cancer (Combination therapy, Late-stage disease) in Germany (PO)

In November 2015, FDA approved osimertinib (Tagrisso™) for the treatment of patients with metastatic EGFR T790M mutation-positive NSCLC, who have progressed on or after EGFR TKI therapy. Based on the clinical performance of the third generation EGFR drugs, more regulatory approvals can be expected.

Nazartinib, also known as EGF816, is an orally available, irreversible, third-generation, mutant-selective epidermal growth factor receptor (EGFR) inhibitor, with potential antineoplastic activity. EGF816 covalently binds to and inhibits the activity of mutant forms of EGFR, including the T790M EGFR mutant, thereby preventing EGFR-mediated signaling. This may both induce cell death and inhibit tumor growth in EGFR-overexpressing tumor cells. EGF816 preferentially inhibits mutated forms of EGFR including T790M, a secondarily acquired resistance mutation, and may have therapeutic benefits in tumors with T790M-mediated resistance when compared to other EGFR tyrosine kinase inhibitors

PATENT

WO 2016016822

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016016822

PATENT

WO 2015081463

http://www.google.co.in/patents/WO2015081463A1?cl=en

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015085482&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

Intermediate 26

1055 (R)-tert-butyl 3-(2-amino-7-chloro- 1 H-benzo[dlimidazol- 1 -yOazepane- 1 -carboxylate

Step A: (R)-tert-butyl 3 -((2-chloro-6-nitrophenyl)amino)azepane-l -carboxylate (I-26a) was prepared following procedures analogous to 1-15, Step A, using the appropriate starting materials. ^JH-NMR (400MHz, CDC1₃): d 8.00-7.91 (m, 1H), 7.58-7.49 (m, 1H), 7.02-6.51

1060 (m, 2H), 4.31-4.03 (m, 1H), 3.84-2.98 (m, 4H), 1.98-1.60 (m, 5H), 1.46-1.39 (m, 10H); MS calculated for Ci₇H₂₅ClN₃0₄ (M+H⁺) 370.15, found 370.10.

Step B: A mixture of I-26a (7.5 g, 19.5 mmol) and Zn (12.8 mg, 195 mmol) in AcOH (22 mL) was stirred at room temperature for 2 h. The reaction was basified with saturated aqueous Na₂C0₃ solution, filtered, and extracted with EtOAc (3 x 80 mL). The combined

1065 organic phase was washed with brine, dried with Na₂S0₄ and concentrated in vacuo to afford (R)-tert-butyl 3-((2-amino-6-chlorophenyl)amino)azepane-l -carboxylate (I-26b). MS calculated for Ci₇H₂₇ClN₃0₂ (M+H⁺) 340.17, found 340.10. The crude was used in the next step without further purification.

Step C: The title compound (Intermediate 26) was prepared from I-26b following

1070 procedures analogous to 1-15, Step C. 1H-NMR (400MHz, CDC1₃): d Ί .34-126 (m, 1H),

7.04-6.97 (m, 2H), 6.05-5.85 (m, 1H), 5.84-5.72 (m, 1H), 5.50-5.37 (m, 0.5H), 5.10-4.80(m, 0.5H), 4.41-4.23(m, 1H), 4.09-3.96(m, 0.5H), 3.94-3.81 (m, 1H), 3.76-3.57 (m, 1H), 3.22-3.14 (m, 0.5H), 2.84-2.63 (m, 1H), 2.34-2.17 (m, 1H), 2.07-1.84 (m, 1H), 1.82-1.64 (m, 2H), 1.53 (s, 9H), 1.48-1.37 (m, 1H); MS calculated for C18H26CIN4O2 (M+H⁺) 365.17,

1075 found 365.10.

Intermediate 27

(R)-N-(l-(azepan-3-yl)-7-chloro-lH-benzo[dlimidazol-2-yl)-2-methylisonicotinamide hydrochloride

Intermediate 27

Step A

1080 Step A: A mixture of 2-methylisonicotinic acid (3.371 g, 24.6 mmol) and 2-(7-aza-lH- benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (9.345 g, 24.6 mmol) in CH2CI2 (120 ml) was treated at room temperature with NEt₃ (4.1 mL, 29.4 mmol). The

reaction was stirred for 1 hour before it was slowly added into a CH₂CI₂ solution (45 ml) of 1-26 (5.98 g, 16.4 mmol). Ten minutes later, more NEt₃ (4.1 mL, 29.4 mmol) was added and 1085 the mixture stirred for 2 h. The mixture was then diluted with CH₂CI₂ (240 mL), washed with H₂0 (2 x 80 mL), saturated aqueous NaHC0₃ solution (70 mL), and brine (70 mL). The organic phase was dried with Na₂SC>4, and concentrated under reduced pressure. The crude material was purified by column chromatography (55% EtOAc/hexanes) to afford

(R)-tert-butyl

1090 3-(7-chloro-2-(2-methylisonicotinamido)-lH-benzo[d]imidazol-l-yl)azepane-l-carboxylate (I-27a) as a light yellow foam. 1H-NMR (400MHz, CDC1₃): d 12.81 (br s, 1H), 8.65-8.62 (m, 1H), 7.95-7.85 (m, 2H), 7.27-7.1 1 (m, 3H), 5.64 – 5.51 (m, 1H), 4.56-4.44 (m, 1H),

4.07-3.92 (m, 1H), 3.79-3.71 (m, 0.5H), 3.41-3.35 (m, 0.5H), 3.29-3.23 (m, 1H), 2.71-2.59 (m, 1H), 2.65 (s, 3H), 2.22-2.00 (m, 3H), 1.93-1.80 (m, 1H), 1.51-1.45 (m, 1H), 1.50 (s,

1095 3.5H), 1.41 (s, 5.5H); MS calculated for C₂5H₃iClN₅0₃ (M+H⁺) 484.20, found 484.20.

Step B: A solution of I-27a (8.62 g, 16.4 mmol) in MeOH (67 mL) was treated with HC1 in dioxane (4M, 67 mL) and the mixture was stirred at room temperature for 7 h. The mixture was then concentrated under reduced pressure to afford the title compound (Intermediate 27). The product was used in the next step without further purification. A sample was treated

1 100 with 1M NaOH, extracted with EtOAc, dried with Na₂SC>4 and concentrated under reduced pressure to afford 1-27 as a free base. 1H-NMR (400MHz, CD₃CN): d 8.49 (d, J=5.0 Hz, 1H), 7.81 (s, 1H), 7.72 (d, J=4.8 Hz, 1H), 7.50 (br d, J=7.52 Hz, 1H), 7.16 – 7.09 (m, 2H), 5.66-5.59 (m, 1H), 3.77 (dd, J = 6.54, 14.3 Hz, 1H), 3.18 (dd, J = 5.3, 14.3 Hz, 1H), 3.05 – 2.98 (m, 1H), 2.76-2.69 (m, 1H), 2.63-2.53 (m, 1H), 2.47 (s, 3H), 2.10-2.03 (m, 1H),