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Trioxacarcin A

March 18, 2016 9:42 am / 1 Comment on Trioxacarcin A

Trioxacarcin A, DC-45A

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;

Molecular Formula:	C₄₂H₅₄O₂₁
Molecular Weight:	894.86556 g/mol

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)

Component-based syntheses of trioxacarcin A, DC-45-A1 and structural analogues

Nature Chemistry5,886–893(2013)

doi:10.1038/nchem.1746

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,

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

ODM-201

March 12, 2016 8:37 am / Leave a comment

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).

Representative binding affinities of ODM-201, ORM-15341, enzalutamide, and ARN-509 measured in competition with [³H]mibolerone using wtAR isolated from rat ventral prostates (C). All data points are means of quadruplicates ±SEM. Ki values are presented in parentheses. D. Antagonism to wtAR was determined using AR-HEK293 cells treated with ODM-201, ORM-15341, enzalutamide, or ARN-509 together with 0.45 nM testosterone in steroid-depleted medium for 24 hours before luciferase activity measurements. All data points are means of triplicates ±SEM. IC₅₀ values are presented in parentheses.

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

str1

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

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O=C(C1=NNC(C(O)C)=C1)N[C@@H](C)CN2N=C(C3=CC=C(C#N)C(Cl)=C3)C=C2

Talazoparib, BMN 673

February 8, 2016 7:25 am / Leave a comment

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

str1

13C NMR DMSOD6

str1

HMBC NMR

str1

HSQC NMR

str1

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

Main article: PARP inhibitor

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

Bing Wang^*, Daniel Chu, Ying Feng, Yuqiao Shen, Mika Aoyagi-Scharber, and Leonard E. Post

BioMarin Pharmaceutical Inc., 105 Digital Drive, Novato, California 94949, United States

J. Med. Chem., 2016, 59 (1), pp 335–357

DOI: 10.1021/acs.jmedchem.5b01498

Publication Date (Web): December 10, 2015

*Phone: 1-415-506-3319. E-mail: bwang@bmrn.com.

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)

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.

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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

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).

Talazoparib (BMN-673) is an orally available poly ADP ribose polymerase (PARP) inhibitor currently in development by Pfizer for the treatment of advanced breast cancer patients with germline BRCA mutations.^[1] Talazoparib is similar to the first in class PARP inhibitor, olaparib.^[2]^[3] However, talazoparib is thought to be more potent than olaparib.^[3]

Mechanism of action

Talazoparib acts as an inhibitor of poly ADP ribose polymerase(PARP) which aids in single strand DNA repair. Cells that have BRCA1/2mutations are susceptible to the cytotoxic effects of PARP inhibitors because of an accumulation of DNA damage.^[1] Talazoparib is theorized to have a higher potency than olaparib due to the additional mechanism of action called PARP trapping. PARP trapping is the mechanism of action where the PARP molecule is trapped on the DNA, which interferes with the cells ability to replicate. Talazoparib is found to be ~100 fold more efficient in PARP trapping than olaparib.^[4] However, this increased potency may not translate directly to clinical effectiveness as many other factors must be considered.^[3]^[4]

Commercialization

Talazoparib was originally developed by BioMarin Pharmaceutical Inc. However, Medivation Inc. acquired all worldwide rights to talazoparib in August 2015 to expand their global oncology franchise.^[5] Medivation acquired talazoparib for $410 million with additional payments of up to $160 million in royalties and milestones. Under this agreement, Medivation assumed all financial responsibilities for the continued development, regulatory, and commercialization of talazoparib.^[5]^[6]

Clinical trials

As of January 2016, talazoparib is in 14 active clinical trials ^[7] including a new arm of I-SPY 2.^[8] These trials cover a variety of cancers types and combination therapies. The most notable clinical trials are the ABRAZO and EMBRACA studies.

ABRAZO

ABRAZO is a phase II study for the safety and efficacy of treatment of BRCA breast cancer patients with Talazoparib monotherapy. This study is for patients who have failed at least two prior chemotherapy treatments for metastatic breast cancer or been previously treated with a platinum regimen.^[6]^[9]^[10] The original target enrollment for the study was 70 patients but Biomarin expanded the trial to 140 patients.^[9]^[10] The estimated completion date is December 2016.^[10]

EMBRACA

EMBRACA is a phase III study for the treatment of BRCA breast cancer patients with Talazoparib.^[11]^[12]^[13] This trial is an open-label, randomized, parallel, 2-arm, multi-center comparison of talazaporib against physician’s preference for the treatment of patients with locally advanced or metastatic breast cancer. Patients must also have received prior chemotherapy regimens for metastatic breast cancer.^[12]^[13] Patients participating in this study are randomly selected for either talazoparib or physician’s choice of chemotherapy at a 2:1 ratio to talazoparib.^[6] The target enrollment for the study was 430 patients ^[12]^[13] and the estimated completion date is June 2017.^[13]

References

^ Jump up to:^a ^b Medivation Inc. “Talazoparib”.
Jump up^ FDA (19 December 2014). “FDA approves Lynparza to treat advanced ovarian cancer”. FDA News Release.
^ Jump up to:^a ^b ^c Jessica Brown, Stan Kaye, Timothy Yap (29 March 2016). “PARP inhibitors: the race is on”. British Journal of Cancer. 114: 713–5. doi:10.1038/bjc.2016.67. PMC 4984871 . PMID 27022824.
^ Jump up to:^a ^b Yuqiao Shen, Mika Aoyagi-Scharber, Bing Wang (June 2015). “Trapping Poly(ADP-Ribose) Polymerase”. Journal of Pharmacology and Experimental Therapeutics.
^ Jump up to:^a ^b Biomarin (24 August 2015). “Medivation to Expand Global Oncology Franchise With the Acquisition of All Worldwide Rights to Talazoparib (BMN 673), a Potent PARP Inhibitor, From BioMarin”.
^ Jump up to:^a ^b ^c Silus Inman (25 August 2015). “Medivation Acquires BioMarin’s PARP Inhibitor Talazoparib”.
Jump up^ BMN 673 trials registered
Jump up^ I-SPY 2 TRIAL: Neoadjuvant and Personalized Adaptive Novel Agents to Treat Breast Cancer (I-SPY 2)
^ Jump up to:^a ^b “BioMarin Provides Program Update for Talazoparib in Metastatic Breast Cancer”. 20 July 2015.
^ Jump up to:^a ^b ^c “A Phase 2, 2-Stage, 2-Cohort Study of Talazoparib (BMN 673), in Locally Advanced and/or Metastatic Breast Cancer Patients With BRCA Mutation (ABRAZO Study)”. ClinicalTrials.gov.
Jump up^ “EMBRACA CLINICAL STUDY IS NOW ENROLLING”.
^ Jump up to:^a ^b ^c “A Study Evaluating Talazoparib (BMN 673), a PARP Inhibitor, in Advanced and/or Metastatic Breast Cancer Patients With BRCA Mutation (EMBRACA Study)”. ClinicalTrials.gov.
^ Jump up to:^a ^b ^c ^d “BioMarin Initiates Phase 3 BMN 673 Trial for Metastatic gBRCA Breast Cancer”. Benzinga.

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

WO2012054698A1	Oct 20, 2011	Apr 26, 2012	Biomarin Pharmaceutical Inc.	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
WO2015069851A1	Nov 6, 2014	May 14, 2015	Biomarin Pharmaceutical Inc.	Triazole intermediates useful in the synthesis of protected n-alkyltriazolecarbaldehydes
US8420650	Mar 31, 2011	Apr 16, 2013	Biomarin Pharmaceutical Inc.	Dihydropyridophthalazinone inhibitors of poly(ADP-ribose)polymerase (PARP)
US8541403	Feb 3, 2011	Sep 24, 2013	Biomarin Pharmaceutical Inc.	Dihydropyridophthalazinone inhibitors of poly(ADP-ribose)polymerase (PARP) for use in treatment of diseases associated with a PTEN deficiency
US8735392	Oct 20, 2011	May 27, 2014	Biomarin Pharmaceutical Inc.	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
US8765945	Feb 8, 2011	Jul 1, 2014	Biomarin Pharmaceutical Inc.	Processes of synthesizing dihydropyridophthalazinone derivatives
US8999987	Mar 6, 2013	Apr 7, 2015	Biomarin Pharmaceutical Inc.	Dihydropyridophthalazinone inhibitors of poly(ADP-ribose)polymerase (PARP)
US9018201	Aug 21, 2013	Apr 28, 2015	Biomarin Pharmaceuticial Inc.	Dihydropyridophthalazinone inhibitors of poly(ADP-ribose)polymerase (PARP) for use in treatment of diseases associated with a PTEN deficiency

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

Talazoparib
Legal status

Legal status	Investigational
Identifiers
IUPAC name [show]
ChemSpider	28637772
UNII	9QHX048FRV
KEGG	D10732
ChEMBL	CHEMBL3137320
Chemical and physical data
Formula	C₁₉H₁₄F₂N₆O
Molar mass	380.35 g/mol
3D model (JSmol)	Interactive image

/////////////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

VAL-083

January 15, 2016 3:46 am / Leave a comment

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.

” … Our research shows that VAL-083 may offer advantages over currently available chemotherapies in a number of tumour types.”

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.

str1

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

str1

SCHEME 5

str1 str1 str1

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

Molecules 2015, 20(9), 17093-17108; doi:10.3390/molecules200917093

Article

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

Dmitry G. Deryabin * and Anna A. Tolmacheva

Microbiological Department, Orenburg State University, 13 Pobedy Avenue, Orenburg 460018, Russia

* Author to whom correspondence should be addressed.

1,2: 5,6-dianhydrogalactitol ** in table 1

Paper

Takano, Seiichi; Iwabuchi, Yoshiharu; Ogasawara, Kunio
Journal of the American Chemical Society, 1991 , vol. 113, 7 pg. 2786 – 2787

http://pubs.acs.org/doi/pdf/10.1021/ja00007a082

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, BÃ¡nos 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, BÃ¡nos 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: BÃ¡nos 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: BÃ¡nos 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: GerÃ¶-Ferencz E, TÃ³th K, Somfai-Relle S, GÃ¡l 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, InstitÃ³ris L. Incorporation of 3H-dibromodulcitol and 3H-dianhydrodulcitol into ascites tumor cells. Autoradiographic study. Neoplasma. 1976;23(1):47-52. PubMed PMID: 1272473.

9: BÃ¡nos 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

WO2013128285A2 *	Feb 26, 2013	Sep 6, 2013	Del Mar Pharmaceuticals	Improved analytical methods for analyzing and determining impurities in dianhydrogalactitol
WO2013128285A3 *	Feb 26, 2013	Dec 27, 2013	Del Mar Pharmaceuticals	Improved analytical methods for analyzing and determining impurities in dianhydrogalactitol
US9029164	Nov 18, 2013	May 12, 2015	Del Mar Pharmaceuticals	Analytical methods for analyzing and determining impurities in dianhydrogalactitol

US3470179 *	Jun 14, 1966	Sep 30, 1969	Sandoz Ag	4-substituted-3,4-dihydroquinazolines
US20020032230 *	May 21, 2001	Mar 14, 2002	Dr. Reddy’s Laboratories Ltd.	Novel compounds having antiinflamatory activity: process for their preparation and pharmaceutical compositions containing them
US20020037328 *	May 31, 2001	Mar 28, 2002	Brown Dennis M.	Hexitol compositions and uses thereof

CN101045542A *	Apr 6, 2007	Oct 3, 2007	中国科学院过程工程研究所	Method for preparing water softening aluminium stone of sodium aluminate solution carbonation resolving
CN101654270A *	Sep 10, 2009	Feb 24, 2010	沈阳工业大学	Method for eliminating periodic thinning of granularity of seed product
CN101775413A *	Mar 23, 2010	Jul 14, 2010	禹城绿健生物技术有限公司	Technique for producing xylitol and dulcitol simultaneously
CN103270035A *	Aug 17, 2011	Aug 28, 2013	德玛医药	Method of synthesis of substituted hexitols such as dianhydrogalactitol

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C1C(O1)C(C(C2CO2)O)O

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

DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO …..FOR BLOG HOME CLICK HERE

FDA approves new oral therapy to treat ALK-positive lung cancer

December 12, 2015 3:19 am / Leave a comment

FDA approves new oral therapy to treat ALK-positive lung cancer

12/11/2015 01:03 PM EST

The U.S. Food and Drug Administration today approved Alecensa (alectinib) to treat people with advanced (metastatic) ALK-positive non-small cell lung cancer (NSCLC) whose disease has worsened after, or who could not tolerate treatment with, another therapy called Xalkori (crizotinib).

December 11, 2015

Release

Lung cancer is the leading cause of cancer death in the United States, with an estimated 221,200 new diagnoses and 158,040 deaths in 2015, according to the National Cancer Institute. An ALK (anaplastic lymphoma kinase) gene mutation can occur in several different types of cancer cells, including lung cancer cells. ALK gene mutations are present in about 5 percent of patients with NSCLC. In metastatic cancer, the disease spreads to new parts of the body. In ALK-positive NSCLC metastatic patients, the brain is a common place for the disease to spread.

“Today’s approval provides a new therapy for a group of patients who would have few treatment options once their disease no longer responds to treatment with Xalkori,” said Richard Pazdur, M.D., director of the Office of Hematology and Oncology Products in the FDA’s Center for Drug Evaluation and Research. “In addition to the primary effect on tumors in the lung, Alecensa clinical trials provide evidence of an effect on tumors that had spread to the brain, which is an important effect for clinicians to understand.”

Alecensa is an oral medication that blocks the activity of the ALK protein, which may prevent NSCLC cells from growing and spreading.

The safety and efficacy of Alecensa were studied in two single-arm clinical trials of patients with metastatic ALK-positive NSCLC whose disease was no longer controlled by treatment with Xalkori. Study participants received Alecensa twice daily to measure the drug’s effect on their lung cancer tumors. In the first study, 38 percent of participants experienced a partial shrinkage of their NSCLC tumors, an effect that lasted for an average of 7.5 months. In the second study, 44 percent of participants experienced a partial shrinkage of their NSCLC tumors, lasting for an average of 11.2 months. The trials also examined Alecensa’s effect on individuals’ brain metastases, a common occurrence in this population. Sixty-one percent of participants in the two trials who had measurable brain metastases experienced a complete or partial reduction in their brain tumors, lasting an average of 9.1 months.

The most common side effects of Alecensa are fatigue, constipation, swelling (edema) and muscle pain (myalgia). Alecensa may cause serious side effects, including liver problems, severe or life-threatening inflammation of the lungs, very slow heartbeats and severe muscle problems. Treatment with Alecensa may cause sunburn when patients are exposed to sunlight.

Alecensa was approved using the accelerated approval regulatory pathway, which allows the FDA to approve products for serious or life-threatening diseases based on evidence that the product has an effect on an outcome that is reasonably likely to predict clinical benefit. In the case of Alecensa, the tumor response to treatment, along with the duration of response, provided this evidence. Under the accelerated approval requirements, a confirmatory study is required to verify and describe the clinical benefit of Alecensa.

The FDA granted the Alecensa application breakthrough therapy designation and priority review status. These are distinct programs intended to facilitate and expedite the development and review of certain new drugs in light of their potential to benefit patients with serious or life-threatening conditions. Alecensa also received orphan drug designation, which provides incentives such as tax credits, user fee waivers and eligibility for exclusivity to assist and encourage the development of drugs for rare diseases.

Alecensa is marketed by Genentech, based in San Francisco, California. Xalkori is marketed by Pfizer, based in New York, New York.

Synthesis

AMG-319

November 17, 2015 4:32 am / Leave a comment

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)

¹H NMR (400 MHz, [D₆]DMSO) δ ppm 12.76 (1 H, br s), 8.69 (1 H, br s), 8.63 (1 H, s), 8.21 (1 H, br s), 7.96–8.12 (4 H, m), 7.93 (1 H, s), 7.76 (1 H, dd, J = 10.4, 2.5 Hz), 7.45–7.57 (2 H, m), 6.00 (1 H, d, J = 1.2 Hz), 1.61 (3 H, d, J = 6.7 Hz). Mass spectrum (ESI) m/e = 386.0 (M + 1).

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

CEP 18770, Delanzomib

November 8, 2015 1:44 pm / Leave a comment

An external file that holds a picture, illustration, etc. Object name is JRPS-8-145-g006.jpg

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

Zhu Y, Zhao X, Zhu X, Wu G, Li Y, Ma Y, et al. Design, synthesis, biological evaluation, and structure−activity relationship (SAR) discussion of dipeptidyl boronate proteasome inhibitors, Part I: Comprehensive understanding of the SAR of á-amino acid boronates. J Med Chem. 2009;52:4192–4199. [PubMed]

Arastu-Kapur S, Anderl JL, Kraus M, Parlati F, Shenk KD, Lee SJ, et al. Nonproteasomal targets of the proteasome inhibitors bortezomib and carfilzomib: A link to clinical adverse events. Clin Cancer Res. 2011;17:2734–2743. [PubMed]

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)

¹H NMR (400 MHz, DMSO-d₆) 8.98 (d,J = 2.99 Hz, 1H), 8.76 (d, J = 8.55 Hz, 1H), 8.2 (m, 3H), 8.11 (t, J = 7.71 Hz, 1H), 8.02 (d, J = 7.54 Hz, 1H), 7.54 (m, 3H), 5.26 (d, J = 4.95 Hz, 1H), 4.49 (dd, J = 4.22, 8.52 Hz, 1H), 4.13 (m, 2H), 2.6 (m, b, 1H), 2.19 (m, b, 1H), 2.02 (br m, 1H), 1.83 (t, J = 5.38 Hz, 1H), 1.75 (br s, 1H), 1.68 (br m, 1H), 1.62 (d, J = 13.9 Hz, 1H), 1.36 (d, J = 10.05 Hz, 1H), 1.3(br m, 3H), 1.22 (d, J = 11.65 Hz, 6H), 1.12 (d, J = 6.26 Hz, 3H), 0.84 (d, J = 6.57 Hz, 6H), 0.79 (s, 3H).

6-(2S,3R)-N-[(1R)-1-(1,3,6,2-dioxoazaborocan-2-yl)-3-methylbutyl]-3-hydroxy-2-[(6-phenylpyridin-2-yl)formamido]butanamide (8)

¹H NMR (400 MHz, DMSO-d₆) 8.8 (d, J = 8.52 Hz, 1H), 8.2 (m, 3H), 8.1 (t, J = 7.68 Hz, 1H), 8.0 (dd, J = 6.7, 0.9 Hz, 1H), 7.5 (m, 3H), 7.2 (br d, 1H), 6.5 (br t, 1H), 5.1 (d, J = 4.92 Hz, 1H), 4.5 (dd, 1H), 4.2 (m, 1H), 3.6 (m, 2H), 3.5 (m, 2H), 3.1 (m, 1H), 3.0 (m, 2H), 2.7 (m, 2H), 1.6 (m, 1H), 1.3 (m, 1H), 1.2 (m, 1H), 1.1 (d, J = 6.32 Hz, 3H), 0.8 (dd, J = 6.68, 6.53 Hz, 6H).

PAPER

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

Bruce D. Dorsey*^†, ^{ET AL}

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

J. Med. Chem., 2008, 51 (4), pp 1068–1072

DOI: 10.1021/jm7010589

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).

References

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.

Patent	Submitted	Granted
Proteasome inhibitors and methods of using the same [US7576206]	2005-05-19	2009-08-18
PROTEASOME INHIBITORS AND METHODS OF USING THE SAME [US7915236]	2009-11-26	2011-03-29
BORONATE ESTER COMPOUNDS AND PHARMACEUTICAL COMPOSITIONS THEREOF [US2009325903]	2009-12-31

Citing Patent	Filing date	Publication date	Applicant	Title
US7442830 *	6 Aug 2007	28 Oct 2008	Millenium Pharmaceuticals, Inc.	Proteasome inhibitors
US7687662 *	2 Jul 2008	30 Mar 2010	Millennium Pharmaceuticals, Inc.	Proteasome inhibitors
US8003819 *	12 Feb 2010	23 Aug 2011	Millennium Pharmaceuticals, Inc.	Proteasome inhibitors
US8962572	4 Oct 2011	24 Feb 2015	Fresenius Kabi Usa, Llc	Bortezomib formulations
WO2012177835A1	21 Jun 2012	27 Dec 2012	Cephalon, Inc.	Proteasome inhibitors and processes for their preparation, purification and use

/////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

Beijing Shenogen Granted Fast Track Status for Novel Cancer Drug, Icaritin

September 21, 2015 3:14 am / Leave a comment

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

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

C₂₁H₂₀O₆
Molecular Weight:	368.3799 g/mol

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 (IC₅₀values 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.

Product Name	Specification	CAS No.
Icariin	HPLC 50%-98%	489-32-7
icaritin	HPLC 98%	118525-40-9
icariside II	HPLC 98%	113558-15-9
3,7-bis(2-hydroxyethyl) icaritin	HPLC 98%	1067198-74-6

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

Figure 1: Structures of icariin (1), icariside I (2) and icaritin (3).

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

Patent	Submitted	Granted
Compositions comprising icariside I and anhydroicaritin and methods for making the same [US6399579]		2002-06-04
COSMETIC COMPOSITION CONTAINING HYDROLYSATES OF ICARIIN [US2009170787]	2009-07-02
COMPOUNDS AND METHODS FOR TREATING ESTROGEN RECEPTOR-RELATED DISEASES [US8252835]	2008-06-19	2012-08-28

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

Chi-Med Says Fruquintinib Successful in Lung Cancer Trial

September 7, 2015 3:56 am / Leave a comment

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-Benzofurancarboxamide, 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).

………..

EP1265874A2 *	Jan 23, 2001	Dec 18, 2002	Gödecke Gmbh	Method for the simplified production of (3-chloro-4-fluoro-phenyl)- 7-(3-morpholino-4-yl-propoxy)-6-nitro-quinazoline-4-yl]-amine or (3-chloro-4-fluoro-phenyl)- 7-(3-morpholino-4-yl-propoxy)-6-amino-quinazoline-4-yl]-amine
US20070208056 *	Jan 23, 2007	Sep 6, 2007	Bristol-Myers Squibb Company	Piperidinyl derivatives as modulators of chemokine receptor activity
US20080033000 *	May 15, 2007	Feb 7, 2008	Senex Biotechnology, Inc.	Identification of CDKI pathway inhibitors


2		See also references of EP2297115A2

Citing Patent	Filing date	Publication date	Applicant	Title
US8212033 *	Sep 29, 2010	Jul 3, 2012	Hutchison Medipharma Enterprises Limited	Use of substituted quinazoline compounds in treating angiogenesis-related diseases
US8497372	Jun 4, 2012	Jul 30, 2013	Hutchison Medipharma Enterprises Limited	Use of substituted quinazoline compounds in treating age-related macular degeneration
US8575184	Sep 1, 2010	Nov 5, 2013	Bristol-Myers Squibb Company	Quinazolines as potassium ion channel inhibitors

Hutchison Medipharma Enterprises Limited

Simon To, M.B.A.
Chairman

Mr To has been a Director since 2000 and an Executive Director and Chairman since 2006. He is also Chairman of the Remuneration Committee and a member of the Technical Committee of the Company. He is managing director of Hutchison Whampoa (China) Limited (“Hutchison China”) and has been with Hutchison China for over thirty years, building its business from a small trading company to a billion dollar investment group. He has negotiated major transactions with multinationals such as Procter & Gamble, Lockheed, Pirelli, Beiersdorf, United Airlines and British Airways.

Mr To’s career in China spans more than thirty years and he is well known to many of the top Government leaders in China. Mr To is the original founder of Hutchison Whampoa Limited’s healthcare business and has been instrumental in the acquisitions made to date. He received a First Class Honours Bachelor’s Degree in Mechanical Engineering from Imperial College, London and an MBA from Stanford University’s Graduate School of Business.

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

Mr Hogg has been an Executive Director and Chief Executive Officer since 2006. He is also a member of the Technical Committee of the Company. He joined Hutchison Whampoa (China) Limited in 2000 and has since led all aspects of the creation, implementation and management of the Company’s strategy, business and listing. This includes the creation of the Company’s start-up businesses and the acquisition and operational integration of assets that led to the formation of the Company’s China joint ventures.

Prior to joining Hutchison China, Mr Hogg spent ten years with Procter & Gamble starting in the US in Finance and then Brand Management in the Laundry and Cleaning Products Division. Mr Hogg then moved to China to manage P&G’s detergent business followed by a move to Brussels to run P&G’s global bleach business. Mr Hogg received a Bachelor’s degree in Civil Engineering from the University of Edinburgh and an MBA from the University of Tennessee.

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

Dr. Su has headed all drug discovery and research since he joined, including creating our R&D strategy, the formation and growth of research platform, and the research and discovery of each and every small molecule drug candidate in the Company’s portfolio.

Prior to joining in 2005, Dr. Su spent 15 years with Pfizer’s US R&D organization. Dr. Su delivered several high quality new drug candidates during his time with Pfizer, most recently as a director in the Medicinal Chemistry Department.

He received his Ph.D. and post-doctoral fellowship in Chemistry from Harvard University under the guidance of Nobel Laureate Professor E. J. Corey, and his Bachelor’s degree in Chemistry from Fudan University in Shanghai, China.

R & D Center Address (A):
Building 4, 720 Cailun Road
Zhangjiang Hi-Tech Park
Pudong, Shanghai, China
Postal Code: 201203, China

Head Office Address (B):
Building 4, 917 Halei Road
Zhangjiang Hi-Tech Park
Pudong, Shanghai, China
Postal Code: 201203, China

Tel: +86 21 2067 3000 Email: BD@hmplglobal.com

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

HMP location

///////

Vintafolide

August 28, 2015 5:46 am / Leave a comment

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.

Vintafolide.
DAVBLH: Desacetylvinblastine hydrazide

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.

: Scheme 1.

Reagents and conditions: (i) a—Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA, RT, 1 h; b—20% piperidine/DMF, rt, 10 min; (ii) a—Fmoc-Arg(Pbf)-OH, PyBOP, DIPEA, rt, 1 h; b—20% piperidine/DMF, rt, 10 min; (iii) a—Fmoc-Glu-OtBu, PyBOP, DIPEA, rt, 1 h; b—20% piperidine/DMF, rt, 10 min; (iv) N¹⁰-TFA-pteroic acid, PyBOP, DIPEA, rt, 1.5 h; (v) TFA/H₂O/TIPS/EDT (92.5:2.5:2.5:2.5), rt, 1 h; (vi) aq NH₄OH, pH 9.3, rt, 1 h.

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

Sridharan, Balaji (Apr 16, 2012). “Endocyte soars on cancer drug deal with Merck”. Reuters.
Statement on a nonproprietary name adopted by the USAN Council, United States Adopted Names (USAN) Council, 6 April 2012
Kuo, Phillip H. (February 2013). “Companion Imaging Diagnostics for Targeted Therapies”. Radiology Today 14 (2): 32.
“Merck, Endocyte in Development Deal”. Drug Development & Discovery magazine. 2012-04-25.
Dosio, F.; Milla, P.; Cattel, L. (2010). “EC-145, a folate-targeted Vinca alkaloid conjugate for the potential treatment of folate receptor-expressing cancers”. Current opinion in investigational drugs (London, England : 2000) 11 (12): 1424–1433. PMID 21154124. edit
“EMA Accepts For Review MAA Filings For Vintafolide And Etarfolatide”. rttnews.com. 2012-11-27.
Garde, Damian (2014-05-02). “Merck halts study of the billion-dollar cancer drug vintafolide”. Fierce Biotech. Retrieved 21 April 2015.
“Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay” 338 (2). March 2005. pp. 284–93. doi:10.1016/j.ab.2004.12.026.PMID 15745749.

WO2008098970A1 *	Feb 13, 2008	Aug 21, 2008	Pf Medicament	Anhydrous crystalline vinflunine salts, method of preparation and use thereof as a drug and means of vinflunine purification
WO2010150100A1 *	Jun 23, 2010	Dec 29, 2010	Entarco Sa	The use of spinosyns and spinosyn compositions against diseases caused by protozoans, viral infections and cancer
WO2011014821A1 *	Jul 30, 2010	Feb 3, 2011	Endocyte, Inc.	Folate-targeted diagnostics and treatment
US20100247669 *		Sep 30, 2010	Cerulean Pharma Inc.	Polymer-agent conjugates, particles, compositions, and related methods of use

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

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