Cycloaddition of epoxides and CO2 catalyzed by bisimidazole-functionalized porphyrin cobalt(III) complexes

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Mar 212016

Cycloaddition of epoxides and CO2 catalyzed by bisimidazole-functionalized porphyrin cobalt(III) complexes

Green Chem., 2016, Advance Article
DOI: 10.1039/C6GC00370B, Paper

Xu Jiang, Faliang Gou, Fengjuan Chen, Huanwang Jing
Bisimidazole-functionalized cobaltoporphyrin acted as efficient bifunctional catalysts to facilitate the synthesis of cyclic carbonates from epoxides and CO₂.

see

http://pubs.rsc.org/en/Content/ArticleLanding/2016/GC/C6GC00370B?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+rss%2FGC+%28RSC+-+Green+Chem.+latest+articles%29#!divAbstract

Cycloaddition of epoxides and CO₂ catalyzed by bisimidazole-functionalized porphyrin cobalt(III) complexes

Xu Jiang,^a Faliang Gou,^a Fengjuan Chen^a and Huanwang Jing*^a^b

*Corresponding authors

^aState Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering Lanzhou University, Gansu 730000, PR China

^bState Key Laboratory of Coal Conversion, Institute of Coal Chemistry, Chinese Academy of Sciences, Taiyuan 030001, P R China
E-mail: hwjing@lzu.edu.cn

Green Chem., 2016, Advance Article

DOI: 10.1039/C6GC00370B

A series of innovative bisimidazole-functionalized porphyrin cobalt(III) complexes have been devised, synthesized and characterized using NMR, MS and elemental analysis. These homogeneous catalysts were applied to the cycloaddition of epoxides and carbon dioxide without organic solvent and co-catalyst. It was found that the performance of the catalysts deeply relies on their structural features. The alkoxyl chain length of the linkage and the imidazole position relative to the phenyl rings of porphyrin evidently affects the catalyst activities. [5,15-Di(3-((8-imidazolyloctyl)oxy)phenyl)porphyrin] cobalt(III) chloride (J-m8) and [5,15-di(2-((6-imidazolylhexyl)oxy)phenyl)porphyrin] cobalt(III) chloride (J-o6) demonstrated excellent activity under optimal reaction conditions. Synchronously, a preliminary kinetic investigation of this reaction was carried out using three catalysts and illustrated the activation energies of cyclic carbonate formation. Furthermore, a tri-synergistic catalytic mechanism has been carefully proposed in light of the features of the new catalysts and experimental results.

References [1] L. Jin, H. Jing, T. Chang, X. Bu, L. Wang and Z. Liu, J. Mol. Catal. A: Chem., 2007, 261, 262. [2] X. Jiang, F. Gou and H. Jing, J. Catal., 2014, 313, 159. [3] B. Li, L. Zhang, Y. Song, D. Bai and H. Jing, J. Mol. Catal. A: Chem., 2012, 363– 364, 26.

///Cycloaddition of epoxides, CO2 catalyzed, bisimidazole-functionalized porphyrin cobalt(III) complexes

Trichloroacetic Acid Removal by a Reductive Spherical Cellulose Adsorbent

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Mar 212016

Chemical Research in Chinese Universities Vol.32 No.1 February 2016 2016 Vol. 32 (1): 0-0 [Abstract] ( 20 ) [HTML 1KB] [PDF 2198KB] ( 11 )

A novel spherical cellulose adsorbent with amide and sulphinate groups was used for a first reduction of trichloroacetic acid(TCAA) and a subsequent adsorption of generated species, haloacetic acids. The removal mechanism involved TCAA reduction by sulphinate groups and the adsorption of the haloacetic acids through electrostatic interaction with amide group. Investigation of product formation and subsequent disappearance reveals that the reduction reactions proceed viasequential hydrogenolysis, and transform to acetate ultimately. Adsorption of haloacetic acids was ascertained by low chloride mass balances(89.3%) and carbon mass balances(75.1%) in solution. The pseudo-first-order rate constant for TCAA degradation was (0.93±0.12) h^-1. Batch experiments were conducted to investigate the effect of pH value on the reduction and adsorption process. The results show that the reduction of TCAA by sulphinate groups requires higher pH values while the electrostatic attraction of haloacetic acids by amino group is favorable in more acidic media.

Trichloroacetic Acid Removal by a Reductive Spherical Cellulose Adsorbent

LIN Chunxiang^1,3, TIAN Chen¹, LIU Yifan^1,3, LUO Wei¹, ZHU Moshuqi¹, SU Qiaoquan¹, LIU Minghua^1,2,3

1. College of Environment & Resources, Fuzhou 350108, P. R. China;
2. State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108, P. R. China;
3. Key Laboratory of Eco-materials Advanced Technology(Fuzhou University), Fujian Province University, Fuzhou 350108, P. R. China

LIU Minghua E-mail: mhliu2000@fzu.edu.cn

Cite this article:

LIN Chunxiang,TIAN Chen,LIU Yifan等. Trichloroacetic Acid Removal by a Reductive Spherical Cellulose Adsorbent[J]. CHEMICAL RESEARCH IN CHINESE UNIVERSITIES, 2016, 32(1): 95-99.

LIN Chunxiang, TIAN Chen, LIU Yifan, LUO Wei, ZHU Moshuqi, SU Qiaoquan, LIU Minghua
Trichloroacetic Acid Removal by a Reductive Spherical Cellulose Adsorbent

2016 Vol. 32 (1): 95-99 [Abstract] ( 9 ) [HTML 1KB] [PDF 0KB] ( 12 )
doi: 10.1007/s40242-016-5304-6

/////Trichloroacetic Acid Removal, Reductive Spherical Cellulose Adsorbent

Trioxacarcin A

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Mar 182016

Trioxacarcin A, DC-45A

CAS No. 81552-36-5

Molecular FormulaC₄₂H₅₂O₂₀
Average mass876.850 Da
17′-[(4-C-Acetyl-2,6-dideoxyhexopyranosyl)oxy]-19′-(dimethoxymethyl)-10′,13′-dihydroxy-6′-methoxy-3′-methyl-11′-oxospiro[oxirane-2,18‘-[16,20,22]trioxahexacyclo[17.2.1.0^2,15.0^5,14.0^7,12.0^17,21 ]docosa[2(15),3,5(14),6,12]pentaen]-8′-yl 4-O-acetyl-2,6-dideoxy-3-C-methylhexopyranoside

(1S,2R,3aS,4S,8S,10S,13aS)-13a-(4-C-Acetyl-2,6-dideoxy-alpha-L-xylo-hexopyranosyloxy)-2-(dimethoxymethyl)-10,12-dihydroxy-7-methoxy-5-methyl-11-oxo-4,8,9,10,11,13a-hexahydro-3aH-spiro[2,4-epoxyfuro[3,2-b]naphtho[2,3-h]-1-benzopyran-1,2′-oxiran]-8-yl 4-O-acetyl-2,6-dideoxy-3-C-methyl-alpha-L-xylo-hexopyranoside
Kyowa Hakko Kirin INNOVATOR

Trioxacarcin B

Trioxacarcin B; Antibiotic DC 45B1; DC-45-B1; Trioxacarcin A, 14,17-deepoxy-14,17-dihydroxy-; AC1MJ5N1; 81534-36-3;

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

Already 13 EMA GMP Non-compliance Reports in 2016 published

regulatory Comments Off

Mar 172016

EudraGMDP is the central database for GMP and GDP compliance. Inspections which have been performed by any of the EU member state inspectorates are published in the database. Please get the details about the GMP non-compliance findings at 11 manufacturers in Europe and abroad.

http://www.gmp-compliance.org/enews_05224_Already-13-EMA-GMP-Non-compliance-Reports-in-2016-published_15159,S-QSB_n.html

EudraGMDP is the central database for GMP and GDP compliance. Inspections which have been performed by any of the EU member state inspectorates are published in the database. If the manufacturing or distribution site has been found in compliance with GMP and or GDP then a certificate is issued in the database as reference for other inspectorates. This information is also available to the public. A negative outcome will lead to a GMP or GDP Non-Compliance Report. In 2016 no GDP Non-Compliance Reports have been published until today but already 13 GMP Non-Compliance Reports until March 15th.

Among the companies concerned there are 5 Chinese, 3 French, 2 Spanish manufacturers as well as one each from Sweden, Romania and Poland. All Non-Compliance Report were either issued in 2016 (12) or updated in 2016 (1).

The GMP non compliance findings reveal severe deviations from EU GMP. In addition some companies are involved in falsification and data manipulations – a serious trend which can be observed in many international inspections (e.g. those performed by FDA, WHO). Data Integrity and falsification issues are highlighted in the findings below.

MINSHENG GROUP SHAOXING PHARMACEUTICAL CO. LTD, China

Overall, 18 deficiencies were observed during the inspection, including 2 Critical and 4 Major deficiencies: [Critical 1]Falsification of source of API (Thiamphenicol): Repackaging, relabeling and selling of purchased API from a non-GMP company (Zhejiang Runkang Pharmaceutical Co.Ltd.) as if manufactured in-house; [Critical 2] Praziquantel manufactured according to CP process/grade was released as USP process/grade without a full traceability of the testing activities ; [Major 1] The maintenance and the cleaning operations of the manufacturing line used for the production of Praziquantel (API) were found deficient; [Major 2] The pipes design of some equipment used for the manufacturing of Praziquantel, the handling of change related to these equipment and the instruction used for the transfer of the intermediate solution using nitrogen were found deficient ; [Major 3] The hoses used for unloading of solvent were not identified, had no cleaning status and were stored on a dirty floor of an area not mentioned in the general layout of the site; [Major 4] There was no procedure in place for audit trail and there was no effective audit trail in place to determine any change or deletion of the chromatographic raw data. The audit trial function including the administrator profiles was enabled for all the QC staff.

DESARROLLOS FARMACÉUTICOS BAJO ARAGÓN, S.L., Spain

The manufacturer has not established a quality management system including adequate controls to ensure the accuracy and completeness of the critical records data.

S.C. IRCON SRL, Romania

During inspection a number of 34 deficiencies were found, out of which 4 were critical and 10 major. Critical deficiencies are related to the quality management system, qualification/validation activities, manufacturing and material management documents and quality control laboratories activity.

Agila Specialties Polska Sp. z o.o, Poland

29 major deficiencies were found in Agila Specialties which pose a risk of microbial and particulate contamination and could not assure the sterility of the final product. Most of these are related to: 1.) design and qualification of HVAC, laminar air flow system and clean areas, 2.) cleaning and maintenance of clean areas. 3.) manufacturing and batch releasing in the conditions not complying with GMP requirements 4.) change control. In December, 2014 the HVAC system of vials and prefilled syringes lines was significantly modified. Since January till July 2015, 49 batches were manufactured in that area without qualification after the change. During the inspection it was found that: 1) pressure differential between clean areas B and C grade were usually below 10 Pa (effective to < 0 Pa) and alarm (generated electronically, non-validated after the change of the system) has triggered at 0 Pa and after reversing the flow; 2.) laminar air flow system did not comply with requirements given in Annex 1; 3.) test of maximum permitted number of particles “in operation” does not perform properly; 4.) technical condition of clean areas and equipment show lack of proper and regular maintenance. In clean areas A/B grade contamination were found on the arm of the filling machine for prefilled syringes and difficult to clean equipment placed without proper SOP. In grade C e.g. crumbling insulation of pipes, peeling teflon on the ports of tanks and pumps, lack of labelling and mixed clean and dirty equipment, chipped glass accessories was found; 5.) the filtration process was not fully validated and during routine process a pressure difference to be used across the filter was not recorded; 6.) lack of confirmation of A grade in a lyophilizer working in a nitrogen atmosphere; 7.) design, installation and use of nitrogen system did not guarantee tightness and can cause contamination of the clean medium.

HUBEI HONGYUAN PHARMACEUTICAL CO., LTD. , China

This inspection was performed in the framework of the CEP dossier for the manufacture of Metronidazole R1-CEP 2007-309-Rev 01. The inspection identified in total 24 deficiencies to EU GMP. One of them was categorized as critical and related to the Company’s Quality Assurance System for production of Metronidazole. 10 deficiencies were categorized as major and were related to: QA, Documentation, Supplier Qualification, Data Integrity, Out-of-Specification handling, Quality Control, Computerised System validation, Change Control.

HUBEI HONGYUAN PHARMACEUTICAL CO., LTD. (Facility 428) , China

The Company’s facility at No. 428 Yishui North Road, Fengshan Town, Luotian County, Huanggang City, Hubei Province, China was subject to a spot check, because this site is mentioned as an intermediate manufacturing site in CEP 2001-450 Metronidazole. The Company clearly stated in their introduction that the site does not follow EU GMP. The following observations were made and together categorized as critical: a. The manufacturing site and it’s equipment was found in a devastated state. b. Huge layers of dust and product indicated that no cleaning was applied to either the facility or the equipment, leading to an extreme risk of cross-contamination. c. The extremely bad shape of the facility and the equipment showed that no maintenance was in place. d. Almost none of the products seen was labelled. e. No batch manufacturing documentation could be seen. Reference: EU GMP Part II was found not implemented at the facility.

SAS JARMAT « LABORATOIRE ADP », France

As a preliminary note, the starting materials repacked by the site were intended for pharmaceutical compounding activity in community pharmacies. The site did not distribute to the industry. Overall, 21 deficiencies were found, including 3 critical deficiencies and 5 major deficiencies: [Critical 1] Important risks of confusion in the repacking operations were identified. [Critical 2] Important risks of cross contamination in the repacking operations by substances of high pharmacological activity or toxicity were identified. [Critical 3] The active substances and excipients batches were not analysed as per the pharmacopoeial specifications. [Major 1] The release of active substances batches was deficient, notably in the absence of batch production records. [Major 2] Several risks of contamination in the sampling operations, notably cross contamination, were identified. [Major 3] The management of active substance suppliers was deficient, notably in the absence of written confirmation. [Major 4] Several risks of contamination in the repacking operations, notably cross contamination, were identified. [Major 5] The transmission of information to pharmacies was incomplete and confusing, notably regarding the analyses actually performed by the site. The inspection’s observations also apply to excipients, which are repacked and distributed under the same conditions as the active substances.

Svenska Bioforce AB, Sweden

During the inspection, 42 deficiencies were found. None of the deficiencies was critical but 17 were major. The 17 major deficiencies related to batch certification, Product Quality Review, change management system, deviation handling system, management responsibility, training, premises and equipment, documentation, line clearance, quality control, complaint handling, and cleaning validation. Re-inspection after implementation of CAPA is required in order to verify that the Pharmaceutical Quality System meets the requirements according to EU-GMP.

CARGILL FRANCE, France

Overall, 14 observations were made, including 1 critical deficiency and 4 major deficiencies: [Critical] The management of semi-finished batches and of the mixing operations was deficient and conformity of the final batches to specifications, notably Ph.Eur. specifications, could not be guaranted. [Major 1] The site had been manufacturing an active substance without ANSM authorisation. [Major 2] The change control related to the suppression of one filtration step in the active substance manufacturing process was deficient. [Major 3] The manufacturing of the active substance had not been made using master production instructions and no batch production records had been established. [Major 4] No review of batch production records of critical process steps had been done before release of the active substance for distribution. 7 observations are related to lack of traceability, risks of contamination induced by the absence of cleanliness in the production environment, very bad condition of the production equipment and insufficient equipment cleaning procedures. The inspection’s observations also apply to the manufacture of pharmaceutical excipients and starting materials that are intended to be used as ingredients in cosmetics and medical devices, which are manufactured under the same conditions as the active substance.

FARMA MEDITERRANIA, S.L., Spain

Critical deficiencies a) Lack of an effective pharmaceutical quality assurance system b) Release of batches of medicinal products produced without completing all of the manufacturing protocols, without being checked quality assurance unit and without the approval of the technical director. c) Use in quality control a non-qualified chromatographic equipment, with operating faults and with an unvalidated computerized management system. As a result, the integrity, reliability, up-to-dateness, originality and authenticity of the data that are obtained cannot be guaranteed. d) Transfer of some of the final analytical quality controls of medicinal products to a third party, without appropriately transferring the control methods and without the authorization of the relevant health authority e) Manufacture of medicinal products using procedures that have not been appropriately validated or have not been periodically revalidated. f) Acceptance of results of repeated analytical controls and sterility tests of finished medicinal products without having undertaken an in-depth investigation to determine the root cause of a previously result obtained which was out of specifications. g) Although a visual inspection of injectable medicinal products reveals a high number of critical quality defects (the presence of visible particles) non deviations are opened and is not investigated. c) Do not do any quality control on a statistical sample of units of injectable medicinal products that have passed the visual inspection. Major deficiencies a) Do not do the annual quality product review of medicinal products manufactured. b) Deviations in the manufacturing processes are not investigated suitably and in-depth. c) The simulation of the aseptic manufacturing process is not performed every six months and samples used in the simulation are not incubated at the right temperature. c) The air treatment system in manufacturing areas is not properly qualified, as it is only checked when it is “at rest” but not “in operation”. e) Medicinal products are manufactured without full compliance with conditions established in the marketing authorisation dossier and/or without carrying out all the established process controls. f) Manufacturing and quality control documents of each batch of medicinal products manufactured are not filed correctly. g) The facilities have been modified considerably without the authorization of the relevant health authority h) Test of growth promotion of culture media, which are used in the sterility testing, in the simulation of the aseptic manufacturing process or in the environmental control of critical manufacturing areas, is not carried out. h) Do not analyse all of the specification parameters for raw materials used in the manufacturing.

Chengdu Okay Pharmaceutical Co. Ltd., China

Overall, 21 deficiencies were observed during the inspection, including 5 critical and 10 major deficiencies. The critical deficiencies were observed in QC Dept. including calculation of impurities of Diosmin and there were no records of standard (used as a reference) for testing in-house standard. Also the data integrity was not guaranteed. In manufacturing Dept. presented measuring methods were inadequate to the results. The condition in clean area was not acceptable for final product. Critical deficiences: Testing of the final product: There was incorrectly way of calculation the impurities and Diosmin content. There were no records of prepared in-house HPLC standard. There was no confirmation of the conditions HPLC analysis. Computerized systems – documentation and control: There was found in HPLC system that the method was changed, without any savings of previous method. There were no logins and passwords to the HPLC system and no procedure for granting permission to access to the HPLC system. There was no register of persons authorized to access the HPLC system. On the same computer station there were two different HPLC software. Manufacturing documentation: Presented measuring methods of pH during the inspection time were inadequate to the results recorded in the batch report. Premises: Crude Diosmin drying was carried out in an area which did not provide the appriopriate coditions during the discharge from the dryer. Qualification of equipment: Some data of HVAC system qualification had been falsified. The major deficiencies were observed among others: in the warehouse, in the manufacturing documentation and in the production area.

Dongying Tiandong Pharmaceutical Co., Ltd., China

This serious Non-Compliance Report refers to a manufacturing site for Heparin. French Inspectors found 2 critical and 3 major deviations. Heparin manufacturing sites were involved in one of the largest counterfeit scandal ever. Therefore it is worrying that critical deviations in Heparin manufacturing have been found again. Read more in our GMP News Chinese Heparin Manufacturer again involved in Falsification and GMP Non-Compliance.

THERAVECTYS – VILLEJUIF, France

Here a manufacturing site for Investigational Medicinal Products (IMPs) is concerned. Overall 45 deficiencies, including 5 critical deficiencies and 17 major deficiencies have been detected. The following critical deviations in sterile production are listed in the agency report:

1) The implementation of exemption SOP for manufacturing operations which is not compliant to GMP principles, for example, Media Fill Test were performed with unqualified equipment.
2) The lack of sample area for incoming materials and their systematic use in quarantine status for manufacturing operations.
3) Appropriate measures in terms of monitoring locations, alert and action limits rationale, were not set for particle and microbiological monitoring in clean rooms grade A and B.
4) No protocol for clean rooms’ qualification was established and clean rooms classification didn’t fulfill ISO14644 requirements.
5) Some analytical methods and process were not validated for the clinical trial EudraCT : 2015-000845-21

All Non-Compliance Reports with the detailed address of the facilities and the product concerned can be found in the EudraGMDP Database.

///////////// 13 EMA, GMP Non-compliance Reports, 2016 published, EudraGMDP, central database

ECA releases Version 18 of GMP Guideline Manager

regulatory Comments Off

Mar 172016

How to access ten thousand pages of GMP Guidelines from FDA, EMA, ICH, PIC/S, ICH, WHO and many other organisations worldwide? You can print them or purchase hundreds of booklets. Or, alternatively, you can take advantage of a software tool developed by the ECA Academy, allowing you to access to the most comprehensive GMP Guideline Database

http://www.gmp-compliance.org/eca_mitt_05241_n.html

How to access ten thousand pages of GMP Guidelines from FDA, EMA, ICH, PIC/S, ICH, WHO and many other organisations worldwide? You can print them or purchase hundereds of booklets. But this will cause a huge amount of paper. And it will be more than difficult to find a specific regulatory requirement in this comprehensive library.

For that reason the ECA Academy has started to set up the largest GMP Guideline Database of its kind worldwide already 18 years ago. And every year a new release is published with all updates. A software was developed to structure the Guidelines in two so called “Guideline Trees”.

1. Guideline Tree structured according to the issuing authorities (e.g. EU, FDA, ICH)

2. Guideline Tree structured according to GMP topics (e.g. GMP for Medicinal Products, GMP for APIs, sterile production, validation etc)

In addition to the two structured libraries the software also allows you to search for certain key words. You may search the entire database for a keyword like “validation”. But you can also limit the search to certain areas (e.g. search in FDA Guidelines only).

If you have no CD drive on your computer you can also access the GMP Guideline Manager via the ECA WebApp. When you register you will receive the login details to access the ECA members area. The same login details will work for the ECA WebApp. With this service you can access the full GMP Guideline Database from your smartphone or tablet via the internet.

The GMP Guideline Manager can not be purchased – it is only available for ECA Members at no costs! This service is unique and not offered by any other organisation. By participating in any of the ECA conferences or courses you become member of the ECA free of charge for 2 years. If you can not attend an ECA course you can also apply for ECA membership for only 190,- Euro via our webpage.

Please find more information about the GMP Guideline Manager 18.0 here.

///////ECA, Version 18, GMP Guideline Manager

Second Revision of USP Chapter <1> Injections and Implanted Drug Products (Parenterals)-Product Quality Tests

regulatory Comments Off

Mar 172016

After the revision of the General Chapter on quality testing of sterile medicinal products in the US American Pharmacopoeia had already been announced last year in the USP 38-NF 33, the USP is planning a new revision. Read more about the revision of Chapter <1>.

http://www.gmp-compliance.org/enews_05240_Second-Revision-of-USP-Chapter–1–Injections-and-Implanted-Drug-Products–Parenterals–Product-Quality-Tests_15090,15160,15266,Z-PEM_n.html

Last year already, the revision of the General Chapter on quality testing of sterile medicinal products was initiated in the USP 38 NF 33. The targeted official date for coming into force was the 1st May 2016. Now, the USP has announced that because of some comments received, there will be a further revision. This is due to the USP’s intention to support in Chapter 1 both existing monographs as well as new monographs to be developed. The new scope should now be focussed again to avoid confusion. The publication is striven for March 2016 as well as the adoption of the changes in the USP 40 NF 35. Furthermore, USP has announced that also the contents of General Chapters <2> to <5> will be examined.

On the USP website you will find further details regarding the revision of Chapter <1>.

///////USP 38-NF 33, revision of Chapter <1>, quality testing of sterile medicinal products, monographs, USP, new revision

New FDA Guidance on Completeness Assessements for Type II API Drug Master Files

regulatory Comments Off

Mar 172016

Since 1st October 2012, special regulations have been applying to the US Type II Drug Master Files. This year in February, the FDA published a new Guidance for Industry. Read here what the DMF holder has to consider when submitting data about the API Drug Master File.

http://www.gmp-compliance.org/enews_05256_New-FDA-Guidance-on-Completeness-Assessements-for-Type-II-API-Drug-Master-Files_15328,15339,S-WKS_n.html

Since the coming into force of the “Generic Drug User Fee Act” (GDUFA) on 1st October 2012, special regulations have been applying to the submission to the FDA of a Drug Master Files for a pharmaceutical API (Type II DMF). The DMF holder must pay a one-time fee when authorising the reference of his/ her DMF in an application for a generic drug (Abbreviated New Drug Application, ANDA). Moreover, the DMF will undergo a completeness assessment through the FDA.

This year in February, the FDA published a Guidance for Industry entitled “Completeness Assessments for Type II API DMFs under GDUFA” which provides DMF holders with comprehensive information regarding the application for a Type II DMF. The document describes the criteria according to which the FDA performs a completeness assessment and which data are expected.

This completeness assessment does not replace the full scientific assessment to be executed at a later time. It serves to find out whether the data contained in the DMF are sufficient for the ANDA. In a completeness assessment, the following elements are examined:

Is the DMF active?
Has the fee been paid?
Has the DMF been previously reviewed?
Does the DMF pertain to a single API?
Does the DMF contain certain administrative information?
Does the DMF contain all the information necessary to enable a scientific review?
Is the DMF written in English?

The Guidance contains a checklist (Appendix 1) listing the criteria according to which the FDA performs the assessment. For the DMF holder, this list is helpful to check the completeness of his/ her data before submitting them to the FDA.

One essential element underlined in this Guidance is to pay the DMF fee in due time (at least 6 months prior to the submission of an ANDA). The FDA won’t continue to process the DMF as long as the fee hasn’t been paid. If the applicant of an ANDA references in his dossier a DMF for which a fee is due, the FDA will inform him. If the fee hasn’t been paid within 20 days after notification, the FDA will stop the further processing of the application.

When submitting a DMF, the form “FDA 3794″ (Generic Drug User Fee Cover Sheet) should be attached. It contains the minimum information required by the FDA to determine whether the DMF holder has satisfied his fee obligations.

After the successful completeness assessment of a DMF, a number will be attributed and posted on a publicly available API DMF list. The FDA has compiled all important information regarding DMFs Type I-V on the Drug Master File webpage. Here, you can also find the list of all active DMFs.

//////API Drug Master File, fda, type2

An Improved Process for the Preparation of Tenofovir Disoproxil Fumarate

MANUFACTURING, PROCESS, SYNTHESIS Comments Off

Mar 152016

VIREAD® (tenofovir disoproxil fumarate) Structural Formula Illustration

Tenofovir Disoproxil Fumarate

For full details see end of page

PAPER

The current three-step manufacturing route for the preparation of tenofovir disoproxil fumarate (1) was assessed and optimized leading to a higher yielding, simpler, and greener process. Key improvements in the process route include the refinement of the second stage through the replacement of the problematic magnesium tert-butoxide (MTB) with a 1:1 ratio of a Grignard reagent and tert-butanol. The development of a virtually solvent-free approach and the establishment of a workup and purification protocol which allows the isolation of a pure diethyl phosphonate ester (8) was achieved

see………….http://pubs.acs.org/doi/abs/10.1021/acs.oprd.5b00364

An Improved Process for the Preparation of Tenofovir Disoproxil Fumarate

Darren L. Riley^*^†^∥, David R. Walwyn^‡^∥, and Chris D. Edlin^§^∥

^† Department of Chemistry, Natural and Agricultural Sciences, University of Pretoria, 2 Lynnwood Road, Hatfield, 0002, Gauteng, South Africa

^‡ Department of Engineering and Technology Management, University of Pretoria, Pretoria, South Africa

^§ Pharmaceutical Manufacturing Technology Centre, University of Limerick, Limerick, V94 T9PX, Republic of Ireland

^∥ iThemba Pharmaceuticals, Modderfontein, 1645, Gauteng South Africa

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.5b00364

Publication Date (Web): March 04, 2016

*E-mail: darren.riley@up.ac.za.

University of Pretoria

Department of Chemistry

Pretoria, South Africa

Department of Chemistry, Natural and Agricultural Sciences, University of Pretoria, 2 Lynnwood Road, Hatfield, 0002, Gauteng, South Africa

Map of Department of Chemistry, Natural and Agricultural Sciences, University of Pretoria, 2 Lynnwood Road, Hatfield, 0002, Gauteng, South Africa

///////

Tenofovir Disoproxil Fumarate

5-[[(1R)-2-(6-Amino-9H-purin-9-yl)-1-methylethoxy]methyl]-2,4,6,8-tetraoxa-5-phosphanonanedioic Acid 1,9-Bis(1-methylethyl) Ester 5-Oxide (2E)-2-Butenedioate; GS 4331-05; PMPA Prodrug; Tenofovir DF; Virea; Viread;

GILEAD-4331-300

201341-05-1 – free base, (Tenofovir Disoproxil

fumarate

202138-50-9

113-115°C (dec.)

Molecular Structure:
CAS No.:	202138-50-9
Name:	Tenofovir disoproxil fumarate

Formula:	C₁₉H₃₀N₅O₁₀P^.C₄H₄O₄
Molecular Weight:	635.51
Synonyms:	TDF;PMPA prodrug;Tenofovir Disoproxil Fumarate [USAN];9-((R)-2-((Bis(((isopropoxycarbonyl)oxy)methoxy)phosphinyl)methoxy)propyl)adenine, fumarate;201341-05-1;Bis(NeopentylOC)PMPA;Viread;GS 4331-05 (1:1 Fumarate salt);Viread (1:1 Fumarate salt);Truvada;Tenofovir DF;[[(2R)-1-(6-aminopurin-9-yl)propan-2-yl]oxymethyl-(propan-2-yloxycarbonyloxymethoxy)phosphoryl]oxymethyl propan-2-yl carbonate;

Usage
tyrosinase inhibitor used for skin lightening and anti-melasma

Usage
An acyclic phosphonate nucleotide analog and selective HIV-1 RT inhibitor

Usage
Acyclic phosphonate nucleotide analogue; reverse transcriptase inhibitor. Used as an anti-HIV agent. Antiviral.

Tenofovir disoproxil is an antiretroviral medication used to prevent and treat HIV/AIDS and to treat chronic hepatitis B.^[1] The active substance is tenofovir, while tenofovir disoproxil is a prodrug that is used because of its better absorption in the gut.

The drug is on the World Health Organization’s List of Essential Medicines, the most important medications needed in a basic health system.^[2] It is marketed by Gilead Sciences under the trade name Viread (as the fumarate, TDF).^[3] As of 2015 the cost for a typical month of medication in the United States is more than 200 USD.^[4]

Medical uses

HIV-1 infection: Tenofovir is indicated in combination with other antiretroviral agents for the treatment of HIV-1 infection in adults and pediatric patients 2 years of age and older.^[5] This indication is based on analyses of plasma HIV-1 RNA levels and CD4 cell counts in controlled studies of tenofovir in treatment-naive and treatment-experienced adults.
Tenofovir is indicated for the treatment of chronic hepatitis B in adults and pediatric patients 12 years of age and older.^[5]^[6]

HIV risk reduction

A Cochrane review examined the use of tenofovir for prevention of HIV before exposure. It found that both tenofovir alone and the tenofovir/emtricitabine combination decreased the risk of contracting HIV.^[7]

The U. S. Centers for Disease Control and Prevention (CDC) conducted a study in partnership with the Thailand Ministry of Public Health to ascertain the effectiveness of providing people who inject drugs illicitly with daily doses of the antiretroviral drug tenofovir as a prevention measure. The results of the study were released in mid-June 2013 and revealed a 48.9%-reduced incidence of the virus among the group of subjects who received the drug, in comparison to the control group who received a placebo. The principal investigator of the study stated: “We now know that pre-exposure prophylaxis can be a potentially vital option for HIV prevention in people at very high risk for infection, whether through sexual transmission or injecting drug use.”^[8]

Adverse effects

The most common side effects associated with tenofovir include nausea, vomiting, diarrhea, and asthenia. Less frequent side effects include hepatotoxicity, abdominal pain, and flatulence.^[9] Tenofovir has also been implicated in causing renal toxicity, particularly at elevated concentrations.^[10]

Tenofovir can cause acute renal failure, Fanconi syndrome, proteinuria, or tubular necrosis.^{[citation needed]} These side effects are due to accumulation of the drug in proximal tubules.^{[citation needed]} Tenofovir can interact with didanosine by increasing didanosine’s concentration.^{[citation needed]} It also decreases the concentration of atazanavir sulfate.^{[citation needed]}

Mechanism of action

Tenofovir is a defective adenosine nucleotide that selectively interferes with the action of reverse transcriptase, but only weakly interferes with mammalian DNA polymerases α, β, and mitochondrial DNA polymerase γ.^[11] Tenofovir prevents the formation of the 5′ to 3′ phosphodiester linkage essential for DNA chain elongation. A phosphodiester bond cannot be formed because the tenofovir molecule lacks an —OH group on the 3′ carbon of its deoxyribose sugar.^[11] Once incorporated into a growing DNA strand, tenofovir causes premature termination of DNA transcription. The drug is classified as a nucleotide analogue reverse transcriptase inhibitor (NRTI), that inhibits reverse transcriptase.^[11] Reverse transcriptase is a crucial viral enzyme in retroviruses such as human immunodeficiency virus (HIV) and in hepatitis B virus infections.^[5]

History

Tenofovir was initially synthesized by Antonín Holý at the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic in Prague. The patent^[12] filed by Holý in 1984 makes no mention of the potential use of the compound for the treatment of HIV infection, which had only been discovered one year earlier.

In 1985, De Clercq and Holý described the activity of PMPA against HIV in cell culture.^[13] Shortly thereafter, a collaboration with the biotechnology company Gilead Sciences led to the investigation of PMPA’s potential as a treatment for HIV infected patients. In 1997 researchers from Gilead and the University of California, San Francisco demonstrated that tenofovir exhibits anti-HIV effects in humans when dosed by subcutaneous injection.^[14]

The initial form of tenofovir used in these studies had limited potential for widespread use because it was not absorbed when administered orally. A medicinal chemistry team at Gilead developed a modified version of tenofovir, tenofovir disoproxil.^[15] This version of tenofovir is often referred to simply as “tenofovir”. In this version of the drug, the two negative charges of the tenofovir phosphonic acid group are masked, thus enhancing oral absorption.

Tenofovir disoproxil was approved by the U.S. FDA on October 26, 2001, for the treatment of HIV, and on August 11, 2008, for the treatment of chronic hepatitis B.^[16]^[17]

Drug forms

Tenofovir disoproxil is a prodrug form of tenofovir. It is also marketed under the brand name Reviro by Dr. Reddy’s Laboratories. Tenofovir is also available in a fixed-dose combination with emtricitabine in a product with the brand name Truvada for once-a-day dosing. Efavirenz/emtricitabine/tenofovir disoproxil (brand name Atripla) — a fixed-dose triple combination of tenofovir, emtricitabine, and efavirenz, was approved by the FDA on 12 July 2006 and is now available, providing a single daily dose for the treatment of HIV.

Therapeutic drug monitoring

Tenofovir may be measured in plasma by liquid chromatography. Such testing is useful for monitoring therapy and to prevent drug accumulation and toxicity in people with kidney or liver problems.^[18]^[19]^[20]

PATENT

http://www.google.com/patents/EP2545063A2?cl=en

Tenofovir Disoproxil is chemically known as 9-[-2-(R)-[[bis [[(isopropoxycarbonyl) oxy]methoxy] phosphinoyl]methoxy]propyl]-adenine, having the following structural formula-I.

Formula-I

Tenofovir is a highly potent antiviral agent, particularly for the therapy or prophylaxis of retroviral infections and belongs to a class of drugs called Nucleotide Reverse Transcriptase Inhibitors (NRTI) which blocks reverse transcriptase an enzyme crucial to viral production in HIV-infected people.

Tenofovir Disoproxil and its pharmaceutically acceptable salts were first disclosed in US 5,922,695. This patent discloses the preparation of Tenofovir Disoproxil by the esterification of Tenofovir with chloromethyl isopropyl carbonate using l-methyl-2- pyrrolidinone and triethylamine. In this patent Tenofovir Disoproxil is converted into its Fumarate salt without isolation. PCT Publication WO 2008007392 discloses process for the preparation of Tenofovir Disoproxil fumarate, wherein the isolated crystalline Tenofovir Disoproxil is converted into fumarate salt.

Tenofovir Disoproxil processes in the prior art are similar to process disclosed in product patent US 5,922,695. According to the prior art processes, Tenofovir Disoproxil fumarate obtained is having low yields and also show the presence of impurities such as dimers.

scheme- 1.

Tenofovir disoproxil chloromethyl isopropyl carbonate

Tenofovir disoproxil fumarate

Example 1 : Process for the preparation of Tenofovir Disoproxil fumarate

Toluene (500 ml) was added to the Tenofovir (100 gm) and stirred at room temperature. To this triethylamine (66.31 gm) was added, temperature was raised to 90° C and water was collected by azeotropic distillation at 110°C. Toluene was completely distilled under vacuum at same temperature. The reaction mixture was cooled to room temperature and to this a mixture of N-methyl pyrrolidine (300 gm), triethylamine (66.31 gm), Tetrabutyl ammonium bromide (52.8 gm) and trimethyl silyl chloride (17.8 gm) were added. The above reaction mixture was heated to 50-55 °C and was added slowly chloromethyl. isopropyl carbonate (CMIC) and maintained the reaction mixture at 50-55°C for 5 hrs. (Qualitative HPLC analysis shows about 85% product formation). The above reaction mixture was cooled to room temperature and filtered. The filtrate was added to DM water at 5-10°C and extract with dichloromethane. The combined dichloromethane layer was concentrated under vacuum and the crude was Co-distilled with cyclohexane and this crude was taken into isopropyl alcohol (1000 ml). To this fumaric acid (38 gm) was added and temperature was raised to 50° C. The reaction mixture was filtered and filtrate was cooled to 5-10° C. The obtained solid was filtered and washed with isopropyl alcohol. The compound was dried under vacuum to yield Tenofovir Disoproxil fumarate (140 gm).

Example-2 : Preparation of Tenofovir

N-methyl-2-pyrrolidone (25 gm) was taken along with toluene (150 gm) into a reaction vessel. l-(6-amino-purin-9-yl)-propan-2-ol (100 gm); toluene-4-sulfonic acid diethoxy phosphoryl methyl ester (200 gm) and magnesium ter-butoxide (71.2 gm) were also taken at’ 25-35°C. Temperature was raised to 74-75 °C and maintained for 5-6hrs. After completion of reaction, acetic acid (60 gm) was added and maintained for 1 hr. Later aq.HBr (332 gm) was taken and heated to 90-95 °C. After reaction completion, salts were filtered and filtrate was subjected to washings with water and extracted into methylene dichloride. Later pH was adjusted using CS lye below 10 °C. Tenofovir product was isolated using acetone.

Yield: 110 gm.

Example 3 : Preparation of Tenofovir disoproxil

(R)-9-[2-(phosphonomethoxy)propyl]adenine (25 gm), triethyl amine (25 ml) and cyclohexane (200 ml) were combined and heated to remove water and the solvent was distilled off under vacuum. The reaction mass was cooled to room temperature N-methyl pyrrolidinone (55 ml), triethyl amine (25 ml) and tetra butyl ammonium bromide(54 gms) were added to the reaction mixture. The reaction mass was heated to 50-60°C and chloromethyl isopropyl carbonate (65 gm) was added and maintained for 4-8 hrs at 50- 60°C and then cooled to 0°C. The reaction mass was diluted with chilled water or ice and precipitated solid product was filtered. The mother liquor was extracted with methylene chloride (150 ml). The methylene chloride layer was washed with water (200 ml). The filtered solid and the methylene chloride layer were combined and washed with water and the solvent was distilled under vacuum. Ethyl acetate was charged to the precipitated solid. The reaction mass was then cooled to 0-5 °C and maintained for 6 hrs. The solid was filtered and dried to produce Tenofovir disoproxil (45 gm).

CLIPS

The reaction of chloromethyl chloroformate (I) with isopropyl alcohol (II) by means of pyridine or triethylamine in ether gives the mixed carbonate (III), which is then condensed with (R)-PMPA (IV) by means of diisopropyl ethyl-amine in DMF.

US 5922695; WO 9804569

CLIP 2

1) The protection of isobutyl D-(+)-lactate (I) with dihydropyran (DHP)/HCl in DMF gives the tetrahydropyranyloxy derivative (II), which is reduced with bis(2-methoxyethoxy)aluminum hydride in refluxing ether/ toluene yielding 2(R)-(tetrahydropyranyloxy)-1-propanol (III). The tosylation of (III) with tosyl chloride as usual affords the expected tosylate (VI), which is condensed with adenine (V) by means of Cs2CO3 in hot DMF, affording 9-[2(R)-(tetrahydropyranyloxy)propyl]adenine (VI). The deprotection of (VI) with sulfuric acid affords 9-[2(R)-hydroxypropyl]adenine (VII), which is N-benzoylated with benzoyl chloride/chlorotrimethylsilane in pyridine to give the benzamide (VIII), which is condensed with tosyl-oxymethylphosphonic acid diisopropyl ester (IX) by means of NaH in DMF to yield 9-[2(R)-(diisopropoxyphosphorylmethoxy)propyl]adenine (X). Finally, this compound is hydrolyzed by means of bromotrimethylsilane in acetonotrile.

2) The reaction of the previously described (R)-2-(2-tetrahydropyranyloxy)-1-propanol (III) with benzyl bromide (XI) by means of NaH in DMF, followed by a treatment with Dowex 50X, gives 1-benzyloxy-2(R)-propanol (XII), which is condensed with tosyloxymethylphosphonic acid diisopropyl ester (IX) by means of NaH in THF, yielding 2-benzyloxy-1(R)-methylethoxymethylphosphonic acid diisopropyl ester (XIII). The hydrogenolysis of (XIII) over Pd/C in methanol affords 2-hydroxy-1(R)-methylethoxymethylphosphonic acid diisopropyl ester (XIV), which is tosylated with tosyl chloride/dimethyl-aminopyridine in pyridine to give the expected tosylate (XV). The condensation of (XV) with adenine (VI) by means of Cs2CO3 in hot DMF yields 9-[2(R)-(diisopropoxyphosphorylmethoxy)propyl]adenine (X), which is finally hydrolyzed as before.

3) The catalytic hydrogenation of (S)-glycidol (XVI) over Pd/C gives the (R)-1,2-propanediol (XVII), which is esterified with diethyl carbonate (XVIII)/NaOEt, yielding the cyclic carbonate (XIX). The reaction of (XIX) with adenine (V) by means of NaOH in DMF affords 9-[2(R)-hydroxypropyl]adenine (VII), which is condensed with tosyloxymethylphosphonic acid diethyl ester (XX) by means of lithium tert-butoxide in THF, giving 9-[2(R)-(diethoxyphosphorylmethoxy)propyl]adenine (XXI). Finally, this compound is hydrolyzed with bromotrimethylsilane as before. Compound (XX) is obtained by reaction of diethyl phosphite (XXII) with paraformaldehyde, yielding hydroxy- methylphosphonic acid diethyl ester (XXIII), which is finally tosylated as usual.

References

1 “Viread”. The American Society of Health-System Pharmacists. Retrieved 31 July 2015.
2
“WHO Model List of EssentialMedicines” (PDF). World Health Organization. October 2013. Retrieved 22 April 2014.
3
Emau P, Jiang Y, Agy MB, et al. (2006). “Post-exposure prophylaxis for SIV revisited: Animal model for HIV infection”. AIDS Res Ther 3: 29. doi:10.1186/1742-6405-3-29. PMC 1687192. PMID 17132170.
4
Hamilton, Richart (2015). Tarascon Pocket Pharmacopoeia 2015 Deluxe Lab-Coat Edition. Jones & Bartlett Learning. p. 66. ISBN 9781284057560.
5
Gilead Sciences, Inc. Prescribing Information. Revised: November 2012.
6
Guidelines for the prevention, care and treatment of persons with chronic hepatitis B infection, WHO, Publication details:Pages: 166 Publication date: March 2015 Languages: English ISBN 978 92 4 154905 9
7
Okwundu CI, Uthman OA, Okoromah CAN (2012). “Antiretroviral pre-exposure prophylaxis (PrEP) for preventing HIV in high-risk individuals”. Cochrane Database Syst Rev 7 (7): CD007189. doi:10.1002/14651858.CD007189.pub3. PMID 22786505.
8
Emma Bourke (14 June 2013). “Preventive drug could reduce HIV transmission among injecting drug users”. The Conversation Australia. The Conversation Media Group. Retrieved 17 June 2013.
9
USPDI. Thompson. 2005. pp. 2741–2.
10
“Viread Prescribing Guidelines” (PDF). U.S. Food and Drug Administration. March 2006. Archived from the original (PDF) on 2007-09-30. Retrieved 2007-02-12.
11
Drugbank: Tenofovir
12
“Patent US4808716 – 9-(phosponylmethoxyalkyl) adenines, the method of preparation and … – Google Patents”.
13
A US 4724233 A, De Clercq, Erik; Antonin Holy & Ivan Rosenberg, “Therapeutical application of phosphonylmethoxyalkyl adenines”, published 1985-04-25
14
Deeks SG, Barditch-Crovo P, Lietman PS, et al. (September 1998). “Safety, pharmacokinetics, and antiretroviral activity of intravenous 9-[2-(R)-(Phosphonomethoxy)propyl]adenine, a novel anti-human immunodeficiency virus (HIV) therapy, in HIV-infected adults”. Antimicrob. Agents Chemother. 42 (9): 2380–4. PMC 105837. PMID 9736567.
15
“Patent US5977089 – Antiviral phosphonomethoxy nucleotide analogs having increased oral … – Google Patents”.
16
FDA letter of approval (regarding treatment of hepatitis B)
17
FDA Clears Viread for Hepatitis B
18
Delahunty T, Bushman L, Robbins B, Fletcher CV (2009). “The simultaneous assay of tenofovir and emtricitabine in plasma using LC/MS/MS and isotopically labeled internal standards”. J. Chrom. B 877 (20–21): 1907–1914. doi:10.1016/j.jchromb.2009.05.029.
19
Kearney BP, Yale K, Shah J, Zhong L, Flaherty JF (2006). “Pharmacokinetics and dosing recommendations of tenofovir disoproxil fumarate in hepatic or renal impairment”. Clin. Pharmacokinet. 45 (11): 1115–24. doi:10.2165/00003088-200645110-00005. PMID 17048975.
20

R. Baselt, Disposition of Toxic Drugs and Chemicals in Man, 8th edition, Biomedical Publications, Foster City, California, 2008, pp. 1490–1492.

External links

Official Viread website

WO2008007392A2	Jul 11, 2007	Jan 17, 2008	Matrix Lab Ltd	Process for the preparation of tenofovir
US5922695	Jul 25, 1997	Jul 13, 1999	Gilead Sciences, Inc.	Antiviral phosphonomethyoxy nucleotide analogs having increased oral bioavarilability

WO2015051874A1	Sep 22, 2014	Apr 16, 2015	Zentiva, K.S.	An improved process for the preparation of tenofovir disoproxil and pharmaceutically acceptable salts thereof
CN103360425A *	Apr 1, 2012	Oct 23, 2013	安徽贝克联合制药有限公司	Synthesis method of tenofovir disoproxil and fumarate thereof
CN103374038A *	Apr 11, 2012	Oct 30, 2013	广州白云山制药股份有限公司广州白云山制药总厂	Preparation method of antiviral medicine
CN103848868A *	Dec 4, 2012	Jun 11, 2014	蚌埠丰原涂山制药有限公司	Method for preparing tenofovir
CN103848869A *	Dec 4, 2012	Jun 11, 2014	上海医药工业研究院	Method for preparing tenofovir
CN103980319A *	Apr 24, 2014	Aug 13, 2014	浙江外国语学院	Preparation method of tenofovir
CN103980319B *	Apr 24, 2014	Dec 2, 2015	浙江外国语学院	一种泰诺福韦的制备方法
EP2860185A1	Oct 9, 2013	Apr 15, 2015	Zentiva, k.s.	An improved process for the preparation of Tenofovir disoproxil and pharmaceutically acceptable salts thereof

The chemical name of tenofovir disoproxil fumarate is 9-[(R)-2[[bis[[(isopropoxycarbonyl)oxy]methoxy]phosphinyl]methoxy]propyl]adenine fumarate (1:1). It has a molecular formula of C₁₉H₃₀N₅O₁₀P • C₄H₄O₄ and a molecular weight of 635.52. It has the following structural formula:

VIREAD® (tenofovir disoproxil fumarate) Structural Formula Illustration

Tenofovir disoproxil fumarate is a white to off-white crystalline powder with a solubility of 13.4 mg/mL in distilled water at 25 °C. It has an octanol/phosphate buffer (pH 6.5) partition coefficient (log p) of 1.25 at 25 °C.

VIREAD is available as tablets or as an oral powder.

VIREAD tablets are for oral administration in strengths of 150, 200, 250, and 300 mg of tenofovir disoproxil fumarate, which are equivalent to 123, 163, 204 and 245 mg of tenofovir disoproxil, respectively. Each tablet contains the following inactive ingredients: croscarmellose sodium, lactose monohydrate, magnesium stearate, microcrystalline cellulose, and pregelatinized starch. The 300 mg tablets are coated with Opadry II Y-3010671-A, which contains FD&C blue #2 aluminum lake, hypromellose 2910, lactose monohydrate, titanium dioxide, and triacetin. The 150, 200, and 250 mg tablets are coated with Opadry II 32K-18425, which contains hypromellose 2910, lactose monohydrate, titanium dioxide, and triacetin.

VIREAD oral powder is available for oral administration as white, taste-masked, coated granules containing 40 mg of tenofovir disoproxil fumarate per gram of oral powder, which is equivalent to 33 mg of tenofovir disoproxil. The oral powder contains the following inactive ingredients: mannitol, hydroxypropyl cellulose, ethylcellulose, and silicon dioxide.

enofovir disoproxil
Systematic (IUPAC) name

Bis{[(isopropoxycarbonyl)oxy]methyl} ({[(2R)-1-(6-amino-9H-purin-9-yl)-2-propanyl]oxy}methyl)phosphonate
Clinical data
Trade names	Viread
AHFS/Drugs.com	monograph
Pregnancy category	AU: B3 US: B (No risk in non-human studies)
Routes of administration	Oral (tablets)
Legal status
Legal status	AU: S4 (Prescription only) CA: ℞-only UK: POM (Prescription only) US: ℞-only
Pharmacokinetic data
Bioavailability	25%
Identifiers
CAS Number	201341-05-1
ATC code	J05AF07 (WHO)
PubChem	CID 5481350
ChemSpider	4587262
UNII	F4YU4LON7I
ChEBI	CHEBI:63717
NIAID ChemDB	080741
Chemical data
Formula	C₁₉H₃₀N₅O₁₀P
Molar mass	519.443 g/mol
SMILES [show]
InChI [show]

Tenofovir
Systematic (IUPAC) name

({[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonic acid
Clinical data
MedlinePlus	a602018
Routes of administration	In form of prodrugs
Pharmacokinetic data
Protein binding	< 1%
Biological half-life	17 hours
Excretion	Renal
Identifiers
CAS Number	147127-20-6
ATC code	None
PubChem	CID 464205
DrugBank	DB00300
ChemSpider	408154
UNII	99YXE507IL
KEGG	D06074
ChEBI	CHEBI:63625
ChEMBL	CHEMBL483
Synonyms	9-(2-Phosphonyl-methoxypropyly)adenine (PMPA)
Chemical data
Formula	C₉H₁₄N₅O₄P
Molar mass	287.213 g/mol

///////

Breaking the symmetry of dibenzoxazines: a paradigm to tailor the design of bio-based thermosets

spectroscopy, SYNTHESIS Comments Off

Mar 132016

Green Chem., 2016, Advance Article

DOI: 10.1039/C5GC03102H, Paper

L. Puchot, P. Verge, T. Fouquet, C. Vancaeyzeele, F. Vidal, Y. Habibi

Asymmetric di-benzoxazine monomers from naturally occurring phenolic compounds – cardanol and vanillin – were synthesized to obtain a processable and self-supported bio-thermoset with valuable properties. Such strategy constitutes an efficient and versatile route for the elaboration of biobased thermoset from a wide range of phenolic compounds derived from renewable resources.

Breaking the symmetry of dibenzoxazines: a paradigm to tailor the design of bio-based thermosets

http://pubs.rsc.org/en/Content/ArticleLanding/2016/GC/C5GC03102H?utm_source=feedburner&utm_medium=feed&utm_campaign=Feed%3A+rss%2FGC+%28RSC+-+Green+Chem.+latest+articles%29#!divAbstract

With the ongoing efforts to promote the development of bio-based dibenzoxazine thermosets, we explore herein a new strategy aiming at the synthesis of asymmetric dibenzoxazine monomers from naturally occurring phenolic compounds, cardanol and vanillin. By taking advantage of the low reactivity of cardanol, a monosubstituted cardanol-based benzoxazine monomer was prepared and further coupled with vanillin to yield vanillin–cardanol di-benzoxazines. The structural features of the resulting products were substantiated by ¹H NMR and HR-MS. The occurrence of the thermally-induced ring-opening polymerization was monitored by rheological measurements and DSC. At 190 °C the new asymmetric monomers showed a moderate gelation time (8 min) compared to 30–31 min revealed for cardanol-based (di-card) dibenzoxazines. Once polymerized, they exhibited a high T_g (129 °C), while the di-card flew under heating because of its low cross-linking density. Asymmetric monomers also exhibited lower melting temperatures than their symmetrical congeners based on vanillin, which significantly enlarge the processing window between the melting and polymerization temperatures up to 126 °C instead of 7 °C for symmetric vanillin-based dibenzoxazines. Therefore, such a strategy constitutes an efficient and versatile route for an easy elaboration of biobased monocomponent thermosets and can be applied to a wide range of phenolic compounds derived from renewable resources.

Breaking the symmetry of dibenzoxazines: a paradigm to tailor the design of bio-based thermosets

L. Puchot,^a^b P. Verge,*^a T. Fouquet,^c C. Vancaeyzeele,^b F. Vidal^b and Y. Habibi*^a

*Corresponding authors

^aLuxembourg Institute of Science and Technology (LIST), 5 avenue des Hauts-Fourneaux, L-4362 Esch-sur-Alzette, Luxembourg

E-mail: Pierre.verge@list.lu, Youssef.habibi@list.lu

^bLaboratoire de Physicochimie des Polymères et des Interfaces (LPPI – EA 2528), I-Mat, Université de Cergy-Pontoise, 5 mail Gay-Lussac, 95031 Cergy-Pontoise, France

^cEnvironmental Measurement Technology Group, Environmental Management and Research Institute (EMRI), National Institute of Advanced Industrial Science and Technology (AIST), Onogawa 16-1, Tsukuba, Japan

Green Chem., 2016, Advance Article

DOI: 10.1039/C5GC03102H

//////////

ITI 214

phase 1 Comments Off

Mar 112016

ITI 214

IC200214; ITI-214

(6aR,9aS)-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-3-(phenylamino)-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4-(2H)-one phosphate

(6aR,9aS)-5-methyl-3-(phenylamino)-2-(4-(6-fluoropyridin-2-yl)-benzyl)-5,6a,7,8,9,9a-hexahydrocyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one…BASE

CAS: 1642303-38-5 (phosphate);

1160521-50-5 (free base).

Chemical Formula: C29H29FN7O5P
Molecular Weight: 605.5672

Takeda Pharmaceutical Company Limited,Intra-Cellular Therapies, Inc.

ITI-214 is an orally active, potent and Selective Inhibitors of Phosphodiesterase 1 for the Treatment of Cognitive Impairment Associated with Neurodegenerative and Neuropsychiatric Diseases. ITI-214 exhibited picomolar inhibitory potency for PDE1, demonstrated excellent selectivity against all other PDE families, and showed good efficacy in vivo. Currently, this investigational new drug is in Phase I clinical development and being considered for the treatment of several indications including cognitive deficits associated with schizophrenia and Alzheimer’s disease, movement disorders, attention deficit and hyperactivity disorders, and other CNS and non-CNS disorders.

Phase I Cognition disorders
- OriginatorIntra-Cellular Therapies
- ClassAntiparkinsonians; Nootropics; Small molecules
- Mechanism of ActionType 1 cyclic nucleotide phosphodiesterase inhibitors

21 Sep 2015Takeda completes a phase I bioavailability trial in Cognition disorders in Japan
21 Sep 2015Takeda completes a phase I trial in Cognition disorders in Japan
21 Sep 2015Takeda initiates enrolment in a phase I bioavailability trial for Cognition disorders in Japan before September 2015

Phosphodiesterase-1 (PDE-1) inhibitor

which is a picomolar PDE1 inhibitor with excellent selectivity against other PDE family members and against a panel of enzymes, receptors, transporters, and ion channels.

It is disclosed in WO 2009/075784 (U.S. Pub. No. 2010/0273754). This compound has been found to be a potent and selective phosphodiesterase 1 (PDE 1) inhibitor useful for the treatment or prophylaxis of disorders characterized by low levels of cAMP and/or cGMP in cells expressing PDE1, and/or reduced dopamine Dl receptor signaling activity (e.g., Parkinson’s disease, Tourette’s Syndrome, Autism, fragile X syndrome, ADHD, restless leg syndrome, depression, cognitive impairment of schizophrenia, narcolepsy); and/or any disease or condition that may be ameliorated by the enhancement of progesterone signaling. This list of disorders is exemplary and not intended to be exhaustive.

Intra-Cellular Therapies logo

PATENT

WO 2013192556

http://www.google.com/patents/WO2013192556A2?cl=en

The method of making the Compound (ea^^a^-S^a ^^^a-hexahydro-S- methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)- cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one is generally described in WO 2009/075784, the contents of which are incorporated by reference in their entirety. This compound can also be prepared as summarized or similarly summarized in the following

CMU PCU PHU PPU (SM2)

PATENT

http://www.google.com/patents/WO2009075784A1?cl=en

1 1. A compound according to claim 1 , wherein said compound is

EXAMPLE 14

(6aJ?,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6- fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]iinidazo[l,2-fl]pyrazolo[4,3- e]pyrimidin-4(2//)-one

This compound may be made using similar method as in example 13 wherein 2-(4-(bromomethyl)phenyl)-6-fluoropyridine may be used instead of 2-(4- (dibromomethyl)phenyl)-5-fluoropyridine.

PATENT

WO 2014205354

https://www.google.co.in/patents/WO2014205354A2?cl=en

EXAMPLES

The method of making the Compound (ea^^a^-S^a ^^^a-hexahydro-S-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one is generally described in WO 2009/075784, the contents of which are incorporated by reference in their entirety. This compound can also be prepared as summarized or similarly summarized in the following

CMU PCU PHU PPU (SM2)

In particular, (6aR,9aS)-3-chloro-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one (Int-5) may be prepared as described or similarly described below. The free base crystals and the mono-phosphate salt crystals of the invention may be prepared by using the methods described or similarly described in Examples 1-14 below.

Preparation of (6aR,9aS)-3-chloro-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one

(4-(6-fluoropyridin-2-yl)phenyl)methanol

The mixture of Na₂C0₃ (121 g), water (500 mL), THF (650 mL), PdCl₂(PPh₃)₂ (997 mg), 2-bromo-6-fluoropyridine (100 g) and 4-(hydroxymethyl)phenylboronic acid (90.7 g) is stirred at 65°C for 4 h under the nitrogen atmosphere. After cooling to room temperature, THF (200 mL) is added. The organic layer is separated and washed with 5% NaCl solution twice. The organic layer is concentrated to 400 mL. After the addition of toluene (100 mL), heptane (500 mL) is added at 55°C. The mixture is cooled to room temperature. The crystals are isolated by filtration, washed with the mixture of toluene (100 mL) and heptane (100 mL) and dried to give (4-(6-fluoropyridin-2-yl)phenyl)methanol (103 g). ^]H NMR (500 MHz, CDC1₃) δ 1.71-1.78 (m, 1H), 4.74-4.79 (m, 2H), 6.84-6.88 (m, 1H), 7.44-7.50 (m, 2H), 7.61-7.65 (m, 1H), 7.80-7.88 (m, 1H), 7.98-8.04 (m, 2H).

2-(4-(chloromethyl)phenyl)-6-fluoropyridine

The solution of thionylchloride (43.1 mL) in AcOEt (200 mL) is added to the mixture of (4-(6-fluoropyridin-2-yl)phenyl)methanol (100 g), DMF (10 mL) and AcOEt (600 mL) at room temperature. The mixture is stirred at room temperature for 1 h. After cooling to 10°C, 15% Na₂C0₃ solution is added. The organic layer is separated and washed with water (500 mL) and 5% NaCl solution (500 mL) twice. The organic layer is concentrated to 500 mL. After the addition of EtOH (500 mL), the mixture is concentrated to 500 mL. After addition of EtOH (500 mL), the mixture is concentrated to 500 mL. After the addition of EtOH (500 mL), the mixture is concentrated to 500 mL. After addition of EtOH (200 mL), water (700 mL) is added at 40°C. The mixture is stirred at room temperature. The crystals are isolated by filtration and dried to give 2-(4-(chloromethyl)phenyl)-6-fluoropyridine (89.5 g). ^]H NMR (500 MHz, CDC1₃) δ 4.64 (s, 2H), 6.86-6.90 (m, 1H), 7.47-7.52 (m, 2H), 7.60-7.65 (m, 1H), 7.82-7.88 (m, 1H), 7.98-8.03 (m, 2H).

6-chloro-l-(4-methoxybenzyl)-3-methylpyrimidine-2,4(lH,3H)-dione

The mixture of 6-chloro-3-methyluracil (100 g), p-methoxybenzylchloride (107 g), K2CO₃ (86.1 g) and DMAc (600 mL) is stirred at 75°C for 4 h. Water (400 mL) is added at 45°C and the mixture is cooled to room temperature. Water (800 mL) is added and the mixture is stirred at room temperature. The crystals are isolated by filtration, washed with the mixture of DMAc and water (1:2, 200mL) and dried to give 6-chloro-l-(4-methoxybenzyl)-3-methylpyrimidine-2,4(lH,3H)-dione (167 g). ^]H NMR (500 MHz, CDC1₃) δ 3.35 (s, 3H), 3.80 (s, 3H), 5.21 (s, 2H), 5.93 (s, 1H), 6.85-6.89 (m, 2H), 7.26-7.32 (m, 2H).

izinyl-l-(4-methoxybenzyl)-3-methylpyrimidine-2,4(lH,3H)-dione

The mixture of 6-chloro-l-(4-methoxybenzyl)-3-methylpyrimidine-2,4(lH,3H)-dione (165 g), IPA (990 mL), water (124 mL) and hydrazine hydrate (62.9 mL) is stirred at room temperature for 1 h. The mixture is warmed to 60°C and stirred at the same temperature for 4 h. Isopropyl acetate (1485 mL) is added at 45°C and the mixture is stirred at the same temperature for 0.5 h. The mixture is cooled at 10°C and stirred for lh. The crystals are isolated by filtration, washed with the mixture of IPA and isopropyl acetate (1:2, 330 mL) and dried to give 6-hydrazinyl-l-(4-methoxybenzyl)-3-methylpyrimidine-2,4(lH,3H)-dione (153 g). ^]H NMR (500 MHz, DMSO-i¾) δ 3.12 (s, 3H), 3.71 (s, 3H), 4.36 (s, 2H), 5.01 (s, 2H), 5.14 (s, 1H), 6.87-6.89 (m, 2H), 7.12-7.17 (m, 2H), 8.04 (s, 1H).

7-(4-methoxybenzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

To the mixture of DMF (725 mL) and 6-hydrazinyl-l-(4-methoxybenzyl)-3-methylpyrimidine-2,4(lH,3H)-dione (145 g) is added POCI₃ (58.5 mL) at 5°C. The mixture is stirred at room temperature for 1 h. Water (725 mL) is added at 50°C and the mixture is stirred at room temperature for 1 h. The crystals are isolated by filtration, washed with the mixture of DMF and water (1:1, 290 mL) and dried to give 7-(4-methoxybenzyl)-5-methyl-

2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (145 g). ^]H NMR (500 MHz, DMSO-i¾) δ 3.23 (s, 3H), 3.71 (s, 3H), 5.05 (s, 2H), 6.82-6.90 (m, 2H), 7.28-7.36 (m, 2H), 8.48 (s, IH), 13.51 (br, IH).

2-(4-(6-fluoropyridin-2-yl)benzyl)-7-(4-methoxybenzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

The mixture of 2-(4-(chloromethyl)phenyl)-6-fluoropyridine (100 g), 7-(4-methoxybenzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (129 g), K2CO₃(62.3 g) and DMAc (1500 mL) is stirred at 45°C for 5 h. Water (1500 mL) is added at 40°C and the mixture is stirred at room temperature for 1 h. The crystals are isolated by filtration, washed with the mixture of DMAc and water (1:1, 500 mL) and dried to give 2-(4-(6-fluoropyridin-2-yl)benzyl)-7-(4-methoxybenzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (207 g). ^]H NMR (500 MHz, DMSO- ) δ 3.21 (s, 3H), 3.66 (s, 3H), 4.98 (s, 2H), 5.45 (s, 2H), 6.77-6.82 (m, 2H), 7.13-7.16 (m, IH), 7.25-7.30 (m, 2H), 7.41-7.44 (m, 2H), 7.92-7.96 (m, IH), 8.04-8.11 (m, 3H), 8.68 (s, IH).

2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione

The mixture of 2-(4-(6-fluoropyridin-2-yl)benzyl)-7-(4-methoxybenzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (105 g), CF₃COOH (300 mL) and

CF₃SO₃H (100 g) is stirred at room temperature for 10 h. Acetonitrile (1000 mL) is added. The mixture is added to the mixture of 25% N¾ (1000 mL) and acetonitrile (500 mL) at 10°C. The mixture is stirred at room temperature for 1 h. The crystals are isolated by filtration, washed with the mixture of acetonitirile and water (1:1, 500 mL) and dried to give the crude product. The mixture of the crude product and AcOEt (1200 mL) is stirred at room temperature for 1 h. The crystals are isolated by filtration, washed with AcOEt (250 mL) and dried to give 2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (75.3 g). ^]H NMR (500 MHz, DMSO-rf₆) δ 3.16 (s, 3H), 3.50-4.00 (br, 1H), 5.40 (s, 2H), 7.13-7.16 (m, 1H), 7.41-7.44 (m, 2H), 7.91-7.94 (m, 1H), 8.04-8.10 (m, 3H), 8.60 (s, 1H).

2-(4-(6-fluoropyridin-2-yl)benzyl)-6-(((lR,2R)-2-hydroxycyclopentyl)amino)-5-methyl-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one

The mixture of BOP reagent (126 g), 2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (80 g), DBU (136 mL) and THF (1120 mL) is stirred at room temperature for 1 h. (lR,2R)-2-Aminocyclopentanol hydrochloride (37.6 g) and THF (80 mL) are added and the mixture is stirred at room temperature for 5 h. After the addition of 5% NaCl (400 mL) and AcOEt (800 mL), the organic layer is separated. The organic layer is washed with 10% NaCl (400 mL), 1M HC1 15% NaCl (400 mL), 5% NaCl (400 mL), 5% NaHC0₃ (400 mL) and 5%NaCl (400 mL) successively. After treatment with active charcoal, the organic layer is concentrated to 400 mL. After the addition of acetonitrile (800 mL), the mixture is concentrated to 400 mL. After the addition of acetonitrile (800 mL), seed crystals are added at 40°C. The mixture is concentrated to 400 mL. Water (800 mL) is added at room temperature and the mixture is stirred for 2 h. The crystals are isolated by filtration, washed with the mixture of acetonitrile and water (1:2, 400 mL) and dried to give 2-(4-(6-fluoropyridin-2-yl)benzyl)-6-(((lR,2R)-2-

hydroxycyclopentyl)amino)-5-methyl-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (81.7 g). ^]H NMR (500 MHz, CDC1₃) δ 1.47-1.59 (m, 1H), 1.68-1.93 (m, 3H), 2.02-2.12 (m, 1H), 2.24-2.34 (m, 1H), 3.42 (s, 3H), 3.98-4.12 (m, 2H), 4.68-4.70 (m, 1H), 5.37 (s, 2H), 6.86-6.90 (m, 1H), 7.36-7.42 (m, 2H), 7.58-7.63 (m, 1H), 7.81-7.88 (m, 1H), 7.89 (s, 1H), 7.97-8.01 (m, 2H).

(6aR,9aS)-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one

The mixture of 2-(4-(6-fluoropyridin-2-yl)benzyl)-6-(((lR,2R)-2-hydroxycyclopentyl)amino)-5-methyl-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (80 g), p-toluenesulfonylchloride (38.6 g), Et₃N (28.2 mL), N,N-dimethylaminopyridine (24.7 g) and THF (800 mL) is stirred at 50°C for 10 h. To the mixture is added 8M NaOH (11.5 mL) at room temperature and the mixture is stirred for 2 h. After the addition of 5% NaCl (400 mL) and AcOEt (800 mL), the organic layer is separated. The organic layer is washed with 5 NaCl (400 mL) twice. The organic layer is concentrated to 240 mL. After the addition of MeOH (800 mL), the mixture is concentrated to 240 mL. After the addition of MeOH (800 mL), the mixture is concentrated to 240 mL. After the addition of MeOH (160 mL), the mixture is stirred at room temperature for 1 h and at 0°C for 1 h. The crystals are isolated by filtration, washed with cold MeOH (160 mL) and dried to give (6aR,9aS)-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one (55.7 g). ^]H NMR (500 MHz, CDC1₃) δ 1.39-1.54 (m, 1H), 1.58-1.81 (m, 3H), 1.81-1.92 (m, 1H), 2.12-2.22 (m, 1H), 3.28 (s, 3H), 4.61-4.70 (m, 2H), 5.20 (s, 2H), 6.79-6.85 (m, 1H), 7.25-7.32 (m, 2H), 7.53-7.58 (m, 1H), 7.68 (s, 1H), 7.75-7.83 (m, 1H), 7.92-7.98 (m, 2H).

(6aR,9aS)-3-chloro-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-5,6a,7,8,9,9a-

hexahydrocyclopenta[4,5]imi ]pyrimidin-4(2H)-one

The mixture of (6aR,9aS)-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one (50 g) and toluene (1000 mL) is concentrated to 750 mL under the nitrogen atmosphere. Toluene (250 mL) and NCS (24 g) is added. To the mixture is added LiHMDS (1M THF solution, 204 mL) at 0°C and the mixture is stirred for 0.5 h. To the mixture is added 20% NH₄C1 (50 mL) at 5°C. The mixture is concentrated to 250 mL. After the addition of EtOH (250 mL), the mixture is concentrated to 150 mL. After the addition of EtOH (250 mL), the mixture is concentrated to 200 mL. After the addition of EtOH (200 mL), the mixture is warmed to 50°C. Water (300 mL) is added and the mixture is stirred at 50°C for 0.5 h. After stirring at room temperature for 1 h, the crystals are isolated by filtration, washed with the mixture of EtOH and water (1:1, 150 mL) and dried to give (6aR,9aS)-3-chloro-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one (51.1 g). ^]H NMR (500 MHz, CDC1₃) δ 1.46-1.61 (m, 1H), 1.67-1.90 (m, 3H), 1.92-2.00 (m, 1H), 2.19-2.27 (m, 1H), 3.37 (s, 3H), 4.66-4.77 (m, 2H), 5.34 (s, 2H), 6.87-6.93 (m, 1H), 7.35-7.41 (m, 2H), 7.59-7.65 (m, 1H), 7.82-7.91 (m, 1H), 7.97-8.05 (m, 2H).

EXAMPLE 1

Crystals of (6a/f,9a5)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one free base mono-ethanol solvate

The mixture of (6a/?,9a5′)-3-chloro-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one (2.5 g), K₂C0₃ (1.53 g), Pd(OAc)₂ (12.5 mg), Xantphos (32 mg), aniline (0.76 mL), and xylene (12.5 mL) is stirred at 125°C for 7 h under nitrogen atmosphere. After addition of water (12.5 mL), the organic layer is separated. The organic layer is washed with water (12.5 mL) twice. The organic layer is extracted with the mixture of DMAc (6.25 mL) and 0.5N HCl (12.5 mL). The organic layer is extracted with the mixture of DMAc (3.2 mL) and 0.5N HCl (6.25 mL). After addition of DMAc (6.25 mL), xylene (12.5 mL) and 25 wt % aqueous NH₃ solution to the combined aqueous layer, the organic layer is separated. The aqueous layer is extracted with xylene (6.25 mL). The combined organic layer is washed with water (12.5 mL), 2.5 wt % aqueous 1 ,2-cyclohexanediamine solution (12.5 mL) twice and water (12.5 mL) successively. After treatment with active charcoal, the organic layer is concentrated. After addition of EtOH (12.5 mL), the mixture is concentrated. After addition of EtOH (12.5 mL), the mixture is concentrated. After addition of EtOH (12.5 mL), n-heptane (25 mL) is added at 70°C. The mixture is cooled to 5°C and stirred at same temperature. The crystals are isolated by filtration and dried to give (ea^^a^-S^a ^^^a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one free base mono-ethanol solvate (2.56 g) as crystals.

^]H NMR (500 MHz, DMSO-d₆) δ 0.98-1.13 (m, 3H), 1.34-1.52 (m, 1H), 1.54-1.83 (m, 4H), 2.03-2.17 (m, 1H), 3.11 (s, 3H), 3.39-3.54 (m, 2H), 4.29-4.43 (m, 1H), 4.51-4.60 (m, 1H), 4.60-4.70 (m, 1H), 5.15-5.35 (m, 2H), 6.71-6.88 (m, 3H), 7.05-7.29 (m, 5H), 7.81-7.93 (m, 1H), 7.94-8.11 (m, 3H), 8.67 (s, 1H).

EXAMPLE 4

Crystals of (6a/f,9a5)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one free

Crystals of (6a«,9a5′)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one free base mono-n-propanol solvate (2.0 g) is dissolved with ethanol (10 mL) at 70°C. Isopropyl ether (20 mL) is added and the mixture is cooled to 45°C. Isopropyl ether (10 mL) is added and the mixture is stirred at 40°C. The mixture is cooled to 5°C and stirred at same temperature. The crystals are isolated by filtration and dried to give (ea/^^a^)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one free base non-solvate (1.7 g) as crystals.

[0082] ^]H NMR (500 MHz, DMSO-d₆) δ 1.32-1.51 (m, 1H), 1.53-1.83 (m, 4H), 1.97-2.20 (m, 1H), 3.11 (s, 3H), 4.49-4.60 (m, 1H), 4.60-4.69 (m, 1H), 5.13-5.37 (m, 2H), 6.70-6.90 (m, 3H), 7.04-7.31 (m, 5H), 7.82-7.93 (m, 1H), 7.93-8.12 (m, 3H), 8.67 (s, 1H).

EXAMPLE 5

Crystals of (6a/f,9a5)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one free base non-solvate

The mixture of (6a/?,9a5′)-3-chloro-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one (25 g), K₂C0₃ (15.4 g), Pd(OAc)₂ (125 mg), Xantphos (321 mg), aniline (7.6 mL), DMAc (6.25 mL) and xylene (125 mL) is stirred at 125°C for 6.5 h under nitrogen atmosphere. After addition of water (125 mL) and DMAc (50 mL), the organic layer is separated. The organic layer is washed with the mixture of DMAc (50 mL) and water (125 mL) twice. The organic layer is extracted with the mixture of DMAc (50 mL) and 0.5N HCl (125 mL). The organic layer is extracted with the mixture of DMAc (50 mL) and 0.5N HCl (62.5 mL). After addition of DMAc (50 mL), xylene (125 mL) and 25 wt % aqueous NH₃ solution (25 mL) to the combined aqueous layer, the organic layer is separated. The aqueous layer is extracted with xylene (62.5 mL). The combined organic layer is washed with the mixture of DMAc (50 mL) and water (125 mL), the mixture of DMAc (50 mL) and 2.5 wt % aqueous 1,2-cyclohexanediamine solution (125 mL) twice and the mixture of DMAc (50 mL) and water (125 mL) successively. After treatment with active charcoal (1.25 g), the organic layer is concentrated to 75 mL. After addition of EtOH (125 mL), the mixture is concentrated to 75 mL. After addition of EtOH (125 mL), the mixture is concentrated to 75 mL. After addition of EtOH (125 mL), n-heptane (250 mL) is added at 70°C. After addition of seed crystals of (6a/?,9a5′)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one non-solvate, the mixture is cooled to room temperature and stirred at room temperature. The crystals are isolated by filtration and dried to give (ea^^a^-S^a ^^^a-hexahydro-S-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo-[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one free base non-solvate (23.8 g) as crystals.

EXAMPLE 8

(6a/f,9a5)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one mono-phosphate salt

[0094] Crystals of (6a«,9a5′)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one free base non-solvate (20 g) are dissolved in acetonitrile (60 mL) at 50°C. After addition of the active charcoal (1 g), the mixture is stirred at same temperature for 0.5 h. The active charcoal is removed by filtration and washed with acetonitrile (40 mL). The filtrate and the washing are combined and warmed to 50°C. A solution of 85 wt. % phosphoric acid (2.64 mL) in acetonitrile (100 mL) is added. After addition of water (20 mL), the mixture is stirred at 50°C for lh. The crystals are isolated by filtration, washed with acetonitrile (60 mL x 3) and dried to give (6a/?,9a5′)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one mono-phosphate salt (20.5 g).

EXAMPLE 9

(6a/f,9a5)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one mono-phosphate salt

[0095] Crystals of (6a«,9a5′)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one free base mono-ethanol solvate (4 g) are dissolved in acetonitrile (12 mL) at 50°C. After addition of active charcoal (0.2 g), the mixture is stirred at same temperature for 0.5 h. Active charcoal is removed by filtration and washed with acetonitrile (8 mL). The filtrate and the washing are combined and warmed to 50°C. A solution of 85 wt. % phosphoric acid (0.528 mL) in acetonitrile (20 mL) is added. After addition of water (4 mL), the mixture is stirred at 50°C for lh. The crystals are isolated by filtration, washed with acetonitrile (12 mL x 3) and dried to give (6a/?,9a5′)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one mono-phosphate salt (4.01 g).

EXAMPLE 10

(6a/f,9a5)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one mono-phosphate salt

Crystals of (6a«,9a5′)-5,6a,7,8,9,9a-Hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one free base non-solvate (20 g) are dissolved in acetone (60 mL) at 32°C. After addition of active charcoal (1 g), the mixture is stirred at same temperature for 0.5 h. Active charcoal is removed by filtration and washed with acetone (40 mL). The filtrate and the washing are combined and warmed to 39°C. A solution of 85 wt. % phosphoric acid (2.64 mL) in acetone (100 mL) is added. After addition of water (20 mL), the mixture is stirred at 40°C for lh. The crystals are isolated by filtration, washed with acetone (60 mL x 3) and dried to give (6a/?,9a5′)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one mono-phosphate salt (22.86 g).

EXAMPLE 11

(6a/f,9a5)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one mono-phosphate salt

Crystals of (6a«,9a5′)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one free base mono-ethanol solvate (20 g) are dissolved in acetone (60 mL) at 38°C. After addition of active charcoal (1 g), the mixture is stirred at same temperature for 0.5 h. Active charcoal is removed by filtration and washed with acetone (40 mL). The filtrate and the washing are combined and warmed to 38°C. A solution of 85 wt. % phosphoric acid (2.64 mL) in acetone (100 mL) is added. After addition of water (20 mL), the mixture is stirred at 40°C for lh. The crystals are isolated by filtration, washed with acetone (60 mL x 3) and dried to give (6a/?,9a5′)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-((4-(6-fluoropyridin-2-yl)phenyl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one mono-phosphate salt (23.2 g).

PAPER

A diverse set of 3-aminopyrazolo[3,4-d]pyrimidinones was designed and synthesized. The structure–activity relationships of these polycyclic compounds as phosphodiesterase 1 (PDE1) inhibitors were studied along with their physicochemical and pharmacokinetic properties. Systematic optimizations of this novel scaffold culminated in the identification of a clinical candidate, (6aR,9aS)-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-3-(phenylamino)-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4-(2H)-one phosphate (ITI-214), which exhibited picomolar inhibitory potency for PDE1, demonstrated excellent selectivity against all other PDE families and showed good efficacy in vivo. Currently, this investigational new drug is in Phase I clinical development and being considered for the treatment of several indications including cognitive deficits associated with schizophrenia and Alzheimer’s disease, movement disorders, attention deficit and hyperactivity disorders, and other central nervous system (CNS) and non-CNS disorders

Discovery of Potent and Selective Inhibitors of Phosphodiesterase 1 for the Treatment of Cognitive Impairment Associated with Neurodegenerative and Neuropsychiatric Diseases

Peng Li^*^†, Hailin Zheng^†, Jun Zhao^†, Lei Zhang^†, Wei Yao^†, Hongwen Zhu^†, J. David Beard^†, Koh Ida^§, Weston Lane^‡, Gyorgy Snell^‡, Satoshi Sogabe^§, Charles J. Heyser^∥, Gretchen L. Snyder^†, Joseph P. Hendrick^†, Kimberly E. Vanover^†, Robert E. Davis^†, and Lawrence P. Wennogle^†

^†Intra-Cellular Therapies, Inc., 430 East 29th Street, Suite 900, New York, New York 10016, United States

^‡ Department of Structural Biology, Takeda California, Inc., 10410 Science Center Drive, San Diego, California 92121,United States

^§ Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan

^∥ Department of Neurosciences, University of California, San Diego, 9500 Gilman Drive, #0608, La Jolla, California 92093,United States

J. Med. Chem., 2016, 59 (3), pp 1149–1164

DOI: 10.1021/acs.jmedchem.5b01751

Publication Date (Web): January 20, 2016

*Phone: 646-440-9388. E-mail: pli@intracellulartherapies.com.

http://pubs.acs.org/doi/full/10.1021/acs.jmedchem.5b01751

Step g. (6aR,9aS)-5-Methyl-3-(phenylamino)-2-(4-(6-fluoropyridin-2-yl)-benzyl)-5,6a,7,8,9,9a-hexahydrocyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one phosphate (3)

………… to give (6aR,9aS)-5-methyl-3-(phenylamino)-2-(4-(6-fluoropyridin-2-yl)-benzyl)-5,6a,7,8,9,9a-hexahydrocyclopent[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one as an off-white solid

BASE FORM

¹H NMR (500 MHz, CDCl₃) δ 7.89 (d, J = 8.3 Hz, 2H), 7.86–7.79 (m, 1H), 7.58 (dd, J = 7.6, 2.5 Hz, 1H), 7.35–7.26 (m, 2H), 7.15–7.08 (m, 1H), 7.05 (d, J = 8.3 Hz, 2H), 6.94 (d, J = 7.6 Hz, 2H), 6.90 (br, 1H), 6.86 (dd, J = 8.1, 3.0 Hz, 1H), 4.96 (s, 2H), 4.88–4.70 (m, 2H), 3.38 (s, 3H), 2.29 (dd, J = 13.0, 6.1 Hz, 1H), 2.15–1.96 (m, 1H), 1.90–1.71 (m, 3H), 1.65–1.52 (m, 1H).

¹³C NMR (126 MHz, CDCl₃) δ 163.4 (d, J_CF = 239 Hz), 159.7, 155.7 (d, J_CF = 13 Hz), 153.0, 147.6, 144.1, 141.7 (d, J_CF = 8 Hz), 140.5, 137.3, 137.1, 129.6, 127.8, 127.1, 124.1, 120.2, 117.3 (d, J_CF = 4 Hz), 107.9 (d, J_CF = 38 Hz), 89.5, 69.9, 62.6, 52.8, 35.4, 32.3, 28.5, 23.2.

MS (ESI) m/z 508.3 [M + H]⁺.

PHOSPHATE SALT

¹H NMR (500 MHz, DMSO-d₆) δ 8.71 (br, 1H), 8.10–8.01 (m, 1H), 7.98 (d, J = 8.3 Hz, 2H), 7.89 (dd, J = 7.6, 2.6 Hz, 1H), 7.23 (d, J = 8.4 Hz, 2H), 7.16 (dd, J = 8.5, 7.3 Hz, 2H), 7.12 (dd, J = 8.1, 2.8 Hz, 1H), 6.86–6.81 (m, 1H), 6.80–6.76 (m, 2H), 5.34–5.19 (m, 2H), 4.77–4.64 (m, 1H), 4.62–4.53 (m, 1H), 3.12 (s, 3H), 2.11 (dd, J = 13.4, 5.7 Hz, 1H), 1.81–1.57 (m, 4H), 1.54–1.41 (m, 1H).

¹³C NMR (126 MHz, CDCl₃) δ 162.6 (d, J_CF = 236 Hz), 155.9, 154.4 (d, J_CF= 13 Hz), 152.4, 146.6, 143.0 (d, J_CF = 8 Hz), 142.5, 141.8, 138.1, 136.0, 128.7, 127.5, 126.7, 120.4, 117.7 (d, J_CF = 4 Hz), 116.0, 108.1 (d, J_CF = 37 Hz), 90.3, 66.3, 62.4, 50.6, 34.2, 31.2, 28.5, 22.5.

MS (ESI) m/z 508.3 [M + H]⁺.

HRMS (ESI) m/z calcd for C₂₉H₂₇N₇OF [M (free base)+H]⁺, 508.2261; found, 508.2272.

HPLC purity, 100.0%; retention time, 13.0 min.

PATENT

WO2015196186

The synthetic methods disclosed in WO 2009/075784 and WO 2013/192556 are particularly applicable, as they include the methods to prepare the compound of Formula I-B. Those skilled in the art will readily see how those methods are applicable to the synthesis of the compounds of the present invention.

Formula I-B

For example, Compounds of the Invention wherein any one or more of R¹ through R⁸ are D, can be prepared from the corresponding aminocyclopentanol, according to the method described in WO 2009/075784 or WO 2013/192556. For example, by reacting said aminocyclopentanol, optionally as its acid salt, with Intermediate A in the presence of a coupling agent, e.g., benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent), and a base, e.g., l,8-diazabicyclo[5.4.0]undec-7-ene (DBU), in a solvent such as tetrahydrofuran (THF). The intermediate alcohol is then cyclized by treatment with toluenesulfonyl chloride (TsCl) in the presence of one or more bases, such as dimethylaminopyridine (DMAP) and triethylamine (TEA) in a solvent, such as THF. The reaction is summarized in the following scheme:

The required aminocyclopentanols can be prepared by methods known to those skilled in the art. For example, the aminocyclopentanol wherein R¹ is D can be prepared via a reductive amination procedure that uses a reducing agent such as sodium triacetoxyborodeuteride or sodium borodeuteride as the reducing agent. For example, an optionally protected (R)-2-hydroxycyclopentanone can be reacted with 4-methoxybenzylamine in the presence of sodium triacetoxyborodeuteride to yield the desired deuterated secondary amine, wherein P is the protecting group. Reaction of the resulting amine with a strong acid such as trifluoromethanesulfonic acid (TMFSA) will result in removal of the 4-methoxybenzyl group and the protecting group to yield the desired aminocyclopentanol. Those skilled in the art will know how to choose a suitable protecting group for the secondary alcohol such that deprotection can take place during the acid treatment step (e.g., a tert-butyldimethylsilyl group or a tert-butoxycarbonyl group). Alternatively, those skilled in the art could choose a protecting group that would survive this step. If desired, the protected intermediate can be purified by chiral HPLC in order to enhance the optical purity of the final

As another example, Compounds of the Invention wherein any one or more of R⁹ to R¹⁵ or R²¹ to R²² are D can be prepared from the corresponding benzyl halide, according to the method described in WO 2009/075784 or WO 2013/192556. For example, by reacting said benzyl halide with the Intermediate B in the presence of suitable base, such as cesium carbonate or potassium carbonate, in a suitable solvent, such as dimethylformamide or dimethylacetamide. The corresponding benzyl halide can be prepared by methods well known to those skilled in the art. The reaction is summarized in the following scheme:

As another example, compounds of the invention wherein any one or more of R¹⁶ to R²⁰ are D can be prepared from the corresponding phenyl

isothiocyanate, according to the method described in WO 2009/075784 or WO

2013/192556. For example, by reacting said phenyl isothiocyanate with Intermediate C in a suitable solvent, such as dimethylformamide. The corresponding phenyl isothiocyanate can be prepared by methods well known to those skilled in the art. The reaction is summarized in the following scheme:

Alternatively, compounds of the invention wherein any one or more of R¹⁶ to R²⁰ are D can be prepared from the corresponding aniline, according to the method described in WO 2009/075784 or WO 2013/192556. For example, by reacting said aniline with Intermediate D and a strong base, such as lithium

hexamethyldisilylazide (LiHMDS), in a suitable solvent, such as THF at elevated temperature. Such a reaction can also be achieved by catalytic amination using a catalyst, such as tris(dibenzylideneacetone)dipalladium (Pd₂(dba)3), and a ligand, such as Xantphos. The corresponding aniline can be prepared by methods well known to those skil

EXAMPLE 1. (6aR,9a5)-5-Methyl-3-(2,3,4,5,6-pentadeuterophenylamino)-2-(4-(6-fluoropyridin-2-yl)-benzyl)-5,6fl,7,8,9,9fl-hexahydrocyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one

To a solution of (6a/?,9a5′)-5,6a,7,8,9,9a-hexahydro-3-chloro-5-methyl-2-(4-(6-fluoropyridin-2-yl)-benzyl)-cyclopent[4,5]irnidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one (200 mg, 0.444 mmol) and 2,3,4,5,6-pentadeuteroaniline (162 μΐ,, 1.8 mmol) in anhydrous 2-methyltetrahydrofuran (3 mL) is added LiHMDS (1.0 M in THF, 0.89 mL) dropwise at room temperature under argon atmosphere. The reaction mixture is gradually heated to 75 °C over a period of 90 min, and then heated at 75 °C for an hour. The mixture is cooled with an ice bath and then quenched by adding 0.2 mL of water. After solvent evaporation, the residue is dissolved in DMF and then filter with a 0.45 m microfilter. The collected filtrated is purified with a semi-preparative HPLC system using a gradient of 0 – 70% acetonitrile in water containing 0.1% formic acid over 16 min to give (6a/?,9a5′)-5-methyl-3-(2,3,4,5,6-pentadeuterophenylamino)-2-(4-(6-fluoropyridin-2-yl)-benzyl)-5,6fl,7,8,9,9fl-hexahydrocyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one as a formate salt, which is dissolved in ethyl acetate, basified with 12.5 mL of 5% sodium carbonate, and then extracted with ethyl acetate three times. The combined organic phase is evaporated to dryness. The residue is dissolved in 4.5 mL of THF and then filter through a 0.45 m microfilter. The filtrate is evaporated to dryness and further dried under vacuum to give (6a/?,9a5′)-5-methyl-3-(2,3,4,5,6-pentadeuterophenylamino)-2-(4-(6-fluoropyridin-2-yl)-benzyl)-5,6fl,7,8,9,9fl-hexahydrocyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one as a white solid (185.8 mg, 81.6% yield). ¾ NMR (400 MHz, CDCb) δ 7.88 (d, / = 8.4 Hz, 2H), 7.88 – 7.77 (m, 1H), 7.58 (dd, J = 7.5, 2.4 Hz, 1H), 7.05 (d, J = 8.3 Hz, 2H), 6.90 – 6.80 (m, 2H), 4.94 (s, 2H), 4.82 – 4.68 (m, 2H), 3.34 (s, 3H), 2.27 (dd, / = 12.4, 5.7 Hz, 1H), 2.09 – 1.91 (m, 1H), 1.91 – 1.67 (m, 3H), 1.67 – 1.49 (m, 1H).MS (ESI) m/z 513.3 [M+H]⁺.

Nov 3, 2014

Intra-Cellular Therapies and Takeda Announce Mutual Termination of Collaboration to Develop Phosphodiesterase (PDE1) Inhibitors for CNS Disorders

NEW YORK and OSAKA, Japan, Nov. 3, 2014 (GLOBE NEWSWIRE) — Intra-Cellular Therapies, Inc. (Nasdaq:ITCI) and Takeda Pharmaceutical Company Limited announced today that they have entered into an agreement to mutually terminate the February 2011 license agreement covering Intra-Cellular Therapies’ proprietary compound ITI-214 and related PDE1 inhibitors and to return the rights for these compounds to Intra-Cellular Therapies.

Under the terms of the agreement, Intra-Cellular Therapies has regained all worldwide development and commercialization rights for the compounds previously licensed to Takeda. Takeda will be responsible for transitioning the compounds back toIntra-Cellular Therapies and will not participate in future development or commercialization activities. After transition of the program, Intra-Cellular Therapies plans to continue the clinical development of PDE1 inhibitors for the treatment of central nervous system, cardiovascular and other disorders.

“We are grateful for Takeda’s substantial efforts in advancing this program into clinical development,” said Dr. Sharon Mates, Chairman and CEO of Intra-Cellular Therapies. “This provides us with the opportunity to unify our PDE1 platform and we look forward to continuing the development of ITI-214 and our other PDE1 inhibitors.”

Intra-Cellular Therapies will discuss the PDE1 program in its previously announced earnings call on Monday, November 3, 2014 at 8:30 a.m. Eastern Time. To participate in the conference call, please dial 844-835-6563 (U.S.) or 970-315-3916 (International) five to ten minutes prior to the start of the call. The participant passcode is 25568442.

About PDE1 Inhibitors

PDE1 inhibitors are unique, orally available, investigational drug candidates being developed for the treatment of cognitive impairments accompanying schizophrenia, Alzheimer’s disease and other neuropsychiatric disorders and neurological diseases and may also treat patients with Attention Deficit Hyperactivity Disorder and Parkinson’s disease. These compounds may also have the potential to improve motor dysfunction associated with these conditions and may also have the potential to treat patients with multiple sclerosis and other autoimmune diseases and pulmonary arterial hypertension. These compounds are very selective for the PDE1 subfamily relative to other PDE subfamilies. They have no known significant off target activities at other enzymes, receptors or ion channels.

About Intra-Cellular Therapies

Intra-Cellular Therapies, Inc. (the “Company”) is developing novel drugs for the treatment of neuropsychiatric and neurodegenerative disease and other disorders of the central nervous system (“CNS”). The Company is developing its lead drug candidate, ITI-007, for the treatment of schizophrenia, behavioral disturbances in dementia, bipolar disorder and other neuropsychiatric and neurological disorders. The Company is also utilizing its phosphodiesterase platform and other proprietary chemistry platforms to develop drugs for the treatment of CNS disorders.

About Takeda Pharmaceutical Company Limited

Located in Osaka, Japan, Takeda is a research-based global company with its main focus on pharmaceuticals. As the largest pharmaceutical company in Japan and one of the global leaders of the industry, Takeda is committed to strive towards better health for people worldwide through leading innovation in medicine. Additional information about Takeda is available through its corporate website, www.Takeda.com.

Source: Intra-Cellular Therapies, Inc.; Takeda Pharmaceutical Company Limited

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