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IRAK4 is responsible for initiating signaling from Toll-like receptors (TLRs) and members of the IL-1/18 receptor family. Kinase-inactive knock-ins and targeted deletions of IRAK4 in mice cause reductions in TLR induced pro-inflammatory cytokines and these mice are resistant to various models of arthritis. Herein we report the identification and optimization of a series of potent IRAK4 inhibitors. Representative examples from this series showed excellent selectivity over a panel of kinases, including the kinases known to play a role in TLR-mediated signaling. The compounds exhibited low nM potency in LPS- and R848-induced cytokine assays indicating that they are blocking the TLR signaling pathway. A key compound (26) from this series was profiled in more detail and found to have an excellent pharmaceutical profile as measured by predictive assays such as microsomal stability, TPSA, solubility, and c log P. However, this compound was found to afford poor exposure in mouse upon IP or IV administration. We found that removal of the ionizable solubilizing group (32) led to increased exposure, presumably due to increased permeability. Compounds 26 and 32, when dosed to plasma levels corresponding to ex vivo whole blood potency, were shown to inhibit LPS-induced TNFα in an in vivo murine model. To our knowledge, this is the first published in vivo demonstration that inhibition of the IRAK4 pathway by a small molecule can recapitulate the phenotype of IRAK4 knockout mice.
In the past decade there has been considerable interest in targeting the innate immune system in the treatment of autoimmune diseases and sterile inflammation. Receptors of the innate immune system provide the first line of defense against bacterial and viral insults. These receptors recognize bacterial and viral products as well as pro-inflammatory cytokines and thereby initiate a signaling cascade that ultimately results in the up-regulation of inflammatory cytokines such as TNFα, IL6, and interferons. Recently it has become apparent that self-generated ligands such as nucleic acids and products of inflammation such as HMGB1 and Advanced Glycated End-products (AGE) are ligands for Toll-like receptors (TLRs) which are key receptors of the innate immune system.
This demonstrates the role of TLRs in the initiation and perpetuation of inflammation due to autoimmunity.
Interleukin-1 receptor associated kinase (IRAK4) is a ubiquitously expressed serine/threonine kinase involved in the regulation of innate immunity. IRAK4 is responsible for initiating signaling from TLRs and members of the IL-1/18 receptor family. Kinase-inactive knock-ins and targeted deletions of IRAK4 in mice lead to reductions in TLR and IL-1 induced pro-inflammatory cytokines. and 7 IRAK-4 kinase-dead knock-in mice have been shown to be resistant to induced joint inflammation in the antigen-induced-arthritis (AIA) and serum transfer-induced (K/BxN) arthritis models. Likewise, humans deficient in IRAK4 also display the inability to respond to challenge by TLR ligands and IL-1
However, the immunodeficient phenotype of IRAK4-null individuals is narrowly restricted to challenge by gram positive bacteria, but not gram negative bacteria, viruses or fungi. This gram positive sensitivity also lessens with age implying redundant or compensatory mechanisms for innate immunity in the absence of IRAK4.These data suggest that inhibitors of IRAK4 kinase activity will have therapeutic value in treating cytokine driven autoimmune diseases while having minimal immunosuppressive side effects. Additional recent studies suggest that targeting IRAK4 may be a viable strategy for the treatment of other inflammatory pathologies such as atherosclerosis.
Indeed, the therapeutic potential of IRAK4 inhibitors has been recognized by others within the drug-discovery community as evidenced by the variety of IRAK4 inhibitors have been reported to-date.12, 13, 14, 15 and 16 However, limited data has been published about these compounds and they appear to suffer from a variety of issues such as poor kinase selectivity and poor whole-blood potency that preclude their advancement into the pre-clinical models. To the best of our knowledge, no in vivo studies of IRAK4 inhibitors have been reported to-date in the literature. Herein we report a new class of IRAK4 inhibitors that are shown to recapitulate the phenotype observed in IRAK4 knockout and kinase-dead mice.
aPfizer Global R&D, 445 Eastern Point Rd., Groton, CT 06340, USA
bPfizer Global R&D, 200 Cambridge Park Dr., Cambridge, MA 02140, USA
cPfizer Global R&D, 87 Cambridgepark Dr., Cambridge, MA 02140, USA
dPfizer Global R&D, 1 Burtt Rd., Andover, MA 01810, USA
Abstract
IRAK4 is responsible for initiating signaling from Toll-like receptors (TLRs) and members of the IL-1/18 receptor family. Kinase-inactive knock-ins and targeted deletions of IRAK4 in mice cause reductions in TLR induced pro-inflammatory cytokines and these mice are resistant to various models of arthritis. Herein we report the identification and optimization of a series of potent IRAK4 inhibitors. Representative examples from this series showed excellent selectivity over a panel of kinases, including the kinases known to play a role in TLR-mediated signaling. The compounds exhibited low nM potency in LPS- and R848-induced cytokine assays indicating that they are blocking the TLR signaling pathway. A key compound (26) from this series was profiled in more detail and found to have an excellent pharmaceutical profile as measured by predictive assays such as microsomal stability, TPSA, solubility, and c log P. However, this compound was found to afford poor exposure in mouse upon IP or IV administration. We found that removal of the ionizable solubilizing group (32) led to increased exposure, presumably due to increased permeability. Compounds 26 and 32, when dosed to plasma levels corresponding to ex vivo whole blood potency, were shown to inhibit LPS-induced TNFα in an in vivo murine model. To our knowledge, this is the first published in vivo demonstration that inhibition of the IRAK4 pathway by a small molecule can recapitulate the phenotype of IRAK4 knockout mice.
2.5 and 5 mg/kg, ig (MRL/lpr mice);
10 mg·kg−1·d−1, ig (NZB/W F1 mice)
Autoimmune diseases; SLE
SM934, an artemisinin derivative, possesses potent antiproliferative and antiinflammatory properties.
In the present study, we investigated the immunosuppressive effects and underlying mechanisms of beta-aminoarteether maleate (SM934), a derivative of artemisinin, against T cell activation in vitro and in vivo. In vitro, SM934 significantly inhibited the proliferation of splenocytes induced by concanavalin A (Con A), lipopolysaccharide (LPS), mixed lymphocyte reaction (MLR), and anti-CD3 plus anti-CD28 (anti-CD3/28). SM934 significantly inhibited interferon (IFN)-gamma production and CD4(+) T cell division stimulated by anti-CD3/28. SM934 also promoted apoptosis of CD69(+) population in CD4(+) T cells stimulated by anti-CD3/28. Furthermore, SM934 inhibited interleukin (IL)-2 mediated proliferation and survival through blocking Akt phosphorylation in activated T cells. In ovalbumin (OVA)-immunized mice, oral administration of SM934 suppressed OVA-specific T cell proliferation and IFN-gamma production. SM934 treatment also significantly inhibited the sheep red blood cell (SRBC)-induced delayed type hypersensitivity (DTH) reactions in mice. Taken together, SM934 showed potent immunosuppressive activities in vitro and in vivo. Our results demonstrated that SM934 might be a potential therapeutic agent for immune-related diseases.
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, People’s Republic of China
bDepartment of Synthetic Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, People’s Republic of China
cLaboratory of Immunology and Virology, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, People’s Republic of China
Hou LF, He SJ, Wang JX, Yang Y, Zhu FH, Zhou Y, et al. SM934, a water-soluble derivative of arteminisin, exerts immunosuppressive functions in vitro and in vivo. Int Immunopharmacol 2009; 9: 1509–17. | Article |
Hou LF, He SJ, Li X, Yang Y, He PL, Zhou Y, et al. Oral administration of artemisinin analog SM934 ameliorates lupus syndromes in MRL/lpr mice by inhibiting Th1 and Th17 cell responses. Arthritis Rheum 2011; 63: 2445–55. | Article
Hou LF, He SJ, Li X, Wan CP, Yang Y, Zhang XH, et al. SM934 treated lupus-prone NZB x NZW F1 mice by enhancing macrophage interleukin-10 production and suppressing pathogenic T cell development. PLoS One 2012; 7: e 32424.
Wu Y, He S, Bai B, Zhang L, Xue L, Lin Z, et al. Therapeutic effects of the artemisinin analog SM934 on lupus-prone MRL/lpr mice via inhibition of TLR-triggered B-cell activation and plasma cell formation. Cell Mol Immunol 2015 Mar 16. doi: 10.1038/cmi.2015.13. [Epub ahead of print].
/////////TLR7/9 signal transduction modulator, SM 934, IND FILED, 133162-25-1, β-Aminoarteether maleate
A MEK1/Raf inhibitor potentially for the treatment of solid tumors and multiple myeloma.
RO-5126766; RG-7304; CH-5126766; CKI-27; R-7304
CAS No. 946128-88-7
Although melanoma is the most aggressive skin cancer, recent advances in BRAF and/or MEK inhibitors against BRAF-mutated melanoma have improved survival rates. Despite these advances, a treatment strategy targeting NRAS-mutated melanoma has not yet been elucidated. We discovered CH5126766/RO5126766 as a potent and selective dual RAF/MEK inhibitor currently under early clinical trials. We examined the activity of CH5126766/RO5126766 in a panel of malignant tumor cell lines including melanoma with a BRAF or NRAS mutation. Eight cell lines including melanoma were assessed for their sensitivity to the BRAF, MEK, or RAF/MEK inhibitor using in vitro growth assays. CH5126766/RO5126766 induced G1 cell cycle arrest in two melanoma cell lines with the BRAF V600E or NRAS mutation. In these cells, the G1 cell cycle arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27 and down-regulation of cyclinD1. CH5126766/RO5126766 was more effective at reducing colony formation than a MEK inhibitor in NRAS- or KRAS-mutated cells. In the RAS-mutated cells, CH5126766/RO5126766 suppressed the MEK reactivation caused by a MEK inhibitor. In addition, CH5126766/RO5126766 suppressed the tumor growth in SK-MEL-2 xenograft model
A method for producing a coumarin derivative of general formula (VII) is disclosed in Patent document 1 or 2. Patent document 1 or 2 discloses a method represented by the scheme below [In the scheme, DMF represents N,N-dimethylformamide, TBS represents a tert-butyldimethylsilyl group, dba represents dibenzylideneacetone, and BINAP represents 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl. Also, the numerical values (%) and “quant.” given below some structural formulas indicate the yields of the respective compounds], for example (see the manufacturing example for “compound 1j-2-16-2K” in Patent document 1 or 2).
Methylamine (158 µL, 317 µmol) and DMAP (38.7 mg, 317 µmol) were added at -78 °C to a solution of sulfuryl chloride (28 µL, 340 µmol) in dichloromethane (2 mL), and the mixture was then stirred at room temperature for 2 hours to yield the corresponding sulfamoyl chloride. 3-(2-Amino-3-fluoropyridin-4-ylmethyl)-7-(pyrimidin-2-yloxy)-4-methyl-2-oxo-2H-1-benzopyran (compound 1h-2-16) (60 mg, 159 µmol), pyridine (65 µL, 795 µmol) and dichloromethane (2 mL) were added to the reaction solution, and the mixture was stirred at room temperature for 4 hours. After addition of water, the organic layer was extracted with dichloromethane. After washing with sodium hydrogen carbonate solution and saturated saline, the organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled away under reduced pressure. The resultant residue was purified by silica gel column chromatography to yield the title compound (32 mg, 43%).
3-(2-(N-Methylsulfamoyl)amino-3-fluoropyridin-4-ylmethyl)-4-methyl-7-(pyrimidin-2-yloxy)-2-oxo-2H-1-benzopyran sodium salt
The title compound was synthesized under the same conditions as in the manufacturing example for compound 1j-1-5-1Na, except that compound 1j-2-16-2 was used instead of compound 1j-1-5-1.
3-(2-(N-Methylsulfamoyl)amino-3-fluoropyridin-4-ylmethyl)-4-methyl-7-(pyrimidin-2-yloxy)-2-oxo-2H-1-benzopyran potassium salt
The title compound was synthesized under the same conditions as in the manufacturing example for compound 1j-1-5-1Na, except that compound 1j-2-16-2 was used instead of compound 1j-1-5-1, and that KOH was used instead of NaOH.
† Research Division, Chugai Pharmaceutical Co., Ltd., 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan
‡ Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
ACS Med. Chem. Lett., 2014, 5 (4), pp 309–314
DOI: 10.1021/ml400379x
Publication Date (Web): January 22, 2014
Substituting a carbon atom with a nitrogen atom (nitrogen substitution) on an aromatic ring in our leads 11a and 13g by applying nitrogen scanning afforded a set of compounds that improved not only the solubility but also the metabolic stability. The impact after nitrogen substitution on interactions between a derivative and its on- and off-target proteins (Raf/MEK, CYPs, and hERG channel) was also detected, most of them contributing to weaker interactions. After identifying the positions that kept inhibitory activity on HCT116 cell growth and Raf/MEK, compound 1(CH5126766/RO5126766) was selected as a clinical compound. A phase I clinical trial is ongoing for solid cancers.
Step 5 Synthesis of 4-methyl-3-(3-fluoro-2-aminopyridin-4-ylmethyl)-7-(pyrimidin-2-yloxy)-2-oxo-2H-1-benzopyran
Under a nitrogen atmosphere, potassium carbonate (2.3 g, 17 mmol) was added to a solution of the solid product of step 4 (3.0 g) and 2-bromopyrimidine (1.6 g, 9.8 mmol) in DMF (48 mL), and the mixture was stirred at 115° C. for 2.5 hours. The reaction mixture was cooled to 28° C., water (48 mL) was added dropwise over a period of 5 minutes at that temperature, and after cooling to 0° C., the mixture was stirred for 2 hours. The precipitated crystals were collected by filtration, washed with water (24 mL) and acetonitrile (24 mL) in that order, and dried under reduced pressure to obtain crude crystals (2.3 g). DMF (65 mL) was added to the crude crystals (2.3 g), and after heating to 60° C. and confirming the dissolution, the mixture was cooled to 25° C. Water (65 mL) was added at 25° C., and the mixture was further cooled to 0° C. and stirred for 4 hours. The precipitated crystals were collected by filtration, washed with water (22 mL) and acetonitrile (22 mL) in that order, and dried under reduced pressure to obtain the title compound (2.1 g). The title compound is a compound disclosed in WO 2007/091736.
Yield (overall yield from the 2-acetylamino-5-chloro-3-fluoropyridine used in step 2): 27%
The title compound was synthesized under the same conditions as in the manufacturing example for compound 1h-2-4 (synthesis scheme 2), except that compound 5d-0-16 was used instead of compound 4a-0-4.
The title compound was synthesized under the same conditions as in the manufacturing example for compound 1j-1-5-2, except that compound 1h-2-4 was used instead of compound 1h-1-5.
One of the CH3 peaks was overlapped with the DMSO peak.
ESI (LC/MS positive mode) m/z: 471 (M+H).
Compound 1j-2-4-2Na:
3-{2-Fluoro-3-(methylaminosulfonyl)aminobenzyl}-4-methyl-7-(pyrimidin-2-yloxy)-2-oxo-2H-1-benzopyran sodium salt
The title compound was synthesized under the same conditions as in the manufacturing example for compound 1j-1-5-1Na, except that compound 1j-2-4-2 was used instead of compound 1j-1-5-1.
3-{2-Fluoro-3-(methylaminosulfonyl)aminobenzyl}-4-methyl-7-(pyrimidin-2-yl-oxy)-2-oxo-2H-1-benzopyran potassium salt
The title compound was synthesized under the same conditions as in the manufacturing example for compound 1j-1-5-1Na, except that compound 1j-2-4-2 was used instead of compound 1j-1-5-1, and that KOH was used instead of NaOH.
Method for producing a coumarin derivative of formula (VII) are described in Patent Documents 1 and 2.Patent Documents 1 and 2, for example, in the following scheme [scheme, DMF is N, represents a N- dimethylformamide, TBS represents a tert- butyldimethylsilyl group, dba represents dibenzylideneacetone, BINAP is 2, I represents a 2′-bis (diphenylphosphino) -1,1′-binaphthyl.Further, numerical values given under the formula (%) or “quant.” Indicates the yield of the compound.Methods have been described that are shown in (see Preparation of “Compound 1j-2-16-2K” in Patent Documents 1 and 2).
WO2007 / 091736WO2009 / 014100
While coumarin derivatives of the general formula (VII) can be prepared by the methods described in Patent Documents 1 and 2, in the method described in Patent Documents 1 and 2, after the formylation reaction and a reduction reaction, and unintended Reaction To suppress, it is necessary to perform the introduction and removal steps of the protecting group for hydroxy group.Also, during the formylation reaction, from the viewpoint of cryogenic conditions of the reaction control (eg, -95 ℃ ~ -65 ℃) is required.Furthermore, the alkylation reaction (the seventh step in the above scheme), it is preferred that an excess amount of use of ethyl acetoacetate in terms of efficient synthesis, in which case, requires complicated operation of removing residual reagents become.
[Example 1] Step 1: Synthesis of 2-acetylamino-5-chloro-3-fluoropyridine:
Under a nitrogen atmosphere, acetamide (94.8g, 1.61mol) in DMF with (200mL) and THF (830mL) was added and heated to 50 ℃.The resulting solution was a THF solution of 40wt% sodium hexamethyldisilazide (629g, 1.37mol) was added dropwise and stirred at the same temperature for 2 hours.5-chloro-2,3-difluoro pyridine (100.0g, 0.67mol) After adding, THF and (20mL), and the mixture was stirred at the same temperature for 3 hours.After cooling to 0 ℃, it is added to 2.8M HCl (500mL) to the reaction mixture, and the organic layer was separated and the temperature was raised to room temperature.The organic layer was washed with 20wt% sodium chloride solution (500mL), and evaporated under reduced pressure.The residue in THF (500mL) was added, and the residue was dissolved by heating at 70 ℃.After confirming the solid precipitated by cooling to room temperature, n- heptane (1500mL) was added and further cooled to 0 ℃, followed by stirring at the same temperature for 3 hours.The The precipitated crystals were collected by filtration, to give after washing with a mixed solvent of THF (100mL) and n- heptane (500mL), and dried under reduced pressure to give the title compound (91.2g). Yield: 72% 1 H-NMR (CDCl 3)δ (ppm): 2.36 (3H, s), 7.49 (1H, dd, J = 2.0,9.5Hz), 7.78 (1H, br), 8.17 (1H, d, J = 2.0Hz). MS (ESI +): 189 [M + 1] +
Step 2: Synthesis of 2-acetylamino-5-chloro-3-fluoro-4-formyl pyridine:
Under a nitrogen atmosphere, and dissolved at room temperature 2-acetylamino-5-chloro-3-fluoropyridine (70.0g, 0.37mol) and 4-formyl-morpholine (128.2g, 1.11mol) to THF (840mL) It was.The solution was cooled to -20 ℃ and was added dropwise a THF solution of 24wt% of lithium hexamethyldisilazide (595g, 0.85mol), and stirred 5.5 hours at the same temperature.The reaction mixture, citric acid monohydrate (257g) and sodium chloride (70g) in an aqueous solution dissolved in water (420mL), and I was added at stirring at 0 ℃.The organic layer was separated and the resulting organic layer was successively washed with 50wt% phosphoric acid aqueous solution of potassium dihydrogen (350mL) and 20wt% sodium chloride solution (350mL) to (1458g).The portion of the organic layer was taken for analysis (292g), and evaporated remainder (1166g) at reduced pressure.The residue in THF (350mL) was added, and the solvent was distilled off under reduced pressure.Again, the residue in THF (350mL) was added to and evaporated under reduced pressure to give a solid (81.4g) containing the title compound.The product was used in the next step without further purification. Some of the organic layer which had been collected (292g) to (29g), and evaporated under reduced pressure.The residue was purified by silica gel column chromatography: subjected to [eluent AcOEt / hexane (1 / 4-9 / 1)], I give the title compound (1.05g, 4.85mmol) as a white powdery solid. Yield: 66% 1 H-NMR (CDCl 3)δ (ppm): 2.40 (3H, s), 7,59 (1H, br), 8.34 (1H, br), 10.42 (1H, s). MS (ESI +): 217 (M + 1)
Under a nitrogen atmosphere to dissolve the solid product of Step 2 (81.4g) in 2,2,2-trifluoroethanol (448mL), piperidine (4.4g, 51.7mmol), acetic acid (3.1g, 51 .7mmol) and 3-oxobutanoic acid ethyl (37.0g, 0.28mol) was added and stirred for 3 hours after raising the temperature to 50 ℃.After cooling the reaction mixture to room temperature, triethylamine (758mL, 5.5mol) and formic acid (172mL, 4.6mol) of 2-propanol (1248mL) solution and 20% Pd (OH) 2 carbon (21.2g, moisture content 46.2%) were added, followed by stirring for 4 hours the temperature was raised to 50 ℃.The reaction mixture was filtered through Celite, and the residue was washed with 2-propanol (679mL).Combined filtrate and washings (2795g), and evaporated under reduced pressure a part of the (399g) (remaining (2396g) I was saved).Ethyl acetate (24.2mL) was added to the residue obtained by evaporation of the solvent, and evaporated under reduced pressure.Again, the residue ethyl acetate (182mL) was added to the washed successively with an organic layer 20wt% brine (61mL), 10wt% of potassium dihydrogen phosphate solution (61mL) and 20wt% sodium chloride solution (61mL), under a reduced pressure The solvent was evaporated.Furthermore, in addition to the residue of 2,2,2-trifluoroethanol (24mL), and the solvent evaporated under reduced pressure to obtain oil containing the title compound (15.0g).The product was used in the next step without further purification. 1 H-NMR (CDCl 3)δ (ppm): 1.24 (3H, t, J = 7.0Hz), 2.27 (3H, s), 2.37 (3H, s), 3.16- 3.26 (2H, m), 3.86 (1H, t, J = 7.5Hz), 4.15-4.22 (2H, m), 6.98 (1H, t, J = 5.0Hz ), 7.68 (1H, br), 8.05 (1H, d, J = 5.0Hz). MS (ESI +): 297 (M + 1)
Step 4: Synthesis of 3- (3-fluoro-2-amino-pyridin-4-ylmethyl) -7-hydroxy-4-methyl-2-oxo -2H-1- benzopyran methanesulphonate:
Under a nitrogen atmosphere, oily product of Step 3 (15.0g) and I were dissolved in 2,2,2-trifluoroethanol (33mL).The solution of resorcinol (5.3g, 47.9mmol) and methane sulfonic acid (11.7mL, 181mmol) was added at 24 ℃, and stirred for 4 hours at 90 ℃.And allowed to stand for 13 hours and cooled to room temperature and ethanol (33mL) and water (11mL), and the mixture was stirred for 4.5 hours at 90 ℃.After adding 2-propanol (105mL) was cooled to 55 ℃, and allowed to stand for 14 hours then cooled to room temperature.The The precipitated crystals were collected by filtration to give 2-propanol was washed twice with (33mL), and dried under reduced pressure to give the title compound (8.2g). (Total from 2-acetylamino-5-chloro-3-fluoropyridine was used in step 2 Yield) Yield: 49% MS (ESI +): 301 [M + 1-MsOH] +
Under a nitrogen atmosphere, 3- (3-fluoro-2-amino-pyridin-4-ylmethyl) -7-hydroxy-4-methyl-2-oxo -2H-1- benzopyran methanesulphonate (7.6g, 19.2mmol) and 2-bromo-pyrimidine (4.0g, 24.9mmol) was dissolved in DMF (122mL), potassium carbonate (5.8g, 42.2mmol) was added, and the mixture was stirred for 3.5 hours at 115 ℃.After cooling the reaction mixture to 28 ℃, water (122mL) was added dropwise over the same temperature for 0.5 hours, and stirred for 2 minutes.In addition, after cooling to 0 ℃, and the mixture was stirred for 1 hour, and the precipitated crystals were collected by filtration.The obtained crystals were washed successively with water (61mL) and acetonitrile (61mL), to give the title compound was dried under reduced pressure and crystals (6.5g). The resultant was taken for analysis a portion of the crystals (0.1g), it was suspended remainder (6.4g) in DMF (70mL).The resulting suspension was stirred 60 ℃ and heated for 5 minutes and stirred for 80 minutes by the addition of acetonitrile (185mL) at the same temperature.Then, it was stirred for 0.5 hours and then cooled to 40 ℃, and the mixture was stirred for 0.5 hours and further cooled to 25 ℃.After a further 1.5 hours with stirring and cooled to 0 ℃, the precipitated crystals were collected by filtration.After washing the resulting crystals in acetonitrile (46mL), was obtained by drying under reduced pressure to the title compound (5.5g).Incidentally, the title compound is a compound described in WO2007 / 091736. Yield: 76%
Under a nitrogen atmosphere, 4-methyl-3- (3-fluoro-2-amino-pyridin-4-ylmethyl) -7- (pyrimidin-2-yloxy) -2-oxo -2H-1- benzopyran (1.7g, 4 the .5mmol) it was suspended in DMF (18mL).To this solution pyridine (0.8mL, 9.9mmol) was cooled to In 10 ℃ added, N- methyl-sulfamoyl chloride (1.05g, 8.1mmol) in acetonitrile (18mL) solution of the internal temperature of 15 ℃ it was dropped so as to maintain below.After stirring for 90 minutes at the same temperature, acetonitrile (3.4mL) was added and further water (50mL), was added dropwise the inner temperature so as to maintain the 20 ℃ below.It was cooled to an external temperature of 0 ℃, and the mixture was stirred for an internal temperature of 5 ℃ 2 hours after arrival.The precipitated crystals were collected by filtration, washed with water (8.5mL), and dried to give the title compound (1.9g, 4.0mmol) was obtained. Yield: 88% MS (ESI +): 472 [M + 1] +
Under a nitrogen atmosphere, 3- {2- (methyl-aminosulfonyl) amino-3-fluoro-pyridin-4-ylmethyl} -4-methyl-7- (pyridin-2-yloxy) -2-oxo -2H-1- benzopyran ( 1.6g, was suspended 3.4mmol) in THF (10mL), water (3mL) was added.The suspension in 2.0M aqueous potassium hydroxide (1.8mL, 3.6mmol) was added dropwise over 10 min at 25 ℃, after raising the temperature to 60 ℃, and the mixture was stirred for 2 hours at the same temperature.After cooling the reaction mixture to 20 ℃, it was added dropwise over a period of THF (8mL) 30 min.After completion of the dropwise addition, the mixture was cooled to an external temperature of -5 ℃, and the mixture was stirred for an internal temperature of 0 ℃ reached after 160 minutes.The precipitated crystals were collected by filtration, then washed with a mixture of THF (14mL) and water (1.6mL) (pre-cooled to 5 ℃), further washed with THF (8mL), and dried to give the title compound (0 .72g, we got 1.4mmol). Yield: 42% MS (ESI +): 472 [M + 2H-K] +
CLIP
RO5126766 (CH5126766) is a first-in-class dual inhibitor of Raf/MEK [1].
The RAS/RAF/MEK/ERK signaling pathway is an important signal transduction system and participates in cell differentiation, movement, division and death. Activated Ras activates RAF kinase, which then phosphorylates and activates MEK (MEK1 and MEK2) [1]. The mutations in BRAF, RAS, and NF1 are associated with many human tumors [2].
RO5126766 (CH5126766) is a first-in-class dual Raf/MEK inhibitor. In cell-free kinase assays, CH5126766 effectively inhibited the phosphorylation of MEK1 protein by RAF and the activation of ERK2 protein by MEK1 with IC50 values of 0.0082-0.056 and 0.16 μM, respectively. In NCI-H460 (KRAS Q61H) human lung large cell carcinoma cell line, RO5126766 induced cell-cycle inhibitor p27Kip1 protein expression and caused G1 arrest. In HCT116 KRAS-mutant colorectal cancer cells, RO5126766 CH5126766 completely inhibited the phosphorylation of MEK and ERK [2].
In Japanese patients with advanced solid tumors, RO5126766 exhibited the maximum tolerable dose (MTD) of 2.25 mg/day once daily [1]. In a HCT116 (G13D KRAS) mouse xenograft model, RO5126766 (1.5 mg/kg) inhibited pERK and ERK signaling and exhibited ED50 value of 0.056 mg/kg [2].
References:
[1]. Honda K, Yamamoto N, Nokihara H, et al. Phase I and pharmacokinetic/pharmacodynamic study of RO5126766, a first-in-class dual Raf/MEK inhibitor, in Japanese patients with advanced solid tumors. Cancer Chemother Pharmacol, 2013, 72(3): 577-584.
[2]. Ishii N, Harada N, Joseph EW, et al. Enhanced inhibition of ERK signaling by a novel allosteric MEK inhibitor, CH5126766, that suppresses feedback reactivation of RAF activity. Cancer Res, 2013, 73(13): 4050-4060.
THIS COMPD HAS ONE FLUORO MISSING, APPLY TO YOUR MK 8876
Step 1 – Synthesis of 2,6-dichloropyridin-3-ol
Η202 (1.60 g, 47.12 mmol) was added slowly to the solution of compound 2,6- dichloropyridin-3-ylboronic acid (3 g, 15.71 mmol) in CH2CI2 (30 mL) at 0 °C. After stirred at room temperature for about 15 hours, the mixture was quenched with sat. Na2S203 aqueous (50 mL) and adjusted to pH < 7 with IN HC1. The mixture was extracted with EtOAc (40 mL x 3). The organic layer was washed with brine (100 mL), dried over Na2S04, filtered and the solvent was evaporated to provide2,6-dichloropyridin-3-ol (2.34 g, yield: 91.4%). 1H-NMR (CDC13, 400 MHz) δ 7.30 (d, / = 8.4 Hz, 1H), 7.19 (d, / = 8.4 Hz, 1H), 5.70 (br, 1H).
– Synthesis of 2,6-dichloro- -methoxypyridine
To a solution of 2,6-dichloropyridin-3-ol (16.3 g, 0.1 mol) and K2C03 (41.4 g, 0.3 mol) in DMF (200 mL) were added Mel (21.3 g, 0.15 mol). The mixture was allowed to stir at 80 °C for 2 hours. The mixture was then diluted with water (200 mL) and extracted with EtOAc (200 mL x 3). The organic layer was washed with brine (200 mL x 3), dried over Na2S04, filtered and the solvent was evaporated to provide 2,6-dichloro-3-methoxypyridine (17.0 g, yield: 96.0%). 1H-NMR (CDC13, 400 MHz) δ 7.12-7.18 (m, 2H), 3.86 (s, 3H). Step 3 – Synthesis of2-(6-chloro-3-methoxypyridin-2-yl)-lH-indole
To a degassed solution of compound 2,6-dichloro-3-methoxypyridine (8.9 g, 0.05 mol), (l-(tert-butoxycarbonyl)-lH-indol-2-yl)boronic acid (13 g, 0.05 mol) and K3PO4 (31.8 g, 3.0 mol) in DMF (100 mL) was added Pd(dppf)Cl2 (3.65 g, 0.005 mol) under N2. The mixture was heated at 60 °C for about 15 hours. The reaction mixture was cooled to room temperature, diluted with EtOAc and filtered. The filtrate was washed with H20, brine, dried over Na2S04. After being concentrated in vacuo, the resulting residue was purified using prep-HPLC to provide the desired product of 2-(6-chloro-3-methoxypyridin-2-yl)-lH-indole (9.0 g, yield:
BBr3 (0.4 mL, 0.39 mmol) was added to the solution of 2-(6-chloro-3- methoxypyridin-2-yl)-lH-indole (50 mg, 0.194 mmol) in CH2C12 (0.5 mL) at -78 °C under N2. The mixture was allowed to stir at room temperature for 3 hours. The mixture was then quenched with CH3OH (10 mL) at -78 °C. After being concentrated in vacuo, the resulting residue was purified using prep-TLC (PE : EtOAc = 2.5 : 1) to afford the desired product of 6- chloro-2-(lH-indol-2-yl)pyridin-3-ol (40 mg, yield: 85.1%). 1H-NMR (CDC13, 400 MHz) δ 10.09 (s, 1H), 9.72 (s, 1H), 7.50 (d, / = 7.9 Hz, 1H), 7.17-7.32 (m, 3H), 7.08-7.14 (m, 1H), 6.87-6.96 (m, 2H).
To a solution of chloroiodomethane (3.51 g, 20.0 mmol) and K2CO3 (1.38 g, 10.0 mmol) in DMF (50 mL) was allowed to stir at 100 °C, 6-chloro-2-(lH-indol-2-yl)pyridin-3-ol (480 mg, 2.0 mmol) in DMF (50 mL) was added dropwise. After addition, the mixture was allowed to stir for another 0.5 hours. The mixture was then diluted with water (100 mL) and extracted with EtOAc (100 mL x 3). The organic layer was washed with brine (100 mL x 3), dried over Na2S04 and concentrated. The residue was purified using prep-TLC (PE : EtOAc = 3 1) to afford the desired product of 2-chloro-6H-pyrido[2′,3′:5,6][l,3]oxazino[3,4-a]indole (260 mg, yield: 50.7%). 1H-NMR (CDC13, 400 MHz) δ 7.63 (d, / = 8.0 Hz, 1H), 7.22-7.27 (m, 3H), 7.19 (d, / = 2.4 Hz, 1H), 7.08-7.12 (m, 2H), 5.86 (s, 2H).
To a degassed solution of 2-(4-fluorophenyl)-N-methyl-6-(N- methylmethylsulfonamido)-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzofuran-3- carboxamide (502 mg, 1.0 mmol), 2-chloro-6H-pyrido[2′,3′:5,6][l,3]oxazino[3,4-a]indole (256 mg, 1.0 mmol) and K3PO4 (636 mg, 3.0 mmol) in dioxane : H20 (1.5 mL : 0.4 mL) was added Pd2(dba)3 (91 mg, 0.1 mmol) and X-phos (91 mg, 0.2 mmol) under N2. The mixture was heated to 110 °C for 3 hours. The reaction mixture was cooled to room temperature, diluted with EtOAc and filtered. The filtrate was washed with H20, brine, dried over Na2S04. After being concentrated in vacuo, the resulting residue was purified using prep-HPLC to provide the desired product of Compound 1 (275 mg, yield: 46.1%). 1H-NMR (CDC13, 400 MHz) δ 7.88-7.94 (m, 3H), 7.61-7.63 (m, 2H), 7.40 (s, 2H), 7.09-7.28 (m, 6H), 5.94 (s, 2H), 5.86 (d, / = 4.4 Hz, 1H), 3.29 (s, 3H), 2.92 (d, / = 5.2 Hz, 3H), 2.65 (s, 3H). MS (M+H)+: 596.
Compounds 2-15, depicted in the table below, were prepared using the method described above.
Synthesis of ethyl 3- 4-fluorophenyl)-3-oxopropanoate
Diethyl carbonate (130 g, 1.1 mol) was dissolved in a suspension ofNaH (60% in oil, 50.2 g, 1.3 mol) in anhydrous tetrahydrofuran (1.5 L), and then l-(4-fluorophenyl)ethanone (150 g, 1.09 mol) was added dropwise at 70 °C. The resulting mixture was stirred at 70 °C for 3 hours. After the reaction mixture was cooled to room temperature and poured into HCl (1 N). The mixture was extracted with EtOAc, the organic phase was dried with anhydrous NaS04 and concentrated in vacuo. The resulting residue was purified using column chromatography (eluted with petroleum ether / EtOAc = 50 / 1) to provide ethyl 3-(4-fluorophenyl)-3-oxopropanoate (217 g, yield: 95%). 1H-NMR (CDC13, 400 MHz) δ 7.92-7.97 (m, 2H), 7.07-7.13 (m, 2H), 4.14-4.20 (m, 2H), 3.93 (s, 2H), 1.22 (d, J= 7.2 Hz, 3H). MS (M+H)+: 211. Step 2 – Synthesis of ethyl 5-bromo-2-(4-fluorophenyl)benzofuran-3-carboxylate
A solution of ethyl 3-(4-fluorophenyl)-3-oxopropanoate (130 g, 0.6 mol), 4- bromophenol (311 g, 1.8 mol) and FeCl3-6H20 (19.5 g, 0.09 mol) in DCE (700 mL) was heated to reflux, and then 2-(tert-butylperoxy)-2-methylpropane (193 g, 1.32 mol) was added dropwise under nitrogen. After 6 hours of refluxing, the mixture was cooled to RT, quenched with saturated NaHS03 and extracted with dichloromethane. The organic phases were washed with water, brine and dried over Na2S04, filtered and concentrated in vacuo. The resulting residue was purified using column chromatography (petroleum ether / dichloromethane = 15 / 1) to provide the crude product, which was crystallized from cold MeOH to provde ethyl 5-bromo-2- (4-fluorophenyl)benzofuran-3-carboxylate (37 g, yield: 14.3%) as solid. 1H- MR (CDC13, 400 MHz) δ 8.12 (s, 1H), 7.97-8.01 (m, 2H), 7.37 (d, J= 4.0 Hz, 1H), 7.32 (d, J= 8.0 Hz, 1H), 7.11 (t, J= 8.0 Hz, 2H), 4.32-4.38 (m, 2H), 1.36 (t, J= 8.0 Hz, 3H). MS (M+H)+: 363 / 365.
Step 3 – Synthesis of eth l 5-bromo-2-(4-fluorophen -6-nitrobenzofuran-3-carboxylate
To a solution of ethyl 5-bromo-2-(4-fluorophenyl)benzofuran-3-carboxylate (50 g,
137.6 mmol) in CHC13 (500 mL), fuming HN03 (50 mL) was added dropwise at -15 °C and the mixture was stirred for 0.5 hour. The reaction mixture was poured into ice water and extracted with CH2C12. The organic layer was washed with a.q. sat. NaHC03 and brine, after removed the most of solvent, the resulting residue was crystallized with petroleum ether / dichloromethane = 20 / 1 to provide product of ethyl 5-bromo-2-(4-fluorophenyl)-6-nitrobenzofuran-3-carboxylate (35 g, yield: 66%). 1H- MR (CDC13, 400 MHz) δ 8.36 (s, 1H), 8.02-8.04 (m, 3H), 7.13-7.18 (m, 2H), 4.36-4.41 (m, 2H), 1.37 (t, J= 4.0 Hz, 3H). MS (M+H)+: 408 / 410.
Step 4 – Synthesis of ethyl 6-amino-5-bromo-2-(4-fluorophenyl)benzofuran-3-carboxylate
A mixture of ethyl 5-bromo-2-(4-fluorophenyl)-6-nitrobenzofuran-3-carboxylate (52 g, 127 mmol), iron filings (21.3 g, 382.2 mmol) and H4C1 (41 g, 764.4 mmol) in MeOH / THF / H20 (2 / 2 / 1, 500 mL) was stirred at reflux for 3 hour. After filtered and concentrated, the resulting residue was purified using column chromatography (petroleum ether / EtOAc / dichloromethane = 20 : 1 : 20) to provide ethyl 6-amino-5-bromo-2-(4-fluorophenyl) benzofuran-3-carboxylate (40 g, yield: 82%). 1H- MR (CDC13, 400 MHz) δ 8.01 (s, 1H), 7.94-7.98 (m, 2H), 7.08 (t, J= 8.0 Hz, 2H), 6.83 (s, 1H), 4.32-4.36 (m, 2H), 4.18 (s, 2H), 1.35 (t, J= 8.0 Hz, 3H). MS (M+H)+: 378 / 380.
Step 5 – Synthesis of 5-Bromo-2-(4-fluoro-phenyl)-6-methanesulfonylamino-benzofuran-3- carboxylic acid eth l ester
MsCI (31.7 g, 277.5 mmol) was added to a solution of ethyl 6-amino-5-bromo-2- (4-fluorophenyl)benzofuran-3-carboxylate (35 g, 92.5 mmol) and pyridine (60 mL) in
dichloromethane (300 mL) at 0 °C. After stirred overnight at room temperature, the mixture was diluted with water and extracted with dichloromethane. The organic layer was washed with brine, dried over Na2S04, filtered and concentrated in vacuo, the resulting residue was purified using crystallized with EtOAc to provde the pure product of ethyl 5-bromo-2-(4-fluorophenyl)-6- (methylsulfonamido)benzofuran-3-carboxylate (35 g, yield: 82%). 1H- MR (CDC13, 400 MHz) δ 8.27 (s, 1H), 8.01-8.05 (m, 2H), 7.87 (s, 1H), 7.15-7.19 (m, 2H), 6.87 (s, 1H), 4.38-4.43 (m, 2H), 3.00 (s, 3H), 1.40 (t, J= 40 Hz, 3H). MS (M+H)+: 456 / 458.
Step 6 – Synthesis of 5-Bromo-2-(4-fluoro-phenyl)-6-methanesulfonylamino-benzofuran-3- carboxylic acid
To a solution of ethyl 5-bromo-2-(4-fluorophenyl)-6-(methylsulfonamido) benzofuran-3-carboxylate (53 g, 0.23 mol) in dioxane / H20 (5 / 1, 600 mL) was added
LiOH-H20 (25 g, 1.17 mol), and the mixture was stirred at 100 °C for 3 hours. After
concentrated, the resulting residue was dissolved in H20, 1 N HCl was added until pH reached 3, and the mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na2S04 and filtered. The solvent was removed to provide the product of 5-bromo-2-(4- fluorophenyl)-6-(methylsulfonamido)benzofuran-3-carboxylic acid (48 g, yield: 96%).1H- MR (DMSO- e, 400 MHz) δ 13.49 (s, 1H), 9.67 (s, 1H), 8.30 (s, 1H), 8.12-8.17 (m, 2H), 7.87 (s, 1H), 7.45-7.50 (m, 2H), 3.16 (s, 3H). MS (M+H)+: 428 / 430. Step 7 – Synthesis of 5-Bromo-2-(4-fluoro-phenyl)-6-methanesulfonylamino-benzofuran-3- carboxylic acid methylamide
A solution of 5-bromo-2-(4-fluorophenyl)-6-(methylsulfonamido) benzofuran-3- carboxylic acid (33 g, 77 mmol), HOBT (15.6 g, 115.5 mmol) and EDCI (22.2 g, 115.5 mmol) in DMF (250 mL) was stirred at room temperature. After 2 hours, Et3N (50 mL) and CH3 H2 (HC1 salt, 17.7 g, 231 mmol) was added to the mixture, and the mixture was stirred overnight. After the solvent was removed, H20 was added and the mixture was extracted with ethyl acetate. The combined organic layer was washed with H20, brine and concentrated in vacuo. The resulting residue was washed with EtOAc to provide the product of 5-bromo-2-(4-fluorophenyl)-N- methyl-6-(methylsulfonamido)benzofuran-3-carboxamide (32 g, yield: 94%). 1H- MR (DMSO- ck, 400 MHz) δ 9.55 (br s, 1H), 8.46-8.48 (m, 1H), 8.12-8.17 (m, 2H), 7.96 (s, 1H), 7.87 (s, 1H), 7.45-7.50 (m, 2H), 3.16 (s, 3H), 2.93 (d, J= 8.4 Hz, 3H). MS (M+H)+: 441 / 443.
Step 8 – Synthesis of 5-bromo-2-(4-fluorophenyl)-N-methyl-6-(N- methylmethylsulfonamido benzofuran-3-carboxamide
CH3I (31.6 g, 223 mmol) was added to a mixture of 5-bromo-2-(4-fluorophenyl)- N-methyl-6-(methylsulfonamido)benzofuran-3-carboxamide (32 g, 74 mmol), K2C03 (25.6 g, 186 mmol) and KI (246 mg, 1.5 mmol) in DMF (150 mL) under N2 protection. The mixture was stirred at 80-90 °C overnight. After concentrated in vacuo, the resulting residue was washed with water (200 mL) and EtOAc (200 mL) to provide the product of 5-bromo-2-(4- fluorophenyl)-N-methyl-6-(N-methylmethylsulfonamido)benzofuran-3-carboxamide (31.5 g, 94%). 1H- MR (CDCI3, 400 MHz) δ 8.16 (s, 1H), 7.88-7.92 (m, 2H), 7.70 (s, 1H), 7.18-7.23 (m, 2H), 5.78 (br s, 1H), 3.34 (s, 3H), 3.09 (s, 3H), 3.00 (d, J= 4.8 Hz, 3H). MS (M+H)+: 455 / 457. Step 9 – Synthesis of 2-(4-fluorophenyl)-N-methyl-6-(N-methylmethylsulfonamido)-5-(4, 4, 5, 5- tetramethyl-1 -dioxaborolan-2-yl)benzofuran-3-carboxamide
a degassed solution of 5-bromo-2-(4-fluorophenyl)-N-methyl-6-(N- methylmethylsulfonamido)benzofuran-3-carboxamide (1.0 g, 2.2 mmol) and pinacol diborane (2.79 g, 11.0 mmol) in 1,4-Dioxane (25 mL) was added KOAc (647 mg, 6.6 mmol) under N2 and stirred for 4 hours at room temperature. Then Pd(dppf)Cl2 (60 mg) was added, and the mixture was stirred for another 30 minutes. Then the mixture was put into a pre-heated oil-bath at 130 °C and stirred for another 1 hour under N2. The reaction mixture was cooled to room
temperatureand concentrated and extracted with EtOAc. The organic layers were washed with brine, dried over Na2S04. After concentrated, the crude product of the boronic ester was purified using column chromatography (petroleum ether / EtOAc = 5 / 1 to 2 / 1) to obtain 2-(4- fluorophenyl)-N-methyl-6-(N-methylmethylsulfonamido)-5-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)benzofuran-3-carboxamide as white solid (700 mg, yield: 64%). 1H- MR (CDCI3, 400 ΜΗζ) δ 8.17 (s, 1H), 7.87-7.91 (m, 2H), 7.52 (s, 1H), 7.11 (t, 7= 7.6 Hz, 2H), 5.81 (d, 7= 2.8 Hz, 1H), 3.30 (s, 3H), 2.97 (d, 7= 5.2 Hz, 3H), 2.90 (s, 3H), 1.31 (s, 12H). MS (M+H)+: 503.
Step 10 – Synthesis of tert-butyl 4-fluoro-lH-indole-l -car boxy late
To a solution of 4-fluoro-lH-indole (5 g, 0.11 mol) and DMAP (150 mg, 3%Wt) in THF (50 mL) was added (Boc)20 (8.5 g, 0.04 mol) dropwise. The mixture was stirred at room temperature for 2 hours. The organic solvent was removed in vacuo, and the resulting residue was purified using column chromatography (pure petroleum ether) to provide tert-butyl 4-fluoro- lH-indole-l-carboxylate (8.3 g, yield: 96%). 1H- MR (CDC13, 400 MHz) δ 7.92 (d, J= 8.4 Hz, 1H), 7.55 (d, J= 3.6 Hz, 1H), 7.23 (m, 1H), 6.90 (m, 1H), 6.66 (d, J= 3.6 Hz, 1H), 1.67 (s, 9H). MS (M+H)+: 236.
Step 11 – Synthesis of (l-(tert-butoxycarbonyl)-4-fluoro-lH-indol-2-yl)boronic acid
To a solution of diisopropylamine (7.5 mL, 0.11 mol) in THF (35 mL) at 0 °C was added «-BuLi (21 mL, 0.055 mol) dropwise. The mixture was stirred at 0 °C for 40 minutes. Then the mixture was cooled to -78 °C. Tert-butyl 4-fluoro-lH-indole-l-carboxylate (5 g, 0.02 mol) in THF (13 mL) was added dropwise slowly. After addition, the mixture was stirred at -78 °C for 2 hours. Then triisopropyl borate (3.29 g, 0.03 mol) was added. The mixture was stirred at -78 °C for another 40 minutes. The reaction was monitored using TLC. When the reaction was completed, the mixture was adjusted to pH = 6 with 1 N HC1. After extracted with EtOAc (25 mL x 3), the combined organic layers were washed with brine (50 mL), dried over Na2S04, filtered and concentrated in vacuo. The obtained solid was recrystallized with EtOAc and petroleum ether to provide (l-(tert-butoxycarbonyl)-4-fluoro-lH-indol-2-yl)boronic acid (4.5 g, yield: 76.7%, which might be unstable at high temp, work up, store in fridge). 1H- MR (CDC13, 400 MHz) δ 7.77 (d, J= 8.4 Hz, 1H), 7.57 (s, 1H), 7.44 (s, 2H), 7.24 (m, 1H), 6.90 (m, 1H), 1.66 (s, 9H). MS (M+H)+: 280.
Step 12 – Synthesis of 6-chloro-2-iodopyridin-3-ol
6-chloropyridin-3-ol (5.0 g, 38.6 mmol) was dissolved in water (50 mL) and placed under an N2 atmosphere. Na2C03 (8.2 g, 77.4 mmol) was added followed by iodine (9.8 g, 38.8 mmol). The reaction mixture was stirred at room temperature for 2 hours. The mixture was poured into 1M Na2S203 and extracted with EtOAc. The combined organic phases were washed with brine, dried over Na2S04 and concentrated to provide the product of 6-chloro-2- iodopyridin-3-ol (7.0 g, yield: 70.9%). 1H- MR (CDC13, 400 MHz) δ 7.17 (d, J= 8.4 Hz, 1H), 7.06 (d, J= 8.4 Hz, 1H). MS (M+H)+: 256 / 258.
Step 13 – Synthesis of 6-chloro-2-(4-fluoro-lH-indol-2-yl)pyridin-3-ol
A mixture of (l-(tert-butoxycarbonyl)-4-fluoro-lH-indol-2-yl)boronic acid (5 g, 18.0 mmol), 6-chloro-2-iodopyridin-3-ol (3.82 g, 15.0 mol) and NaHC03 (3.78 g, 45.0 mol) in 1, 4-dioxane (76 mL) and water (7 mL) was stirred at room temperature for 15 minutes. Then Pd(PPh3)2Cl2 (527 mg, 0.75 mmol) was added under nitrogen atmosphere, and the mixture was heated at 100 °C under N2 for 16 hours. The reaction mixture was cooled to room temperature, diluted with EtOAc (50 mL), filtered and concentrated in vacuo. The resulting residue was diluted with H20 (60 mL) and EtOAc (30 mL), and the layer was separated, the aqueous layer was extracted with EtOAc (3*30 mL). The combined organic layers were washed with brine (50 mL), dried over Na2S04, filtered and concentrated in vacuo. The resulting residue was purified using column chromatography (petroleum ether / EtOAc = 20 / 1 ~ 3 / 1) to provide 6-chloro-2- (4-fluoro-lH-indol-2-yl)pyridin-3-ol (3 g, yield: 76.5%). 1H- MR (MeOD, 400 MHz) δ 7.36 (s, 1H), 7.23-7.27 (m, 2H), 7.03-7.11 (m, 2H), 6.63-6.68 (m, 1H). MS (M+H)+: 263 / 265.
Ste 14 – Synthesis of 2-chloro-ll-fluoro-6H-pyrido[2′,3′:5, 6][l,3]oxazino[3,4-a]indole
A solution of 6-chloro-2-(4-fluoro-lH-indol-2-yl)pyridin-3-ol (2 g, 7.6 mmol) and Cs2C03 (7.46 g, 22.89 mmol) in DMF (100 mL) was stirred at 100 °C (internal temperature) for 15 min, and then chloroiodomethane (2.85 g, 15.3 mmol) in DMF (2 mL) was added dropwise. After the reaction was completed, the mixture was filtered and concentrated in vacuo. The resulting residue was diluted with water (50 mL) and extracted with ethyl acetate (30 mL x 3). The organic layer was washed with brine, dried over Na2S04 and concentrated in vacuo. The resulting residue was purified using column chromatography (petroleum ether:EA=10: l) to provde 2-chloro-l l-fluoro-6H-pyrido[2′,3′:5,6][l,3]oxazino[3,4-a]indole (1.8 g, yield: 86.1%). 1H- MR (DMSO-i¾, 400 MHz) δ 7.64 (d, J= 8.8 Hz, 1H), 7.39-7.46 (m, 2H), 7.21-7.25 (m, 1H), 7.06 (s, 1H), 6.88-6.92 (m, 1H), 6.18 (s, 2H). MS (M+H)+: 275 / 277. Step 15 – Synthesis of5-(ll-fluoro-6H-pyrido[2 3′:5, 6][l,3]oxazino[3,4-a]indol-2-yl)-2-(4- fluorophenyl)-N-methyl-6-(N-methylmethylsulfonamido)benzofuran-3-carboxam
To a degassed solution of 2-(4-fluorophenyl)-N-methyl-6-(N- methylmethylsulfonamido)-5-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)benzofuran-3- carboxamide (100 mg, 0.199 mmol), 2-chloro-l l-fluoro-6H-pyrido[2′,3′:5,6][l,3]oxazino[3,4- a]indole (56 mg, 0.199 mmol) and Κ3Ρ04·3Η20 (159 mg, 0.597 mmol) in dioxane / H20 (0.8 mL / 0.2 mL) was added Pd2(dba)3 (9 mg, 0.01 mmol) and X-Phos (9 mg, 0.02 mmol) under N2. The mixture was heated at 80 °C for 1 hour. The mixture was then diluted with water (30 mL) and extracted with EtOAc (15 mL x 3). The organic layer was washed with brine (20 mL), dried over Na2S04 and concentrated in vacuo. The resulting residue was purified using prep-TLC (petroleum ether / EtOAc = 1 : 1.5) to provde the pure product of 5-(l l-fluoro-6H- pyrido [2′, 3 ‘ : 5 , 6] [ 1 , 3 ]oxazino [3 ,4-a]indol-2-yl)-2-(4-fluorophenyl)-N-methyl-6-(N- methylmethylsulfonamido)benzofuran-3-carboxamide (60 mg, 48.8%). 1H- MR (CDC13, 400 MHz) δ: 7.99 (s, 1H), 7.93-7.96 (m, 2H), 7.65 (s, 1H), 7.45-7.50 (m, 2H), 7.17-7.21 (m, 4H), 7.10 (d, J= 8.0 Hz, 1H), 6.81-6.85 (m, 1H), 5.98 (s, 3H), 3.35 (s, 3H), 2.98 (d, J= 4.8 Hz, 3H), 2.72 (s, 3H). MS (M+H)+: 615.
Paper
We describe the route development and multikilogram-scale synthesis of an HCV NS5B site D inhibitor, MK-8876. The key topics covered are (1) process improvement of the two main fragments; (2) optimization of the initially troublesome penultimate step, a key bis(boronic acid) (BBA)-based borylation; (3) process development of the final Suzuki–Miyaura coupling; and (4) control of the drug substance form. These efforts culminated in a 28 kg delivery of the desired active pharmaceutical ingredient.
Process Development of the HCV NS5B Site D Inhibitor MK-8876
Using the Teasdale method, purge factor estimates for six impurities identified as mutagenic alerts in the synthesis of MK-8876 are compared to actual measured amounts of these impurities determined via appropriate analytical methods. The results from this comparison illustrate the conservative nature of purge factor estimates, meaning that overprediction of mutagenic impurity purging is unlikely when using this method. Industry and regulatory acceptance of the purge factor estimation method may help minimize analytical burden in pharmaceutical development projects.
Evaluation and Control of Mutagenic Impurities in a Development Compound: Purge Factor Estimates vs Measured Amounts
*Pharmaceutical Division, Tokyo New Drug Research Laboratories, Kowa Co., Ltd., 2-17-43, Noguchicho, Higashimurayama, Tokyo 189-0022, Japan
Abstract
A practical synthesis of novel Rho-kinase inhibitor, (S)-4-fluoro-5-(2-methyl-1,4-diazepan-1-ylsulfonyl)isoquinoline hydrochloride dihydrate (1) was achieved in a pilot-scale production. We have demonstrated the regioselective chlorosulfonylation of 4-fluoroisoquinoline in an one-pot reaction to afford 4-fluoroisoquinoline-5-sulfonyl chlorideand the asymmetric construction of the (S)-2-methyl-1,4-diazepane moiety as key steps.
White crystalline solid.: mp 258-259 °C (decomp);
[]20D –8.82 (c1.00, H2O);
IR (KCl) 3406, 2983, 2763, 1588, 1324, 1146, 1129 cm-1;
K-115, an isoquinolinesulfonamide compound, is a highly selective and potent (IC50 = 31 nM) Rho-kinase inhibitor; is in Phase II clinical development in patients with POAG or ocular hypertension.Ripasudil hydrochloride hydrate (Glanatec® ophthalmic solution 0.4 %; hereafter referred to as ripasudil) is a small-molecule, Rho-associated kinase inhibitor developed by Kowa Company, Ltd. for the treatment of glaucoma and ocular hypertension. This compound, which was originally discovered by D. Western Therapeutics Institute, Inc., reduces intraocular pressure (IOP) by directly acting on the trabecular meshwork, thereby increasing conventional outflow through the Schlemm’s canal.
Ripasudil hydrochloride hydrate was first approved by Pharmaceuticals and Medical Devices Agency of Japan (PMDA) on Sept 26, 2014. It was developed and marketed as Glanatek® by Kowa Pharmaceuticals.
Ripasudil hydrochloride hydrate is the first drug that can inhibit the rho-associated, coiled-coil containing protein kinase (ROCK). It is indicated for the treatment of glaucoma and ocular hypertension.
Glanatek® is available as solution (0.4%) for ophthalmic use, containing 4 mg of free Ripasudil per millimeter, and the recommended dose is one drop twice daily.
As a result of this mechanism of action, ripasudil may offer additive effects in the treatment of glaucoma and ocular hypertension when used in combination with agents such as prostaglandin analogues (which increase uveoscleral outflow) and β blockers (which reduce aqueous production).
The eye drop product has been approved in Japan for the twice-daily treatment of glaucoma and ocular hypertension, when other therapeutic agents are not effective or cannot be administered. Phase II study is underway for the treatment of diabetic retinopathy.
K-115 is a Rho-kinase inhibitor as ophthalmic solution originally developed by Kowa and D Western Therapeutics Institute (DWTI). The product candidate was approved and launched in Japan for the treatment of glaucoma and ocular hypertension in 2014.
In 2002, the compound was licensed to Kowa Pharmaceutical by D Western Therapeutics Institute (DWTI) in Japan for the treatment of glaucoma. The compound is currently in phase II clinical trials at the company for the treatment of age-related macular degeneration and diabetic retinopathy.
Use of (S)-(-)-1-(4- fluoro-5-isoquinoline-sulfonyl)-2-methyl-1,4-homopiperazine (ripasudil hydrochloride, first disclosed in WO9920620), in the form of eye drops, for the treatment of retinal diseases, particularly diabetic retinopathy or age-related macular degeneration.
Follows on from WO2012105674 by claiming a combination of the same compound. Kowa, under license from D Western Therapeutics Institute, has developed the Rho kinase inhibitor ripasudil hydrochloride hydrate (presumed to be Glanatek) as an eye drop formulation for the treatment of glaucoma and ocular hypertension which was approved in Japan in September 2014..
The company is also developing the agent for the treatment of diabetic retinopathy, for which it is in phase II trial as of October 2014.
Ripasudil (Glanatec) is a drug used for the treatment of glaucoma and ocular hypertension. It is approved for use in Japan as a 0.4% ophthalmic solution.[1]
A Practical Synthesis of (S)-tert-butyl 3-methyl-1,4-diazepane-1-carboxylate, the key intermediate of Rho-kinase inhibitor K-115
Synthesis (Stuttgart) 2012, 44(20): 3171
practical synthesis of (S)-tert-butyl 3-methyl-1,4-diazepane-1-carboxylate has been established for supplying this key intermediate of Rho–kinase inhibitor K-115 in a multikilogram production. The chiral 1,4-diazepane was constructed by intramolecular Fukuyama–Mitsunobu cyclization of a N-nosyl diamino alcohol starting from the commercially available (S)- or (R)-2-aminopropan-1-ol. In the same manner, an enantiomeric pair of a structural isomer were prepared for demonstration of the synthetic utility.
The including prevention and treatment cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, cerebrovascular disorders such as cerebral edema, the present invention relates to a salt thereof or isoquinoline derivatives useful as therapeutic agents, particularly glaucoma.
(S) – (-) -1 – (4 – fluoro-iso-5 – yl) sulfonyl – 2 – methyl -1,4 – diazepane the following formula (1):
It is a compound represented by the particular it is a crystalline water-soluble, not hygroscopic, because it is excellent in chemical stability, it is useful as a medicament has been known for its hydrochloride dihydrate ( refer to Patent Documents 1 and 2). -5 Isoquinoline of these – the sulfonamide compounds, that prophylactic and therapeutic agents for cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage, cerebrovascular disorders such as cerebral edema, is useful as a therapeutic agent for preventing and glaucoma in particular is known (1-5 see Patent Document 1).
Conventionally, for example, a method of manufacturing by the method described in Patent Document 1, as shown in the following production process has been reported preparation of said compound (Production Method 1-A).
That is, (S)-1-tert-butoxycarbonyl – 3 – by reacting the presence of triethylamine in methylene chloride-fluoro-isoquinoline (2) – methyl -1,4 – diazepane and 5 (3) – chloro-sulfonyl -4 by adding trifluoroacetic acid in methylene chloride compound (the first step), obtained following (4) to synthesize a compound (4) by deprotection to (second step) the desired compound (1) This is a method of manufacturing.
It is also an important intermediate for preparing the compound (1) (S)-1-tert-butoxycarbonyl – 3 – methyl-1 ,4 – diazepane to (3), for example, in the following manner (; see JP Production Process 1-B) that can be produced is known.
Further, on the other hand, the compound (1) (see Patent Document 1) to be manufactured manufacturing routes such as: Any (Process 2) are known.
WO 1999/20620 pamphlet WO 2006/057397 pamphlet WO 1997/028130 pamphlet JP Patent Publication No. 2006-348028 JP Patent Publication No. 2006-290827
However, it is possible to produce in the laboratory of a small amount scale, but you place the point of view for mass industrial production, environmentally harmful halogenated hydrocarbon solvent in the compound of the above-mentioned process for producing 1-A is ( problem because it is carried out coupling step (3) and 2), giving significant adverse environmental exists. Therefore, solvent of halogenated hydrocarbon other than those listed to the specification of the patent document 1, for example, I tried actually dioxane, tetrahydrofuran and the like, but the present coupling reaction will be some progress indeed, Problems reaction is not completed raw material remained even after prolonged reaction time, yield undesirably stays in at most 30% was found. Furthermore, it is hard to decompose in the environment, elimination is also difficult to dioxane is not preferred irritating to humans, and are known as compounds that potentially harmful brain, kidney and liver .
When we actually produced compound (3) by the above production method 1-B, can be obtained desired compound in good yield merged with reproducibility is difficult has further been found that. That is, in the production path, 1,4 – and is used sodium hydride with dimethyl sulfoxide in forming a diazepane ring, except that I actually doing this step, Tsu than the reproducibility of the desired compound It could not be obtained in high yield Te. Also, that this is due to the synthetic route through the unstable intermediate, that it would be converted into another compound easily found this way. limitations and potential problems of the present production process is exposed since this stability may affect the reproducibility of the reaction.
Meanwhile, an attempt to carry out mass production is actually in the Process 2, it encounters various problems. For example, it is stored as an impurity whenever I repeat step, by-products formed in each stage by tandem production process ranging from step 8 gave more complex impurity profile. Depending, it is necessary to repeat a complicated recrystallization purity obtained as a medicine until the purification, the yield in the laboratory be a good overall yield is significantly reduced in the mass production of actual example be away, it does not have industrial utility of true was found. It can be summarized as follows: Considering from the viewpoint of GMP process control required for pharmaceutical production these problems.
Requires control process and numerous complex ranging 1) to 8 step, 3 2) third step – amino-1 – in the step of reacting a propanol, a difficult to remove positional isomers are mixed, 3) The fourth step water is mixed by the minute liquid extraction operation at the time of return to the free base from oxalate require crystallization purification by oxalate in the removal of contaminants of positional isomers, in 4) fifth step, 5) sixth step The Mitsunobu by reproducibility poor require water control in the Mitsunobu reaction used in the ring closure compounds to (1) compounds in (6), 6) ring closure reaction, departing management of the reagent added or the like is generated, in 7) Seventh Step it takes a complicated purification in impurity removal after the reaction, resulting in a decrease in isolated yield. These are issues that must be solved in order to provide a stable supply of raw material for pharmaceuticals high chemical purity is required.
Thus, gentle salt thereof, or the environment isoquinoline derivative comprising a compound represented by the formula (1), the present invention provides a novel production method having good reproducibility and high purity easily and in high yield I intended.
As a result of intensive studies in view of such circumstances, the present inventors, in the manufacturing process of the final target compound shown by the following expression
(Wherein represents a fluorine, chlorine, bromine or iodine,may,R 3 and 1, R 2 R represents a C 1-4 alkyl group be the same or different from each other, and P, X 1 is a protecting group shows a, 0 to m represents an integer of 3, 0 to n is. represents an integer of 3)
Is a urea-based solvents nitrile solvents, amide solvents, sulfoxide or solvents, the solvent may be preferably used in the coupling step of the compound (III) and (II) are generally very short time With these solvents It has been found that can be converted to the desired product quantitatively. It is possible to carry out the coupling step Volume scale while maintaining a high yield by using these solvents, there is no need to use a halogenated hydrocarbon solvent to give significant adverse environment. In consideration of the process such as removal of the solvent after the reaction was further found that acetonitrile is the best among these solvents. Also, since by using hydrochloric acid with ethyl acetate solvent in step deprotection can be isolated as crystal of hydrochloride desired compound (I), without going through the manipulation of solvent evaporation complicated , it has been found that it is possible to obtain the object compound (I) is a simpler operating procedure. Since there is no need to use a halogenated hydrocarbon solvent in this deprotection step further, there is no possibility of harming the environment.
It has been found that it is possible in mass production of (II), leading to the target compound purity, in high yield with good reproducibility as compared with the conventional method compounds are important intermediates in the coupling step further. That is, was it possible to lead to the intermediate high purity and in high yield by eliminating the production of a harmful halogenated hydrocarbon solvent to the environment in this manner. 1,4 addition – in order to avoid the problems encountered in the reaction using sodium hydride in dimethyl sulfoxide in forming the diazepane ring, in order to allow the cyclization reaction at mild conditions more, as a protecting group By performing the Mitsunobu reaction using Noshiru group instead of the carbobenzyloxy group, in addition to one step shorten the manufacturing process of the whole, without deteriorating the optical purity was successfully obtained the desired compound desired.
Ripasudil hydrochloride hydrate (Glanatec)
Ripasudil hydrochloride hydrate (Glanatec) was approved in Japan in 2014 for the treatment of glaucoma and ocular hypertension.
219 Originally discovered by D. Western Therapeutics Institute,Inc. and licensed by the Kowa Company, Ltd, ripasudil
functions as a selective Rho-kinase inhibitor and reduces intraocular pressure by stimulation of aqueous humour drainage of the
trabecular meshwork.219–221
While this recent approval allows for use of ripasudil as a twice-daily monotherapy treatment when
other drugs cannot be used or are not effective, clinical trials using ripasudil as a combination therapy with other glaucoma
drugs have shown promising results in the treatment of primary open-angle glaucoma or ocular hypertension.222,223 Currently, the
Kowa Company is also pursuing trials focused on the use of ripasudil for the treatment of diabetic retinopathy and diabetic macular edema.224
While initial synthetic routes to ripasudil were carried out via a stepwise functionalization of 4-fluoroisoquinoline-5-sulfonylchloride (238),225,226 more recent reports describe an efficient route to ripasudil employing a late stage-coupling of Boc-diazepane
(237) with 4-fluoroisoquinoline-5-sulfonyl chloride (238), enabling synthesis on multi-kilogram scale and isolation of the
drug in high purity (Scheme 40).221,227,228 This optimized route to ripasudil begins with 2-nitrobenzene sulfonyl chloride (NsCl)-
mediated protection of (S)-2-amino-1-propanol (234) in 82% yield.
In this case, use of the NaHCO3/THF/H2O conditions were essential for preventing bis-nosylation.228 Alcohol activation with methanesulfonyl chloride (MsCl) in N-methyl morpholine (NMM) took place smoothly to give the corresponding mesylate 235 in 91%
yield. Direct mesylate displacement with 3-aminopropanol and subsequent amine protection as the carbamate ((Boc)2O) in a
one-pot fashion provided the corresponding Boc-amino propanol product 236 in 95% yield over 2 steps.
With the acyclic diazepane precursor 236 in hand, employment of the intramolecular Fukuyama-Mitsunobu N-alkyl cyclization conditions (diisopropylazodicarboxylate (DIAD)/PPh3) allowed generation of the diazepane in 75% yield. Nosyl group cleavage with thiophenol/K2CO3provided the Boc-diazepane 237 in 65% overall yield and 98% purity following a pH-controlled aqueous workup.
Finally, 4-fluoroisoquinoline- 5-sulfonyl chloride (238)—prepared via subjection of 4- fluoroisoquinoline (239, Scheme 41)229 to sulfur trioxide and sulfuric acid followed by treatment with thionyl chloride and finally 4 N HCl in ethyl acetate—was involved in a 1-pot, two-step procedure in which this sulfonyl chloride was coupled with diazepane 237 (TEA/MeCN) to access the ripasudil framework in quantitative yield.
Synthesis of the final drug target by deprotection with 4 MHCl in ethyl acetate followed by neutralization with aqueoussodium hydroxide provided the free base of ripasudil in 93% yield and 99.8% purity. Conversion to the more stable hydrochloride dihydrate form could be performed by treatment of the free base with 1 M HCl/EtOH and subsequent heating of the hydrochloride in H2O/acetone to provide ripasudil hydrochloride dihydrate XXIX in 83% yield.230,231
219. Garnock, J. P. K. Drugs 2014, 74, 2211.
220. Isobe, T.; Mizuno, K.; Kaneko, Y.; Ohta, M.; Koide, T.; Tanabe, S. Curr. Eye Res.2014, 39, 813.
221. Sumi, K.; Inoue, Y.; Nishio, M.; Naito, Y.; Hosoya, T.; Suzuki, M.; Hidaka, H.
Bioorg. Med. Chem. Lett. 2014, 24, 831.
222. Mizuno, K. WO Patent 2,012,105,674, 2012.
223. Mizuno, K.; Matsumoto, J. WO Patent 2,007,007,737, 2007.
224. http://clinicaltrials.jp/user/cteDetail.jsp.
225. Gomi, N.; Ohgiya, T.; Shibuya, K. WO Patent 2,012,026,529, 2012.
226. Hidaka, H.; Nishio, M.; Sumi, K. US Patent 20,080,064,681, 2008.
227. Gomi, N.; Kouketsu, A.; Ohgiya, T.; Shibuya, K. Synthesis 2012, 44, 3171.
228. Gomi, N.; Ohgiya, T.; Shibuya, K.; Katsuyama, J.; Masumoto, M.; Sakai, H.Heterocycles 2011, 83, 1771.
229. Sakai, H.; Masunoto, M.; Katsuyama, J.; Onogi, K. WO Patent 2006090783A1,2006.
230. Hidaka, H.; Matsuura, A. WO Patent 1999020620A1, 1999.
231. Ohshima, T.; Hidaka, H.; Shiratsuchi, M.; Onogi, K.; Oda, T. US Patent7858615B2, 2008.
H-NMR spectral analysis
CAS NO. 223645-67-8, 4-fluoro-5-[[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl]isoquinoline H-NMR spectral analysis
C-NMR spectral analysis
CAS NO. 223645-67-8, 4-fluoro-5-[[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl]isoquinoline C-NMR spectral analysis
Tanihara, H; Inoue, T; Yamamoto, T; Kuwayama, Y; Abe, H; Suganami, H; Araie, M; the K-115 Clinical Study Group (2014). “Intra-ocular pressure-lowering effects of a Rho kinase inhibitor, ripasudil (K-115), over 24 hours in primary open-angle glaucoma and ocular hypertension: A randomized, open-label, crossover study”. Acta Ophthalmologica: n/a. doi:10.1111/aos.12599. PMID25487877.
To a solution of 4-bromobenzene-l,2-diamine (2.5 g, 13.37 mmol) in DCM (30 mL) was added (S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoic acid (3.09 g, 13.37 mmol), DIPEA (2.334 mL, 13.37 mmol) and HATU (5.08 g, 13.37 mmol). The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with water and extracted with DCM. The organic phase was washed with brine, dried over Na2S04, filtered and concentrated. The crude material was purified by ISCO using 40 g Redisep silica column, CHCl3/MeOH as eluant to obtain (S)-tert-butyl ( 1 -((2-amino-4-bromophenyl)amino)-3 ,3 -dimethyl- 1 -oxobutan-2-yl) carbamate (1.82 g) as yellow solid. LC (Condition 1): Rt = 2.13 min. LC/MS: Anal. Calcd. for [M+H20]+ Ci7H27BrN204 : 402.12; found 402.2. 1H NMR (DMSO-d6, δ = 2.50 ppm, 400 MHz): δ 9.35 – 9.21 (m, 1 H), 7.07 (d, J= 8.5 Hz, 1 H), 6.91 (d, J= 2.0 Hz, 1 H), 6.80 – 6.60 (m, 1 H), 5.25 – 5.01 (m, 2 H), 4.07 – 3.89 (m, 1 H), 1.52 – 1.34 (m, 9 H), 1.02 – 0.86 (m, 9 H).
Example B-l, Step b
Acetic acid (15 mL) was added to (S)-tert-butyl (l-((2-amino-4-bromo phenyl)amino)-3,3-dimethyl-l-oxobutan-2-yl)carbamate (1.8 g, 4.50 mmol) and the reaction mixture was heated to 65 °C for overnight. The volatile component was removed in vacuo, and the residue was co-evaporated with dry CH2C12 (2 x 15 mL). The organic phase was washed with saturated NaHC03 solution, brine, dried over Na2S04 and concentrated to obtain (S)-tert-butyl (l-(6-bromo-lH-benzo[d] imidazol-2-yl)-2,2-dimethyl propyl)carbamate (1.68 g) as yellow solid. LC (Condition 1): Rt = 2.19 min. LC/MS: Anal. Calcd. for [M+H]+ Ci7H25BrN302 : 381.11; found 382.2. 1H NMR (DMSO-dg, δ = 2.50 ppm, 300 MHz): δ 12.46 – 12.27 (m, 1 H), 7.82 – 7.65 (m, 1 H), 7.59 – 7.41 (m, 1 H), 7.29 (dt, J= 1.9, 8.5 Hz, 1 H), 7.12 – 6.90 (m, 1 H), 4.64 (d, J= 9.8 Hz, 1 H), 1.44 – 1.27 (m, 9 H), 0.88 (br. s., 9 H).
-1 Step c
To a solution of (S)-tert-butyl (l-(6-bromo-lH-benzo[d]imidazol-2-yl)-2,2-dimethyl propyl) carbamate (1.57 g, 4.11 mmol) in dioxane (25 mL) was added bis (pinacolato)diboron (1.564 g, 6.16 mmol) and potassium acetate (1.209 g, 12.32 mmol). The reaction mixture was purged with argon for 10 min then PdCl2(dppf) (0.150 g, 0.205 mmol) was added to the above reaction mixture and again purged with argon for 5 min. The reaction mixture was heated to 90 °C for overnight. The reaction mixture was diluted with water (15 ml) and extracted with EtOAc (2 x 25 ml). The combined organic phase was washed with brine, dried over Na2S04 and concentrated in vacuo. The crude material was purified by ISCO using 40 g Redisep column, hexane/ethyl acetate as eluant to afford (S)-tert-butyl (2,2-dimethyl-l-(6-(4,4,5 ,5-tetramethyl- 1 ,3 ,2-dioxaborolan-2-yl)- 1 H-benzo[d]imidazol-2-yl)propyl) carbamate (1.35 g) as yellow solid. LC (Condition 1): Rt = 2.21 min. LC/MS: Anal. Calcd. for [M+H]+ C23H37BN304 : 430.29; found 430.4. 1H NMR (CD3OD, δ = 3.34 ppm, 400 MHz): δ 7.98 (s, 1 H), 7.65 (dd, J= 1.0, 8.5 Hz, 1 H), 7.53(d, J= 8.5 Hz, 1 H), 4.73 (br. s., 1 H), 1.37 (s, 12 H), 1.24 (m, 9 H), 1.01 (s, 9 H).
-1 Step d
To a solution of (S)-tert-butyl (2,2-dimethyl-l-(6-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-lH-benzo[d]imidazol-2-yl)propyl)carbamate (1.114 g, 2.59 mmol) and 4,16-dibromo[2,2]paracyclophane (0.38g, 1.038 mmol) in dioxane (10 mL) was added Cs2C03 (0.845 g, 2.59 mmol) in water (2 mL) and degassed for 10 min.
PdCl2(dppf) (0.038 g, 0.052 mmol) was added to the above reaction mixture and again degassed for 5 min. The reaction mixture was heated to 90 °C for 12 h. Then the reaction mixture was filtered to get Example B-1 Step d which was taken for next step without further purification. LC (Condition 1): Rt = 2.54 min. LC/MS: Anal. Calcd. for [M+H]+ ^0Η63Ν6Ο4 : 811.49; found 811.6. 1H NMR (DMSO-d6, δ = 2.50 ppm, 300 MHz): δ 12.36 (br. s., 2 H), 7.85 – 7.52 (m, 4 H), 7.32 (d, J= 7.9 Hz, 2 H), 7.05 (br. s., 2 H), 6.89 – 6.67 (m, 4 H), 6.54 (br. s., 2 H), 4.72 (d, J= 8.7 Hz, 2 H), 3.57 – 3.44 (m, 2 H), 3.07 (br. s., 2 H), 2.83 (br. s., 2 H), 2.65 (br. s., 2 H), 1.36 (s, 18 H), 1.08 – 0.91 (m, 18 H).
-1 Step e
HC1 in dioxane (4 mL, 24.00 mmol) was added to Example B-1 Step d (0.1 g,
0.102 mmol), and the reaction mixture was allowed to stir at RT for 2 h. Completion of the reaction was monitored by LCMS. The volatile component was removed in vacuo and the residue was washed with diethyl ether and dried to afford Example B-1 Step e (0.07 g) as yellow solid. LC (Condition 1): R, = 2.54 min. LC/MS: Anal.
To a solution of Example B-1 Step e (0.04 g, 0.053 mmol) in DMF (5 mL) was added 4,4-difluorocyclohexanecarboxylic acid (0.017 g, 0.106 mmol), DIPEA (0.055 mL, 0.317 mmol) and HATU (0.030 g, 0.079 mmol). After being stirred for 2 h at room temperature, the volatile component was removed in vacuo and the residue was dissolved in DCM (10 mL), washed with saturated solution of NH4C1, 10% NaHC03 solution, brine, dried over Na2S04 and concentrated in vacuo. The crude was purified by reverse phase HPLC purification to give Example B-1 as a white solid. LC (Condition 1): R, = 2.37 min. LC/MS: Anal. Calcd. for [M+H]+
Starting materials can be obtained from commercial sources or prepared by well-established literature methods known to those of ordinary skill in the art. Acid precursors for the final step can be prepared according to the methods described in U.S. Patent Application Serial No. 13/933495, filed July 2, 2013.
LC/MS Condition 1
Column = Ascentis Express C18, 2.1 X 50 mm, 2.7 um
Solvent A = CH3CN (2%) + 10 mM NH4COOH in H20 (98%)
Solvent B = CH3CN (98%) + 10 mM NH4COOH in H20 (2%)
Start %B = 0; Final %B = 100
Gradient time = 1.4 min; Stop time = 4 min
Stop time = 4 min
Flow Rate = 1 mL/min; Wavelength = 220 nm
LC/MS Condition 2
Column = Waters BEH CI 8, 2.0 x 50 mm, 1.7 μιη
Slovent A = ACN (5%) + H20 (95%) containing 10 mM NH4OAc
Solvent B = ACN (95%) + H20 (5%) containing 10 mM NH4OAc
Start %B = 0; Final %B = 100
Gradient time = 3 min
Flow Rate = 1 mL/min
Wavelength = 220 nm
Temperature = 50 °C
LC/MS Condition 3
Column: Waters Phenomenex CI 8, 2.0 x 30 mm, 3 μιη particle
Mobile Phase A: 10% MeOH:90% Water :0.1%TFA
Mobile Phase B: 90% MeOH: 10% Water :0.1%TFA
Gradient: 0%B, 0-100% B over 3 minutes, then a 1 -minute hold at 100% B Flow: 0.8mL/min
Detection: 220 nm
Temperature: 40 °C
LC/MS Condition 4
Column: Waters BEH CI 8, 2.0 x 50 mm, 1.7 μιη particle
Mobile Phase A: 5:95 acetonitrile: water with 10 mM ammonium acetate Mobile Phase B: 95:5 acetonitrile: water with 10 mM ammonium acetate Gradient: 0%B, 0-100% B over 3 minutes, then a 0.5-minute hold at 100% B Flow: 1 mL/min
Phase II clinical development at AbbVie (previously Abbott) for the treatment of chronic hepatitis C infection in combination with ABT-450/ritonavir and, in combination with peginterferon alpha-2a/ribavirin (pegIFN/RBV) in treatment naïve Hepatitis C virus (HCV) genotype 1 infected patients.
Ombitasvir is Dimethyl ([(2S,5S)-1-(4-tert-butylphenyl) pyrrolidine-2,5diyl]bis{benzene-4,1-diylcarbamoyl(2S)pyrrolidine-2,1-diyl[(2S)-3-methyl-1-oxobutane-1,2diyl]})biscarbamate hydrate. The molecular formula is C50H67N7O8•4.5H2O (hydrate) and the molecular weight for the drug substance is 975.20 (hydrate).
Ombitasvir is in phase II clinical development at AbbVie (previously Abbott) for the treatment of chronic hepatitis C infection in combination with ABT-450/ritonavir and, in combination with peginterferon alpha-2a/ribavirin (pegIFN/RBV) in treatment naïve Hepatitis C virus (HCV) genotype 1 infected patients.
Ombitasvir is part of a fixed-dose formulation with ABT-450/ritonavir that is approved in the U.S. and the E.U.
In January 2013, Abbott spun-off its research-based pharmaceutical business into a newly-formed company AbbVie. In 2013, breakthrough therapy designation was assigned in the U.S. for the treatment of genotype 1 hepatitis C in combination with ABT-450, ritonavir and ABT-333, with and without ribavirin.
Ombitasvir acts by inhibiting the HCV protein NS5A.[5]
Ombitasvir is an orally available inhibitor of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) replication complex, with potential activity against HCV. Upon oral administration and after intracellular uptake, ombitasvir binds to and blocks the activity of the NS5A protein. This results in the disruption of the viral RNA replication complex, blockage of HCV RNA production, and inhibition of viral replication. NS5A, a zinc-binding and proline-rich hydrophilic phosphoprotein, plays a crucial role in HCV RNA replication. HCV is a small, enveloped, single-stranded RNA virus belonging to the Flaviviridae family; HCV infection is associated with the development of hepatocellular carcinoma (HCC).
Ombitasvir hydrate is a NS5A non-nucleoside polymerase inhibitor which is approved as part of a four drug combination for the
treatment of adults with genotype 1 hepatitis C virus infection including those with compensated cirrhosis.REF 6,7
The four drug combination treatment consists of ombitasvir, paritaprevir (XXVII), ritonavir, and dasabuvir (X). This combination treatment is marketed as Viekira PakTM and was developed by Abbvie as an all oral treatment that eliminates the need for pegylated interferon-a injections.
The synthesis of ombitasvir hydrate is shown in Scheme 34.REF 8 Alkylation of 1-(4-nitrophenyl)ethanone (209)
with 2-bromo-1-(4-nitrophenyl)ethanone (208) in the presence of zinc chloride produced diketone 210 in 61% yield.
Asymmetric reduction of the diketone using N,N-diethylaniline borane with (S)-()-a,a-diphenyl-2-pyrrolidinemethanol (211) and trimethoxyborate gave diol 212 in 61% yield and 99.3% ee.
The diol was then treated with methanesulfonic anhydride to generate the corresponding bis-mesylate which was reacted with 4-tert-butylaniline to give pyrrolidine 213 in 51% yield over the two steps.
Hydrogenolysis of the nitro groups was accomplished using Raney nickel catalyst to give bis-aniline 214.
Separately, (L)-valine (216,Scheme 35) was reacted with methyl chloroformate to give the corresponding methyl carbamate in 90% yield which was coupled to L-proline benzyl ester in the presence of EDC and HOBt to give the corresponding dipeptide in 90% yield.
Hydrogenolysis of the benzyl ester group of the protected dipeptide using Pd/alumina catalyst produced dipeptide acid 215. Aniline 214 was treated with two equivalents of acid 215 in the presence of 1-propanephosphonic acid cyclic anhydride (T3P). The crude product was recrystallized from ethanol and heptane to give ombitasvir hydrate (XXV). No yields were provided to the final steps of this synthesis.
7. DeGoey, D. A.; Randolph, J. T.; Liu, D.; Pratt, J.; Hutchins, C.; Donner, P.;Krueger, A. C.; Matulenko, M.; Patel, S.; Motter, C. E.; Nelson, L.; Keddy, R.;Tufano, M.; Caspi, D. D.; Krishnan, P.; Mistry, N.; Koev, G.; Reisch, T. J.;Mondal, R.; Pilot-Matias, T.; Gao, Y.; Beno, D. W.; Maring, C. J.; Molla, A.;Dumas, E.; Campbell, A.; Williams, L.; Collins, C.; Wagner, R.; Kati, W. M. J.
Med. Chem. 2014, 57, 2047. 8. DeGoey, D. A.; Kati, W. M.; Hutchins, C. W.; Donner, P. L.; Krueger, A. C.;Randolph, J. T.; Motter, C. E.; Nelson, L. T.; Patel, S. V.; Matulenko, M. A.;Keddy, R. G.; Jinkerson, T. K.; Soltwedel, T. N.; Liu, D.; Pratt, J. K.; Rockway, T.W.; Maring, C. J.; Hutchinson, D. K.; Flentge, C. A.; Wagner, R.; Tufano, M. D.;Betebenner, D. A.; Lavin, M. J.; Sarris, K.; Woller, K. R.; Wagaw, S. H.; Califano,
J. C.; Li, W.; Caspi, D. D.; Bellizzi, M. E. US Patent 2010317568A1, 2010.
We describe here N-phenylpyrrolidine-based inhibitors of HCV NS5A with excellent potency, metabolic stability, and pharmacokinetics. Compounds with 2S,5S stereochemistry at the pyrrolidine ring provided improved genotype 1 (GT1) potency compared to the 2R,5Ranalogues. Furthermore, the attachment of substituents at the 4-position of the central N-phenyl group resulted in compounds with improved potency. Substitution with tert-butyl, as in compound 38 (ABT-267), provided compounds with low-picomolar EC50 values and superior pharmacokinetics. It was discovered that compound 38 was a pan-genotypic HCV inhibitor, with an EC50 range of 1.7–19.3 pM against GT1a, -1b, -2a, -2b, -3a, -4a, and -5a and 366 pM against GT6a. Compound 38 decreased HCV RNA up to 3.10 log10 IU/mL during 3-day monotherapy in treatment-naive HCV GT1-infected subjects and is currently in phase 3 clinical trials in combination with an NS3 protease inhibitor with ritonavir (r) (ABT-450/r) and an NS5B non-nucleoside polymerase inhibitor (ABT-333), with and without ribavirin.
Dimethyl (2S,2′S)-1,1′-((2S,2′S)-2,2′-(4,4′-((2S,5S)-1-(4-tert-Butylphenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl)bis(oxomethylene)bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-2,1-diyl)dicarbamate (38)…desired and Dimethyl (2S,2′S)-1,1′-((2S,2′S)-2,2′-(4,4′-((2R,5R)-1-(4-tert-Butylphenyl)pyrrolidine-2,5-diyl)bis(4,1-phenylene))bis(azanediyl)bis(oxomethylene)bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-2,1-diyl)dicarbamate (39)…….undesired
…………….. The resulting mixture was stirred at room temperature for 16 h. The mixture was partitioned between ethyl acetate and water, and the organic layer was washed with saturated aqueous NaHCO3, brine (2×) and dried with Na2SO4. The drying agent was filtered off and the solution was concentrated in vacuo to give a crude product that was purified by column chromatography on silica gel, eluting with a solvent gradient of 2–8% methanol in dichloromethane to give a 1:1 mixture of trans-pyrrolidine isomers (290 mg, 96%). The mixture was separated on a Chiralpak AD-H column, eluting with a mixture of 1 part (2:1 isopropanol/ethanol) and 2 parts hexanes (0.1% TFA).
Example 34A l-(4-fer?-butylphenyl)-2,5-bis(4-nitrophenyl)pyrrolidine The product from Example 1C (3.67 g, 7.51 mmol) and 4-tert-butylaniline (11.86 ml, 75 mmol) in DMF (40 ml) was stirred under nitrogen at 50 °C for 4 h. The resulting mixture was diluted into ethyl acetate, treated with IM HCl, stirred for 10 minutes and filtered to remove solids. The filtrate organic layer was washed twice with brine, dried with sodium sulfate, filtered and evaporated. The residue was purified by chromatography on silica gel eluting with ethyl acetate in hexane (5% to 30%) to give a solid. The solid was triturated in a minimal volume of 1 :9 ethyl acetate/hexane to give a light yellow solid as a mixture of trans and cis isomers (1.21 g, 36%).
Example 34B 4,4′-((2S,5S)-1-(4-tert-butylphenyl)pyrrolidine-2,5-diyl)dianiline and 4,4′-((2R,5R)-1-(4-fert- butylphenyl)pyrrolidine-2,5-diyl)dianiline To a solution of the product from Example 34A (1.1 g, 2.47 mmol) in ethanol (20 ml) and
THF (20 ml) was added PtC>2 (0.22 g, 0.97 mmol) in a 50 ml pressure bottle and stirred under 30 psi hydrogen at room temperature for 1 h. The mixture was filtered through a nylon membrane and evaporated. The residue was purified by chromatography on silica gel eluting with ethyl acetate in hexane (20% to 60%). The title compound eluted as the first of 2 stereoisomers (trans isomer, 0.51 g, 54%).
Example 34C
(2S,2’S)-tert-Butyl 2,2′-(4,4′-((2S,5S)-1-(4-fer/’-butylphenyl)pyrrolidine-2,5-diyl)bis(4,l- phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine- 1 -carboxylate and (2S,2’S)-tert-Butyl 2,2′- (4,4′-((2R,5R)-1-(4-tert-butylphenyl)pyrrolidine-2,5-diyl)bis(4,l- phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-carboxylate To a mixture of the product from Example 34B (250 mg, 0.648 mmol), (S)-1-(tert- butoxycarbonyl)pyrrolidine-2-carboxylic acid (307 mg, 1.427 mmol) and HATU (542 mg, 1.427 mmol) in DMSO (10 ml) was added Hunig’s base (0.453 ml, 2.59 mmol). The reaction mixture was stirred at room temperature for 1 h. The mixture was partitioned with ethyl acetate and water. The organic layer was washed with brine, dried with sodium sulfate, filtered and evaporated. The residue was purified by chromatography on silica gel eluting with ethyl acetate in hexane (10% to 50%) to give the title compound (500 mg, 99%).
Example 34D
(2S,2’S)-N,N’-(4,4′-((2S,5S)-1-(4-ter/’-butylphenyl)pyrrolidine-2,5-diyl)bis(4,l- phenylene))dipyrrolidine-2-carboxamide and (2S,2’S)-N,N’-(4,4′-((2R,5R)-1-(4-tert- butylphenyl)pyrrolidine-2,5-diyl)bis(4,l-phenylene))dipyrrolidine-2-carboxamide To the product from Example 34C (498 mg, 0.638 mmol) in dichloromethane (4 ml) was added TFA (6 ml). The reaction mixture was stirred at room temperature for 1 h and concentrated in vacuo. The residue was partitioned between 3: 1 CHCl3dsopropyl alcohol and saturated aq. NaHCO3. The aqueous layer was extracted by 3: 1 CHCl3:isopropyl alcohol again. The combined organic layers were dried over
filtered and concentrated to give the title compound (345 mg, 93%).
Example 34E Dimethyl (2S,2’S)-l,r-((2S,2’S)-2,2′-(4,4′-((2S,5S)-1-(4-fert-butylphenyl)pyrrolidine-2,5-diyl)bis(4,l- phenylene))bis(azanediyl)bis(oxomethylene)bis(pyrrolidine-2, 1 -diyl))bis(3-methyl- 1 -oxobutane-2, 1 – diyl)dicarbamate and
The product from Example 34D (29.0 mg, 0.050 mmol), (S)-2-(methoxycarbonylamino)-3- methylbutanoic acid (19.27 mg, 0.110 mmol), EDAC (21.09 mg, 0.110 mmol), HOBT (16.85 mg,
0.110 mmol) and N-methylmorpholine (0.027 ml, 0.250 mmol) were combined in DMF (2 ml). The reaction mixture was stirred at room temperature for 3 h. The mixture was partitioned with ethyl acetate and water. The organic layer was washed with brine twice, dried with sodium sulfate, filtered and evaporated. The residue was purified by chromatography on silica gel eluting with ethyl acetate in hexane (50% to 80%) to give a solid. The solid was triturated with ethyl acetate/hexane to give the title compound (13 mg, 29%). 1H NMR (400 MHz, DMSO-D6) δ ppm 0.85 – 0.95 (m, 12 H) 1.11 (s, 9 H) 1.59 – 1.65 (m, 2 H) 1.79 – 2.04 (m, 8 H) 2.10 – 2.18 (m, 2 H) 2.41-2.46 (m, 2H) 3.52 (s, 6 H)
Hz, 2 H) 7.50 (d, J=8.24 Hz, 4 H) 9.98 (s, 2 H); (ESI+) m/z 895 (M+H)+. The title compound showed an EC50 value of less than about 0.1 nM in HCV lb-Conl replicon assays in the presence of 5% FBS.
The product from Example 34E was purified by chiral chromatography on a Chiralpak AD-H semi-prep column eluting with a 2:1 mixture of hexane:(2: l isopropyl alcohol: EtOH). The title compound was the second of 2 diastereomers to elute. 1H NMR (400 MHz, DMSO-D6) δ ppm 0.87
Example 37A (S)-2,5-dioxopyrrolidin-1-yl 2-(methoxycarbonylamino)-3-methylbutanoate To a mixture of (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid (19.66 g, 112 mmol) and N-hydroxysuccinimide (13.29g, 116 mmol) was added ethyl acetate (250 ml), and the mixture was cooled to 0-5 °C. Diisopropylcarbodiimide (13.88 g, 110 mmol) was added and the reaction mixture was stirred at 0-5 °C for about 1 hour. The reaction mixture was warmed to room temperature. The solids (diisopropylurea by-product) were filtered and rinsed with ethyl acetate. The filtrate was concentrated in vacuo to an oil. Isopropyl alcohol (200 ml) was added to the oil and the mixture was heated to about 50 °C to obtain a homogeneous solution. Upon cooling, crystalline solids formed. The solids were filtered and washed with isopropyl alcohol (3 x 20 ml) and dried to give the title compound as a white solid (23.2 g, 77% yield).
Example 37B
(S)- 1 -((S)-2-(methoxycarbonylamino)-3-methylbutanoyl)pyrrolidine-2-carboxylic acid To a mixture of L-proline (4.44g, 38.6 mmol), water (20 ml), acetonitrile (20 ml) and DIEA (9.5 g, 73.5 mmol) was added a solution of the product from Example 37A (1Og, 36.7 mmol) in acetonitrile (20 inL) over 10 minutes. The reaction mixture was stirred overnight at room temperature. The solution was concentrated under vacuum to remove the acetonitrile. To the resulting clear water solution was added 6N HCl (9 ml) until pH ~ 2 .The solution was transferred to a separatory funnel and 25% NaCl (10 ml) was added and the mixture was extracted with ethyl acetate (75 ml), and then again with ethyl acetate (6 x 20 ml), and the combined extracts were washed with 25% NaCl (2 x 10ml). The solvent was evaporated to give a thick oil. Heptane was added and the solvent was evaporated to give a foam, which was dried under high vacuum. Diethyl ether was added and the solvent was evaporated to give a foam, which was dried under high vacuum to give the title compound (10.67g) as a white solid.
The compound of Example 37B can also be prepreared according to the following procedure: To a flask was charged L- valine (35 g, 299 mmol), IN sodium hydroxide solution (526 ml,
526 mmol) and sodium carbonate (17.42 g, 164 mmol). The mixture was stirred for 15 min to dissolve solids and then cooled to 15 °C. Methyl chloroformate (29.6 g, 314 mmol) was added slowly to the reaction mixture. The mixture was then stirred at rt for 30 min. The mixture was cooled to 15 °C and pH adjusted to -5.0 with concentrated HCl solution. 100 inL of 2-methytetrahydrofuran (2- MeTHF) was added and the adjustment of pH continued until the pH reached ~ 2.0. 150 mL of 2- MeTHF was added and the mixture was stirred for 15 min. Layers were separated and the aqueous layer extracted with 100 mL of 2-MeTHF. The combined organic layer was dried over anhyd Na2SC^ and filtered, and Na2SC^ cake was washed with 50 mL of 2-MeTHF. The product solution was concentrated to ~ 100 mL, chased with 120 mL of IPAc twice. 250 mL of heptanes was charged slowly and then the volume of the mixture was concentrated to 300 mL. The mixture was heated to 45 °C and 160 mL of heptanes charged. The mixture was cooled to rt in 2h, stirred for 30 min, filtered and washed with 2-MeTHF/heptanes mixture (1:7, 80 inL). The wetcake was dried at 55 °C for 24 h to give 47.1 g of Moc-L- VaI-OH product as a white solid (90%).
Moc-L- VaI-OH (15O g, 856 mmol), HOBt hydrate (138 g, 899 mmol) and DMF (1500 ml) were charged to a flask. The mixture was stirred for 15 min to give a clear solution. EDC hydrochloride (172 g, 899 mmol) was charged and mixed for 20 min. The mixture was cooled to 13
°C and (L)-proline benzyl ester hydrochloride (207 g, 856 mmol) charged. Triethylamine (109 g,
1079 mmol) was then charged in 30 min. The resulting suspension was mixed at rt for 1.5 h. The reaction mixture was cooled to 15 °C and 1500 mL of 6.7% NaHCO3 charged in 1.5 h, followed by the addition of 1200 mL of water over 60 min. The mixture was stirred at rt for 30 min, filtered and washed with water/DMF mixture (1 :2, 250 mL) and then with water (1500 mL). The wetcake was dried at 55 °C for 24 h to give 282 g of product as a white solid (90%).
The resulting solids (40 g) and 5% Pd/ Alumina were charged to a Parr reactor followed by THF (160 mL). The reactor was sealed and purged with nitrogen (6 x 20 psig) followed by a hydrogen purge (6 x 30 psig). The reactor was pressurized to 30 psig with hydrogen and agitated at room temperature for approximately 15 hours. The resulting slurry was filtered through a GF/F filter and concentrated to approximately 135 g solution. Heptane was added (120 mL), and the solution was stirred until solids formed. After an addition 2 – 3 hours additional heptane was added drop-wise (240 mL), the slurry was stirred for approximately 1 hour, then filtered. The solids were dried to afford the title compound.
The product from Example 32 (5.01 g, 13.39 mmol) was combined with 2- methyltetrahydrofuran (70 mL) and cooled to -5 °C, and N,N-diisopropylethylamine (6.81 g, 52.7 mmol) was added over 30 seconds. Separately, a solution of methanesulfonic anhydride (6.01 g, 34.5 mmol) in 2-methyltetrahydrofuran (30 mL) was prepared and added to the diol slurry over 3 min., maintaining the internal temperature between -15 °C and -25 °C. After mixing for 5 min at -15 °C, the cooling bath was removed and the reaction was allowed to warm slowly to 23 °C and mixed for 30 minutes. After reaction completion, the crude slurry was carried immediately into the next step.
To the crude product solution from Example 37C (7.35 g, 13.39 mmol) was added 4-tert- butylaniline (13.4 g, 90 mmol) at 23 °C over 1 minute. The reaction was heated to 65 °C for 2 h. After completion, the reaction mixture was cooled to 23 °C and diluted with 2-methyltetrahydrofuran (100 mL) and 1 M HCl (150 mL). After partitioning the phases, the organic phase was treated with 1 M HCl (140 mL), 2-methyltetrahydrofuran (50 mL), and 25 wt% aq. NaCl (100 mL), and the phases were partitioned. The organic phase was washed with 25 wt% aq. NaCl (50 mL), dried over MgSO/t, filtered, and concentrated in vacuo to approximately 20 mL. Heptane (30 mL) and additional 2- methyltetrahydrofuran were added in order to induce crystallization. The slurry was concentrated further, and additional heptane (40 mL) was slowly added and the slurry was filtered, washing with 2- methyltetrahydrofuran:heptane (1:4, 20 mL). The solids were suspended in MeOH (46 mL) for 3 h, filtered, and the wet solid was washed with additional MeOH (18 mL). The solid was dried at 45 °C in a vacuum oven for 16 h to provide the title compound (3.08 g, 51% 2-step yield).
To a 160 ml Parr stirrer hydrogenation vessel was added the product from Example 37D (2 g, 4.49 mmol), followed by 60 ml of THF, and Raney Nickel Grace 2800 (1 g, 50 wt% (dry basis)) under a stream of nitrogen. The reactor was assembled and purged with nitrogen (8 x 20 psig) followed by purging with hydrogen (8 x 30 psig). The reactor was then pressurized to 30 psig with hydrogen and agitation (700 rpm) began and continued for a total of 16 h at room temperature. The slurry was filtered by vacuum filtration using a GF/F Whatman glass fiber filter. Evaporation of the filtrate to afford a slurry followed by the addition heptane and filtration gave the crude title compound, which was dried and used directly in the next step.
Example 37F dimethyl (2S,2’S)-l,r-((2S,2’S)-2,2′-(4,4′-((2S,5S)-1-(4-tert-butylphenyl)pyrrolidine-2,5-diyl)bis(4, l- phenylene)bis(azanediyl)bis(oxomethylene))bis(pyrrolidine-2, 1 -diyl))bis(3-methyl- 1 -oxobutane-2, 1 – diy 1) die arb amate To a solution of the product from Example 37E (1.64 g, 4.25 mmol) in DMF (20 ml), the product from Example 37B (2.89 g, 10.63 mmol), and HATU (4.04 g, 10.63 mmol) in DMF (15OmL) was added triethylamine (1.07 g, 10.63 mmol), and the solution was stirred at room temperature for 90 min. To the reaction mixture was poured 20 mL of water, and the white precipitate obtained was filtered, and the solid was washed with water (3×5 mL). The solid was blow dried for Ih. The crude material was loaded on a silica gel column and eluted with a gradient starting with ethyl acetate/ heptane (3/7), and ending with pure ethyl acetate. The desired fractions were combined and solvent distilled off to give a very light yellow solid, which was dried at 45 °C in a vacuum oven with nitrogen purge for 15 h to give the title compound (2.3 g, 61% yield). 1H NMR (400 MHz, DMSO- D6) δ ppm 0.88 (d, J=6.61 Hz, 6 H) 0.93 (d, J=6.72 Hz, 6 H) 1.11 (s, 9 H) 1.63 (d, J=5.42 Hz, 2 H) 1.80 – 2.04 (m, 8 H) 2.09 – 2.19 (m, 2 H) 2.44 – 2.47 (m, 2 H) 3.52 (s, 6 H) 3.59 – 3.66 (m, 2 H) 3.77 – 3.84 (m, 2 H) 4.02 (t, J=8.40 Hz, 2 H) 4.42 (dd, J=7.86, 4.83 Hz, 2 H) 5.14 (d, J=6.18 Hz, 2 H) 6.17 (d, J=8.67 Hz, 2 H) 6.94 (d, J=8.78 Hz, 2 H) 7.13 (d, J=8.46 Hz, 4 H) 7.31 (d, J=8.35 Hz, 2 H) 7.50 (d, J=8.35 Hz, 4 H) 9.98 (s, 2 H).
Alternately, the product from example 37E (11.7 g, 85 wt%, 25.8 mmol) and the product from example 37B (15.45 g, 56.7 mmol) are suspended in EtOAc (117 mL), diisopropylethylamine (18.67 g, 144 mmol) is added and the solution is cooled to 0 °C. In a separate flask, 1-propanephosphonic acid cyclic anhydride (T3P®) (46.0 g, 50 wt% in EtOAc, 72.2 mmol) was dissolved in EtOAc (58.5 mL), and charged to an addition funnel. The T3P solution is added to the reaction mixture drop-wise over 3-4 h and stirred until the reaction is complete. The reaction is warmed to room temperature,and washed with IM HCl/7.5 wt% NaCl (100 mL), then washed with 5% NaHCO3 (100 mL), then washed with 5% NaCl solution (100 mL). The solution was concentrated to approximately 60 mL, EtOH (300 mL) was added, and the solution was concentrated to 84 g solution.
A portion of the EtOH solution of product (29 g) was heated to 40 °C, and added 134 g 40 w% EtOH in H2O. A slurry of seeds in 58 wt/wt% EtOH/H2O was added, allowed to stir at 40 °C for several hours, then cooled to 0 °C. The slurry is then filtered, and washed with 58wt/wt% EtOH/H2O. The product is dried at 40 – 60 °C under vacuum, and then rehydrated by placing a tray of water in the vacuum oven to give the title compound. The title compound showed an EC50 value of less than about 0.1 nM in HCV lb-Conl replicon assays in the presence of 5% FBS.
Example 34A l-(4-fer?-butylphenyl)-2,5-bis(4-nitrophenyl)pyrrolidine The product from Example 1C (3.67 g, 7.51 mmol) and 4-tert-butylaniline (11.86 ml, 75 mmol) in DMF (40 ml) was stirred under nitrogen at 50 °C for 4 h. The resulting mixture was diluted into ethyl acetate, treated with IM HCl, stirred for 10 minutes and filtered to remove solids. The filtrate organic layer was washed twice with brine, dried with sodium sulfate, filtered and evaporated. The residue was purified by chromatography on silica gel eluting with ethyl acetate in hexane (5% to 30%) to give a solid. The solid was triturated in a minimal volume of 1 :9 ethyl acetate/hexane to give a light yellow solid as a mixture of trans and cis isomers (1.21 g, 36%).
Example 34B 4,4′-((2S,5S)-1-(4-tert-butylphenyl)pyrrolidine-2,5-diyl)dianiline and 4,4′-((2R,5R)-1-(4-fert- butylphenyl)pyrrolidine-2,5-diyl)dianiline To a solution of the product from Example 34A (1.1 g, 2.47 mmol) in ethanol (20 ml) and
THF (20 ml) was added PtC>2 (0.22 g, 0.97 mmol) in a 50 ml pressure bottle and stirred under 30 psi hydrogen at room temperature for 1 h. The mixture was filtered through a nylon membrane and evaporated. The residue was purified by chromatography on silica gel eluting with ethyl acetate in hexane (20% to 60%). The title compound eluted as the first of 2 stereoisomers (trans isomer, 0.51 g, 54%).
Example 34C
(2S,2’S)-tert-Butyl 2,2′-(4,4′-((2S,5S)-1-(4-fer/’-butylphenyl)pyrrolidine-2,5-diyl)bis(4,l- phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine- 1 -carboxylate and (2S,2’S)-tert-Butyl 2,2′- (4,4′-((2R,5R)-1-(4-tert-butylphenyl)pyrrolidine-2,5-diyl)bis(4,l- phenylene))bis(azanediyl)bis(oxomethylene)dipyrrolidine-1-carboxylate To a mixture of the product from Example 34B (250 mg, 0.648 mmol), (S)-1-(tert- butoxycarbonyl)pyrrolidine-2-carboxylic acid (307 mg, 1.427 mmol) and HATU (542 mg, 1.427 mmol) in DMSO (10 ml) was added Hunig’s base (0.453 ml, 2.59 mmol). The reaction mixture was stirred at room temperature for 1 h. The mixture was partitioned with ethyl acetate and water. The organic layer was washed with brine, dried with sodium sulfate, filtered and evaporated. The residue was purified by chromatography on silica gel eluting with ethyl acetate in hexane (10% to 50%) to give the title compound (500 mg, 99%).
Example 34D
(2S,2’S)-N,N’-(4,4′-((2S,5S)-1-(4-ter/’-butylphenyl)pyrrolidine-2,5-diyl)bis(4,l- phenylene))dipyrrolidine-2-carboxamide and (2S,2’S)-N,N’-(4,4′-((2R,5R)-1-(4-tert- butylphenyl)pyrrolidine-2,5-diyl)bis(4,l-phenylene))dipyrrolidine-2-carboxamide To the product from Example 34C (498 mg, 0.638 mmol) in dichloromethane (4 ml) was added TFA (6 ml). The reaction mixture was stirred at room temperature for 1 h and concentrated in vacuo. The residue was partitioned between 3: 1 CHCl3dsopropyl alcohol and saturated aq. NaHCO3. The aqueous layer was extracted by 3: 1 CHCl3:isopropyl alcohol again. The combined organic layers were dried over
filtered and concentrated to give the title compound (345 mg, 93%).
Example 34E Dimethyl (2S,2’S)-l,r-((2S,2’S)-2,2′-(4,4′-((2S,5S)-1-(4-fert-butylphenyl)pyrrolidine-2,5-diyl)bis(4,l- phenylene))bis(azanediyl)bis(oxomethylene)bis(pyrrolidine-2, 1 -diyl))bis(3-methyl- 1 -oxobutane-2, 1 – diyl)dicarbamate and
The product from Example 34D (29.0 mg, 0.050 mmol), (S)-2-(methoxycarbonylamino)-3- methylbutanoic acid (19.27 mg, 0.110 mmol), EDAC (21.09 mg, 0.110 mmol), HOBT (16.85 mg,
0.110 mmol) and N-methylmorpholine (0.027 ml, 0.250 mmol) were combined in DMF (2 ml). The reaction mixture was stirred at room temperature for 3 h. The mixture was partitioned with ethyl acetate and water. The organic layer was washed with brine twice, dried with sodium sulfate, filtered and evaporated. The residue was purified by chromatography on silica gel eluting with ethyl acetate in hexane (50% to 80%) to give a solid. The solid was triturated with ethyl acetate/hexane to give the title compound (13 mg, 29%). 1H NMR (400 MHz, DMSO-D6) δ ppm 0.85 – 0.95 (m, 12 H) 1.11 (s, 9 H) 1.59 – 1.65 (m, 2 H) 1.79 – 2.04 (m, 8 H) 2.10 – 2.18 (m, 2 H) 2.41-2.46 (m, 2H) 3.52 (s, 6 H)
Hz, 2 H) 7.50 (d, J=8.24 Hz, 4 H) 9.98 (s, 2 H); (ESI+) m/z 895 (M+H)+. The title compound showed an EC50 value of less than about 0.1 nM in HCV lb-Conl replicon assays in the presence of 5% FBS.
The product from Example 34E was purified by chiral chromatography on a Chiralpak AD-H semi-prep column eluting with a 2:1 mixture of hexane:(2: l isopropyl alcohol: EtOH). The title compound was the second of 2 diastereomers to elute. 1H NMR (400 MHz, DMSO-D6) δ ppm 0.87
Example 37A (S)-2,5-dioxopyrrolidin-1-yl 2-(methoxycarbonylamino)-3-methylbutanoate To a mixture of (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid (19.66 g, 112 mmol) and N-hydroxysuccinimide (13.29g, 116 mmol) was added ethyl acetate (250 ml), and the mixture was cooled to 0-5 °C. Diisopropylcarbodiimide (13.88 g, 110 mmol) was added and the reaction mixture was stirred at 0-5 °C for about 1 hour. The reaction mixture was warmed to room temperature. The solids (diisopropylurea by-product) were filtered and rinsed with ethyl acetate. The filtrate was concentrated in vacuo to an oil. Isopropyl alcohol (200 ml) was added to the oil and the mixture was heated to about 50 °C to obtain a homogeneous solution. Upon cooling, crystalline solids formed. The solids were filtered and washed with isopropyl alcohol (3 x 20 ml) and dried to give the title compound as a white solid (23.2 g, 77% yield).
Example 37B
(S)- 1 -((S)-2-(methoxycarbonylamino)-3-methylbutanoyl)pyrrolidine-2-carboxylic acid To a mixture of L-proline (4.44g, 38.6 mmol), water (20 ml), acetonitrile (20 ml) and DIEA (9.5 g, 73.5 mmol) was added a solution of the product from Example 37A (1Og, 36.7 mmol) in acetonitrile (20 inL) over 10 minutes. The reaction mixture was stirred overnight at room temperature. The solution was concentrated under vacuum to remove the acetonitrile. To the resulting clear water solution was added 6N HCl (9 ml) until pH ~ 2 .The solution was transferred to a separatory funnel and 25% NaCl (10 ml) was added and the mixture was extracted with ethyl acetate (75 ml), and then again with ethyl acetate (6 x 20 ml), and the combined extracts were washed with 25% NaCl (2 x 10ml). The solvent was evaporated to give a thick oil. Heptane was added and the solvent was evaporated to give a foam, which was dried under high vacuum. Diethyl ether was added and the solvent was evaporated to give a foam, which was dried under high vacuum to give the title compound (10.67g) as a white solid.
The compound of Example 37B can also be prepreared according to the following procedure: To a flask was charged L- valine (35 g, 299 mmol), IN sodium hydroxide solution (526 ml,
526 mmol) and sodium carbonate (17.42 g, 164 mmol). The mixture was stirred for 15 min to dissolve solids and then cooled to 15 °C. Methyl chloroformate (29.6 g, 314 mmol) was added slowly to the reaction mixture. The mixture was then stirred at rt for 30 min. The mixture was cooled to 15 °C and pH adjusted to -5.0 with concentrated HCl solution. 100 inL of 2-methytetrahydrofuran (2- MeTHF) was added and the adjustment of pH continued until the pH reached ~ 2.0. 150 mL of 2- MeTHF was added and the mixture was stirred for 15 min. Layers were separated and the aqueous layer extracted with 100 mL of 2-MeTHF. The combined organic layer was dried over anhyd Na2SC^ and filtered, and Na2SC^ cake was washed with 50 mL of 2-MeTHF. The product solution was concentrated to ~ 100 mL, chased with 120 mL of IPAc twice. 250 mL of heptanes was charged slowly and then the volume of the mixture was concentrated to 300 mL. The mixture was heated to 45 °C and 160 mL of heptanes charged. The mixture was cooled to rt in 2h, stirred for 30 min, filtered and washed with 2-MeTHF/heptanes mixture (1:7, 80 inL). The wetcake was dried at 55 °C for 24 h to give 47.1 g of Moc-L- VaI-OH product as a white solid (90%).
Moc-L- VaI-OH (15O g, 856 mmol), HOBt hydrate (138 g, 899 mmol) and DMF (1500 ml) were charged to a flask. The mixture was stirred for 15 min to give a clear solution. EDC hydrochloride (172 g, 899 mmol) was charged and mixed for 20 min. The mixture was cooled to 13
°C and (L)-proline benzyl ester hydrochloride (207 g, 856 mmol) charged. Triethylamine (109 g,
1079 mmol) was then charged in 30 min. The resulting suspension was mixed at rt for 1.5 h. The reaction mixture was cooled to 15 °C and 1500 mL of 6.7% NaHCO3 charged in 1.5 h, followed by the addition of 1200 mL of water over 60 min. The mixture was stirred at rt for 30 min, filtered and washed with water/DMF mixture (1 :2, 250 mL) and then with water (1500 mL). The wetcake was dried at 55 °C for 24 h to give 282 g of product as a white solid (90%).
The resulting solids (40 g) and 5% Pd/ Alumina were charged to a Parr reactor followed by THF (160 mL). The reactor was sealed and purged with nitrogen (6 x 20 psig) followed by a hydrogen purge (6 x 30 psig). The reactor was pressurized to 30 psig with hydrogen and agitated at room temperature for approximately 15 hours. The resulting slurry was filtered through a GF/F filter and concentrated to approximately 135 g solution. Heptane was added (120 mL), and the solution was stirred until solids formed. After an addition 2 – 3 hours additional heptane was added drop-wise (240 mL), the slurry was stirred for approximately 1 hour, then filtered. The solids were dried to afford the title compound.
The product from Example 32 (5.01 g, 13.39 mmol) was combined with 2- methyltetrahydrofuran (70 mL) and cooled to -5 °C, and N,N-diisopropylethylamine (6.81 g, 52.7 mmol) was added over 30 seconds. Separately, a solution of methanesulfonic anhydride (6.01 g, 34.5 mmol) in 2-methyltetrahydrofuran (30 mL) was prepared and added to the diol slurry over 3 min., maintaining the internal temperature between -15 °C and -25 °C. After mixing for 5 min at -15 °C, the cooling bath was removed and the reaction was allowed to warm slowly to 23 °C and mixed for 30 minutes. After reaction completion, the crude slurry was carried immediately into the next step.
To the crude product solution from Example 37C (7.35 g, 13.39 mmol) was added 4-tert- butylaniline (13.4 g, 90 mmol) at 23 °C over 1 minute. The reaction was heated to 65 °C for 2 h. After completion, the reaction mixture was cooled to 23 °C and diluted with 2-methyltetrahydrofuran (100 mL) and 1 M HCl (150 mL). After partitioning the phases, the organic phase was treated with 1 M HCl (140 mL), 2-methyltetrahydrofuran (50 mL), and 25 wt% aq. NaCl (100 mL), and the phases were partitioned. The organic phase was washed with 25 wt% aq. NaCl (50 mL), dried over MgSO/t, filtered, and concentrated in vacuo to approximately 20 mL. Heptane (30 mL) and additional 2- methyltetrahydrofuran were added in order to induce crystallization. The slurry was concentrated further, and additional heptane (40 mL) was slowly added and the slurry was filtered, washing with 2- methyltetrahydrofuran:heptane (1:4, 20 mL). The solids were suspended in MeOH (46 mL) for 3 h, filtered, and the wet solid was washed with additional MeOH (18 mL). The solid was dried at 45 °C in a vacuum oven for 16 h to provide the title compound (3.08 g, 51% 2-step yield).
To a 160 ml Parr stirrer hydrogenation vessel was added the product from Example 37D (2 g, 4.49 mmol), followed by 60 ml of THF, and Raney Nickel Grace 2800 (1 g, 50 wt% (dry basis)) under a stream of nitrogen. The reactor was assembled and purged with nitrogen (8 x 20 psig) followed by purging with hydrogen (8 x 30 psig). The reactor was then pressurized to 30 psig with hydrogen and agitation (700 rpm) began and continued for a total of 16 h at room temperature. The slurry was filtered by vacuum filtration using a GF/F Whatman glass fiber filter. Evaporation of the filtrate to afford a slurry followed by the addition heptane and filtration gave the crude title compound, which was dried and used directly in the next step.
Example 37F dimethyl (2S,2’S)-l,r-((2S,2’S)-2,2′-(4,4′-((2S,5S)-1-(4-tert-butylphenyl)pyrrolidine-2,5-diyl)bis(4, l- phenylene)bis(azanediyl)bis(oxomethylene))bis(pyrrolidine-2, 1 -diyl))bis(3-methyl- 1 -oxobutane-2, 1 – diy 1) die arb amate To a solution of the product from Example 37E (1.64 g, 4.25 mmol) in DMF (20 ml), the product from Example 37B (2.89 g, 10.63 mmol), and HATU (4.04 g, 10.63 mmol) in DMF (15OmL) was added triethylamine (1.07 g, 10.63 mmol), and the solution was stirred at room temperature for 90 min. To the reaction mixture was poured 20 mL of water, and the white precipitate obtained was filtered, and the solid was washed with water (3×5 mL). The solid was blow dried for Ih. The crude material was loaded on a silica gel column and eluted with a gradient starting with ethyl acetate/ heptane (3/7), and ending with pure ethyl acetate. The desired fractions were combined and solvent distilled off to give a very light yellow solid, which was dried at 45 °C in a vacuum oven with nitrogen purge for 15 h to give the title compound (2.3 g, 61% yield). 1H NMR (400 MHz, DMSO- D6) δ ppm 0.88 (d, J=6.61 Hz, 6 H) 0.93 (d, J=6.72 Hz, 6 H) 1.11 (s, 9 H) 1.63 (d, J=5.42 Hz, 2 H) 1.80 – 2.04 (m, 8 H) 2.09 – 2.19 (m, 2 H) 2.44 – 2.47 (m, 2 H) 3.52 (s, 6 H) 3.59 – 3.66 (m, 2 H) 3.77 – 3.84 (m, 2 H) 4.02 (t, J=8.40 Hz, 2 H) 4.42 (dd, J=7.86, 4.83 Hz, 2 H) 5.14 (d, J=6.18 Hz, 2 H) 6.17 (d, J=8.67 Hz, 2 H) 6.94 (d, J=8.78 Hz, 2 H) 7.13 (d, J=8.46 Hz, 4 H) 7.31 (d, J=8.35 Hz, 2 H) 7.50 (d, J=8.35 Hz, 4 H) 9.98 (s, 2 H).
Alternately, the product from example 37E (11.7 g, 85 wt%, 25.8 mmol) and the product from example 37B (15.45 g, 56.7 mmol) are suspended in EtOAc (117 mL), diisopropylethylamine (18.67 g, 144 mmol) is added and the solution is cooled to 0 °C. In a separate flask, 1-propanephosphonic acid cyclic anhydride (T3P®) (46.0 g, 50 wt% in EtOAc, 72.2 mmol) was dissolved in EtOAc (58.5 mL), and charged to an addition funnel. The T3P solution is added to the reaction mixture drop-wise over 3-4 h and stirred until the reaction is complete. The reaction is warmed to room temperature,and washed with IM HCl/7.5 wt% NaCl (100 mL), then washed with 5% NaHCO3 (100 mL), then washed with 5% NaCl solution (100 mL). The solution was concentrated to approximately 60 mL, EtOH (300 mL) was added, and the solution was concentrated to 84 g solution.
A portion of the EtOH solution of product (29 g) was heated to 40 °C, and added 134 g 40 w% EtOH in H2O. A slurry of seeds in 58 wt/wt% EtOH/H2O was added, allowed to stir at 40 °C for several hours, then cooled to 0 °C. The slurry is then filtered, and washed with 58wt/wt% EtOH/H2O. The product is dried at 40 – 60 °C under vacuum, and then rehydrated by placing a tray of water in the vacuum oven to give the title compound. The title compound showed an EC50 value of less than about 0.1 nM in HCV lb-Conl replicon assays in the presence of 5% FBS.
To (S)-(-)-α,α-diphenyl-2-pyrrohdinemethanol (2 71 g, 10 70 mmol) was added THF (80 mL) at 23 °C The very thin suspension was treated with t11methyl borate (1 44 g, 13 86 mmol) over 30 seconds, and the resulting solution was mixed at 23 °C for 1 h The solution was cooled to 16-19 °C, and N,N-diethylanilme borane (21 45 g, 132 mmol) was added dropwise via syringe over 3-5 mm (caution vigorous H2 evolution), while the internal temperature was maintained at 16-19 °C After 15 mm, the H2 evolution had ceased To a separate vessel was added the product from Example IA (22 04 g, 95 wt%, 63 8 mmol), followed by THF (80 mL), to form an orange slurry After cooling the slurry to 11 °C, the borane solution was transferred via cannula into the dione slurry over 3-5 min During this period, the internal temperature of the slurry rose to 16 °C After the addition was complete, the reaction was maintained at 20-27 °C for an additional 2 5 h After reaction completion, the mixture was cooled to 5 °C and methanol (16 7 g, 521 mmol) was added dropwise over 5-10 mm, maintaining an internal temperature <20 °C (note vigorous H2 evolution) After the exotherm had ceased (ca 10 mm), the temperature was adjusted to 23 °C, and the reaction was mixed until complete dissolution of the solids had occurred Ethyl acetate (300 mL) and 1 M HCl (120 mL) were added, and the phases were partitioned The organic phase was then washed successively with 1 M HCl (2 x 120 mL), H2O (65 mL), and 10% aq NaCl (65 mL) The orgamcs were dried over MgSO4, filtered, and concentrated in vacuo Crystallization of the product occurred during the concentration The slurry was warmed to 50 °C, and heptane (250 inL) was added over 15 min. The slurry was then allowed to mix at 23 °C for 30 min and filtered. The wet cake was washed with 3: 1 heptane:ethyl acetate (75 mL), and the orange, crystalline solids were dried at 45 °C for 24 h to provide the title compound (15.35 g, 99.3% ee, 61% yield), which was contaminated with 11% of the meso isomer (vs. dl isomer).
Jordan J. Feld; Kris V. Kowdley; Eoin Coakley; Samuel Sigal; David R. Nelson; Darrell Crawford; Ola Weiland; Humberto Aguilar; Junyuan Xiong; Tami Pilot-Matias; Barbara DaSilva-Tillmann; Lois Larsen; Thomas Podsadecki & Barry Bernstein (2014). “Treatment of HCV with ABT-450/r–Ombitasvir and Dasabuvir with Ribavirin”. N Engl J Med370: 1594–1603. doi:10.1056/NEJMoa1315722.
UncategorizedComments Off on Metoprolol lactose adduct
Jun302016
. Mass spectrometry was performed for this isolated impurity by ESI technique in positive mode. The positive DI-MS spectrum of isolated impurity exhibited molecular ion peak as [M + H]+ at m/z 592.29 and as sodium adduct [M + Na]+ at m/z 614.28. The MS/MS data displayed a dominant fragment at m/z 574.28 which is 17 amu less than the molecular ion peak indicating a removal of the hydroxyl group.
This indicates that the impurity is Metoprolol-lactose adduct as proposed. The high resolution mass proposed
the probable molecular formula C27H45NO13.
The 1H NMR spectrum of this impurity displayed signals at δ = 1.27–1.30(6H), δ = 2.73–2.76 (2H), δ = 2.95–2.98 (1H), δ = 3.12–3.18(1H), δ = 3.23 (3H), δ = 3.44–3.68(11H), δ = 3.73–3.77(2H), δ = 3.83–3.84(2H), δ = 4.15–4.17(3H), δ = 4.41–4.43(1H), δ = 4.64–4.67 (1H), δ = 6.68–6.90 (2H), δ = 7.15–7.17(2H)
corresponding to 37 protons, indicating the Metoprolol adduct impurity possibility as it contains total 45 protons out of which 8 protons are of hydroxyl groups of lactose.
The 1H and 13C NMR spectra of Metoprolol adduct impurity and Metoprolol tartrate was compared and significant changes were observed. In 1H NMR spectrum of impurity additional 13 protons in aliphatic region were observed. While in 13C NMR, additional 12 carbon signals can be seen. Methylene carbons C21 and C16 were observed at 60.5 and 61.8 ppm respectively and 10 carbon signals were observed between 68.5 ppm to 103 ppm. These signals
confirmed the presence of both lactose as well as Metoprolol moieties in the impurity.
Further to confirm the exact structure of Metoprolol adduct impurity, the 2D NMR HSQC has also been reviewed (see
Supplementary Fig. S-5). It was observed that the proton in aliphatic region showing doublet at (4.41–4.43) ppm corresponds to C22 which appeared at 103 ppm. This confirms the presence of anomeric carbon of pyranose ring. Also C17 appeared at 95.4 ppm found to be quaternary carbon which confirms the presence of furanose anomeric carbon. Proton corresponding to C20 found to shown multiplet in the region of (4.15–4.17) ppm which confirms that C20 is from furanose ring. Apart from these interactions, carbon signals appeared at 70.7, 72.2, 74.5, 61.8 ppm confirming the arabinosyl moiety.
Based on the above observations it has been confirmed that the impurity is Metoprolol lactose adduct and the ‘glucose moiety’ of lactose present in adduct exists in furanose form
The 1H Metoprolol tartrate
The 13C NMR spectra Metoprolol tartrate
.
The 1H spectra of Metoprolol adduct impurity
The13C NMR spectra of Metoprolol adduct impurity
HSQC spectra of Metoprolol adduct impurity
Identification, synthesis, isolation and characterization of new impurity in metoprolol tartrate tablets
Ataluren, formerly known as PTC124, is a pharmaceutical drug for the treatment of Duchenne muscular dystrophy and potentially other genetic disorders. It was designed by PTC Therapeutics and is sold under the trade name Translarna in the European Union.
Ataluren was approved by European Medicine Agency (EMA) on July 31, 2014. It was developed and marketed as Translarna® by PTC Therapeutics.
Ataluren was regulator of nonsense mutations indicated for the treatment of Duchenne muscular dystrophy resulting from a nonsense mutation in the dystrophin gene, in ambulatory patients aged 5 years and older.
Translarna® is available as granules for oral use, containing 125 mg, 250 mg or 1000 mg of free Ataluren. The recommended dose is 10 mg/kg body weight in the morning, 10 mg/kg body weight at midday, and 20 mg/kg body weight in the evening.
Medical uses
Ataluren has been tested on healthy humans and humans carrying genetic disorders caused by nonsense mutations,[1][2] such as some people with cystic fibrosis and Duchenne muscular dystrophy. It is approved for the use in Duchenne in the European Union.
Mechanism of action
Ataluren makes ribosomes less sensitive to premature stop codons (referred to as “read-through”). This may be beneficial in diseases such as Duchenne muscular dystrophy where the mRNA contains a mutation causing premature stop codons or nonsense codons. Studies have demonstrated that PTC124 treatment increases expression of full-length dystrophin protein in human and mouse primary muscle cells containing the premature stop codon mutation for Duchenne muscular dystrophy and rescues striated muscle function.[3] Studies in mice with the premature stop codon mutation for cystic fibrosis demonstrated increased CFTR protein production and function.[4] The European Medicines Agency review on the approval of ataluren concluded that “the non-clinical data available were considered sufficient to support the proposed mechanism of action and to alleviate earlier concerns on the selectivity of ataluren for premature stop codons.” [5]
In cystic fibrosis, early studies of ataluren show that it improves nasal potential difference.[6] Ataluren appears to be most effective for the stop codon ‘UGA’.[1]
History
Clinical trials
In 2010, PTC Therapeutics released preliminary results of its phase 2b clinical trial for Duchenne muscular dystrophy, with participants not showing a significant improvement in the six minute walk distance after the 48 weeks of the trial.[7] This failure resulted in the termination of a $100 million deal with Genzyme to pursue the drug.
Phase 2 clinical trials were successful for cystic fibrosis in Israel, France and Belgium.[8] Multicountry phase 3 clinical trials are currently in progress for cystic fibrosis in Europe and the USA.[9]
Approval
On 23 May 2014 ataluren received a positive opinion from the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA).[10]Translarna was first available in Germany, the first EU country to launch the new medicine.[11]
In August 2014, ataluren received market authorization from the European Commission to treat patients with nonsense mutation Duchenne muscular dystrophy. A confirmatory phase III clinical trial is ongoing.[11] The drug does not yet have approval by the US Food and Drug Administration.
In October 2015, NICE asked for further evidence of benefit to justify the “very high cost”.[12] NICE estimated that for a typical patient, treatment would cost £220,256 per year.
In February 2016, FDA declined to approve or even discuss PTC Therapeutics application for ataluren because it deemed the data presented by the developer “insufficient to warrant a review”.[13]
Samples were analyzed for purity on an Agilent 1200 series LC/MS equipped with a Luna® C18 reverse phase (3 micron, 3 x 75 mm) column having a flow rate of 0.8-1.0 mL/min. The mobile phase was a mixture of acetonitrile (0.025% TFA) and H2O (0.05% TFA), and temperature was maintained at 50 °C. A gradient of 4% to 100% acetonitrile over 7 minutes was used during analytical analysis. Purity of final compounds was determined to be >95%, using a 5 μL injection with quantitation by AUC at 220 and 254 nM. High resolution mass spectra were obtained with an Agilent 6210 Time-of-Flight LC/MS with a 3.5 um Zorbax SB-C18 column (2.1 x 30 mm) (solvents are Water and ACN with 0.1% Formic Acid). A 3 minute gradient at 1 mL/min from 5% to 100% acetonitrile was used.
Ataluren (Translarna) Ataluren is a drug marketed under the trade name Translarna which was developed by PTC Therapeutics and approved by the European Union in May 2014 for the treatment of Duchenne’s muscular dystrophy (DMD) and potentially other genetic disorders.50
Ataluren renders ribosomes less sensitive to premature stop or ‘read-through’ codons, which are thought to be beneficial in diseases such as DMD and cystic fibrosis.51 Of the reported synthetic approaches to ataluren,52–55 the most likely process-scale approach consists of the sequence described in Scheme 7, which reportedly has been exemplified on kilogram scale.56
The sequence to construct ataluren, which was described by the authors at PTC Therapeutics, commenced with commercially available methyl 3-cyanobenzoate (38).56 This ester was exposed to hydroxylamine in aqueous tert-butanol and warmed gently until the reaction was deemed complete.
Then this mixture was treated with 2-fluorobenzoyl chloride dropwise and subsequently triethylamine dropwise. To minimize exotherm and undesired side products, careful control of the addition of reagents was achieved through slow dropwise addition of these liquid reagents.
Upon complete consumption of starting materials and formation of amidooxime 39, the aqueous reaction mixture was then heated to 85 C to facilitate 1,2,4-oxadiazole formation, resulting in the tricyclic ester 40 in excellent yield across the three steps.
Finally,saponification of ester 40 through the use of sodium hydroxide followed by acidic quench gave ataluren (V) in 96% over the two-step sequence.57
50. Welch, E. M.; Barton, E. R.; Zhuo, J.; Tomizawa, Y.; Friesen, W. J.; Trifillis, P.;Paushkin, S.; Patel, M.; Trotta, C. R.; Hwang, S.; Wilde, R. G.; Karp, G.; Takasugi,J.; Chen, G.; Jones, S.; Ren, H.; Moon, Y. C.; Corson, D.; Turpoff, A. A.; Campbell,J. A.; Conn, M. M.; Khan, A.; Almstead, N. G.; Hedrick, J.; Mollin, A.; Risher, N.;Weetall, M.; Yeh, S.; Branstrom, A. A.; Colacino, J. M.; Babiak, J.; Ju, W. D.;Hirawat, S.; Northcutt, V. J.; Miller, L. L.; Spatrick, P.; He, F.; Kawana, M.; Feng,H.; Jacobson, A.; Peltz, S. W.; Sweeney, H. L. Nature 2007, 447, 87.
51. Hirawat, S.; Welch, E. M.; Elfring, G. L.; Northcutt, V. J.; Paushkin, S.; Hwang,S.; Leonard, E. M.; Almstead, N. G.; Ju, W.; Peltz, S. W.; Miller, L. L. J. Clin.Pharmacol. 2007, 47, 430.
52Karp, G. M.; Hwang, S.; Chen, G.; Almstead, N. G. US Patent 2004204461A1,2004.
53. Andersen, T. L.; Caneschi, W.; Ayoub, A.; Lindhardt, A. T.; Couri, M. R. C.;Skrydstrup, T. Adv. Synth. Catal. 2014, 356, 3074.
54. Gupta, P. K.; Hussain, M. K.; Asad, M.; Kant, R.; Mahar, R.; Shukla, S. K.; Hajela,K. New J. Chem. 2014, 38, 3062.
55. Lentini, L.; Melfi, R.; Di Leonardo, A.; Spinello, A.; Barone, G.; Pace, A.; PalumboPiccionello, A.; Pibiri, I. Mol. Pharm. 2014, 11, 653.
56. Almstead, N. G.; Hwang, P. S.; Pines, S.; Moon, Y. -C.; Takasugi, J. J. WO Patent2008030570A1, 2008.
57. Almstead, N. G.; Chen, G.; Hirawat, S.; Hwang, S.; Karp, G. M.; Miller, L.; Moon,Y. C.; Ren, H.; Takasugi, J. J.; Welch, E. M.; Wilde, R. G. WO Patent2007117438A2, 2007.
Last week the newspaper NRC Handelsblad reported on a court case in which the parents of two young boys sued a pharmaceutical company over access to one of their developmental drugs. The drug in question wasAtaluren, the pharmaceutical companyPTC Therapeutics. The boys suffer from Duchenne muscular dystrophyand had taken part in a clinical trial. Whereas the results of this trial on the whole were inconclusive the boys did seriously benefit from the drug. Hardly any wonder the parents took action when the whole development program was canceled.
And the judge? He threw the case out arguing that doctors do not make the compound themselves and arguing that the compound is not commercially available. Are these arguments valid? and do the boys have options?
It is not that ataluren is a complex molecule. To judge from one of the patents, synthesis is straightforward starting from 2-cyanobenoic acid and 2-fluorobenzoyl chloride, both commercially available. The synthetic steps are methylation of 2-cyanobenoic acid (iodomethane), nitrile hydrolysis with hydroxylamine, esterification with the fluoro acid chloride using DIPEA, high-temperature dehydration to the oxadiazole and finally ester hydrolysis (NaOH).
Except for the fluorine atom in it the compound is unremarkable. If you have to believe the Internet many Chinese companies produce and sell it. Ataluren is also still in the running as a potential treatment for some other diseases. So if need be the compound will be around for some time to come.
CLIP
Ataluren [3-[5-(2-Fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid] is an orally available, small molecule compound that targets nonsense mutation. It is the first drug in its class and appears to allow cellular machinery to read through premature stop codons in mRNA, and thus enables the translation process to produce full-length, functional proteins.
Ataluren is developed and approved for the treatment of nonsense mutation Duchenne muscular dystrophy (nmDMD) by EU in July 2014 [1].
Ataluren: 2D and 3D Structure
Nonsense Mutations as Target for DMD
A single nucleotide change in the DNA sequence that introduces a premature stop codon is known as a nonsense mutation, a subset of a major class of premature termination codon (PTC) mutations. Nonsense mutations cause premature termination of translation resulting in the production of truncated polypeptides, which in turn halts the ribosomal translation process at an earlier site than normal, producing a truncated, non-functional protein [1].
Nonsense mutations are implicated in 5-70 % of individual cases of most inherited diseases, including Duchenne muscular dystrophy (DMD) and cystic fibrosis. Ataluren appears to allow cellular machinery to read through premature stop codons in mRNA, enabling the translation process to produce full length, functional proteins.
Ataluren Synthesis
New J Chem 2014, 38, 3062-3070: The text reports one pot synthesis of Ataluren with an overall yield of 40%. It also reports few interesting and potent derivatives too.
WO 2007117438A2: It appears to be the industrial process. The patent also reports various pharmaceutically relevant assay and their results wrt Ataluren. Identifications:
It is not that ataluren is a complex molecule. To judge from one of the patents, synthesis is straightforward starting from 2-cyanobenoic acid and 2-fluorobenzoyl chloride, both commercially available. The synthetic steps are methylation of 2-cyanobenoic acid (iodomethane), nitrile hydrolysis with hydroxylamine, esterification with the fluoro acid chloride using DIPEA, high-temperature dehydration to the oxadiazole and finally ester hydrolysis (NaOH).
Carcinogenicity bioassays in transgenic mice (26 weeks) and in rats (24 months):
● For Tg.rasH2 mouse: Ataluren did not increase the incidence of tumors up to the HDs in males (600 mg/kg/day) and in females (300 mg/kg/day). The non-neoplastic findings included endometrial hyperplasia and nephropathy in females.
● For rats: Urinary bladder tumors (benign urothelial cell papilloma [2 rats] and malignant urothelial cell carcinoma [1 rat]) were observed in 3/60 female rats dosed at 300 mg/kg/day. In addition, one case of malignant hibernoma was observed in 1/60 male rats at the dose of 300 mg/kg/day. The non-neoplastic toxicity consisted of a decrease of body weight.
Method ataluren according to Patent Document 2 is described in Example W02004091502A2 prepared.
Specific methods of preparation:
To a solution of 0.6 l of DMF was 44. 14g3- cyano acid 62.19 g of potassium carbonate was added, followed by stirring at room temperature for 30 minutes. 20 minutes To the suspension was added 28 ml of methyl iodide (450mmol), and the reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was poured into 1.2 l of ice water, stirred for 30 minutes, the precipitate was filtered out thereof. The white cake was dissolved in 70 ml of methanol, and then reprecipitated in cold water. To give 79% yield of 3-cyano-benzoic acid methyl ester.
50 g of 3-cyano-benzoic acid methyl ester was dissolved in 500 ml of ethanol, to which was added 41 ml of 50% aqueous hydroxylamine (620mmol). 100 ° C and the reaction mixture was stirred for 1 hour, the solvent was removed under reduced pressure. So that the oily residue is dissolved in 100 ml of 20/80 ethanol / toluene, concentrated again. To give 61 g 3- (N- hydroxy amidino (carbamimidoyl)) – benzoic acid methyl ester.
60 g of 3- (N- hydroxy amidino (carbamimidoyl)) – benzoic acid methyl ester was dissolved in 200 ml of anhydrous tetrahydrofuran, followed by adding thereto 75 ml of diisopropylethylamine (434 mmol), and then 20 minutes this mixture was added 48.1 ml 2- fluorobenzoyl chloride (403mmol). The reaction mixture was stirred at room temperature for 1 hour. The precipitate was filtered off, the filtrate was concentrated under reduced pressure. The residue was dissolved in 400 ml of ethyl acetate, washed with 400 ml of water and then twice. The solvent was removed under reduced pressure, containing 60% ethyl acetate in hexane to give the desired product, generating 81 g 3- (N-2- amidino-fluorobenzoyl) – benzoate.
at 130 ° C with a Dean-Stark apparatus was dissolved in 500 ml of toluene was heated under reflux in 44 g of 3- (N-2- fluorobenzoyl) -1,2,3,4-_ benzoate 4 hours. 5 ° C and the reaction mixture was stirred for 18 hours. The white precipitate was filtered off, the filtrate was concentrated, recrystallized in toluene. To give 38 g of 3- [5- (2-fluorophenyl) – [1,2,4] oxadiazol-3-yl] – benzoic acid methyl ester.
33 g of 3- [5- (2-fluorophenyl) – [1,2,4] oxadiazol-3-yl] – benzoic acid methyl ester was dissolved in 400 ml of tetrahydrofuran, to which was added 100 ml of 1. 5M aqueous sodium hydroxide solution. At 100 ° C and the reaction mixture was heated at reflux for 2 hours. The solvent was removed under reduced pressure at 5 ° C the solution was stirred for 2 hours. The organic solvent was removed, washed with 50 mL of water. The aqueous solution was then acidified with hydrochloric acid to pH 1. The white precipitate was filtered off, the filter cake washed with cold water, then dried with a freeze dryer. To give 3.0 g of 3- [5- (2-fluorophenyl) – [1,2,4] oxadiazol-3-yl] benzoic acid. 1H-NMR (500MHz, d6-DMS0): 8. 31 (1H), 8 18 (2H), 8 08 (1H), 7 88 (2H), 7 51 (2H)….. Display: ataluren- Sample Preparation Example 1 prepared in Preparation Example 2 and TO2004091502A2 induced.
Each prepared in Example 2 (prepared according to known Form A)
Method [0084] A known polymorph according to Patent Document W02008039431A2 Example 5. 1. 1.1 prepared as described. Specifically: ataluren be prepared 1 100 mg Preparation Example, 60 ° C add 16.2 ml of isopropanol ultrasound clear solution, the solution by 2 square micron filter and the filtrate was kept covered with aluminum foil having a small hole. vial, 60 ° C and evaporated. The solid formed was isolated to give ataluren the A polymorph.
as needles.
its XRPD shown in Figure 1, the display ataluren polymorph A disclosed in Patent Document W02008039431A2 consistent.
SEE
European Journal of Organic Chemistry (2016), 2016(3), 438-442
Russian Chemical Bulletin (2015), 64(1), 142-145.
European Journal of Medicinal Chemistry (2015), 101, 236-244.
[l,2,4]oxadiazol-3-yl]-benzoic acid, which has the following chemical structure (I):
(I)
In particular, crystalline forms of 3-[5-(2-fluorophenyl)-[l,2,4]oxadiazol-3-yl]- benzoic acid are useful for the treatment, prevention or management of diseases ameliorated by modulation of premature translation termination or nonsense-mediated mRNA decay, as described in U.S. Patent No. 6,992,096 B2, issued January 31, 2006, which is incorporated herein by reference in its entirety. In addition, the present provides a crystalline form of 3-[5-(2-fluorophenyl)-[l,2,4]oxadiazol-3-yl]-benzoic acid which is substantially pure, i.e., its purity greater than about 90%.
Processes for the preparation of 3-[5-(2-fluorophenyl)-[l,2,4]oxadiazol-3-yl]- benzoic acid are described in U.S. Patent No. 6,992,096 B2, issued January 31, 2006, and U.S. patent application no. 1 1/899,813, filed September 9, 2007, both of which are incorporated by reference in their entirety.
The 3-cyano-benzoic acid methyl ester (105 kg) and t-butanol was added molten drying reactor.Under an inert atmosphere for about 2 hours 48 minutes, 50./.Aqueous hydroxylamine (43L, 47.4 kg) was added to a clear solution of 3-cyano benzoic acid methyl ester in a molten in t-butanol.The addition of a 50% aqueous solution of hydroxylamine period, the maximum temperature batch of about 43 ° C.50% aqueous solution of hydroxylamine addition rate of from about 9L / h when the changes start adding to about 30L / hr.To maintain the temperature of the batch by varying the reactor jacket set point.In particular, the set value is about 40.5 ° C, with the addition of a rate increase at the beginning join, change the setting to about 29.6 ° C.After about 40-45t stirred for about 4 hours, the reaction was deemed complete (i.e., less than about 0.5% ester).
The batch was transferred to a drying reactor, additional (chased through) approximately 10L molten tert-butanol.Jacket setpoint from about 33 when the batch was received when dried reactor.C is reduced to about 27 after the completion of the transfer.C.Batch crystallization was observed part, which does not adversely affect stirring.The batch was cooled to about 34.4 ° C, triethylamine (72.6 kg, IOOL) added to the reactor.The jacket temperature set value from about 20.4.C is increased to about 31.0 ° C, in order to maintain the batch temperature in the range of about 30-35t.With molten tert-butanol (IO L) was washed with a linear (line rinse) After the batch was added to the 2-fluorobenzoyl chloride (113.7 kg, 86.0L).Charge is added to the first third of the rate of about 25L / hr.In the meantime, the jacket inlet temperature was lowered to about 15 ° C, the batch temperature is maintained at about 34.6 ° C.In about 5.5 hours after the addition was complete.During the addition, the maximum temperature of the batch was about 38.8 ° C.Near the end of the addition, the addition rate slowed to about 11L / hr was added last 27 liters of 2-fluoro-benzoyl chloride.30-35.C After stirring for about 2 hours, that the reaction was complete (i.e., less than about 0.5% of methyl 3-amidinophenoxy).Then, after about 1 hour 42 minutes, the batch was heated to reflux temperature (about 82 ° C), and then stirred for about 18 hours.During the stirring, a number of product partially crystallized to form a slurry.The slurry was cooled to about 40.C thus sampled, during which complete crystallization occurs.The batch was then heated to reflux temperature and stirred for about 1 hour 50 minutes.Then, after about two hours, the batch was cooled to about 69 ° C, and after about four hours and 15 minutes, slowly added 630L of pure water, while maintaining the batch temperature at about 66-69 ° C.After about 3 hours 14 minutes, the slurry was cooled to about 22.4 ° C, and transferred to 2x200L ceramic filter, the ceramic filter equipped 25-30n polypropylene mesh filter cloth.In about 55 minutes after the completion of material from the container to the filter transfer.With 50n /.The tert-butanol solution (210L) was washed cake was washed for about 10 minutes so that the cleaning liquid can penetrate into each cake.Then, the cake was dried in a vacuum for about 5-10 minutes.The purified water as a second washing (158L / cake) applied to the filter cake to remove residual t-butanol and triethylammonium chloride salt.Dried in a vacuum for about 5 minutes, the solution was removed.In vacuo and then the cake was dried for about 2 hours, and then sampled using liquid chromatography.The filter cake was measured by liquid chromatography purity of about 99.6%.
The filter cake was dried in vacuo for about 8 hours 25 minutes later, the wet cake (207.4kg) is transferred to an air oven.At about 50-55.C, the oven dried in air for about 52 hours.The product was isolated in a total yield of about 89.9% (174.65kg), in the calculation of cost of materials sampling, you can adjust the overall yield of about 90.7%.
Batch 2
The 3-cyano-benzoic acid methyl ester (105 kg) and t-butanol was added molten drying reactor.Under an inert atmosphere for about 3 hours 29 minutes, 50% aqueous solution of hydroxylamine (47.85 kg) was added to the reactor.During the addition, the temperature is maintained at about 40-45 ° C.At about 40-45.C After stirring for about 3 hours 16 minutes, that the reaction was complete (i.e., less than about 0.5% ester).As for the drying reactor, the batch was transferred to one of the batch in.The batch was cooled
To about 34.4 ° C, and triethylamine (72.6 kg, 100 L).During about 45 minutes was added, while maintaining the batch temperature between about 30-35 ° C.During the addition, the jacket inlet temperature of from about 31.4.C increased to about 32.6.C.After the molten tert-butanol linear washed, was added to the batch 2- fluorobenzoyl chloride (l 13.7 kg, 86.0 L).After about 3 hours, 27 minutes, add the acid chloride.35.C under stirring for about 8 hours, that the reaction is not complete (i.e., more than about 0.5% residual 3-amidino-benzoyl ester).Then, 1.5% by weight of the original charge of triethylamine and 2-fluorobenzoyl chloride was added to the batch.Linear washed with tert-butanol (IO L) associated with each additional charge.During the addition of the acid chloride, no additional cooling.The batch was maintained at a temperature of about 30-35 ° C, the jacket inlet temperature range was maintained at about 30.3.C to about 33.0 ° C.After stirring for about 2 hours at 30-35t, that reaction was complete (i.e., less than 0.5% of methyl 3-amidinophenoxy).
After about 1 hour and 44 minutes, the batch was heated to reflux temperature (about 83 ° C), and stirred for about 18 hours.The same batch 1, during cooling the sample, the solid was completely crystallized.The batch was then heated to reflux temperature and stirred for about 1 hour and 2 minutes.Then, after about 2 hours and 20 minutes, the batch was cooled to about 69.2 ° C, and after about four hours and 30 minutes, slowly added 630 L of pure water, while the temperature of the batch was maintained at about 65.6-69.2 ° C.After about 3 hours and 30 minutes, the slurry was cooled to about 23.4 ° C, and, as for, the contents were transferred to one of the double batch of the ceramic filter.About 5 hours and 6 minutes, to complete the transfer of the material.With about 50% of t-butanol (2 volumes / cake) was washed filter cake was washed with 10 minutes to allow the cleaning liquid to penetrate into each cake, then dried in vacuo.About 1 hour and 40 minutes, the filter is completed.The purified water was added to a final wash the filter cake.The liquid was removed by drying under vacuum for about 10 minutes.In vacuo and then the cake was dried for about 2 hours and 5 minutes, and then sampled using liquid chromatography.The cake purity liquid chromatography were about 99.5% and 99.6%.After the cake was then dried in vacuo for about 2 hours and 5 minutes, the wet cake (191.5 kg) is transferred to an air oven.At about 50-55.C under dry in an air oven for about 48 hours.The product was isolated in a total yield of about 92.5% (179.7 kg).
Lot 3
The 3-cyano-benzoic acid methyl ester (52.5 kg) and molten tert-butanol (228 kg) added to the reaction vessel.The vessel was sealed, the batch temperature set of about 40-45 ° C, and the stirrer is started.Under an inert atmosphere, after 2 hours 40 minutes, 50% of the shoes amine solution (24 kg) was added to the reactor.During the addition, the temperature is maintained at about 40-45 ° C.In about 42.Under C, then further stirred for about 5 hours to complete the reaction.
The batch was cooled to 30-35 ° C, and after 15 minutes, was added triethylamine (36 kg).After about 2 hours 44 minutes, was added 2-fluorobenzoyl chloride (57 kg).During the addition, batch temperature was maintained at about 30-35 ° C.Under the 32t, the batch was stirred for 2 hours 10 minutes to complete the reaction.
After about 50 minutes, the batch was heated to reflux temperature (about 83-86 ° C), at about 8rc, stirred for about 18 hours.Then, over about two hours, the batch was cooled to about 65-70 ° C, and after about 6 hours 25 minutes, slowly added to purified water (315 L), while the batch temperature was maintained at about 65- 70 ° C.After about 2 hours and 15 minutes, the slurry was cooled to about 22 ° C, and the contents were transferred to a centrifuge filter (2 batches).About 1 hour and 40 minutes, the filter is completed.After about 20 minutes, with about 50% aqueous solution of tert-butyl alcohol (90 kg / cake), dried cake.The purified water (79 kg / cake) as the last added to the filter cake washed.At about 900 rpm drying the cake for about 1 hour and 5 minutes, then filled cylinder.Liquid chromatography wet cake (91.5 kg, LOD = 5% w / w) of a purity of about 99.75% area.
3- [5- (2-fluorophenyl) – [l, 2,4] oxadiazol-3-yl] – benzoic acid methyl ester (74.0kg) added to the reaction vessel, the vessel is sealed, evacuated and purification.Jacket set value of about 35.C, start the stirrer in the container.Molten tert-butanol (222 L, 3 volumes) and purified water (355 L, 4.8 vol) was added to the vessel.After the addition was added 25.1% w / w aqueous sodium hydroxide solution (43.5 kg, 1.1 molar equivalents), and with additional purified water (100L, 1.35 mol) was washed linear.During the addition, the batch temperature from about 39.0t reduced to about 38.8 ° C.After about 1 hour and 54 minutes, the batch temperature to about 63-67.C, and then, after about 30 minutes, which was adjusted to about 68-72.C.About 68-72t, stirring the mixture for about 3 hours.Then, after about five hours 11 minutes, the solution was cooled to about 40-45 ° C.Then, after the above process, after about three hours 33 minutes, the solution was then heated to about 68-72.C.
Jacket temperature of the reaction vessel was set to about 60 ° C, the stirrer started, and at about 70 ° C, a slightly positive pressure of nitrogen (1.5 to 5.6 psig), the heat transfer liquid through a micron filter .During the transfer, the product temperature is reduced to about 64.3 ° C, the transfer is completed in about 45 minutes.Was added to the purified water container (61 L, 0.82 vol) and the contents were heated to about 68-72.C.
The batch temperature was adjusted to about 69.4 ° C, and after about four hours and 18 minutes, with 13.9% w / w sulfuric acid (100.7 kg, 1.15 mol equiv.).During the addition, batch temperature was maintained at about 68.0-70.8 ° C.After the addition of the acid, with purified water (50 L, 0.68 vol) line wash at about 68-72 ° C, the stirring was continued for 31 minutes.
After about 4 hours and 10 minutes, the batch in a linear fashion from about 69.2t cooled to about 41.2 ° C.The stirrer Rosenmund filter / dryer was elevated to the highest position and jacket set value is set at about 40 ° C.The slurry was transferred to the two portions of the filter / drier.Applying a constant nitrogen pressure to the first portion (less than about 15 psig).During the transfer, a pressure of about 23.9 to about 28.8 psi, the transfer is complete in about 1 hour and 5 minutes.The second part of the slurry was transferred onto the filter cake, and the composite was stirred briefly to homogenize the batch.Use about 26.1 to about 29.1 psi nitrogen pressure filters the second part, after about three hours, squeeze the cake so that it does not contain liquid.With about 38-42 ° C hot tert-butanol solution (352 kg, 5 volumes) and about 65-70 ° C in 3x hot purified water (370 L, 5 volumes) and the filter 々.
Said filter / dryer jacket temperature was set to about 43 ° C, the product was dried under vacuum for about 26 hours while stirring periodically.Determination of purity of about 99.7%.The product was isolated in a total yield of about 74.4% (52.45 kg).
Batch 2
Was added to the reactor vessel 3- [5- (2-fluorophenyl) – [1,2,4] oxadiazol-3-yl] – benzoic acid methyl ester (47 kg, wet cake) and melt-hyun tert-butyl alcohol (111.4 kg).A sealed container, and the batch temperature was set at 30-40t, and start the stirrer.The purified water (51.6 kg) was added to the vessel.After the addition was added 3.47% w / w aqueous sodium hydroxide solution (202.4 kg).After about l hour, the batch temperature to about 67-73.C, then, at about 7 (under TC, stirred for about three hours.
Under a slight positive pressure of nitrogen, with a 1 micron polypropylene bag filter the batch, and then transferred to the new reactor.Was added to the vessel pure water (146 kg), and heating the batch to about 68-72.C.
After about four hours, the 10.7% aqueous hydrochloric acid was added to the batch.During the addition, batch temperature was maintained at about 68-72 ° C.PH was measured by using the batch pH of about 2.2, and then stirring was continued at about 7 (under TC about 1 hour.
After about two hours, the batch in a linear fashion from about 70.C is cooled to about 60 ° C.After about two hours, about 60.C of the batch in a linear fashion from about 6 (TC was cooled to about 40 ° C. In 40t, the batch was stirred for 2 hours, and the slurry was transferred to a centrifuge filter. After about 30 minutes, filtered completion . After about 30 minutes, with about 42Mw / w in t-butanol solution (165kg) cake was washed. The purified water (118kg, 4 (TC) as the last added to the filter cake was washed. The filter cake was dried at about 900rpm about 1 hour, then filled cylinder.
The wet cake was transferred to a paddle dryer (a double cone drier also suitable for this step), the jacket temperature was set to about 70.C.At about 70.C, the product was dried under vacuum for about 48 hours while stirring periodically.Determination of purity of about 99.8%.The product was isolated overall yield of about 74% (68.5 kg).
Lot 3
To the reaction vessel was added 3- [5- (2-fluorophenyl) – [1,2,4] oxadiazol-3-yl] – benzoic acid methyl ester (10 g) and t-butanol fused (128mL ).The batch temperature was set to 30-40 ° C, and the stirrer is started.After about 30 minutes, the aqueous sodium hydroxide solution 4.48% w / w of (32.5 g) was added to the vessel.The batch was maintained at a temperature of about 40-50 ° C.After about l hour, the batch temperature is raised to about 78-82 ° C, and then, at about 78-82t, and then stirred for about one hour.Under positive pressure of nitrogen, a polyethylene bag with a 5 micron filter the batch, and then transferred to a new reaction vessel.The batch was maintained at a temperature of about 78-82 ° C.
It was added to a new vessel 37% aqueous hydrochloric acid (4 mL) and tert-butanol molten (8 mL).The temperature was maintained at about 30-40.Under C, and stirring the mixture for about 30 minutes.
After about four hours, using a metering pump was added to the batch of hydrochloric acid in tert-butanol.After about SO-SO minutes before adding half filled.The stirrer speed is set at about 200rpm.After about 3.5 hours, add the remaining charge.The stirrer speed is set at about 100 rpm.During the addition, batch temperature was maintained at about 78-82 ° C.PH meter with a final batch pH was adjusted to about 1.2, at about 78-82t, then continue stirring for about l hour.After about one hour, the batch in a linear fashion from about 78-82.C is cooled to about 70 ° C.After about four hours, about 7 (TC batches in a linear fashion from 70.C cooled to about 50 ° C, and the stirrer speed was set at about 80 rpm. After about four hours, about 50 ° C Batch linearly cooled from 50 ° C to about 40t, and stirrer speed was set at approximately 60rpm. In 40.C, the batch was stirred for a further 4 hours.
The temperature of the filter is set to about 40-45 ° C.The slurry was transferred to the filter.After about one minute to complete filtration.After about two minutes, with tert-butanol (50 mL, 50.C) washing the filter cake.The pure water (IOO mLx2, 60.C) as the last wash was added to the cake.Under vacuum at about 60-70 ° C the cake was dried for about 12 hours, and then loaded into the container.
Determination of HPLC purity of about 99.9% of the area.The yield of isolated product was about 94% (9.0g).
The methyl 3-cyanophenyl Yue (7.35 g) and tert-butanol molten (100 mL) added to the reactor vessel.Sealed containers, the batch temperature was set to 60 ° C, and the stirrer is started.The suspension was stirred for 1 hour and then the batch temperature was set to 40.C.Under an inert atmosphere, after three hours, 50% aqueous solution of hydroxylamine (3.63 g) was added to the reactor.During the addition, batch temperature was maintained at 38-41 ° C.40.C After stirring for 18 hours, to complete the reaction.
The batch was cooled to 27 ° C, and after two minutes, triethylamine (5.56 g).After 3 hours, was added 2-fluorobenzoyl chloride (7.82 g).During the addition, batch temperature was maintained at 24-27 ° C.40.C, the batch was stirred for a further 4 hours.
After 30 minutes, the batch was heated to 79 ° C, and at about 79.C was stirred for 16 hours.After 3 hours, the white suspension was added to the water (IOO mL), while the batch temperature was maintained at 70 ° C.After 20 minutes, a 37% aqueous hydrochloric acid were added to the batch.PH was measured by using the batch pH of about 2.2, stirring was continued at about 70t for about 1 hour.
After three hours, the batch in a linear manner from 7 (TC cooled to 30 ° C, and the slurry is transferred to the filter. After 5 minutes, the filtering is done. After five minutes, with tert-butanol (50mL, 40 .C) filter cake was washed. The purified water (IOO mL, 60.C) is added to a final wash the filter cake. In 70.C of the filter cake was dried in a vacuum oven for 18 hours and then removed. Determination of purity approximately 98.68%. The total yield of isolated product of about 76% (10.8g).
PICS
A large-scale, multinational, phase 3 trial of the experimental drug ataluren has opened its first trial site, in Cincinnati, Ohio.
The trial is recruiting boys with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) caused by anonsense mutation — also known as a premature stop codon — in the dystrophin gene. This type of mutation causes cells to stop synthesizing a protein before the process is complete, resulting in a short, nonfunctional protein. Nonsense mutations are believed to cause DMD or BMD in approximately 10 to 15 percent of boys with these disorders.
Ataluren — sometimes referred to as a stop codon read-through drug — has the potential to overcome the effects of a nonsense mutation and allow functional dystrophin — the muscle protein that’s missing in Duchenne MD and deficient in Becker MD — to be produced.
The orally delivered drug is being developed by PTC Therapeutics, a South Plainfield, N.J., biotechnology company, to whichMDA gave a $1.5 million grant in 2005.
Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P, Paushkin S, Patel M, Trotta CR, Hwang S, Wilde RG, Karp G, Takasugi J, Chen G, Jones S, Ren H, Moon YC, Corson D, Turpoff AA, Campbell JA, Conn MM, Khan A, Almstead NG, Hedrick J, Mollin A, Risher N, Weetall M, Yeh S, Branstrom AA, Colacino JM, Babiak J, Ju WD, Hirawat S, Northcutt VJ, Miller LL, Spatrick P, He F, Kawana M, Feng H, Jacobson A, Peltz SW, Sweeney HL (May 2007). “PTC124 targets genetic disorders caused by nonsense mutations”. Nature447 (7140): 87–91. Bibcode:2007Natur.447…87W.doi:10.1038/nature05756. PMID17450125.
Hirawat S, Welch EM, Elfring GL, Northcutt VJ, Paushkin S, Hwang S, Leonard EM, Almstead NG, Ju W, Peltz SW, Miller LL (Apr 2007). “Safety, tolerability, and pharmacokinetics of PTC124, a nonaminoglycoside nonsense mutation suppressor, following single- and multiple-dose administration to healthy male and female adult volunteers”. Journal of clinical pharmacology47 (4): 430–444.doi:10.1177/0091270006297140. PMID17389552.
Nature. 2007 May 3;447(7140):87-91.
Proc Natl Acad Sci U S A. 2008 Feb 12;105(6):2064-9.
Neuromuscul Disord. 2015 Jan;25(1):5-13.
Wilschanski, M. (2013). “Novel therapeutic approaches for cystic fibrosis”. Discovery Medicine15 (81): 127–133. PMID23449115.
Wilschanski, M.; Miller, L. L.; Shoseyov, D.; Blau, H.; Rivlin, J.; Aviram, M.; Cohen, M.; Armoni, S.; Yaakov, Y.; Pugatsch, T.; Cohen-Cymberknoh, M.; Miller, N. L.; Reha, A.; Northcutt, V. J.; Hirawat, S.; Donnelly, K.; Elfring, G. L.; Ajayi, T.; Kerem, E. (2011). “Chronic ataluren (PTC124) treatment of nonsense mutation cystic fibrosis”. European Respiratory Journal38 (1): 59–69. doi:10.1183/09031936.00120910. PMID21233271.Sermet-Gaudelus, I.; Boeck, K. D.; Casimir, G. J.; Vermeulen, F.; Leal, T.; Mogenet, A.; Roussel, D.; Fritsch, J.; Hanssens, L.; Hirawat, S.; Miller, N. L.; Constantine, S.; Reha, A.; Ajayi, T.; Elfring, G. L.; Miller, L. L. (November 2010). “Ataluren (PTC124) induces cystic fibrosis transmembrane conductance regulator protein expression and activity in children with nonsense mutation cystic fibrosis”. American Journal of Respiratory and Critical Care Medicine182 (10): 1262–1272. doi:10.1164/rccm.201001-0137OC. PMID20622033.
References:
1. Ryan, N. J. Ataluren: first global approval. Drugs 2014, 74(14), 1709-14. (FMO only)
2. Gupta, P. K.; et. al. A metal-free tandem approach to prepare structurally diverse N-heterocycles: synthesis of 1,2,4-oxadiazoles and pyrimidinones. New J Chem 2014, 38, 3062-3070 (FMO only)
3. Almstead, N. G.; et. al. Methods for the production of functional protein from dna having a nonsense mutation and the treatment of disorders associated therewith. WO2007117438A2
THIS COMPD HAS ONE FLUORO MISSING, APPLY TO YOUR MK 8876
![4-fluoro-5-[[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl]isoquinoline NMR spectra analysis, Chemical CAS NO. 223645-67-8 NMR spectral analysis, 4-fluoro-5-[[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl]isoquinoline H-NMR spectrum](https://i0.wp.com/pic11.molbase.net/nmr/nmr_image/2014-11-28/002/466/2466109_1h.png?resize=695%2C612)
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. Mass spectrometry was performed for this isolated impurity by ESI technique in positive mode. The positive DI-MS spectrum of isolated impurity exhibited molecular ion peak as [M + H]+ at m/z 592.29 and as sodium adduct [M + Na]+ at m/z 614.28. The MS/MS data displayed a dominant fragment at m/z 574.28 which is 17 amu less than the molecular ion peak indicating a removal of the hydroxyl group.
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