Making small-molecule drugs usually goes something like this: set up a reaction, purify the intermediate, change a solvent, and repeat, repeat, repeat to get the final product. But there’s a lot of waste involved, which is why chemists stress the environmental benefits of an alternate approach: biocatalysis. Engineering enzymes to make reactions happen saves a lot of materials, minimizes chemical and hazardous waste, and even uses less plasticware and glassware. And not having to isolate intermediates saves time.
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DRUG APPROVALS BY DR ANTHONY MELVIN CRASTO
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DR ANTHONY MELVIN CRASTO Ph.D ( ICT, Mumbai)
, INDIA
36Yrs Exp. in the feld of Organic Chemistry,Working for AFRICURE PHARMA as ADVISOR earlier with GLENMARK PHARMA at Navi Mumbai, INDIA. Serving chemists around the world. Helping them with websites on Chemistry.Million hits on google,
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Islatravir (MK-8591, EFdA)
Islatravir (MK-8591, EFdA)
2′-Deoxy-4′-ethynyl-2-fluoroadenosine
- Molecular FormulaC12H12FN5O3
- Average mass293.254 Da
- 865363-93-5
2′-Deoxy-4′-ethynyl-2-fluoroadenosine
4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine
Adenosine, 2′-deoxy-4′-C-ethynyl-2-fluoro-
(2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol
2′-deoxy-4′-c-ethynyl-2-fluoroadenosine
865363-93-5 [RN]
9H-Purin-6-amine, 9-(2-deoxy-4-C-ethynyl-β-D-erythro-pentofuranosyl)-2-fluoro-
E2FDA
(2R,3S,5R)-5-(6-amino-2-fluoropurin-9-yl)-2-ethynyl-2-(hydroxymethyl)oxolan-3-ol
MK-8591
UNII:QPQ082R25D
Islatravir is known to be a nucleoside reverse transcriptase inhibitor, useful for treating HIV-1 and -2 infection and AIDS.
Islatravir (MK-8591, EFdA), useful for the treatment of eg HIV, AIDS and related diseases.
Merck & Co and Idenix , under license from Yamasa Shoyu , are developing islatravir, a nucleoside reverse transcriptase inhibitor, for the oral prevention and treatment of HIV-1 and HIV-2 infection; in July 2019, data from a phase IIb trial in patients with HIV-1 infection were presented.In August 2015, Merck licensed Codexis ‘ CodeEvolver® protein engineering platform technology to develop enzymes for use in the manufacture of the pharmaceutical products such as islatravir.
Islatravir (4′-ethynyl-2-fluoro-2′-deoxyadenosine, EFdA, or MK-8591) is an investigational drug for the treatment of HIV infection.[1]It is classified as a nucleoside reverse transcriptase translocation inhibitor (NRTTI).[2] Merck is developing a subdermal drug-eluting implant to administer islatravir.[3][4]
Biological activity
Islatravir has activity against HIV in animal models,[5] and is being studied clinically for HIV treatment and prophylaxis.[6] Islatravir is a nucleoside analog reverse transcriptase translocation inhibitor that unlike other such inhibitors, inhibits HIV through multiple mechanisms,[5] providing rapid suppression of the virus, when tested in macaques and mice.[7] Nevertheless, there are HIV strains resistant to islatravir and research is ongoing.[8]
PATENTS
WO2020014046 ,
PATENT
WO2020014047
PATENT
WO2020014050 (assigned to Codexis ), covering engineered phosphopentomutase (PPM) enzymes, useful in the synthesis of pharmaceutical compounds including islatravir.
PATENT
WO-2020014041
4’-Ethynyl-2’-deoxy nucleoside analogs are known for activity against HIV, AIDS and related diseases.
One example of a 4’-ethynyl nucleoside analog is 4’-ethynyl-2-fluoro-2’-deoxyadenosine (EFdA, also known as MK-8591) which is a nucleoside reverse transcriptase translocation inhibitor that blocks HIV-l and SIV viral replication in vitro (Kawamoto, A., Kodama, E., Sarafianos S. F. et al, Int. J. Biochem. Cell Biol.; 40(l l):24lO-2O [2008]; Ohrui, H., Kohgo, S., Hayakawa, H. et al, Nucleosides, Nucleotides & Nucleic Acids, 26, 1543-1546
[2007]) and in vivo (Hattori, S., Ide, K., Nakata, H. et al. Antimicrobial. Agents and
Chemotherapy, 53, 3887-3893 [2009]). EFdA is claimed in US Patent No. 7,339,053 (referred to in the‘053 patent as 2,-deoxy-4’-C-ethynyl-2-fluoroadenosine). EFdA has the following chemical structure:
EFdA is metabolized in cells to its active triphosphate anabolite which inhibits HIV reverse transcriptase. In contrast to nucleoside reverse transcriptase inhibitors (NsRTIs) and nucleotide reverse transcriptase inhibitors (NtRTIs) currently available for the treatment of HIV infection which lack a 3′-OH group to block incorporation of incoming nucleotide, EFdA retains a 3′ OH group and acts as a chain terminator by preventing translocation of the primer template in the reverse transcriptase (RT) active site and preventing binding of incoming
deoxyribonucleotide triphosphates (dNTPs). In addition, the pucker of the modified ribose ring of EFdA is believed to contribute to inhibition of reverse transcriptase by placing the 3′-OH in a vector in which phosphotransfer from the incoming nucleotide is inefficient. (Michailidis E, et ak, Mechanism of inhibition of HIV-l reverse transcriptase by 4’-ethynyl-2-fluoro-2’-deoxyadenosine triphosphate, J Biol Chem 284:35681-35691 [2009]; Michailidis E, et ak, 4’-Ethynyl-2-fluoro-2’-deoxyadenosine (EFdA) inhibits HIV-l reverse transcriptase with multiple mechanisms, J Biol Chem 289:24533-24548 [2014] ).
In in-vitro HIV replication assays, EFdA is a potent antiretroviral and exhibits comparable antiviral activity against clinical isolates across all subtypes that have been evaluated. It is rapidly anabolized in both lymphoid derived cell lines and in peripheral blood mononuclear cells to the active triphosphate in vitro, and the intracellular half-life of EFdA Triphosphate (EFdA- TP) exceeds 72 hrs. (Stoddart, C. A., Galkina, et ak, Oral Administration of the Nucleoside EFdA (4’-Ethynyl-2-Fluoro-2’-Deoxyadenosine) Provides Rapid Suppression of HIV Viremia in Humanized Mice and Favorable Pharmacokinetic Properties in Mice and the Rhesus Macaque, Antimicrob Agents Chemother, 2015 Jul; 59(7): 4190-4198, Published online 2015 May 4).
EFdA has been shown to have efficacy in animal models of HIV infection including humanized mouse models and an SIV infected rhesus macaque model. Pharmacokinetic studies of orally administered EFdA in mouse and rhesus monkey have demonstrated rapid absorption and high plasma concentrations. A long intracellular half-life was demonstrated by the fact that isolated peripheral blood mononuclear cells from the rhesus macaque were refractory to SIV infection 24 hr after drug administration. (Ibid.)
Previous syntheses of 4’-ethynyl nucleoside analogs including EFdA suffer from modest stereoselectivity in the formation of the C-N bond between the ethynyl-deoxyribose sugar and the 2-fluoroadenine (also referred to as 2-fluoro-9H-purin-6-amine) nucleobase. The previous syntheses also require protecting groups to carry out the glycosylation reaction which reduces the efficiency of the syntheses.
The synthesis described in Kei Fukuyama, et ak, Synthesis of EFdA via a
Diastereoselective Aldol Reaction of a Protected 3-Keto Furanose, Organic Letters 2015, 17(4), pp. 828-831; DOI: 10.102 l/ol5036535) is a l4-step synthesis from D-glucose diacetonide that uses diastereoselective reactions to set the three stereocenters. The stereochemistry of the anomeric center is controlled by having a 2′-acetoxy directing group that is subsequently removed by hydrolysis and deoxygenation. This route requires 4 chromatographic purifications, and the stoichiometric use of a toxic organotin reagent for late-stage deoxygenation.
In another route (see Mark McLaughlin, et al., Enantioselective Synthesis of 4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) via Enzymatic Desymmetrization, Organic Letters 2017, 19 (4), pp. 926-929), the fully-substituted 4′- carbinol is generated stereoselectively with an enzymatic desymmetrization. The 3 ‘-stereocenter is set with a catalytic asymmetric transfer hydrogenation, and the anomeric 1 ‘-linkage is established in modest stereoselectivity using substrate control, with an upgrade in stereochemical purity achieved by crystallization of an intermediate. This process requires 15 steps, requires the use of several protecting groups and generates the glycosyl linkage between the nucleobase and sugar fragments in low
stereoselectivity (1.8: 1).
A l2-step synthesis for making EFdA from R-glyceraldehyde acetonide is described in Kageyama, M., et al., Concise Synthesis of the Anti-HIV Nucleoside EFdA, Biosci. Biotechnol. Biochem, 2012 , 76, pp. 1219 -1225; and Enantioselective Total Synthesis of the Potent Anti-HIV Nucleoside EFdA, Masayuki Kageyama, et al., Organic Letters 2011 13 (19), pp. 5264-5266 [DOL 10.1021 / ol202116k] . The syntheses use the chiral starting material to set the 3′-stereocenter with moderate diastereoselectivity. After chromatographic separation of stereoisomers, the new stereocenter is used to guide a diastereoselective alkyne addition to set the fully-substituted 4’-stereocenter. The anomeric 1 ‘-position is established with little stereocontrol and requires chromatography to separate the anomers. This route requires chromatographic separation of diastereoisomers at two different stages and starts from an expensive chiral starting material.
Kohgo, S., et al., Design, Efficient Synthesis, and Anti-HIV Activity of 4′-C-Cyano- and 4′-C-Ethynyl-2′-deoxy Purine Nucleosides, Nucleosides, Nucleotides and Nucleic Acids, 2004, 23, pp. 671-690 [ DOL 10.1081/NCN-120037508] describes a synthetic route that starts from an existing nucleoside and modifies both the sugar and nucleobase portions. It is an 18-step synthesis starting from 2-amino-2’-deoxy adenosine with a low 2.5% overall yield.
It is known that enzymes such as purine nucleoside phosphorylase (PNP, EC 2.4.2.1) can form the glycosyl linkage in nucleosides and nucleoside analogs in high stereoselectivity and without the use of protecting groups. See for example the review: New Trends in Nucleoside Biotechnology, Mikhailopulo, I. A., Miroshnikov, A.I,. Acta Naturae 2010, 2, pp. 36-58.
However, the current scope of the sugar fragments capable of undergoing reaction catalyzed by PNP has been limited to the a- 1 -phosphates of natural ribose and deoxyribose along with a small number of analogs with small H, NH2, or F substituents at the C2’ and C3’ positions and replacements of the C5’ OH group. There have been no reports of successful glycosylation catalyzed by PNP using sugars with carbon substituents on the ring or any substitution at the C4’ position.
Access to the ribose and deoxyribose a- 1 -phosphate substrates for the PNP-catalyzed glycosylation has been demonstrated by translocation of the phosphate group from the 5’-hydroxyl to G -hydroxyl position with the enzyme phosphopentomutase (PPM, EC 5.4.2.7) (see Mikhailopulo, I. A., et al. supra). However, the scope of the sugars for which PPM is capable of catalyzing this reaction has been limited to ribose, arabinose, 2-deoxyribose, and 2,3-dideoxyribose. No examples have been reported of successful reaction with sugar phosphates containing any additional substituents.
Deoxyribose phosphate aldolase (DERA, EC 4.1.2.4) enzymes are known to catalyze the aldol addition of acetaldehyde to other short-chain aldehydes (see review: Stephen M. Dean, et al., Recent Advances in Aldolase-Catalyzed Asymmetric Synthesis, Adv. Synth. Catal. 2007, 349, pp. 1308 – 1320; DOI: 10. l002/adsc.200700115). However, no examples have been reported with aldehydes bearing a fully substituted carbon a to the aldehyde.
ETS Patent 7,229, 797 describes the formation of deoxyribonucleosides from the natural unsubstituted deoxyribose 1 -phosphate by use of purine nucleoside phosphorylase (PNP) and additionally using enzymes such as sucrose phosphorylase to remove the inorganic phosphate byproduct and drive the equilibrium. It does not disclose enzyme engineering for the creation of PNP enzymes that can generate nucleosides from the unnatural 4-ethynyl-D-2-deoxyribose 1-phosphate, nor that through engineering of PPM and DERA enzymes to act on unnatural substrates, 4-ethynyl-D-2-deoxyribose 1 -phosphate can be generated.
In view of the difficult and lengthy synthetic options developed to date for producing 4’-ethynyl nucleoside analogs, it would be desirable to develop an improved enzymatic synthesis for 4’-ethynyl nucleoside analogs such as EFdA that reduces the number of process steps, minimizes the use of protecting groups, improves the stereoselectivity of glycosylation and avoids the use of toxic materials.
Surprisingly, it has been found that PPM enzymes have some activity with the 3-atom ethynyl substituent at the 4’ position on ribose and that the PPM enzyme activity could be improved by introducing mutations into the enzymes to successfully develop a reaction for
isomerization of
4-ethynyl-D-2-deoxyribose 5-phosphate (6) to 4-ethynyl-D-2-deoxyribose 1 -phosphate (6.5) catalyzed by PPM to enable a more efficient method for production of 4’-ethynyl-2’-deoxy nucleosides.
Additionally, PNP enzymes have also been found to have some activity with the 3-atom ethynyl substituent at the 4 position on deoxyribose and that the PNP enzyme activity could be improved by introducing mutations into the enzymes to successfully develop a glycosylation reaction catalyzed by PNP to enable a more efficient method for production of 4’ -ethynyl -2’-deoxy nucleosides.
Even further improvement to the overall synthetic method came from the finding that
DERA enzymes, particularly the DERA from Shewanella halifaxensis, have activity for aldol reaction with 2-ethynyl-glyceraldehyde 3-phosphate which has a fully substituted a-carbon. This discovery allowed for the efficient synthesis of 4-ethynyl-D-2-deoxyribose 5-phosphate, a precursor to 4’-ethynyl-2’-deoxy nucleoside analogs, e.g., including EFdA.
SUMMARY OF THE INVENTION
The present invention involves the use of engineered enzymes in a novel enzymatic synthesis of 4’-ethynyl-2’-deoxy nucleoside analogs, including EFdA, that eliminates the use of protecting groups on intermediates, improves the stereoselectivity of glycosylation and greatly reduces the number of process steps needed to make said compounds compared to prior methods, among other process improvements. It further relates to novel intermediates which are an integral part of the enzymatic process.
The overall process is summarized in the following Scheme 1 and Scheme 2; the latter scheme provides an alternative method for making compound 5:
Scheme 1
kinase
p p y
Scheme 1A
kinase galactose oxidase
3 2X+ 9
2
p p y
It has been discovered that 4’-ethynyl-2’-deoxy nucleoside analogs such as EFdA can be synthesized employing a final step one-pot process by combining 4-ethynyl-D-2-deoxyribose 5-phosphate (6) with two enzymes, phosphopentomutase (PPM) [for example but not limited to SEQ ID NO.: 8] and purine nucleoside phosphorylase (PNP) [for example but not limited to SEQ ID NO.: 9, SEQ ID NO.: 15], as shown in Scheme 2.
Scheme 2
Scheme 2A
Several upstream intermediates used in the present process for the synthesis of the final product 4’-ethynyl-2’-deoxy nucleosides and analogs thereof are also made using enzymatic reaction methods as shown in Scheme 3; Scheme 3 A and Scheme 3B
Scheme 3
Scheme 3A
o2
pTsOH
deoxyribose
aldolase
Scheme 3B
Experimental Procedures
Preparation of 2-ethynyl-2-hvdroxypropane-l,3-diyl diacetate 12)
Method A:
To a -35 °C solution of diacetoxyacetone (1) (159 g, 914.0 mmol) in THF (1000 mL) was added 1600 mL of a 0.5 M solution of ethynyl magnesium chloride in THF maintaining the temperature below -20 °C. After the reaction reached completion, acetic acid (78 mL) in 400 mL methyl tert-butyl ether (MTBE) was added dropwise keeping the temperature below -20 °C. MTBE (800 mL) was then added and the mixture was warmed to room temp. Saturated NaCl in water (1000 mL) was added followed by saturated NH4CI solution in water (1050 mL). The organic layer was separated, dried over Na2SC>4 and evaporated to give compound (2) as an oil (160 g, 88%). 1H NMR (CDCI3, 500 MHz): d 4.26 (dd, 4 H), 2.55 (s, 1H), 2.14 (s, 6H).
Preparation of 2-ethynyl-propane-l,2,3-triol 13)
Method B:
To a solution of 2-ethynyl-2-hydroxypropane-l,3-diyl diacetate (2) (70 g, 350 mmol) in ethanol was added a 0.5M solution of sodium methoxylate in methanol (69.9 mL, 35.0 mmol) at room temperature (rt). The reaction was stirred at rt for 2 hours (h) to reach completion. The solvents were evaporated and the residue was re-dissolved in 100 mL water and extracted with 3 x 50 mL MTBE. The aqueous layer was sparged with nitrogen to remove residual solvents to give a 40.9% solution of 2-ethynyl-propane-l,2,3-triol (3) (108 g , 100% yield) as determined by nuclear magnetic resonance (NMR) (maleic acid as internal standard). lH NMR (D2O, 500 MHz): d 3.60 (dd, 4 H), 2.85 (s, 1H).
Alternate Preparations o ethynyl-glvcer aldehyde 14)
Method Cl:
In a stirred reactor, 2-ethynyl-propane-l,2,3-triol (3) (1.1 g, 9.47 mmol) in sodium phosphate buffer (30 mL, 100 mM, pH 7.0) containing antifoam 204 (Sigma A6426, 1 drop ~ 20 pL) was warmed to 30 °C with air sparging at 12.5 seem. Galactose oxidase (GOase, SEQ ID NO.: 1) (250 mg), Horseradish Peroxidase* (Type I, 5 mg) and bovine catalase** (5 mg) dissolved in sodium phosphate buffer (5 mL 100 mM, pH 7.0) were added to the reactor, followed by the addition of CuS04 aq. solution (100 mM, 150 pL). The reaction mixture was stirred at 600 rpm with air sparging for 47h to give (f?)-2-ethynyl-glyceraldehyde (4) in 47% conversion (by NMR) and 72% e.e. . (The product was not isolated). lH NMR (D2O, 500 MHz): d 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H).
* Horse Radish Peroxidase: wild type peroxidase from horseradish Type I, commercially available from SIGMA (P8125), isolated from horseradish roots (Amoracia rusticana).
** Bovine catalase: heme-dependent catalase from bovine source, commercially available from Sigma (C1345)
Method C2:
In a stirred 100 L jacketed reactor charged with deionized water (56.2 kg), sodium dihydrogen phosphate (1.212 kg, 10 moles) was added. The pH was adjusted to 7.02 using 10 N sodium hydroxide solution (852.6 g) at 25 °C. The reactor was charged with Antifoam 204 (A6426, 10 mL), followed CuS04*5H20 (6.5 g). Galactose oxidase (451.2 g) (SEQ ID NO.: 10) was added and stirred for 15 min while sparged with air. Horseradish peroxidase* (200.2 g) and catalase** (502.6 g) were added and the reactor was rinsed with water (2.0 kg). Next 2-ethynyl-propane- 1,2, 3 -triol (3) solution in water (9.48%, 30.34 kg, 24.72 mol) was added followed by an additional portion of Antifoam 204 (A6426, 10 mL). The reaction was sparged with air and
stirred overnight to give 94.0 kg of (A)-2-ethynyl-glyceraldehyde (4) in 66% conversion (by NMR) and 84% e.e. Assay yield 60%: 1H NMR (D20, 500 MHz): d 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H).
* Horse Radish Peroxidase: wild type peroxidase from horseradish purified, commercially available from Toyobo (PEO-301), isolated from horseradish roots (Amoracia rusticana).
** Bovine catalase: heme-dependent catalase from bovine source, commercially available from Sigma (C1345).
The above reaction was also performed using the galactose oxidase (SEQ ID NO.: 11) and the product (4) was obtained in 67% conversion (by NMR) and 88% e.e. and assay yield 59%: 1H NMR (D2O, 500 MHz): d 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H).
Method C3:
In a 100 mL Easy Max vessel equipped with sparger and flow controller, water (82 mL) and PIPES potassium buffer (5mL, 0.5 M) were charged. The pH was adjusted to 7.5 using 5 M KOH solution at 25 °C. Antifoam 204 (200 pL) was added, followed by evolved galactose oxidase (SEQ ID NO.: 17, 450 mg enzyme powder) and copper(II) sulfate pentahydrate (100 pL, 100 mM). The reaction mixture was sparged with air at 125 standard cubic centimeters per minute (seem) for 15 min. Bovine catalase (Cl 345, Sigma-Aldrich, 150 mg, 2000-5000 U/mg, 0.75 MU) was charged, followed by horseradish peroxidase (HRP, Toyobo PEO-301, 100 mg,
130 U/mg, 1.3 kU) and the aqueous solution of 2-ethynyl-propane-l,2,3-triol (3) (25 wt%, 12 mL, 25.8 mmol). The reaction mixture was stirred at 30 °C with aeration at 125 seem and sampled using EasySampler over 20h to give 70% conversion and form compound (4) ((A)- 2-ethynyl-glyceraldehyde) in 58% assay yield and 99% e.e. lH NMR (D2O, 500 MHz): d 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H). The crude reaction stream was carried directly into the subsequent phosphorylation step.
Method C4: Oxidation with immobilized galactose oxidase
Galactose
Oxidase
immobilized
3
Enzyme immobilization procedure:
Nuvia IMAC Ni-charged resin (16 mL based on settled volume) was added to a filter funnel and washed with binding buffer (10 column volumes, 160 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0) to remove the resin storage solution. In a vessel evolved galactose oxidase (SEQ ID NO.: 17, 2.00 g) lyophilized powders were resuspended in copper (II) sulphate solution (100 mM; 5.00 mL), followed by addition of binding buffer (50 mL) and the resin. The solution was mixed using rotating mixer at 20 °C for 5h. The resin was filtered and washed with binding buffer (10 column volumes, 160 mL) and potassium PIPES buffer (10 column volumes, 160 mL; 50 mM, pH 7.5) and it was used directly in a reaction. Reaction procedure:
In a 100 mL Easy Max vessel equipped with sparger and flow controller, water (82 mL) and PIPES potassium buffer (5mL, 1 M) were charged. The pH was adjusted to 7.5 using 5 M KOH solution at 25 °C. Antifoam 204 (200 pL) was added, followed by evolved galactose oxidase immobilized on the resin (SEQ ID NO.: 17, 750 mg enzyme powder per 6 mL resin) and copper(II) sulfate pentahydrate (100 pL, 100 mM). The reaction mixture was sparged with air at 125 standard cubic centimeters per minute (seem) for 15 min. Bovine catalase (C1345, Sigma-Aldrich, 210 mg, 2000-5000 U/mg, 1.05 MU) was charged, followed by horseradish peroxidase (HRP, Toyobo PEO-301, 100 mg, 130 U/mg, 1.3 kU) and the aqueous solution of 2-ethynyl-propane- 1,2, 3 -triol (3) (25 wt%, 13 mL, 29.4 mmol). The reaction mixture was stirred at 25 °C with aeration at 125 seem. After 22h the reaction reached 91% conversion to give 200 mM (//)-2-ethynyl-glyceraldehyde (4) solution (100 mL, 68% assay yield, 97% e.e. lH NMR (D2O, 500 MHz): d 4.29 (s, 1H), 3.65 (dd, 2H), 2.83 (s, 1H). The crude reaction stream was carried directly into the subsequent phosphorylation step.
Method C5: Optional Isolation of aldehyde via formation of aminal (8)
Step 1: Preparation of (S)-2-( \ .3-dibenzylimidazolidin-2-yl )but-3-yne- l 2-diol
A 100 L jacketed cylindrical vessel equipped with nitrogen bubbler, mechanical stirrer and thermocouple was charged with crude oxidase reaction stream containing (f?)-2-ethynyl-glyceraldehyde ((4), 26.0 kg, 1.85 wt% aldehyde, 3.64 mol) and inerted with N2 atmosphere. The aqueous solution was warmed to 20 °C and Af,A-di methyl dodecan- 1 -ami ne oxide (DDAO) (30 wt% in water, 798 g, 0.96 mol;) was added, followed by MTBE (55.3 kg, 76 L) and N,N -dibenzylethane-l, 2-diamine (1.55 kg, 6.43 mol). The brown, biphasic mixture was stirred overnight at 20 °C under nitrogen atmosphere. After 17 hours the stirring was stopped and the organic phase was removed and discarded. A light brown MTBE solution of fV)-2-( l ,3-dibenzylimidazolidin-2-yl)but-3-yne-l,2-diol (56.5 kg, 2.02 wt% aminal, 3.39 mmol, 93% assay yield) was obtained.
Six similar MTBE solutions were processed together in a single distillation and crystallization step (in total 374.4 kg of solution, containing 7.91 kg aminal).
A 50 L jacketed cylindrical vessel equipped with mechanical stirrer, distillation head (condenser at -20 °C) and thermocouple was charged with aminal solution (45 L). Vacuum was applied to the vessel (65-95 torr) and the jacket was set to 40 °C. Solvent was removed by distillation until a volume of 35 L had been reached. At this point, the internal temperature was 6.1 °C and an off-white solid had begun to crystallize. The remaining MTBE solution was slowly added, maintaining a constant volume of 35-40 L and an internal temperature of 0-10 °C. Once all the MTBE solution had been added the volume was decreased to 25 L. Distillation was halted, the vessel was inerted with nitrogen and the jacket temperature was decreased to 10 °C. The resulting pale yellow suspension was aged at this temperature for 2 hours and the solids were collected by filtration. The filter cake was washed with cold (-2 °C) MTBE (12.7 kg) and then dried under nitrogen flow for 7 hours. (5)-2-(l,3-dibenzylimidazolidin-2-yl)-but-3-yne-l,2-diol was obtained as an off-white crystalline solid (5.75 kg) lff NMR (500 MHz, DMSO-i¾) d 7.42 – 7.35 (m, 4H), 7.32 (td, J= 7.5, 1.6 Hz, 4H), 7.27 – 7.21 (m, 2H), 5.10 (t, J= 5.6 Hz, 1H), 5.03 (s, 1H), 4.28 (d, J= l3.3Hz, 1H), 4.16 (d, J= 13.3 Hz, 1H), 3.76 (s, 1H), 3.70 – 3.58 (m, 4H), 3.21 (d, J= 0.9 Hz, 1H), 2.90 – 2.80 (m, 2H), 2.60 – 2.51 (m, 2H).13C NMR (126 MHz, DMSO-i¾) d 140.0, 140.0, 128.5, 128.3, 128.2, 128.1, 126.8, 126.8, 88.6, 86.9, 75.0, 74.0, 66.4, 60.7, 60.5, 50.4, 50.3, 39.5. HR-MS (ESI) Aminal (M + H+) C21H25N202+ calculated 337.1911; found 337.1922.
Step 2: Prep l (8)
A 4 L jacketed cylindrical vessel equipped with nitrogen bubbler and mechanical stirrer was charged with of TsOH»H20 (12.0 g, 63.1 mmol), water (60 mL), (ri)-2-(l,3-dibenzylimidazolidin-2-yl)but-3-yne-l,2-diol (110 g, 327 mmol) and MTBE (1700 mL). The biphasic mixture was placed under nitrogen and the jacket temperature was set to 15 °C. A solution of TsOH»H20 (114 g, 599.3 mmol) in water (600 mL) was added dropwise over 1.5 hours with overhead stirring (200 rpm). After addition had completed, the jacket temperature was lowered to 5 °C and the resulting slurry was aged for 1 hour. The solids were removed by filtration and washed with cold water (270 mL). The biphasic solution was transferred to a separating funnel and the organic phase was removed and discarded. The aqueous phase was treated with DOWEX™ MARATHON™ A resin (hydroxide form, 11.0 g) and AMBERLYST® 15 resin (hydrogen form, 11.0 g) while sparging with N2 at a rate of 200 seem for 24 hours to remove residual MTBE. The resins were removed by filtration to give a colorless aqueous solution of (f?)-2-hydroxy-2-(hydroxymethyl)but-3-ynal (774 g, 4.6 wt% aldehyde, 82% yield). lH MR (500 MHz, D2O) d 5.01 (s, 1H), 3.77 (d, J= 11.7 Hz, 1H), 3.73 (d, J= 11.7 Hz, 1H), 2.92 (s, 1H). 13C NMR (126 MHz, D2O) d 129.4, 125.4, 90.3, 81.0, 76.0, 73.9, 65.3. HRMS
(ESI) Aldehyde dimer (2M + Na+) CioHi2Na06+ calculated 251.0526; found 251.0530.
Alternate Preparations o ethvnyl-glvceraldehvde 3-phosphate (5):
Method Dl: Acetate kinase: ATP -regeneration system
Pantothenate kinase PanK
ATP
Acetate kinase
4 Acetate phosphate
5
In a stirred reactor, to a solution of adenosine diphosphate disodium salt (40 mg, 0.087 mmol) and magnesium chloride (38 mg, 0.400 mmol) in HEPES buffer (66 mM, pH 7.5, 30 mL) was added (i?)-2-ethynyl-glyceraldehyde (4) (1.9 mL, 210 g/L solution in water, 3.51 mmol), followed by acetate kinase (SEQ ID NO.: 3) (40 mg), and pantothenate kinase (SEQ ID NO.: 2) (120 mg). The reaction mixture was warmed to 25 °C and a solution of acetyl phosphate lithium potassium salt (1.3 g, 7.01 mmol) in HEPES buffer (50 mM, pH 7.5, 10 mL) was added dropwise over 4 hours, with pH maintained at 7.5 using 5M sodium hydroxide. The reaction was stirred for 18 hours to give (i?)-2-ethynyl-glyceraldehyde 3-phosphate (5) in 85% conversion (by HPLC) (The product was not isolated). iH NMR (D2O, 400 MHz): d 5.02 (s, 1H), 4.00 (dq, 2 H), 2.88 (s, 1H). LC-MS: (ES, m/z): calculated for C5H7O6P (M-H): 193.1; found 193.0.
Method D2: Pyruvate oxidase ATP -regeneration system
Pan
Pyruvate oxidase
Pyruvate
Phosphate
02
In a stirred reactor, a solution of sodium pyruvate (3.11 g, 28 mmol) and phosphoric acid (0.523 mL, 7.71 mmol) in 76 mL water pH 7.5 was charged with (i?)-2-ethynyl-glyceraldehyde (4) (3.8 mL, 210 g/L solution in water, 7.01 mmol), adenosine diphosphate disodium salt (80 mg, 0.174 mmol), thiamine pyrophosphate (40 mg, 0.086 mmol), flavin adenine dinucleotide disodium salt hydrate (64 mg, 0.077 mmol), and magnesium chloride (400 pL, 1 M solution in water, 0.4 mmol). The pH was re-adjusted to 7.5 with 5M aq sodium hydroxide and the reaction volume was re-adjusted to 80 mL with water. Acetate kinase (SEQ ID NO.: 3) (80 mg), pyruvate oxidase (SEQ ID NO.: 4) (80 mg, lyophilized cell free extract), pantothenate kinase (SEQ ID NO.: 2) (400 mg), and catalase (800 pL, ammonium sulfate suspension CAT-101, Biocatalytics) were added. The reaction was stirred at 500 rpm and 30 °C with air sparging for 72 hours to give (//)-2-ethynyl-glyceraldehyde 3 -phosphate 5 in 95% conversion (by HPLC) (The product was not isolated). lH NMR (D2O, 400 MHz): d 5.02 (s, 1H), 4.00 (dq, 2 H), 2.88 (s, 1H). LC-MS: (ES, m/z): calculated for C5H7O6P (M-H): 193.1; found 193.0.
The above reaction was also performed using the pantothenate kinase (SEQ ID NO.: 13) and the product 5 was obtained in 66% conversion. (The product was not isolated). iH NMR (D2O, 400 MHz): d 5.02 (s, 1H), 4.00 (dq, 2 H), 2.88 (s, 1H).
Method D3: Acetate kinase: ATP -regeneration system using immobilized enzymes
Panth
Acetate phosphate
Enzyme immobilization procedure:
NUVIA™ Immobilized Metal-ion Affinity Chromatography (IMAC) nickel-charged resin (168 mL based on settled volume) was added to a filter funnel and washed with binding buffer (1.6 L; 500 mM sodium chloride, 50 mM sodium phosphate, pH 8.0). In a vessel, pantothenate kinase
(8.4 g) (SEQ ID NO.: 12) and acetate kinase (2.8 g) (SEQ ID NO.: 3) were dissolved in binding buffer (500 mL). The washed resin was charged to the vessel and the solution was stirred for 4 hours at 20 °C. The resin was filtered and washed first with binding buffer (1.6 L) followed by piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) buffer (840 mL; 50 mM, pH 6.5). The washed resin was used directly in the next step.
Reaction procedure:
To a 1 L reactor, a solution of (f?)-2-ethynyl-glyceraldehyde (4) in water (608.7 g, 4.6 wt%, 212 mmol) was charged and cooled to 5 °C. To the cooled solution piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) buffer (32.7 mL, 1 M, pH 6.5, 32.7 mmol), magnesium chloride (9.33 mL, 1 M, 9.33 mmol), acetyl phosphate diammonium salt (51.8 g, 265 mmol), adenosine diphosphate disodium salt hydrate (1.17 g, 2.12 mmol), and water (192 mL) were added. The solution was allowed to stir and pH was adjusted to 6.4 using 5 N KOH. The reaction was warmed to 20 °C and 168 mL of resin with co-immobilized pantothenate kinase (SEQ ID NO.: 12) and acetate kinase (SEQ ID NO.: 3) was added. The reaction was stirred for 10 hours with 5 N KOH used to maintain a pH of 6.4 to give (f?)-2-ethynyl-glyceraldehyde 3-phosphate (5) in
92% conversion (by HPLC) and 91% yield (by 3 lp NMR with tetraphenylphosphonium chloride as internal standard) (the product was not isolated). lH NMR (D2O, 400 MHz): d 5.02 (s, 1H), 4.00 (dq, 2 H), 2.88 (s, 1H). LC-MS: (ES, m/z): calculated for C5H7O6P (M-H): 193.1; found 193.0.
Preparation of 4-ethynyl-D-2-deoxyribose 5-phosphate 16)
Method E:
To a solution of (f?)-2-ethynyl-glyceraldehyde 3-phosphate (5) (5, 20 mL, 5.3 mmol) in water, a solution of acetaldehyde in water (40 wt.%, 2.02 mL, 15.9 mmol) was added at room
temperature, followed by the addition of Deoxyribose-phosphate aldolase (DERA) (SEQ ID NO. : 6), 25 mg solution in triethanolamine hydrochloride buffer (1 mL, 1 M, pH 7.0). The reactor was sealed and the mixture was stirred overnight at 30 °C and 600 rpm to give 4-ethynyl-D-2-deoxyribose 5-phosphate (6) in 99% conv. and 99% e.e., 99% d.e. as a 1 : 1 anomer mixture (The product was not isolated) a-anomer: lH NMR (D2O, 600 MHz) 5 5.31 (t, 1H), 4.13 (t, 1H), 3.81-3.72 (m, 2H), 2.89 (s, 1H), 2.42-2.34 (m, 1H), 1.87-1.79 (m, 1H); 13c NMR (D2O, 151 MHz) 5 97.7 (s), 81.4 (d), 79.4 (s), 78.9 (s), 71.1 (s), 67.7 (d), 39.6 (s). b-anomer: 1H NMR
(D2O, 600 MHz) 5 5.40 (dd, 1H), 4.28 (t, 1H), 3.88-3.80 (m, 2H), 2.87 (s, 1H), 2.13-2.06 (m,
1H), 2.04-1.97 (m, 1H); 13C NMR (D20, 151 MHz) 5 97.3 (s), 82.2 (d), 78.7 (s), 78.5 (s), 71.3 (s), 68.4 (d), 39.6 (s). LC-MS: (ES, m/z): calculated for C7H10O7P (M-H): 237.0; found 237.0
Alternate Preparations of (2 ?,3A,5 ?)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol monohydrate (7) [alternative name 4’-ethynyl-2-fluoro- 2’-deoxvadenosine or EFdAI
Method FI:
Ammonium ((2f?,3ri)-2-ethynyl-3,5-dihydroxytetrahydrofuran-2-yl)m ethyl hydrogen phosphate (1.00 g, 3.91 mmol) was dissolved in 10 mL of pH 7.5 buffer (100 mM triethanolamine ΉO containing 5 mM MnCl2). The solution pH was adjusted to 7.3 with 5 N NaOH. To the solution was added 2-fluoroadenine (0.599 g, 3.91 mmol) and sucrose (2.68 g, 7.82 mmol). The enzyme solution was prepared by dissolving phosphopentomutase (SEQ ID NO. : 8) (100 mg), purine nucleoside phosphorylase (SEQ ID NO.: 9) (50 mg), and sucrose phosphorylase (SEQ ID NO. :
7) (10 mg) in 10 mL of the pH 7.5 buffer. The enzyme solution was added to the reagent mixture and the resulting suspension was shaken at 40 °C. After 20 h, the suspension was cooled to 0 °C and filtered, rinsing with cold water. The solid was suction dried to give the title compound (1.12 g, 92%) as a single isomer.
iH NMR: (300 MHz, DMSO-d6, ppm): d 7.68 (br s, 2H), 7.32 (d, J = 2.0 Hz, 1H), 6.44 (t, J =
5.8 Hz, 1H), 5.52 (d, J = 5.6 Hz, 1H), 5.27 (t, J = 6.0 Hz, 1H), 4.44 (q, J = 6.4 Hz, 1H), 3.60 (q, J = 6.0 Hz, 1H), 3.53 (q, J = 6.4 Hz, 1H), 3.48 (s, 1H), 2.48-2.41 (m, 1H), 2.37-2.30 (m, 1H). 13c NMR (150.92 MHz, DMSO-d6, ppm) d 158.5 (d, JCF = 203.5), 157.6 (d, JCF = 21.2), 150.2 (d, JCF = 20.2), 139.7 (d, JCF = 2.4), 117.4 (d, JCF = 4.0), 85.1, 82.0, 81.4, 78.7, 70.1, 64.2, 38.1. LC-MS: (ES, m/z): calculated for C12H12FN5O3 (M+Na): 316.0822; found 316.0818.
The PPM and PNP enzymes used in this step were each derived from mutations starting from the enzymes from E. coli ( Escherichia coli). The sucrose phosphorylase (SP) used in this step was derived from Alloscardovia omnicolens ; SP derived from other organisms could also be used.
Method F2:
To an aqueous solution of (f?)-2-ethynyl-glyceraldehyde 3-phosphate (5) (950 mL, 157 mmol) containing piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) buffer at a pH from about 5.5 to 6.0 was added triethanolamine (7.09 g, 47.5 mmol). The pH of the solution was adjusted from 7.1 to 7.6 using potassium hydroxide (8 mL, 8M). Manganese(II) chloride hydrate (0.592 g, 4.70 mmol) was added followed by sucrose (161 g, 470 mmol), giving a pH of 7.5 To the solution
was added the following enzymes: deoxyribose-phosphate aldolase (SEQ ID NO. : 14) (461 mg), sucrose phosphorylase (SEQ ID NO. : 7) (494 mg), phosphopentomutase (SEQ ID NO.: 8)(2.63 g), and purine nucleoside phosphorylase (SEQ ID NO. : 15) (659 mg). Once the enzymes were dissolved, 2-fluoroadenine (19.80 g, 125 mmol) was added. The reaction was heated to 35 °C and acetaldehyde was added (40 wt% in isopropyl alcohol, 29.8 mL, 235 mmol). After reacting for 2h, the mixture was seeded with EFdA crystalline product (0.96 g, 2 mol%). After reacting over 26 h at 35 °C, the slurry was cooled to 0 °C, and the solids were collected by filtration, washing with water two times (40 mL ea.). The solids were dried under a nitrogen sweep. Yield 43.2 g, 92 wt%, 96.2% corrected. ¾ NMR: (300 MHz, DMSO-d6, ppm): d 7.68 (br s, 2H), 7.32 (d, J = 2.0 Hz, 1H), 6.44 (t, J = 5.8 Hz, 1H), 5.52 (d, J = 5.6 Hz, 1H), 5.27 (t, J = 6.0 Hz, 1H), 4.44 (q, J = 6.4 Hz, 1H), 3.60 (q, J = 6.0 Hz, 1H), 3.53 (q, J = 6.4 Hz, 1H), 3.48 (s, 1H), 2.48-2.41 (m, 1H), 2.37-2.30 (m, 1H). 13C NMR (150.92 MHz, DMSO-d6, ppm) d 158.5 (d, JCF = 203.5), 157.6 (d, JCF = 21.2), 150.2 (d, JCF = 20.2), 139.7 (d, JCF = 2.4), 117.4 (d, JCF = 4.0), 85.1, 82.0, 81.4, 78.7, 70.1, 64.2, 38.1. LC-MS: (ES, m/z): calculated for C12H12FN5O3 (M+Na): 316.0822; found 316.0818.
Alternate Preparations of -2-ethvnyl-propane-l,2,3-triol 1 1-phosphate 19) :
Method Gl: Acetate kinase: ATP-regeneration system using enzymes SEQ. ID No.: 2 and SEQ. ID No.: 3
Panthotenate kinase PanK
ATP
Acetate kinase
Acetate phosphate
A 50 mL reactor was charged with a solution of 2-ethynyl-propane-l,2,3-triol (3) in water (9.29 g, 9.46 wt%, 7.57 mmol) potassium PIPES buffer (1.02 mL, 1 M, pH 6.5, 1.02 mmol), magnesium chloride (292 pL, 1 M, 0.292 mmol), acetyl phosphate diammonium salt (1.851 g, 89 wt%, 9.46 mmol), adenosine diphosphate disodium salt hydrate (ADP, 42 mg, 0.076 mmol, 0.01 eq), and water (28 mL). The pH was adjusted to 6.4 using 5 M KOH, the solution was warmed to 20 °C and evolved pantothenate kinase PanK SEQ. ID No.: 2 (264 mg) and acetate kinase AcK SEQ. ID No. : 3 (88 mg) were added. The reaction was stirred for 16 hours with pH maintained at 6.4 using 5 N KOH. The final reaction contents provided C.V)-2-ethynyl -propane- 1 ,2,3-triol 1-phosphate (9) in >95% e.e. and 99% conversion (by 31P NMR). The product was not isolated. ¾ NMR (D2O, 500 MHz) d 3.89 (m, 2H), 3.72 (d, 7= 11.6 Hz, 1 H), 3.65 (d, J= 11.6 Hz, 1H),
2.93 (s, 1H). 13C NMR (D2O, 126 MHz) d 82.9 (s), 75.1 (s), 71.0 (d, J= 6.9 Hz), 67.0 (d, J= 4.5 Hz), 64.7 (s). 31P NMR (D2O, 202 MHz) d 3.39. HRMS: (ESI, m/z): calculated for [M-l] CsHsOeP: 195.0058; Found 195.0068 [M-H] : 195.0058.
Method G2: Acetate kinase: ATP-regeneration system using enzyme SEQ. ID No.: 20 and enzyme SEQ. ID No.: 21
Panthotenate kinase PanK
– – ATP
Acetate kinase
Acetate phosphate
To a jacketed reactor aqueous solution 2-ethynyl-propane-l,2,3-triol (3) (11.47 kg, 8.7% wt, 8.61 mol) and water (7.5kg) was charged, followed by 1M BIS-TRIS methane buffer pH 6.5 (1L) and magnesium chloride (41.4 g). ATP (48g, 0.086 mol, 0.01 equivalent) and diammonium acetyl phosphate (2.021 kg, 89%, 10.33 mmol) were added, the solution was warmed up to 20 °C and the pH of the solution was re-adjusted to 6.8 using KOH (270.4 g). Evolved pantothenate kinase SEQ. ID No.: 20 (20.4 g) and evolved acetate kinase SEQ. ID No.: 21 (3 g) were then charged as solids. The reaction was stirred for at 20 °C for l6h during which pH dropped to 5.5.
Quantitative conversion of 2-ethynyl-propane-l,2,3-triol (3) was obtained as judged by ‘H and 31P NMR. Such prepared (ri)-2-ethynyl-propane-l,2,3-triol l-phosphate (9) solution (397 mM, 22.5 kg, 98% yield) was used in subsequent oxidation step without any further purification. ‘H NMR (D2O, 500 MHz) d 3.89 (m, 2H), 3.72 (d, 7= 11.6 Hz, 1 H), 3.65 (d, J= 11.6 Hz, 1H),
2.93 (s, 1H).
Method G3: Acetate kinase: ATP-regeneration system using enzyme SEQ. ID No.: 20 and enzyme SEQ. ID No.: 21 with deuterated compound (3) to assign absolute stereochemistry and demonstrate desymmetrizing phosphorylation.
Acetate phosphate
Z-d2, 95:5 er
Evolved pantothenate kinase SEQ. ID No. : 20 (100 pL of 10 g/L solution in water ) and evolved acetate kinase SEQ. ID No. : 21 (100 pL of 2g/L solution in water) were added to a solution containing diammonium acetyl phosphate (41 mg), 2-ethynyl-propane-l, l-72-l,2,3-triol ((A)- 3-d2, 20 mg, 170 pmol), magnesium chloride (10 pL of 1 M solution in water), ADP (10 pL of 100 g/L solution in water), and sodium phosphate buffer (10 pL of 1 M solution in water) in water (800 pL) at pH 6.5. The reaction was incubated for 24h at rt to give deuterated 2-ethynyl-propane-l,2,3-triol l-phosphate analogs (S)-9-(3,3-d2) and (S)-9-(l,l-d2) in 95:5 ratio and 99% overall yield. The ratio of phosphorylated compounds was determined by 31P NMR to be -95:5, confirming stereoselective phosphorylation of the 2-ethynyl-propane-l,2,3-triol (3) at the pro-(S) hydroxyl group (i.e. a desymmetrizing phosphorylation). 1H NMR (D2O, 500 MHz) d 3.89 (m, 2H), 3.72 (d, 7= 11.6 Hz, 1 H), 3.65 (d, J= 11.6 Hz, 1H), 2.93 (s, 1H). 13C NMR (D20, 126 MHz) d 82.9 (s), 75.1 (s), 71.0 (d, J= 6.9 Hz), 67.0 (d, J= 4.5 Hz), 64.7 (s).
Method G4: Acetate kinase: ATP-regeneration system using immobilized enzymes SEQ. ID No. : 20 and enzyme SEQ. ID No. : 21
Panthotenate kinase PanK
– – ATP
Acetate kinase
Acetate phosphate
Enzyme immobilization procedure:
Nuvia IMAC Ni-charged resin (75 mL based on settled volume) was added to a filter funnel and washed with water (9 column volumes, 3 x 225 mL) and binding buffer (1 column volume, 75mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0). In a vessel pantothenate kinase (SEQ ID NO. : 20, 6.0 g) lyophilized powder was resuspended in binding buffer (200 mL) and the washed resin was added. The solution was mixed using rotating mixer at 25 °C for 6h. The resin was filtered and washed with binding buffer (6 column volumes, 6 x 225 mL) and BIS-TRIS buffer (8 column volumes, 600 mL; 50 mM, pH 6.2).
Reaction procedure:
An aqueous solution of 2-ethynyl-propane-l,2,3-triol (3) (574 g, 8.7% wt, 0.430 mol) and water (350 mL) was charged to a jacketed reactor, followed by 1M BIS-TRIS methane buffer pH 6.5 (50 mL) and magnesium chloride (2.033 g, 0.01 mol). ATP (2.37g, 0.0043 mol, 0.01 equivalent) and diammonium acetyl phosphate (101 g, 89%, 0.530 mmol, 1.2 eq) were added, the solution was warmed up to 20 °C and the pH of the solution was re-adjusted to 6.8 using 5 M KOH.
Resin with immobilized pantothenate kinase SEQ. ID No. : 20 (25 mL) and evolved acetate kinase SEQ. ID No. : 21 (0.15 g) were then charged as solids. The reaction was stirred for at 20 °C for l6h during which the pH dropped to 5.5. Quantitative conversion of 2-ethynyl-propane- I,2,3-triol (3) to (ri)-2-ethynyl-propane-l,2,3-triol l-phosphate (9) was obtained as judged by ¾ and 31P NMR. ¾ NMR (D20, 500 MHz) d 3.89 (m, 2H), 3.72 (d, J= 11.6 Hz, 1 H), 3.65 (d, J =
I I .6 Hz, 1H), 2.93 (s, 1H).
Alternate Preparations of (i?V2-ethvnyl-glvceraldehvde 3-phosphate 15):
Method HI: Immobilized galactose oxidases SEP ID No.: 16
Enzyme immobilization procedure:
Nuvia IMAC Ni-charged resin (10 mL based on settled volume) was added to a filter funnel and washed with binding buffer (10 column volumes, 100 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0) to remove the resin storage solution and give 16 g of washed resin. In a vessel evolved galactose oxidase (SEQ ID NO.: 16, 750 mg) lyophilized powders were resuspended in copper (II) sulphate solution (100 mM; 5.00 mL), followed by addition of binding buffer (20 mL) and the washed resin (3.0g). The solution was mixed using rotating mixer at 20 °C for 5h. The resin was filtered and washed with binding buffer (10 column volumes, 100 mL) and BIS-TRIS buffer (10 column volumes, 100 mL; 50 mM, pH 7.5) and it was used directly in the glycosylation reaction.
Reaction procedure:
The resin with immobilized galactose oxidase SEQ ID NO.: 16 (3.0 g) was added to a solution of S)-2-ethynyl-propane-l,2,3-triol l-phosphate (9, 5.4 mmol, 270 mM, 20 mL) in BIS-TRIS methane buffer (35 mM, pH adjusted to 7.2), followed by addition of copper (II) sulphate solution in water (30 pL, 100 mM) and horseradish peroxidase (PEO-301, 18 mg) and bovine catalase (C1345, 120 mg) resuspended in water (600 pL). The reaction was sealed with gas permeable membrane and shaken vigorously at 22 °C for 4 days to reach final conversion of 77% and give (f?)-2-ethynyl-glyceraldehyde 3 -phosphate (5) in 95% e.e. The enzyme resin was filtered off and the solution of the(f?)-2-ethynyl-glyceraldehyde 3-phosphate (5) was used
directly in the glycosylation reaction. iH NMR (D2O, 400 MHz): d 5.02 (s, 1H), 4.00 (dq, 2 H), 2.88 (s, 1H). LC-MS: (ES, m/z): calculated for C5H7O6P (M-H): 193.1; found 193.0.
Method H2: Immobilized galactose oxidases SEP ID No.: 17
Enzyme immobilization procedure:
Nuvia IMAC Ni-charged resin (10 mL based on settled volume) was added to a filter funnel and washed with binding buffer (10 column volumes, 100 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0) to remove the resin storage solution and give l6g of washed resin. In a vessel, evolved galactose oxidase (SEQ ID NO.: 16, 750 mg) lyophilized powders were resuspended in copper (II) sulphate solution (100 mM; 5.00 mL), followed by addition of binding buffer (20 mL) and the washed resin (3.0g). The solution was mixed using rotating mixer at 20 °C for 5h. The resin was filtered and washed with binding buffer (10 column volumes, 100 mL) and BIS-TRIS methane buffer (10 column volumes, 100 mL; 50 mM, pH 7.5) and it was used directly in the reaction.
Reaction procedure:
The resin with immobilized evolved galactose oxidase SEQ ID NO.: 17 (3.0 g) was added to a solution of (ri)-2-ethynyl-propane-l,2,3-triol l-phosphate (9, 5.4 mmol, 270 mM, 20 mL) in BIS-TRIS methane buffer (35 mM, pH adjusted to 7.2), followed by addition of copper (II) sulphate solution in water (30 pL, 100 mM) and horseradish peroxidase (PEO-301, 18 mg) and bovine catalase (C1345, 120 mg) resuspended in water (600 pL). The reaction was sealed with gas permeable membrane and shaken vigorously at 22 °C for 4 days to reach final conversion of 77% and give (i?)-2-ethynyl-glyceraldehyde 3-phosphate (5) in 95% e.e. The enzyme resin was filtered off and the solution of the (i?)-2-ethynyl-glyceraldehyde 3 -phosphate (5) was used directly in the glycosylation reaction. lH NMR (D2O, 400 MHz): d 5.02 (s, 1H), 4.00 (dq, 2 H), 2.88 (s, 1H). LC-MS: (ES, m/z): calculated for C5H7O6P (M-H): 193.1; found 193.0.
Method H3: Immobilized galactose oxidases SEQ ID No.: 18
Enzyme immobilization procedure:
Nuvia IMAC Ni-charged resin (3 mL based on settled volume) was added to a filter funnel and washed with binding buffer (10 column volumes, 30 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0) to remove the resin storage solution and give 2.4 g of washed resin. In a vial evolved galactose oxidase (SEQ ID NO.: 18, 75mg) lyophilized powders were resuspended in copper (II) sulphate solution (100 mM; 1.00 mL), followed by addition of binding buffer (5 mL) and the washed resin (400 mg). The solution was mixed using rotating mixer at 20 °C for 5h. The resin was filtered and washed with binding buffer (10 column volumes, 4 mL) and BIS-TRIS methane buffer (10 column volumes, 4 mL; 50 mM, pH 7.5) and it was used directly in a reaction.
Reaction procedure:
Immobilized evolved GOase SEQ ID NO.: 18 was added (400 mg) to a solution of (5)-2-ethynyl-propane-l,2,3-triol l-phosphate solution ((9), 5.4 mmol, 270 mM, 1 mL) in BIS-TRIS methane buffer (35 mM, pH adjusted to 7.2), , followed by addition of horseradish peroxidase (PEO-301, 1 mg) and catalase from Corynebacterium glutamicum (Roche, lyophilizate, #11650645103, 3 mg) resuspended in water (100 pL). The reaction was sealed with gas permeable membrane and shaken vigorously at 30 °C for 48h. Final conversion after 2 days reached 90% conversion and the (i?)-2-ethynyl-glyceraldehyde 3-phosphate (5) >99% e.e. The enzyme resin was filtered off and the solution of the (i?)-2-ethynyl-glyceraldehyde 3-phosphate (5) was used directly without further purification. lH NMR (D2O, 400 MHz): d 5.02 (s, 1H),
4.00 (dq, 2 H), 2.88 (s, 1H). LC-MS: (ES, m/z): calculated for C5H7O6P (MΉ): 193.1; found 193.0.
Method H4: Immobilized galactose oxidases SEP ID No.: 19
Enzyme immobilization procedure:
Nuvia IMAC Ni-charged resin (3 mL based on settled volume) was added to a filter funnel and washed with binding buffer (10 column volumes, 30 mL; 500 mM sodium chloride, 50 mM sodium phosphate, 15 mM imidazole, pH 8.0) to remove the resin storage solution and give 2.4 g of washed resin. In a vial evolved galactose oxidase (SEQ ID NO.: 19, 75mg) lyophilized powders were resuspended in copper (II) sulphate solution (100 mM; 1.00 mL), followed by addition of binding buffer (5 mL) and the washed resin (400 mg). The solution was mixed using rotating mixer at 20 °C for 5h. The resin was filtered and washed with binding buffer (10 column volumes, 4 mL) and BIS-TRIS methane buffer (10 column volumes, 4 mL; 50 mM, pH 7.5) and it was used directly in a reaction.
Reaction procedure:
Immobilized evolved GOase SEQ ID NO.: 18 was added (400 mg) to a solution of (5)-2-ethynyl-propane-l,2,3-triol l-phosphate solution (9, 5.4 mmol, 270 mM, 1 mL) in BIS-TRIS methane buffer (35 mM, pH adjusted to 7.2), , followed by addition of horseradish peroxidase (PEO-301, 1 mg) and catalase from Corynebacterium glutamicum (Roche, lyophilizate, #11650645103, 3 mg) resuspended in water (100 pL). The reaction was sealed with gas permeable membrane and shaken vigorously at 30 °C for 48h. Final conversion after 2 days reached 100% conversion and (i?)-2-ethynyl-glyceraldehyde 3 -phosphate (5) was obtained in >99% e.e. The enzyme resin was filtered off and the solution of the (i?)-2-ethynyl-glyceraldehyde 3-phosphate (5) was used directly without further purification. lH NMR (D2O, 400 MHz): d 5.02 (s, 1H), 4.00 (dq, 2 H), 2.88 (s, 1H). LC-MS: (ES, m/z): calculated for C5H7O6P (M-H): 193.1; found 193.0.
PATENT
CA 2502109
WO 2017053216
US 20200010834
US 20200010868
PAPER
Organic letters (2017), 19(4), 926-929.
Organic Letters (2017), 19(4), 926-929.
Journal of medicinal chemistry (2018), 61(20), 9218-9228.
Bioscience, Biotechnology, and Biochemistry (2020), 84(2), 217-227.
PAPER
Organic letters (2011), 13(19), 5264-6.
A concise enantioselective total synthesis of 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA), an extremely potent anti-HIV agent, has been accomplished from (R)-glyceraldehyde acetonide in 18% overall yield by a 12-step sequence involving a highly diastereoselective ethynylation of an α-alkoxy ketone intermediate.
Processes for preparing islatravir and its analogs comprising the reaction of a substituted tetrahydrofuran compound with purine nucleoside phosphorylase and a nucleobase, followed by stereochemical synthesis, glycosylation, reduction, oxidation and isolation are claimed. Also claimed are novel intermediates of islatravir and processes for their preparation and their use for the preparation of islatravir.
(2R,3S,5R)-5-(6-Amino-2-fluoropurin-9-yl)-2-ethynyl-2-(hydroxymethyl)- tetrahydrofuran-3-ol (1). To a stirred solution of 16 (66.2 mg, 0.115 mmol) in MeOH/CH2Cl2 (2:1, 1.5 mL) was added NH4F (85.1 mg, 2.30 mmol) at room temperature. After 16 h, MeOH (0.5 mL) was added, and the resulting mixture was stirred for an additional 27 h. To the mixture was added 10% NaOH in MeOH (1.5 mL) to adjust the pH of the mixture to ca. 10. After 10 min, Dowex 50W×8 (200– 400 mesh (H)) was added until the pH of the mixture reached ca. 4. To the resulting mixture was added CaCO3 (259 mg, 2.59 mmol), and the mixture was stirred for 30 min. The mixture was filtered through a pad of Celite, and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (CHCl3/MeOH = 10:1) to give 29.3 mg (87%) of 1. Mp: 220.0–221.4 °C (dec.); [α] 25 D +12.4 (c 0.97, MeOH); IR: νmax 3315 (br m), 3179 (br m), 1690 (vs), 1356 (vs); 1 H NMR (600 MHz, DMSO-d6): δ 2.43 (1H, ddd, J = 13.2, 7.3, 7.3 Hz), 2.70 (1H, ddd, J = 13.2, 6.8, 5.1 Hz), 3.52 (1H, s), 3.54 (1H, dd, J = 11.7, 6.4 Hz), 3.65 (1H, dd, J = 11.7, 5.0 Hz), 4.57 (1H, m), 5.32 (1H, m), 5.60 (1H, m), 6.24 (1H, dd, J = 7.2, 5.1 Hz), 7.82 (1H, br s), 7.92 (1H, br s), 8.31 (1H, s); 13C NMR (150 MHz): δ 38.3, 64.4, 70.3, 79.2, 81.7, 82.2, 85.4, 117.6, 140.0, 150.4 (d, JCF = 20.7 Hz), 157.8 (d, JCF = 21.2 Hz), 158.8 (d, JCF = 203.4 Hz); HRMS (FAB): m/z calcd for C12H13FN5 O3, 294.1002; found, 294.1000 ([M+H]+ ).
https://pubs.acs.org/doi/suppl/10.1021/ol202116k/suppl_file/ol202116k_si_001.pdf
PAPER
Organic Letters (2011), 13(19), 5264-5266.
PAPER
Bioscience, biotechnology, and biochemistry (2012), 76(6), 1219-25.
https://www.tandfonline.com/doi/abs/10.1271/bbb.120134
EFdA (4′-ethynyl-2-fluoro-2′-deoxyadenosine), a nucleoside reverse transcriptase inhibitor with extremely potent anti-HIV activity, was concisely synthesized from (R)-glyceraldehyde acetonide in an 18% overall yield by a 12-step sequence involving highly diastereoselective ethynylation of an α-alkoxy ketone intermediate. The present synthesis is superior, both in overall yield and in the number of steps, to the previous one which required 18 steps from an expensive starting material and resulted in a modest overall yield of 2.5%.
PAPER
Bioscience, Biotechnology, and Biochemistry (2012), 76(6), 1219-1225.
Organic letters (2015), 17(4), 828-31.
Organic Letters (2015), 17(4), 828-831.
PAPER
https://cen.acs.org/pharmaceuticals/process-chemistry/Scientists-made-HIV-drug-using/97/web/2019/12
Some pharmaceutical companies are investigating biocatalysis at different points in their drug development pipelines, but mostly at one or two steps into the making of a small molecule. Scientists at Merck & Co. have taken this further—they are reporting an entire drug synthesis using a chain of nine enzymes, five of which had been engineered, to produce an experimental HIV drug at high yield in just a few steps (Science 2019, DOI: 10.1126/science.aay8484).
This biocatalytic cascade is turning heads. For the most part, scientists aren’t using biocatalysis to manufacture a compound so much as to develop it, says Princeton University chemist Todd Hyster. The Merck process stitches together nine enzymes to get good yields of the final product, which Hyster says is no small feat.
It literally took my breath away.
Alison Narayan, assistant professor, University of Michigan
“I was blown away,” Hyster says of the first time he saw Merck scientists talk about this work. “It’s something that was very complicated.”
Mark Huffman, a chemist who led the work at Merck with Anna Fryszkowska, says they turned to biocatalysis in order to overcome a couple of key hurdles in synthesizing some molecules. One is stereochemistry. Islatravir is a nucleoside that blocks the HIV enzyme reverse transciptase and traditionally, in medicinal chemistry, it’s been hard to get the stereochemistry of nucleosides right, Huffman says. But this is something enzymes are designed by nature to do. The other is preventing unwanted side reactions. A number of steps in the traditional chemical synthesis of islatravir put the compound’s functional groups at risk of being lopped off, so they must be protected. Huffman says enzymes are specific in the types of reactions they catalyze, so there’s little to no risk of an unwanted side reaction.
Scientists at Merck & Co. use nine enzymes, five of which are engineered, to biocatalytically make an HIV drug called islatravir without having to purify intermediates. Enzymes not engineered by Merck are not pictured.
On top of that, Huffman says, they are doing these reactions at neutral pH, in aqueous solvents, and at room temperature, which cuts down on electricity and the need for multiple bioreactors running under different conditions. Islatravir normally takes between 12 and 18 steps to make. With biocatalysis, the team has cut this down to three.
“You don’t have rigorous equipment requirements,” he says. “You’re usually running [these reactions] under much milder conditions.”
To run the cascade, the team started with 2-ethynylglycerol, and added a mixture of three enzymes to run one group of reactions. They then added more enzymes to drive a second set of reactions. Then, they remove the enzymes from the solution, which are immobilized and easy to filter out, and use four more enzymes to drive the final reactions that lead to islatravir. There are no intermediate purification steps. The overall yield is about 51% using biocatalysis, compared to yields of 7% and 15% using two more traditional syntheses.
To make their biocatalysts, the team surveyed natural enzymes, mostly from microbes, that interacted with the different intermediates in islatravir production. One of the reasons why Huffman says islatravir is an ideal small molecule to produce using biocatalysis is that most organisms have to make and break down nucleosides, so there are several natural enzymes found across multiple species. This gave the team a lot of starting material from which to alter amino acids and build the enzymes they needed to do their syntheses. By making adjustments to active sites and other areas of the enzymes, the team built five of the nine enzymes needed to make islatravir biochemically.
Huffman says that while islatravir is a good molecule to show that scientists can build large biocatalytic cascades, Merck is also looking at biocatalysis to make other small molecules and biologic drugs.
Alison Narayan, a biocatalysis chemist at the University of Michigan, calls Merck “bold” for putting the time, money, and people behind this change in production—it takes a lot of resources to try an entire synthesis via biocatalysis. And, she says, they’ve succeeded spectacularly. “It literally took my breath away,” Narayan says of her first exposure to this project in 2018. “I think it’s a huge accomplishment.”
She says that Merck’s islatravir work shows that industry is starting to appreciate what biocatalysis can do for their drug pipelines and their financial bottom lines. Alongside Merck, companies like GlaxoSmithKline and Pfizer are also exploring biocatalysis at different points in drug development and manufacturing.
“It’s an important proof of concept,” Narayan says. “This is a practical way to build molecules, and this will be the way that people will build molecules when you take into consideration efficiency, green-ness, and constructing an effective synthesis. Biocatalysis has a lot to offer.”
PAPER
Biocatalytic cascades go viral
IN SECTION: PERSPECTIVE | BIOCATALYSIS
An investigational drug targeting the HIV virus is synthesized with nine enzymes
Natural biosynthesis assembles a vast array of complex natural products starting from a limited set of building blocks, under physiological conditions, and in the presence of numerous other biomolecules. Organisms rely on the extraordinary selectivity of enzymes and their ability to operate under similar reaction conditions, meaning that these catalysts are perfectly adapted to mediate cascade reactions. In these multistep processes, the product of one biocatalytic step becomes the substrate for the next transformation (Display footnote number:1-3). On page 1255 of this issue, Huffman et al. (Display footnote number:4) report the development of an impressive nine-enzyme biocatalytic cascade for the synthesis of the investigational drug islatravir for the treatment of human HIV.
This study represents a partnership between scientists from Merck and Codexis. These two companies have a history of successfully collaborating to develop biocatalysts for the synthesis of important pharmaceuticals. Almost a decade ago, they developed a chemoenzymatic route for the synthesis of the type 2 diabetes drug sitagliptin (Januvia), relying on a key enzyme-catalyzed transamination with a highly engineered (R)-selective transaminase (Display footnote number:5). The work was considered a landmark example of directed evolution and functioned to highlight the potential application of biocatalysis to revolutionize industrial chemical processes.
The cascade for synthesizing islatravir was inspired by the bacterial nucleoside salvage pathway, which recycles precious nucleosides by using three key enzymes: a purine nucleoside phosphorylase (PNP), a phosphopentomutase (PPM), and a deoxyribose-5-phosphate aldolase (DERA) (see the figure). However, to achieve the synthesis of the target molecule, Huffman et al. required the natural nucleoside degradative cascade to run in reverse. The reversible nature of enzymes is central to the design of this cascade and is one of the important features that sets biocatalysts apart from the majority of traditional chemical catalysts.
The success of the cascade developed by the team also relied on all three enzymes accepting non-natural substrates bearing a fully substituted carbon at the C-4 position of the 2-deoxyribose ring. The authors reconstructed the reverse nucleoside salvage pathway from a PNP and PPM found in Escherichia coli and a DERA from Shewanella halifaxensis. The native E. coli enzymes required engineering to improve their activity. The DERA displayed existing high activity and stereoselectivity for the formation of the desired sugar phosphate enantiomer, but it required engineering to improve its ability to operate at high substrate concentration.
One of the many advantages of performing biocatalytic cascade reactions is the effective displacement of unfavorable reaction equilibria that can be achieved through product removal. However, despite performing the PNP and PPM steps in tandem, the reaction proceeded with poor conversion, and the inorganic phosphate by-product inhibits the enzymes. An elegant solution to these issues was the inclusion of an auxiliary sucrose phosphorylase, along with its sugar substrate, which removed free phosphate and effectively displaced the reaction equilibrium toward product formation.
Having assembled enzymes for the three key steps in the cascade, Huffman et al. sought to develop a biocatalytic route for the synthesis of the DERA substrate 2-ethynylglyceraldehyde 3-phosphate. Extensive screening of a broad range of kinases resulted in the identification of pantothenate kinase (PanK) from E. coli, which displayed low levels of activity (∼1% conversion) toward the (R)-enantiomer of the target aldehyde. Despite the modest initial activity, directed evolution was successfully used to substantially improve the productivity and stability of this enzyme. Finally, after 12 rounds of evolution, the authors reversed the enantioselectivity and improved the activity, stability, and expression of a galactose oxidase variant for the desymmetrization of the starting substrate, 2-ethynylglycerol.
Opens in modal lightbox
Viewable Image – engineering a biocatalytic cascade
Image Caption
GRAPHIC: A. KITTERMAN/SCIENCE
Advancements in protein engineering, rapid gene sequencing, and the availability of low-cost DNA synthesis have made it possible to alter the properties of enzymes and fine-tune them for biocatalytic applications (Display footnote number:6-8). The work by Huffman et al. is a milestone in cascade design, largely because of the number of biocatalysts operating in tandem and the engineering feat required to optimize five of the nine enzymes involved in the synthesis. It also highlights how biosynthetic or degradative pathways can be a source of inspiration for the design of efficient biocatalytic cascades and how sequences can be reconstituted using enzymes recruited from multiple sources—in this case, of bacterial, fungal, plant, and mammalian origin. The diverse role that biocatalysts can play is also exemplified in this work, where five engineered enzymes are directly involved in the synthesis of the target molecule, and four additional enzymes function to recycle coenzyme, remove inhibitory by-products, and maintain the correct oxidation state of the copper cofactor.
Previous approaches reported for the synthesis of islatravir relied on multistep syntheses and require protecting group manipulations and intermediate purification steps (Display footnote number:9, 10). The incorporation of a key biocatalytic step or steps has the potential to revolutionize synthetic design strategies by making possible transformations that are not accessible using solely chemical approaches (Display footnote number:11, 12). The application of enzymes in industry and the development of chemoenzymatic routes to complex molecules is now well established. However, multistep syntheses exclusively comprising biocatalytic transformations are rare (Display footnote number:13), and this contribution sets a new standard for the synthesis of complex molecules with enzymatic cascades.
School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland. Email: elaine.oreilly@ucd.ie.
REFERENCES AND NOTES
1. S. P. France, L. J. Hepworth, N. J. Turner, S. L. Flitsch, ACS Catal. 7, 710 (2017).
2. S. Gandomkar, A. Żadło-Dobrowolska, W. Kroutil, ChemCatChem 11, 225 (2019).
3. P. Both et al., Angew. Chem. Int. Ed. 55, 1511 (2016).
4. M. A. Huffman et al., Science 366, 1255 (2019).
5. C. K. Savile et al., Science 329, 305 (2010).
6. F. H. Arnold, Angew. Chem. Int. Ed. 57, 4143 (2018).
7. C. Zeymer, D. Hilvert, Annu. Rev. Biochem. 87, 131 (2018).
8. C. A. Denard, H. Ren, H. Zhao, Curr. Opin. Chem. Biol. 25, 55 (2015).
9. M. McLaughlin et al., Org. Lett. 19, 926 (2017).
10. M. Kageyama, T. Nagasawa, M. Yoshida, H. Ohrui, S. Kuwahara, Org. Lett. 13, 5264 (2011).
11. N. J. Turner, E. O’Reilly, Nat. Chem. Biol. 9, 285 (2013).
12. M. Hönig, P. Sondermann, N. J. Turner, E. M. Carreira, Angew. Chem. Int. Ed. 56, 8942 (2017).
13. S. Wu et al., Nat. Commun. 7, 11917 (2016).
ACKNOWLEDGMENTS
J.R. acknowledges the School of Chemistry, University College Dublin, for support.
References
- ^ Kawamoto, A; Kodama, E; Sarafianos, SG; Sakagami, Y; Kohgo, S; Kitano, K; Ashida, N; Iwai, Y; Hayakawa, H; Nakata, H; Mitsuya, H; Arnold, E; Matsuoka, M (2008). “2′-deoxy-4′-C-ethynyl-2-halo-adenosines active against drug-resistant human immunodeficiency virus type 1 variants”. The International Journal of Biochemistry & Cell Biology. 40 (11): 2410–20. doi:10.1016/j.biocel.2008.04.007. PMID 18487070.
- ^ Roy M. Gulick (2018). “Investigational Antiretroviral Drugs: What is Coming Down the Pipeline”. Top Antivir Med. 25 (4): 127–132. PMC 5935216. PMID 29689540.
- ^ “Someday, an Arm Implant May Prevent H.I.V. Infection for a Year”. New York Times. July 23, 2019.
- ^ “Merck Presents Early Evidence on Extended Delivery of Investigational Anti-HIV-1 Agent Islatravir (MK-8591) via Subdermal Implant”(Press release). July 23, 2019.
- ^ Jump up to:a b Michailidis, Eleftherios; Huber, Andrew D.; Ryan, Emily M.; Ong, Yee T.; Leslie, Maxwell D.; Matzek, Kayla B.; Singh, Kamalendra; Marchand, Bruno; Hagedorn, Ariel N.; Kirby, Karen A.; Rohan, Lisa C.; Kodama, Eiichi N.; Mitsuya, Hiroaki; Parniak, Michael A.; Sarafianos, Stefan G. (2014). “4′-Ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) Inhibits HIV-1 Reverse Transcriptase with Multiple Mechanisms”. Journal of Biological Chemistry. 289 (35): 24533–48. doi:10.1074/jbc.M114.562694. PMC 4148878. PMID 24970894.
- ^ Grobler, Jay (February 22–25, 2016). Long-Acting Oral and Parenteral Dosing of MK-8591 for HIV Treatment or Prophylaxis. Boston, Massachusetts. Conference on Retroviruses and Opportunistic Infections. 98.
- ^ Stoddart, Cheryl A.; Galkina, Sofiya A.; Joshi, Pheroze; Kosikova, Galina; Moreno, Mary E.; Rivera, Jose M.; Sloan, Barbara; Reeve, Aaron B.; Sarafianos, Stefan G.; Murphey-Corb, Michael; Parniak, Michael A. (2015). “Oral Administration of the Nucleoside EFdA (4′-Ethynyl-2-Fluoro-2′-Deoxyadenosine) Provides Rapid Suppression of HIV Viremia in Humanized Mice and Favorable Pharmacokinetic Properties in Mice and the Rhesus Macaque”. Antimicrobial Agents and Chemotherapy. 59 (7): 4190–8. doi:10.1128/AAC.05036-14. PMC 4468726. PMID 25941222.
- ^ Bruno Marchand. “The Crystal Structure of EFdA‐Resistant HIV‐1 Reverse Transcriptase Reveals Structural Changes in the Polymerase Active Site” (PDF).
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| Names | |
|---|---|
| IUPAC name
2′-Deoxy-4′-ethynyl-2-fluoroadenosine
|
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| Other names
EFdA; MK-8591
|
|
| Identifiers | |
|
3D model (JSmol)
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| ChemSpider | |
|
PubChem CID
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|
| UNII | |
| Properties | |
| C12H12FN5O3 | |
| Molar mass | 293.258 g·mol−1 |
|
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
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////////////////Islatravir, MK-8591, EFdA, PHASE 2, HIV-1 , HIV-2,
C#CC1(C(CC(O1)N2C=NC3=C(N=C(N=C32)F)N)O)CO
LNS-8801 (SRR G-1)
LNS-8801 (SRR G-1)
CAS 925419-53-0
Chemokine receptor-like 2 agonist
C21 H18 Br N O3, 412.28
Ethanone, 1-[(3aS,4R,9bR)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-
1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one
- 1-[(3aS,4R,9bR)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]ethanone
- G 1
- G 1 (estrogen receptor agonist)
Linnaeus Therapeutics Inc
NOTE 1-((3aR,4S,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one (RSS G-1 or LNS8812)
Novel crystalline forms of an enantiomerically purified LNS-8801 (SRR G-1), or a derivative useful for treating cancer, hearing disorder, depression and myocardial infarction.
Linnaeus Therapeutics is developing LNS-8801, a G-protein coupled estrogen receptor (GPER) agonist, an oral capsule formulation for the treatment of cancer including melanoma, pancreatic ductal adenocarcinoma, non-small cell lung cancer, solid tumor, hematological cancer, advanced cancer and colon carcinoma.
In October 2019, a phase I trial was initiated in patients with locally advanced or metastatic cancer.
PATENT
WO-2020023391
Novel crystalline and amorphous forms of 1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one (here referred to as SRR-G-1 or LNS-8801) and 1-((3aS,4R,9bR)-4-(6-bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-1-one (RSS G-1 or LNS8812) and its derivative (having chiral purity of >90%; substantially free of its opposite enantiomer; designated as Form A-C) and their co-crystal (eg benzenesulfonic acid) and compositions and combinations comprising them are claimed. Also claimed is their use for treating cancers (disorder expresses GPER), depression, myocardial infarction, osteoporosis, rheumatoid arthritis, insomnia, inflammatory bowel disease, Crohn’s disease, thymic atrophy and hearing disorder and further type-2 diabetes. Further claimed are methods for reducing the likelihood of pregnancy after intercourse, increasing skin pigmentation and skin protection and preventing cancer, reoccurrence of cancer and inhibiting the progression of cancer
Embodiments of the present invention relate to an enantiomerically purified agonist of the G-protein coupled estrogen receptor (GPER), pharmaceutical compositions comprising an enantiomerically purified SRR G-l, or a derivative thereof, and methods of treating disease states and conditions in subjects in need thereof, and methods of treating disease states and conditions mediated through GPER receptors.
[0003] Estrogens mediate multiple complex physiological responses throughout the body. The responses are in turn mediated through the binding of estrogen to receptors. The classical receptors bind steroids, such as estrogen, and are soluble cytoplasmic/nuclear proteins that function as transcription factors. These receptors are known as estrogen receptor alpha and beta (two closely related proteins) that mediate transcriptional activity. GPER is a 7-transmembrane G protein-coupled receptor that also binds to estrogen with high affinity (Kd~6 nM) and mediates rapid cellular responses including cyclic adenosine monophosphate signaling, calcium mobilization and phosphatidylinositol 3,4,5 trisphosphate production.
[0004] Diseases whose development, progression, and or response to therapy, may be influenced by endogenous, and/or pharmacologic activation of GPER signaling include cancer (including the prevention of cancer, prevention of the reoccurrence of cancer, and the inhibition of the progression of cancer; and particularly melanoma, pancreatic, lymphomas, uveal melanoma, non-small cell lung cancer, breast, reproductive and other hormone-dependent cancers, leukemia, colon cancer, prostate, bladder cancer), reproductive (genito-urological) including endometritis, prostatitis, polycystic ovarian syndrome, bladder control, hormone-related disorders, hearing disorders, cardiovascular conditions including hot flashes and profuse sweating, hypertension, stroke, obesity, diabetes, osteoporosis, hematologic diseases, vascular diseases or conditions such as venous thrombosis, atherosclerosis, among numerous others and disorders of the central and peripheral nervous system, including depression, insomnia, anxiety, neuropathy, multiple sclerosis, neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease, as well as inflammatory bowel disease, Crohn’s disease, coeliac (celiac) disease and related disorders of the intestine.
Embodiments of the present invention encompass compounds comprising enantiomerically purified G-l and methods of use in the treatment of diseases. G-l is a racemic mixture of the enantiomers l-((3aS,4R,9bR)-4-(6-bromobenzo[if][l,3]dioxol-5-yl)-3a.4.5.9b-tetrahydro-3H-cyclopenta|c |quinolin-8-yl)ethan- 1 -one (henceforth referred to as “SRR G-l” or “LNS8801”) and l-((3aR,4S,9bS)-4-(6-bromobenzo[if][l,3]dioxol-5-yl)-3a.4.5.9b-tetrahydro-3H-cyclopenta|c |quinolin-8-yl)ethan- 1 -one (henceforth referred to as “RSS G-l” or“LNS8812”).
Enantiomerically purified G-l has been purified in favor of its l-((3aS,4R,9bR)-4-(6-bromobenzo[ri][l,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-l-one enantiomer over the corresponding l-((3aR,4S,9bS)-4-(6-bromobenzo[ri][l,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-l-one enantiomer. Unless specifically described, SRR Gl, or a derivative thereof includes, any physical form, including an amorphous form or any crystalline solid forms such as A, B, C or combinations thereof.
Experimental Section
Scheme 1
[0166] A synthesis of G-l is described in Org. Biomol Client., 2010,8, 2252-2259, which is hereby incorporated by reference, and depicted in Scheme 1. A catalytic amount of Sc(OTf)3 (0.492 g, 1.0 mmol) in anhydrous acetonitrile (2.0 cm3) was added to the mixture of 6-bromopiperonal (2.30 g, 10.0 mmol). / aminoacetophenone (1.30 g, 10.0 mmol) and cyclopentadiene (3.30 g, 50.0 mmol) in acetonitrile (25 cm3). The reaction mixture was stirred at ambient temperature (~23 °C) for 2.0 h. The volatiles were removed in vacuo. The residue was purified by preparative silica gel column chromatography using ethyl acetate-hexanes (10 : 90) to provide G-l (4.03 g, 98%, dr. = 94 : 6) as a white solid. The minor diastereomer was substantially removed by recrystallization to yield a racemic mixure of SRR G-l and RSS G-l.
Example 1: Isolation of the SRR G-l and RSS G-l Enantiomers
[0167] Starting with a highly purified sample of G-l, (±)l-(4-(6-bromobenzo[ri][l,3]dioxol-5-yl)-(3aS*,4R*,9bR*)-tetrahydro-3H-cyclopenta[c]quinolin-8-yl)ethan-l-one, (99.4% purity) purchased from Tocris Bioscience, the material was dissolved in 90: l0:0. l(v/v/v) methyl tert-butyl ether / ethanol / diethyl amine and subjected to preparative chromatography using a column packed with Chrialpak 1A resin. Elution was conducted with 90: l0:0. l(v/v/v) methyl tert-butyl ether / ethanol / diethyl amine and the fractions corresponding to each enantiomer were collected and concentered to a solid. The early eluting enantiomer was determined to be the SRR G-l enantiomer by single crystal x-ray structural analysis.
Example 2: SRR G-l Polymorph Screen
[0168] Starting with SRR G-l prepared according to Example 1, a polymorph screening study was conducted analyzing the solids isolated from slurry of the solid, or from fast and slow evaporation and cooling of solutions (Table 1). Two crystal forms were identified, an anhydrous form designated Form A and mono dichloromethane solvate designated Form B. On exposure to elevated temperature the Form B crystal form desolvates to form the Form C crystal form. Amorphous material was generated from purified SRR G-l by two different methods; quick evaporating a diethyl ether solution of SRR G-l or rotary evaporating from a solution of a dichloromethane solution of SRR G-l.
Table 1
PAPER
Anti-Cancer Agents in Medicinal Chemistry (2019), 19(6), 760-771.
/////////RSS G-1, LNS8812, phase I, Locally advanced, metastatic cancer, LNS-8801, SRR G-1, Linnaeus Therapeutics
CC(=O)c1cc4c(cc1)N[C@@H](c2cc3OCOc3cc2Br)[C@H]5CC=C[C@@H]45
CC-90010
CC-90010
C21 H21 N O4 S, 383.46
CAS 1706738-98-8
1(2H)-Isoquinolinone, 4-[2-(cyclopropylmethoxy)-5-(methylsulfonyl)phenyl]-2-methyl-
- 4-[2-(Cyclopropylmethoxy)-5-(methylsulfonyl)phenyl]-2-methyl-1(2H)-isoquinolinone
- 4-[2-(Cyclopropylmethoxy)-5-(methanesulfonyl)phenyl]-2-methylisoquinolin-1(2H)-one
- 4-[2-(Cyclopropylmethoxy)-5-methylsulfonylphenyl]-2-methylisoquinolin-1-one
Quanticel Pharmaceuticals Inc, Michael John BennettJuan Manuel BetancortAmogh BoloorStephen W. KaldorJeffrey Alan StaffordJames Marvin Veal
Celgene (now a wholly owned subsidiary of Bristol-Myers Squibb ) , following its acquisition of Quanticel , is developing CC-90010, an oral inhibitor of BET (bromodomain and extraterminal) proteins, for the potential treatment of solid tumors and non-Hodgkin’s lymphoma. In August 2019, a phase I trial for diffuse astrocytoma, grade III anaplastic astrocytoma and recurrent glioblastoma was planned
PATENT
WO2018075796 claiming solid composition comprising a bromodomain inhibitor, preferably 4-[2-(cyclopropylmethoxy)-5-methylsulfonylphenyl]-2-methylisoquinolin-1-one in crystalline form A.
PATENT
WO2015058160 (compound 89, page 103).
Example 89: 4-[2-(cyclopropylmethoxy)-5-methylsulfonylphenyl]-2-methylisoquinolin-l-one
Step 1 : 2-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)isoquinolin-l-one
[00344] A suspension of 4-bromo-2-methylisoquinolin-l-one (100 mg, 0.42 mmol), bis(pinacolato)diboron (214 mg, 0.84 mmol), Pd(dppf)Cl2 (31 mg, 0.04 mmol) and potassium acetate (104 mg, 1.05 mmol) in dioxane (2 mL) under nitrogen was warmed up to 90 °C for 135 minutes. It was then cooled down to room temperature and diluted with ethyl acetate (8 mL). The mixture was washed with aqueous saturated solution of NaHC03 (8 mL) and brine (8 mL). The organic phase was separated, dried over Na2S04, filtered and concentrated under reduced pressure. The residue was purifed by normal phase column chromatography (10-90% EtOAc/Hexanes) to give the title compound (44 mg, 37%). 1H NMR (CDC13, 400 MHz) δ 8.43 (d, J = 7.9 Hz, 1 H), 8.40 (dd, J = 8.2 Hz, 0.9 Hz, 1 H), 7.68 (s, 1 H), 7.65 (ddd, J = 8.2, 8.2, 1.1 Hz, 1 H), 7.46 (t, J = 7.5 Hz, 1 H), 3.63 (s, 3H), 1.38 (s, 12H). LCMS (M+H)+ 286. Step 2: 4-[2-(cyclopropylmethox -5-methylsulfonylphenyl]-2-methylisoquinolin-l-one
[00345] The title compound was prepared in a manner similar to Example 18, step 3, substituting 2-bromo-l-(cyclopropylmethoxy)-4-methylsulfonylbenzene for 4-bromo-2-methylisoquinolin-l(2H)-one and 2-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)isoquinolin-l-one for N-benzyl-2-methoxy-5-(tetramethyl-l,3,2-dioxaborolan-2-yl)benzamide. 1H NMR (DMSO-d6, 400 MHz) δ 0.09 (m, 2 H), 0.29 (m, 1H), 0.35 (m, 1H),
0.94 (m, 1H), 3.22 (s, 3H), 3.57 (s, 3H), 3.95 (m, 2H), 7.16 (d, J = 7.9 Hz, 1H), 7.37 (d, J =
8.8 Hz, 1H), 7.53 (m, 2H), 7.65 (t, J = 7.6 Hz, 1H), 7.81 (d, J = 2.4 Hz, 1H), 7.97 (dd, J = 8.8,
2.4 Hz, 1H), 8.30 (d, J = 8.1 Hz, 1H). LCMS (M+H)+ 384.
[00346] Alternatively, 4-[2-(cyclopropylmethoxy)-5-methylsulfonylphenyl]-2-methylisoquinolin-l-one can be prepared as described below.
Step 1 : 2-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)isoquinolin-l-one
[00347] A mixture of 4-bromo-2-methylisoquinolin-l-one (8.0 g, 33.6 mmol),
bis(pinacolato)diboron (17.1 g, 67.2 mmol), KOAc (6.6 g, 67.2 mmol), Pd2(dba)3 (3.1 g, 3.36 mmol) and X-Phos (1.6 g, 3.36 mmol) in anhydrous dioxane (200 mL) was stirred at 60 °C for 12 h. The reaction mixture was concentrated and the residue was purified by column chromatography on silica gel (PE : EA = 15 : 1) to give the title compound (6.0 g, 62 %) as a solid.
Step 2: 4-[2-(cyclopropylmethoxy)-5-methylsulfonylphenyl]-2-methylisoquinolin-l-one
[00348] The title compound from Step 1 (5.0 g, 17.5 mmol), 2-bromo-l-(cyclopropylmethoxy)-4-methylsulfonylbenzene (6.4 g, 21 mmol), K3PO4 (9.3 g, 43.9 mmol) and Pd(dppf)Cl2 (1.4 g, 1.75 mmol) in a dioxane/water (100 mL / 10 mL) mixture were stirred at 60 °C for 12 hrs. The reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (EA : DCM = 1 : 4).
Appropriate fractions were combined and concentrated under reduce pressure. The resultant solid was recrystallized from DCM / MTBE (1 : 1, 50 mL) to give the title compound (4.0 g, 60 %) as a white solid. 1H NMR: (CDC13, 400 MHz) δ 8.51 (dd, Ji = 8.0 Hz, J2 = 0.8 Hz, 1 H), 7.98 (dd, Ji = 8.4 Hz, J2 = 2.4 Hz, 1 H), 7.86 (d, J = 2.4 Hz, 1 H), 7.53 (m, 2 H), 7.16 (d, J = 7.6 Hz, 1 H), 7.10 (m, 2 H), 3.88 (m, 2 H), 3.66 (s, 3 H), 3.09 (s, 3 H), 1.02-0.98 (m, 1 H), 0.44-0.38 (m, 2 H), 0.11-0.09 (m, 2 H). LCMS: 384.1 (M+H)+
Patent
WO-2020023438
A process for preparing bromodomain inhibitor, particularly 4-[2(cyclopropylmethoxy)-5-methylsulfonylphenyl]-2-methylisoquinolin-1-one (having HPLC purity of 99%; compound 1; (hereafter referred to as C-90010)) and its hydrates, solvates, prodrugs and salts comprising the reaction of a substituted 4-(methylsulfonyl)phenol compound with a quinoline derivative, followed by purification is claimed. Also claimed are novel intermediates of CC-90010 and their processes for preparation. Further claimed are novel crystalline form of CC-90010. CC-90010 is known and disclosed to be a bromodomain containing protein inhibitor, useful for treating cancer.
Scheme 10: Synthesis of Compound 1
[0090] Acetonitrile (1.6L) was charged to a mixture of Compound 2 (156.7g, 460 mmol), Compound 3 (lOOg, 420 mmol) and potassium phosphate tribasic (223g, l.OSmol). Agitation
was begun and water (400mL) charged to the batch. The system was vacuum purged three times with nitrogen and charged with Pd(PPh3)2Cl2 (2.9g, 4 mmol) and the system vacuum purged three times with nitrogen. The batch was heated to about 65 to about 75 °C (or any temperature in between and including these two values) and contents stirred for at least about 16 hours until reaction was complete by HPLC analysis. The batch was cooled to about 60 to about 70 °C (or any temperature in between and including these two values), agitation halted and the mixture allowed to settle. The bottom aqueous layer was removed. Water (150mL) and acetonitrile (700mL) were charged at about 60 to about 70°C (or any temperature in between and including these two values). Ecosorb C-941 (15g) and Celite (lOg) were charged to the reaction vessel at about 60 to about 70°C (or any temperature in between and including these two values). After lh, the mixture was filtered to remove solids. The solids were washed twice each with 18% water in acetonitrile (500 mL) at about 60 to about 70°C (or any temperature in between and including these two values). The filtrates were combined and concentrated under atmospheric pressure to a final volume of 1.5L. The batch was cooled to about 60 to about 65°C (or any temperature in between and including these two values) and seeded with Compound 1 (1 g). After lh, water (500 mL) was charged over at least 1 hour at about 60 to about 65°C (or any temperature in between and including these two values). The slurry was cooled to about 15 to about 25°C (or any temperature in between and including these two values) over 4 hours. The product was collected by suction filtration. The wet cake was washed with 45% water in acetonitrile (500mL) twice. The product was dried under vacuum at about 40°C with nitrogen purge. Yield: 139g of 1.
[0091] The above procedure for coupling Compound 3 and Compound 2 to produce
Compound 1 may be modified in any of the ways that follow. Reaction solvents: Different reaction solvents from acetonitrile can be used, including tetrahydrofuran, 2-methyl tetrahydrofuran, toluene, and isopropanol. Boronic ester: Different boronic esters from Compound 2 can be used, including pinacolato ester compound 7, and the free boronic acid of Compound 2. Examples of boronic esters can be found in Lennox et al., Chem. Soc. Rev., 43: 412 (2014). Carbon treatment: Different carbon treatments from Ecosorb C-941 could be used. Different amounts of carbon, from 0.01 to 0.5X weight can be used. The carbon can be eliminated. Different amounts of Celite, from 0.01 to 0.5X weight can be used.
Crystallization: Different amounts of water, including 5 volumes to 50 volumes can be used.
The crystallization can also proceed without the addition of seeds. Different water addition times and final hold times can be used. Different wash procedures can be used. Drying: A temperature range of 10 to 60 °C could be used for drying. Catalysts: Different metal and ligand combination could be used. Examples of metal/ligand combinations can be found in Maluenda, Irene; Navarro, Oscar, Molecules, 2015, 20, 7528. Various catalysts can be including: XPhos-3G (cas# 1445085-55-1); cataCXium® A Pd 3G (CAS# 1651823-59-4); PdCk(DtBPF) (CAS# 95408-45-0); SPhos 3G (Cas# 1445085-82-4); AmPhos 3G (Cas# 1820817-64-8); PCy3 3G (Cas# 1445086-12-3); Pd PEPPSI IPent Cas#l 158652-41-5);
Pd(PPh3)2Cb (Cas# 13965-03-2). Examples of catalyst systems that have been demonstrated to afford Compound 1 are listed below in Table 4 using boronic esters 2 or 7 in coupling to 3.
Table 4: Catalyst screen summary
VI. Purification of Compound 1 fCC-900101 bv crystallization from formic acid and water
[0092] Described herein are methods of purifying Compound 1 by crystallization from formic acid and water. Also described are methods for obtaining three different polymorphs of Compound 1, including the most stable form, Form 1 and two metastable forms, Form 4
The crystallization can also proceed without the addition of seeds. Different water addition times and final hold times can be used. Different wash procedures can be used. Drying: A temperature range of 10 to 60 °C could be used for drying. Catalysts: Different metal and ligand combination could be used. Examples of metal/ligand combinations can be found in Maluenda, Irene; Navarro, Oscar, Molecules, 2015, 20, 7528. Various catalysts can be including: XPhos-3G (cas# 1445085-55-1); cataCXium® A Pd 3G (CAS# 1651823-59-4); PdCh(DtBPF) (CAS# 95408-45-0); SPhos 3G (Cas# 1445085-82-4); AmPhos 3G (Cas# 1820817-64-8); PCy3 3G (Cas# 1445086-12-3); Pd PEPPSI IPent Cas#l 158652-41-5);
Pd(PPh3)2Cl2 (Cas# 13965-03-2). Examples of catalyst systems that have been demonstrated to afford Compound 1 are listed below in Table 4 using boronic esters 2 or 7 in coupling to 3.
Table 4: Catalyst screen summary
VI. Purification of Compound 1 (CC-90010! bv crystallization from formic acid and water
[0092] Described herein are methods of purifying Compound 1 by crystallization from formic acid and water. Also described are methods for obtaining three different polymorphs of Compound 1, including the most stable form, Form 1 and two metastable forms, Form 4
33 -a
and Form 5. Supporting data (XRPD, DSC, photomicroscopy) for all three forms is provided in the examples below.
[0093] The stmcture of Compound 1 (CC-90010) is shown below:
Example 1: Synthesis of Compound 1
[0217] Synthesis of compound 1 was accomplished according to Scheme 1 below. Referring to Scheme 1, synthesis commenced with bromination of starting material 4-(methylsulfonyl)phenol 4, to produce compound 5. Compound 5 was O-alkylated with (bromomethyl)cyclopropane to produce compound 6. Boronate Compound 2 was then formed by borylation of Compound 6 with Pd catalyst and bis(pinacolato)diboron to produce transient Compound 7, which was subsequenctly treated with diethanolamine (DBA) to afford cross-coupling partner Compound 2. Cross-coupling partner Compound 3 was formed in one pot starting from commercially available Compound 8. Compound 8 was N-methylated and brominated to afford Compound 3. Compounds 2 and 3 were cross-coupled (Norio, M. and Suzuki, A., Chem. Rev., 95(7), 2457-2483 (1995)) to afford the target compound 1.
Scheme 1: Synthesis of compound 1
1.1: Bromination of 4
[0218] The bromination of Compound 4 to produce Compound 5 itself is simple, however stopping at the mono-brominated Compound 5 was challenging. The bis-brominated Compound 5-a (see Scheme 2 below) is a particularly pernicious impurity as it couples downstream to form a di ffi cult-to-purge impurity.
Scheme 2: Bromination of Compound 4
[0219] The key to high purity with reasonable yield was to exploit the solubility differences of the starting material Compound 4 (46 mg/ml at 20 °C) and the product Compound 5 (8 mg/ml) in CH2CI2. These solubility differences are summarized in Table 3 below.
[0220] This solubility difference is exploited by performing the reaction at a high
concentration to drive Compound 5 out of solution once formed, thereby minimizing its ability to react further with the brominating reagent to form Compound 5-a diBr. The reaction is seeded with Compound 5 to initiate its crystallization.
[0221] In Fig. 22 (Conversion of Compound 4 to Compound 5: Effect of Sulfuric Acid) it can be seen that in the absence of acid the initial reaction to Compound 5 is rapid, however the conversion plateaus at about 30% Compound 5. The main side product was found to be the impurity Compound 5-a diBr (see Fig. 23: Conversion of Compound 5 and Compound 5-a diBr: No H2SO4). Addition of increasing amounts of sulfuric acid leads to a higher conversion to desired Compound 5.
[0222] Fig. 24 (Compound 4 to Compound 5 Reaction Profile: Portion-wise Addition of NBS, Seeding) depicts further reaction control. The portion-wise addition ofNBS after addition of catalytic sulfuric acid minimizes the temperature rise, and the addition of Compound 5 after an initial NBS charge promotes the reactive crystallization of Compound 5. After about 6 to 7 hours of reaction it can be seen that the major product is Compound 5, with only a small (<5%) of the di-brominated impurity formed. In contrast, in a reaction where Compound 4 and all of the NBS were charged followed by the addition of 4 volumes of methylene chloride, a rapid exotherm resulted and undesired Compound 5-a diBr was found to be the major product.
[0223] Thus, the reaction was run under a high concentration in CH2CI2 with a portion-wise solid addition of NBS (to control both availability of the electrophile and the exotherm). An end of reaction slurry sample typically showed not more than 5% of the starting material Compound 4 remaining. After filtration the crude cake was washed with cold CH2CI2 and the OkCk-washed filter cake contained not more than 0.5% by weight dibrominated Compound 5-a. It also contained a large amount of HPLC-silent succinimide.
[0224] The following procedure was carried out: Compound 4 (25g, 145mmol) followed by CH2CI2 (lOOmL) were added to a reaction vessel and agitated. The batch was adjusted to 17 °C to 23 °C. Sulfuric acid was charged (2.7mL, Slmmol) to the batch maintaining 17 °C to 23°C. The batch was stirred at 17 °C to 23 °C for 10 minutes to 20 minutes. The first portion of A-bromosuccimide (NBS) was charged (6.5g, 36.5 mmol) to the batch at 17 °C to 23°C and stirred for at least 30 min. The second portion of NBS was charged (6.5g, 36.5 mmol) to the batch at 17 °C to 23°C and stirred for at least 30 min. The batch was seeded with
Compound 5 (0.02wt) and stirred for ca. 30 min at 17 °C to 23 °C to induce crystallization.
[0225] The third portion of NBS was charged (6.5g, 36.5 mmol) to the batch at 17 °C to 23 °C and stirred for at least 30 min. NBS (6.5g, 36.5 mmol) was charged to the batch at 17 °C to 23 °C and stirred for at least 30 min. Additional CH2CI2 was charged (50mL) to the batch while maintaining 17 °C to 23 °C to aid in agitation and transfer for filtration. The batch was stirred at 17 °C to 23 °C until complete by HPLC analysis (~20 – 40 h). The product was collected by suction filtration. The filter cake was slurry washed with CH2CI2 (3 x 50mL) at 17 °C to 23 °C (target 20 °C). The filter cake was slurry washed with purified water (3.0vol) at 65 °C to 75 °C for 2 to 3 hours. Then, the filter cake was slurry washed with purified water (3 x 1.0 vol, 3 x 1.0 wt) at 17 °C to 23°C. The wet cake was dried under vacuum with nitrogen bleed at 60 °C. Yield: 27g 5 (74% molar) >97% by weight. ¾ NMR (500 MHz, de-DMSO) 8.01 (1H, d, 4J = 2.1 Hz, RO-Ar meta- H ), 7.76 (1H, dd, J = 8.6 and 4J = 2.1 Hz, RO-Ar meta-H ), 7.14 (1H, d, J = 8.6 Hz, RO-Ar ortho- H), 3.38 (1H, br s, OH), 3.20 (3H, s,
CHJ); MS (ES-) calc. 249/251; found 249/251. Melting point (MP): (DSC) 188 °C.
[0226] The above procedure allowed for the following modifications. Solvents: Alternative solvents could be used. Examples include chlorinated solvents, such as chloroform or 1,2 dichloroethane, and non-chlorinated solvents such as acetonitrile, tetrahydrofuran, or 2- methyltetrahydrofuran. Reaction concentration: The reaction concentration can be varied from about 2X vol to about 20 X vol (with respect to Compound 4). Brominating agents: Additional brominating reagents include bromine and l,3-dibromo-5,5-dimethylhydantoin. Bromination reagent stoichiometry: Different amounts of the brominating reagent can be used, from about 0.8 equiv to about 1.9 equiv. Bromination reagent addition: The brominating reagent can be added all at once, portion wise in about 2 to about 20 portions, or continuously. The addition times can vary from about 0 to about 72 hours. Temperature: Reaction temperatures from about 0 °C to about 40 °C could be used. Acids: Different acids can be envisioned, including benzenesulfonic acid, para-toluenesulfonic acid, triflic acid, hydrobromic acid, and trifluoroacetic acid. Isolation: Instead of directly filtering the product and washing with methylene chloride and water, at the end of reaction an organic solvent capable of dissolving Compound 5 could be charged, followed by an aqueous workup to remove succinimide, and addition of an antisolvent or solvent exchange to an appropriate solvent to crystallize Compound 4. Drying: A temperature range of about 10 to about 60 °C could be used for drying.
[0227] An alternative process to Compound 5 has also been developed. This process is advantageous in that it does not use a chlorinated solvent, and provides additional controls over the formation of the Compound 5-a dibromo impurity. See Oberhauser, T. J Org. Chem 1997, 62, 4504-4506. The process is as follows. Compound 4 (10 g, 58 mmol) and acetonitrile (100 ml) were charged to the reactor and agitated. The batch was cooled to -20 °C. Triflic acid (CF3SO3H or TfOH, 5.5 mL, 62 mmol) was charged while maintaining a batch temperature of -10 to -25 °C. N-bromosuccinimide was charged (NBS, 11.4 g, 64 mmol), stirred at -10 to -25 °C for 30 minutes, then warmed to 20 °C over 3 to 4 hours. Agitation was continued at 15 °C to 25 °C until reaction completion. If the reaction conversion plateaued before completion, the reaction was cooled to -5 to -15 °C, and additional NBS was added, the amount based off of unreacted starting material, followed by warming to 15 °C to 25 °C and reacting until complete.
[0228] After reaction completion, the batch was warmed to 40 °C to 50 °C and concentrated under reduced pressure to 40 mL. The batch was cooled to -5 °C to -15 °C and the resulting product solids were filtered off. The solids were slurry washed three times, each with 20 mL water, for at least 15 minutes. The final cake was dried at 50 °C to 60 °C under reduced pressure to furnish 10 g of 5 containing less than 0.1% MeCN, 0.07% water, and 0.1% triflic acid (TfOH) by weight.
[0229] Alternatives to the above procedure employing MeCN and TfOH are as follows. Brominating agents: Additional brominating reagents include bromine and l,3-dibromo-5,5-dimethylhydantoin. Bromination Reagent Stoichiometry: Different amounts of the brominating reagent can be used, from about 0.8 equiv to about 2 equiv. Drying: A temperature range of about 10 °C to about 60 °C could be used for drying.
[0230] The impurity 5-a is was prepared and characterized as follows. 10 g of Compound 4 and sulfuric acid (35 mol%) were dissolved in MeOH (10 vol). The mixture was set to stir at 20 °C to 25 °C for 5-10 min and 2.0 equivalents of NBS were charged in one portion. The resulting yellow mixture was stirred for three days at 20-25 °C. The batch was concentrated under reduced pressure and the resulting solid was slurried in water at 95-100 °C for 3 hours. After a second overnight slurry in CH2CI2 at room temperature, the batch was filtered and dried to give a white solid 5-a (15.0 g, 78%). ¾ NMR (500 MHz, de-DMSO), 8.05 (2H, s, ArH), 3.40 (1H, br s, HO-Ar), 3.28 (3H, s, CH3); MS (ES‘) calc. 327/329/331; found
327/329/331; MP (DSC): 226 °C (onset 221 °C, 102 J/g); lit. 224-226 °C.
1.2: O-alkylation of 5 to produce 6
[0231] Compound 6 was prepared according to Scheme 7 below.
Scheme 7: O-alkylation of 5 to produce 6
[0232] Compound 5 (100 g, 398 mmol) and methyl ethyl ketone (MEK, 700 mL) were charged to the reaction vessel and agitated. Potassium carbonate (K2CO3, 325 mesh 82.56 g, 597 mmol) was then charged to the stirred reaction vessel at 15 °C to 25 °C.
Bromomethylcyclopropane (64.4 mL, 664 mmol) was charged to the reaction vessel over at least 1 hour, maintaining the temperature between 15 °C to 25 °C. MEK (200 mL) was added into the reactor and the reactor heated to 65 to 75 °C. The contents of the reaction vessel were stirred at 65 to 75°C for approximately 10 hours until reaction was complete by HPLC analysis. Water (3.0 vol, 3.0wt) was charged to the vessel maintaining the temperature at 65 to 75 °C. The batch was stirred at 65 to 75 °C. The phases were allowed to separate at 65°C to 75 °C and the lower aqueous phase was removed. Water (300 mL) was charged to the vessel maintaining the temperature at 65 °C to 75 °C. The batch was agitated for at least 10 minutes at 65 to 75 °C. The phases were allowed to separate at 65 °C to 75 °C and the lower aqueous phase was removed. The water wash was repeated once. The temperature was adjusted to 40 to 50°C. The mixture was concentrated to car. 500 mL under reduced pressure. The mixture was distilled under reduced pressure at up to 50 °C with MEK until the water content was <1.0% w/w. n-heptane (500mL) was charged to the vessel maintaining the temperature at 40 to 50 °C. The mixture was continuously distilled under vacuum with n-heptane (300mL), maintaining a 1L volume in the reaction vessel. Compound 6 seeds (0.0 lwt) were added at 40 to 50 °C. The mixture was continuously distilled under reduced pressure at up to 50 °C with n-heptane (300mL) while maintaining 1L volume in the reactor. The batch was cooled to 15 to 25 °C and aged for 2 hours. The product was collected by suction filtration. The filter cake was washed with a solution of 10% MEK in n-heptane (5vol) at 15 to 25°C. The filter cake was dried under reduced pressure at up to 40 °C under vacuum with nitrogen flow to afford 95g of 6. 1H NMR (500 MHz, de-DMSO) 8.07 (1H, d, 4J = 2.2 Hz, ArH), 7.86 (1H, d, J = 8.7 Hz, meta-ArH), 7.29 (1H, d, J = 8.8 Hz, ortho-AiK),
4.04 (2H, d, J = 6.9 Hz, OCH2CH), 3.21 (3H, s, CH3), 1.31-1.24 (1H, m, OCH), 0.62- 0.58 (2H, m, 2 x CHCHaHb), 0.40-0.37 (2H, m, 2 x CHC¾Hb); MS (ES+) calc. 305/307; found 305/307; MP: (DSC) 93 °C.
[0233] The following modifications of the above reaction, synthesis of 6 from 5, may be employed as well. Solvent: Different solvents could be used, for example acetone, methyl isobutyl ketone, ethyl acetate, isopropyl acetate, acetonitrile, or 2-methyl tetrahydrofuran. Reaction volume: Reaction volumes of 3 to 30 volumes with respect to 3 could be used. Base: Different inorganic bases, such as cesium carbonate or phosphate bases (sodium, potassium, or cesium) could be used. Also, organic bases, such as trimethylamine or diisopropyldiimide could be used. Base particle size: Different particle sizes of potassium carbonate from 325 mesh could be used. Reaction temperature: A lower temperature, such
as 50 °C could be used. A higher temperature, such as about 100 °C could be used. Any temperature above the boiling point of the solvent could be run in a pressure vessel.
Isolation: Different solvent ratios of MEK to n-heptane could be used. Different amounts of residual water can be left. Different amounts of seeds, from 0 to 50% could be used.
Seeding could take place later in the process and/or at a lower temperature. An un-seeded crystallization can be employed. A different isolation temperature, from 0 °C to 50 °C could be used. A different wash could be used, for example a different ratio of MEK to n-heptane. A different antisolvent from n-heptane could be used, such as hexane, pentane, or methyl tert-butyl ether. Alternatively, the batch could be solvent exchanged into a solvent where Compound 3 has a solubility of less than 100 mg/ml and isolated from this system. Drying: A temperature range of 10 to 60 °C could be used for drying.
[0234] Compound 10, shown below may also be formed as a result of O-alkylation of unreacted 4 present in product 5, or alternatively from or via a palladium mediated proteodesbromination or proteodesborylation in subsequent chemistry discussed in Example 1.3 below.
[0235] Preparation of methylsulfonylphenyl(cyclopropylmethyl) ether 10: Compound 4 (0.86 g, 5.0 mmol) and K2CO3 (1.04 g, 7.5 mmol) were slurried in acetone (17 mL, 20 vols). Cyclopropylmethyl bromide (0.73 mL, 7.5 mmol) was added in several small portions over ~1 minute and the reaction mixture heated to 50 °C for 48 hours, then cooled to 25 °C. Water (5.0 mL) was added with stirring and the acetone was evaporated on a rotary evaporator from which a fine white solid formed which was filtered off and returned to a vessel as a damp paste. A 1 : 1 mixture of MeOH/ water (8 mL) was added and heated to 40 °C with stirring. After 1 hour, the white solid was filtered off. Some residual solid was washed out with fresh water that was also rinsed through the cake, which was then isolated and left to air dry over the two days to give a dense white solid 10 (1.00 g, 88%). ¾ NMR (500 MHz, CDCb) 7.85
(2H, d, J = 8.8 Hz, RO-Ar ortho-H), 7.00 (2H, d, J = 8.8 Hz, RO-Ar meta- H), 3.87 (2H, d, J = 7.0 Hz, OCH2CH), 3.02 (3H, s, CHs), 1.34-1.23 (1H, m, OCH2CH), 0.72-0.60 (2H, m, 2 x CHCHflHb), 0.42-0.31 (2H, m, 2 x CHCH^.
1.3: Synthesis and Isolation Coupling Partner Boronic Ester 2
[0236] The final bond forming step to Compound 1 is a Suzuki-Miyaura coupling between Compounds 2 and 3, as shown in Scheme 3 below (Norio, M. and Suzuki, A., Chem. Rev., 95(7), 2457-2483 (1995)). Early studies demonstrated that the boronic ester of the isoquinolinone Compound 3-a had poor physical attributes and solid phase stability (Kaila, N. et al., J. Med Chem., 57: 1299-1322 (2014)). The pinacolatoboronate of the O-alkyl phenol, Compound 7, had acceptable solid phase stability and could be isolated via crystallization.
Scheme 3: Suzuki-Miyaura coupling between 2 and 3
[0237] Process robustness studies for the isolation of Compound 7, however, indicated that Compound 7 has poor solution stability, decomposing primarily to the proteodeborylated compound 10, as shown in Scheme 4 below. This was particularly problematic as the isolation process involved a solvent exchange from 2-MeTHF (2-methyl tetrahydrofuran) to iPrOAc (isopropyl acetate), which is not a fast unit operation on scale.
Scheme 4: Modification of 7
[0238] A search for a more stable boronic ester was undertaken. Early attempts targeted making N-methyliminodiacetic acid (MID A) boronate Compound 2-a (E. Gilis and M. Burke,“Multi step Synthesis of Complex Boronic Acids from Simple MIDA Boronates,” J Am. Chem. Soc., 750(43): 14084-14085 (2008)), however, all attempts resulted in product decomposition. Applicant then turned to a relatively obscure boronate formed by the addition of diethanolamine to Compound 7 (Bonin et al., Tetrahedron Lett., 52: 1132-1135 (2011)). Addition of diethanolamine to a solution of Compound 7 led to rapid ester formation and concomitant crystallization of Compound 2.
[0239] The discovery of boronic ester Compound 2 allowed for a simple, fast, high-yielding, high-purity process comprising the following procedure. Tetrahydrofuran (THF, 1500mL) was charged to a flask containing Compound 6 (100g, 328 mmol), bis(pinacolato)diboron (90.7g, 357 mmol) and cesium acetate (CsOAc, 158g, 822 mmol). The system was vacuum purged three times with nitrogen. Pd(PPh3)2Cl2 (13.8g, 20 mmol) was charged to the reaction and the system was vacuum purged three times with nitrogen. The reaction was then heated to 55 to 65°C.
[0240] The batch was stirred for approximately 8 hours until reaction was complete by HPLC analysis. The batch was cooled to 15 to 25 °C (target 20 °C ) and charged with silica gel (20g) and Ecosorb C-941 (20g). After lh, the mixture was filtered to remove solid. The residual solids were washed twice, each with THF (300mL). The filtrate and washes were combined. In a separate vessel, diethanolamine (34.5mL, 360 mmol) was dissolved in THF (250 mL). The diethanolamine solution in THF (25mL) was then charged to the batch. After 10 minutes, the batch was seeded with 2 (1 g) and aged for 1 to 2 hours. The remaining of the diethanolamine solution in THF was charged to the batch over at least 2 hours and the slurry was stirred for at least 2 hours. The product 2 was collected by suction filtration. The wet cake was washed thrice with THF (200mL). The material was dried under vacuum at 40 °C with nitrogen purge yielding 94.6g of 2.
[0241] The reaction to synthesize Compound 2 from Compound 6 described above may be modified as follows. Solvent: Different solvents from THF could be used, such as 1,4 dioxane or 2-methyltetrahydrofuran. Reaction volume: The reaction volume can be varied from 4 to 50 volumes with respect to compound 2. Catalyst and base: Different palladium catalyst and bases can be used for the borylation. Examples can be found in Chow et al., RSC Adv., 3 : 12518-12539 (2013). Borylation reaction temperature: Reaction temperatures from room temperature (20 °C) to solvent reflux can be used. Carbon/ Silica treatment:
The treatment can be performed without silica gel. The process can be performed without a carbon treatment. Different carbon sources from Ecosorb C-941 can be used. Different amounts of silica, from 0.01X to IX weight equivalents, can be used. Different amounts of Ecosorb C-941, from 0.01X to IX weight equivalents, can be used. Crystallization: A different addition rate of diethanolamine can be used. Different amounts of diethanolamine, from 1.0 to 3.0 molar equivalents can be used. A different cake wash with more or less THF can be used. Different amount of seeds from 0.0001X wt to 50X wt can be used.
Alternatively, the process can be unseeded. Drying: A temperature range of 10 °C to 60 °C could be used for drying.
[0242] The subsequent Suzuki-Miyaura coupling between Compounds 2 and 3 also proceeded well, providing over 20 kg of crude compound 1 with an average molar yield of 80% and LCAP of 99.7%.
1.4: Synthesis of Coupling Partner 3
[0243] Cross-coupling partner 3 was prepared by two different processes corresponding to Schemes 8 and 9 shown below.
Scheme 8: Process A for preparation of 3
[0244] According to Process A, Compound 9 (100g, 628 mmol) was dissolved in acetonitrile (450 mL) at room temperature. In a separate vessel, N-bromosuccinimide (NBS, 112g, 628 mmol) was suspended in acetonitrile (1 L). Compound 9 in acetonitrile was charged to the NBS slurry over at least 45 minutes. The contents of the reaction vessel were warmed to 45 °C to 55 °C and the batch stirred until the reaction was complete by HPLC analysis. The batch was cooled to 35 °C to 45 °C and ensured dissolution. Norit SX plus carbon (lOg) was charged to the mixture and the reaction mixture adjusted to 55 °C to 60 °C. The mixture was stirred at 55 °C to 60 °C for about lh and the mixture filtered at 55 °C to 60 °C to remove solids. The solids were washed with acetonitrile (500mL) at 55 °C to 60 °C. The volume of the combined filtrate was reduced to 900 mL by distilling off acetonitrile under reduced pressure. The batch with Compound 3 (lg) and stirred at 35 °C to 45 °C for at least 60 minutes. The contents of the reaction vessel were cooled to 15 °C to 25 °C over at least 1 hour. Water (2000 mL) was charged to the reaction vessel over at least 90 minutes and the slurry aged for at least 60 minutes. The product was collected by suction filtration. The cake was washed with a premixed 5% solution of acetonitrile in water (300mL). The wet cake was dried under vacuum at 40 °C with nitrogen purge. Yield: 120g of 3.
[0245] The above procedure, Process A for this synthesis of 3, may be practiced with alternative reagents and conditions as follows. Solvents: Alternative solvents could be used. Examples include chlorinated solvents, such as methylene chloride, chloroform or 1,2 dichloroethane, and non-chlorinated solvents such as tetrahydrofuran, or 2-methyltetrahydrofuran. Reaction concentration: The reaction concentration can be varied from 2X vol to 40 X vol (with respect to Compound 9). Brominating agents: Additional brominating reagents include bromine and l,3-dibromo-5,5-dimethylhydantoin. Bromination reagent Stoichiometry: Different amounts of the brominating reagent can be used, from 0.8 equiv to 2 equiv. Crystallization: Different amounts of water, including 5 volumes to 50 volumes can be used. The crystallization can also proceed without the addition of seeds. Different water addition times and final hold times can be used. Different wash procedures can be used. Drying: A temperature range of 10 °C to 60 °C could be used for drying.
Scheme 9: Process B for preparation of 3
[0246] According to Process B, Compound 3 can be formed starting from 8 via non-isolated compound 9 as follows. Compound 8 (80 g, 55 mmol), cesium carbonate (CS2CO3, 215 g, 66 mmol), and acetonitrile (800 mL) were charged to the reactor. The temperature was adjusted from 15 to 25 °C and iodomethane charged to the reactor (Mel, 86 g, 0.61 mol) while maintaining a batch temperature below 25 °C. The batch was heated to 40 °C and agitated for 10 hours to form Compound 9. The batch was cooled to 25 °C, filtered into a fresh reactor to remove solids, and the solids washed twice with acetonitrile. The combined organic layers were concentrated via atmospheric distillation to about 320 mL.
[0247] In a separate reactor N-bromosuccinimide (NBS, 98.1 g, 0.55 mol) was charged to acetonitrile (800 mL) and agitated. The batch containing Compound 9 was transferred to the NBS solution while maintaining a batch temperature of 15 to 25 °C. The batch was heated to 45 to 55 °C and agitated for at least 4 hours to allow for reaction completion to Compound 3. Upon reaction completion, Norit SX Plus activated carbon (8 g) was charged, and agitated at 45 to 55 °C for one hour. The batch was filtered into a fresh vessel, the Norit SX plus cake was washed with 400 ml of 45 to 55 °C acetonitrile. The acetonitrile layers were combined, cooled to 35 to 45 °C, and distilled under reduced pressure to 720 mL. The batch was adjusted to a temperature of 40 °C, charged with Compound 3 seeds (0.8 g), agitated for one hour, cooled to 15 to 25 °C over at least on hour, then charged with water (1600 mL) over at least two hours. The mixture was agitated for an additional one to two hours, filtered, the cake washed with a premixed 5% solution of acetonitrile in water (240 mL). The wet cake was dried under vacuum at 40°C with nitrogen purge. Yield: 52 g of 3.
[0248] Process B to synthesize Compound 3, described above, may be modified as follows. Solvents: Alternative solvents could be used. Examples include chlorinated solvents, such as methylene chloride, chloroform or 1,2 dichloroethane, and non-chlorinated solvents such as tetrahydrofuran, or 2-methyltetrahydrofuran. Reaction concentration: The reaction concentration can be varied from 2X vol to 40 X vol (with respect to Compound 8).
Alkylating reagent: Alternative methylating reagents to methyl iodide can be used such as dimethylsulfate. Alkylating reagent stoichiometry: 1 to 10 molar equivalents of methyl iodide may be used. Base: Different inorganic bases, such as potassium carbonate or phosphate bases (sodium, potassium, or cesium) could be used. Brominating agents:
Additional brominating reagents include bromine and l,3-dibromo-5,5-dimethylhydantoin. Bromination reagent stoichiometry: Different amounts of the brominating reagent can be used, from 0.8 equiv to 2 equiv. Crystallization: Different amounts of water, including 5 volumes to 50 volumes can be used. Seeding levels from 0.0001% to 50% can be used. The crystallization can also proceed without the addition of seeds. Different water addition times and final hold times can be used. Different wash procedures can be used. Drying: A temperature range of 10 to 60 °C could be used for drying.
1.5: Cross-coupling of 2 and 3 to Produce Target Compound 1
[0249] 1 is synthesized by Suzuki cross-coupling of 3 and 2 according to Scheme 10 and as described below.
Scheme 10: Synthesis of 1
[0250] Acetonitrile (1.6L) was charged to a mixture of Compound 2 (156.7g, 460 mmol), Compovmd 3 (lOOg, 420 mmol) and potassium phosphate tribasic (223 g, l.OSmol). Agitation was begun and water (400mL) charged to the batch. The system was vacuum purged three times with nitrogen and charged with Pd(PPh3)2Cl2 (2.9g, 4 mmol) and the system vacuum
purged three times with nitrogen. The batch was heated to 65 to 75°C and contents stirred for at least 16 hours until reaction was complete by HPLC analysis. The batch was cooled to 60 to 70°C, agitation halted and the mixture allowed to settle. The bottom aqueous layer was removed. Water (150mL) and acetonitrile (700mL) were charged at 60 to 70°C. Ecosorb C-941 (15g) and Celite (lOg) were charged to the reaction vessel at 60 to 70°C. After lh, the mixture was filtered to remove solids. The solids were washed twice each with 18% water in acetonitrile (500 mL) at 60 to 70°C. The filtrates were combined and concentrated under atmospheric pressure to a final volume of 1.5L. The batch was cooled to 60 to 65°C and seeded with Compound 1 (1 g). After lh, water (500 mL) was charged over at least 1 hour at 60 to 65°C. The slurry was cooled to 15 to 25°C over 4 hours. The product was collected by suction filtration. The wet cake was washed with 45% water in acetonitrile (500mL) twice. The product was dried under vacuum at 40°C with nitrogen purge. Yield: 139g of 1.
[0251] The above procedure for coupling Compound 3 and Compound 2 to produce
Compound 1 may be modified in any of the ways that follow. Reaction solvents: Different reaction solvents from acetonitrile can be used, including tetrahydrofuran, 2-methyl tetrahydrofuran, toluene, and isopropanol. Boronic ester: Different boronic esters from Compound 2 can be used, including pinacolato ester compound 7, and the free boronic acid of Compound 2. Examples of boronic esters can be found in Lennox, Alister, J.J., Lloyd-Jones, Guy C. Chem. Soc. Rev., 2014, 43, 412. Carbon treatment: Different carbon treatments from Ecosorb C-941 could be used. Different amounts of carbon, from 0.01 to 0.5X weight can be used. The carbon can be eliminated. Different amounts of Celite, from 0.01 to 0.5X weight can be used. Crystallization: Different amounts of water, including 5 volumes to 50 volumes can be used. The crystallization can also proceed without the addition of seeds. Different water addition times and final hold times can be used. Different wash procedures can be used. Drying: A temperature range of 10 to 60 °C could be used for drying. Catalysts: Different metal and ligand combination could be used. Examples of metal/ligand combinations can be found in Maluenda, Irene; Navarro, Oscar, Molecules, 2015, 20, 7528. Various catalysts can be including: XPhos-3G (cas# 1445085-55-1);
cataCXium® A Pd 3G (CAS# 1651823-59-4); PdCk(DtBPF) (CAS# 95408-45-0); SPhos 3G (Cas# 1445085-82-4); AmPhos 3G (Cas# 1820817-64-8); PCy3 3G (Cas# 1445086-12-3); Pd PEPPSI IPent Cas#l 158652-41-5); Pd(PPh3)2Cl2 (Cas# 13965-03-2). Examples of
catalyst systems that have been demonstrated to afford Compound 1 are listed below in Table 4 using boronic esters 2 or 7 in coupling to 3.
Table 4: Catalyst screen summary
1.6: Crystallization of 1
[0252] The final isolation of Compound 1 requires a polish filtration. For this, the batch must be completely soluble. Unfortunately, Compound 1 has low solubility in almost all
International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Class 3 and common Class 2 (e.g. THF, MeCN) solvents (ICH
Harmonized Guideline“Impurities: Guideline for Residual Solvents Q3C(R6)” October 20, 2016). A reasonable solubility was obtained in a warm MeCN-water mix, but this is not an optimal system (requires a heated filtration, MeCN has a residual solvent limit of only 410 ppm). Additional solvents with reasonable solubility (>50 mg/ml) include N-methyl-2- pyrrolidone (NMP) and dimethylacetamide (DMAc); but the development of isolations from these solvents required large volumes and raised residual solvent limit concerns (530 ppm or less for NMT and 1090 ppm or less for DMAc).
catalyst systems that have been demonstrated to afford Compound 1 are listed below in Table 4 using boronic esters 2 or 7 in coupling to 3.
Table 4: Catalyst screen summary
1.6: Crystallization of 1
[0252] The final isolation of Compoxmd 1 requires a polish filtration. For this, the batch must be completely soluble. Unfortunately, Compound 1 has low solubility in almost all
International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Class 3 and common Class 2 (e.g. THF, MeCN) solvents (ICH
Harmonized Guideline“Impurities: Guideline for Residual Solvents Q3C(R6)” October 20, 2016). A reasonable solubility was obtained in a warm MeCN-water mix, but this is not an optimal system (requires a heated filtration, MeCN has a residual solvent limit of only 410 ppm). Additional solvents with reasonable solubility (>50 mg/ml) include N-methyl-2- pyrrolidone (NMP) and dimethylacetamide (DMAc); but the development of isolations from these solvents required large volumes and raised residual solvent limit concerns (530 ppm or less for NMT and 1090 ppm or less for DMAc).
[0253] Formic acid is one ICH Class 3 solvent in which Compound 1 is highly soluble, having a solubility greater than 250 mg/ml at 20 °C. The solubility curve of Compound 1 in formic acid-Water is quite steep (see Figure 7), which enables a volumetrically efficient process.
[0254] Initial attempts to recrystallize crude Compound 1 involved dissolving in formic acid, polish filtering, and charging polish filtered water to about 20% supersaturation, followed by seeding with the thermodynamically most stable form (Form 1), followed by slow addition of water to the final solvent ratio, filtration, washing, and drying. Applicant observed that during the initial water charge, if the batch self-seeded it formed a thick slurry. X-ray diffraction (XRD), differential scanning calorimetry (DSC), and photomicroscopy demonstrated that a metastable form was produced. Once seeded with Form 1, the batch converted to the desired form (Form 1) prior to the addition of the remaining water. This process worked well during multiple lab runs, consistently delivering the desired form and purity with about 85% yield.
[0255] Unfortunately, upon scale-up, the batch did not convert to Form 1 after seeding. Additional water was charged and the batch began to convert to the desired form (mix of Form 1 and the metastable form by X-ray powder diffraction (XRPD)). When additional water was charged, the XRPD indicated only the metastable form. After a few hours with no change, Applicant continued the water charge to the final solvent ratio, during which time the batch eventually converted to Form 1. This process is summarized in Figure 8.
[0256] It was subsequently found by closer analysis of the plant and laboratory retains that a new metastable form was formed during scale up, with a similar, but different XRPD pattern. This form (metastable B) could be reproduced in the laboratory, but only when the batch has a high formic acid:water ratio and is seeded with Form 1. Without Form 1 seeds, metastable A is the kinetic form. Both metastable forms converted to Form 1 with additional water and/or upon drying, leading Applicant to believe that the metastable forms are formic acid solvates. These findings are summarized in Figure 9.
[0257] While there is little risk in not being able to control the final form, there is a risk of forming a difficult-to-stir slurry which can lead to processing issues. The crystallization procedure was therefore modified to keep a constant formic acid-water ratio. This was performed by charging 2.4X wt. formic acid and 1.75X wt. water (final solvent composition)
to the crystallizer with 0.03X wt. Form 1 seeds, and performing a simultaneous addition of Compound 1 in 6. IX wt. formic acid and 4.4X wt. water. The batch filtered easily and was washed with formic acid/water, then water, and dried under reduced pressure to yield 8.9 kg of Compound 1 (92% yield) with 99.85% LCAP and N.D. formic acid.
Example 2: Exemplary high throughput experimentation reaction
[0258] The following procedure is an exemplary high throughput experimentation reaction.
[0259] An overview of the reaction is shown below in Scheme 5:
Scheme 5: Reaction conditions tested for cross-coupling reaction of 2 and 3
[0260] Pd catalysts were dosed into the 24-well reactor vial as solutions (100 pL of 0.01 M solution in tetrahydrofuran (THF) or dichloroethane (DCE) depending upon the solubility of the ligand). Plates of these ligands are typically dosed in advance of the reaction, the solvent is removed by evacuation in an evaporative centrifuge and plates are stored in the glovebox. The catalysts screened in the coupling are the following: XPhos, SPhos, CataCXium A, APhos, P(Cy)3, PEPPSI-IPent. For the first five ligands, these were initially screened as the Buchwald Pd G2/G3 precatalysts.
[0261] To the plates was then added a stock solution of Compound 3 (10 pmol) and Compound 2 (12 pmol) dissolved in the following solvents: dimethylformamide (DMF),
tetrahydrofuran (THF), butanol (/r-BuOH), and toluene. The base was then added as a stock solution (30 mmol) in 20 mL of water.
[0262] A heatmap summarizing catalyst performance is shown in Figs. 10A and 10B. High performance liquid chromatography (HPLC) yields for this screening span from <5% up to -85%. Larger circles indicate higher yield. Lighter circles indicate higher cleanliness.
[0263] A similarly designed screening of base and solvent also indicate that a range of alcoholic solvents (methanol, ethanol, propanol, 2-butanol, 2-propanol, and /-amyl alcohol) are also all viable in this coupling chemistry. Bases such as potassium phosphate, potassium carbonate, potassium acetate, and potassium hydroxide were all successful in achieving the coupling. Fig. 10B shows a heatmap with HPLC yields ranging from -50 – 95%. Larger, darker circles indicate higher yield.
[0264] This chemistry from microvial screening has been scaled to a laboratory process. To a 3 -necked jacketed 250 mL flask equipped with overhead stirring, nitrogen inlet, and thermocouple was added Compound 3 (1.0 eq, 4.00 grams), Compound 2 (1.2 eq, 1.71 x wt), potassium carbonate (3.0 eq, 1.74 x wt). The reactor was inerted three times and then degassed 2-propanol (24 x vol.) followed by degassed water (6 x vol) was then added.
Stirring was then initiated at 300 rpms. The reactor was then stirred and blanketed with nitrogen for 1 hour. The catalyst was then added (0.01 eq, 0.028 x wt) and stirring continued (300 rpms) and the reactor was heated into the Tj = 65 °C.
[0265] After 2 hours, with full conversion confirmed analytically, trioctylphosphine (0.1 eq, 0.16 x wt) dosed, and reaction mixture allowed to cool slowly to room temperature hours.
The reaction mixture was then filtered, washed with 2-propanol (4 x vol), 2-propanol: water (4: 1, 4 x vol), and then with water (4 x vol). Note: If 2 is dimer present in cake, an additional ethyl acetate (EtOAc) wash (4 x vol) can be added for purging. The cake was then transferred to a vacuum oven to dry overnight at 40 °C, -40 cm Hg, under nitrogen flow. After transfer to a bottle, 6.03 grams of 1 were isolated, 98.6% assay, 91% overall yield.
Scheme 6: Alternative reagents and solvents for cross-coupling
[0266] Based on the previously delineated results, it was expected that a variety of monodentate (PPI13 [triphenylphosphine], PBu3 [tributylphosphine], etc) and bidentate phosphines (dppf [1,1 ‘-bis(diphenylphosphino)ferrocene], BINAP [2,2 -bis(diphenylphosphino)- 1 , 1 -binaphthyl], Xantphos [4,5-bis(diphenylphosphino)-9,9-dimethylxanthene], dppe [l,2-bis(diphenylphosphino)ethane], etc) ligated to any number of Pd sources (Pd halides, Pd(H) precatalyts, Pd(0) sources) could reasonably be employed to arrive at the Compound 1 crude material. A range of organic solvents ranging from non-polar (heptane, benzene), protic (alcohols), polar aprotic (dimethylsulfoxide, dimethylformamide, dimethylacetamide, acetonitrile) as well as a variety of esters and ketones (acetone, 2-butanone, ethylacetate) should also serve as effective solvents for this reactivity. Finally, inorganic bases of varying strength (phosphates, carbonates, acetates, etc) along with organic variants such as triethylamine, l,8-diazabicyclo(5.4.0)undec-7-ene, and others in a wide pKa range are viable as stoichiometric basic additives.
Example 3: Exemplary Compound 5 process
[0267] The purpose of this example was to describe an exemplary process for making Compound 5.
[0268] Charge 4 (lOg, 58mmol) and acetonitrile (lOOmL) to a reaction vessel and start the stirrer. Adjust the batch to -18 °C to -22 °C (target -20 °C). Charge triflic acid (5.5mL, 62mmol) to the batch maintaining -10 °C to -25 °C (target -20 °C). Stir the batch at -10 °C to -25 °C (target -20 °C) for 10 to 20 minutes. Charge NBS (11.38g, 64mmol) to the batch at -10 °C to -25 °C (target -20 °C) and stir for ca. 30 min at -10 °C to -25 °C (target -20 °C). Warm the batch to 20 °C over 3-4 hours (reaction will occur when internal temp is between 5 °C and 15 °C). Stir the batch at 15 °C to 25 °C (target 20 °C) for approximately 1 hour and sample for reaction completion.
[0269] If Compound 4 relative to Compound 5 is more than 5%:
[0270] Cool the bath to -5 °C to -15 °C (target -10 °C) (cooling below 0 °C to ensure selectivity). Charge NBS to the batch according to the follow formula: Mass of NBS = (% Compound 4 x lOg). Warm the batch to 20 °C over 1-2 hours. Stir the batch at 15 °C to 25 °C (target 20 °C) for approximately 1 hour and check reaction for completion. Proceed to next line.
[0271] If Compound 4 relative to Compound 5 is less than 5%:
[0272] Warm the batch to 40 °C to 50 °C (target 48 °C). Concentrate the batch under reduced pressure to a final volume of ~40mL. Cool the batch to -15 °C to -5 °C (target -10 °C) and stir for ca. lh. Filter the batch by suction filtration. Slurry wash the filter cake with purified water (3 x 20mL) at 15 °C to 25 °C (target 20 °C) for 10 to 15 minutes each wash. Remove a sample of the filter cake for analysis by ¾ NMR. Continue washing cake until the residual succimide is below 1.0%mol% relative to 5. Dry the filter cake at up to 60°C under vacuum and nitrogen purge. Analyse the 5 by HPLC analysis (97%w/w to 99%w/w). Expected yield: 60-85% theory (90-110% w/w).
Example 4: Purification of Compound 1 (CC-90010) by crystallization from formic acid and water.
[0273] This example describes a method for the purification of Compound 1 by
crystallization from formic acid and water. Also detailed are methods for obtaining three different polymorphs of Compound 1, including the most stable form, Form 1.
[0274] Figure 11 shows XH NMR of Compound 1 (CC-90010). Solvent: d6DMSO; and Figure 12 shows microscopy of Compound 1 (CC-90010) Form I. Figure 13 shows XRPD of Compound 1 (CC-90010) Form I, with peak information detailed in Table 6:
PATENT
US 20190008852
WO 2018081475
US 20180042914
WO 2016172618
WO 2015058160
/////////CC-90010, solid tumors , non-Hodgkin’s lymphoma, PHASE 1, CANCER, QUANTICEL
CS(=O)(=O)c4cc(C1=CN(C)C(=O)c2ccccc12)c(OCC3CC3)cc4
Delgocitinib
Delgocitinib
デルゴシチニブ
3-[(3S,4R)-3-methyl-7-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,7-diazaspiro[3.4]octan-1-yl]-3-oxopropanenitrile
1,6-Diazaspiro(3.4)octane-1-propanenitrile, 3-methyl-beta-oxo-6-(7H-pyrrolo(2,3-d)pyrimidin-4-yl)-, (3S,4R)-
3-((3S,4R)-3-methyl-6-(7H-pyrrolo(2,3-d)pyrimidin-4-yl)-1,6-diazaspiro(3.4)octan-1-yl)-3-oxopropanenitrile
| Formula |
C16H18N6O
|
|---|---|
| CAS |
1263774-59-9
|
| Mol weight |
310.3537
|
Approved, Japan 2020, Corectim, 2020/1/23, atopic dermatitis, Japan Tobacco (JT)
Torii
7/23/2025 fda approved, Anzupgo
| To treat moderate-to-severe chronic hand eczema when topical corticosteroids are not advisable or produce an inadequate response |
UNII-9L0Q8KK220, JTE-052, LP-0133, ROH-201, 9L0Q8KK220, LEO 124249A, LEO 124249, HY-109053
CS-0031558, D11046, GTPL9619, JTE-052A, JTE052
Delgocitinib, also known as LEO-124249 and JTE052, is a potent and selective JAK inhibitor. JTE-052 reduces skin inflammation and ameliorates chronic dermatitis in rodent models: Comparison with conventional therapeutic agents. JTE-052 regulates contact hypersensitivity by downmodulating T cell activation and differentiation.
Delgocitinib is a JAK inhibitor first approved in Japan for the treatment of atopic dermatitis in patients 16 years of age or older. Japan Tobacco is conducting phase III clinical trials for the treatment of atopic dermatitis in pediatric patients. Leo is developing the drug in phase II clinical trials for the treatment of inflammatory skin diseases, such as atopic dermatitis, and chronic hand eczema and for the treatment of discoid lupus erythematosus. Rohto is evaluating the product in early clinical development for ophthalmologic indications.
In 2014, the drug was licensed to Leo by Japan Tobacco for the development, registration and marketing worldwide excluding Japan for treatment of inflammatory skin conditions. In 2016, Japan Tobacco licensed the rights of co-development and commercialization in Japan to Torii. In 2018, Japan Tobacco licensed the Japanese rights of development and commercialization to Rohto for the treatment of ophthalmologic diseases.
Delgocitinib, sold under the brand name Corectim among others, is a medication used for the treatment of autoimmune disorders and hypersensitivity, including inflammatory skin conditions.[3] Delgocitinib was developed by Japan Tobacco and approved in Japan for the treatment of atopic dermatitis.[3] In the United States, delgocitinib is in Phase III clinical trials and the Food and Drug Administration has granted delgocitinib fast track designation for topical treatment of adults with moderate to severe chronic hand eczema.[4]
Delgocitinib works by blocking activation of the JAK-STAT signaling pathway which contributes to the pathogenesis of chronic inflammatory skin diseases.[5]
PATENTS
WO 2018117151
IN 201917029002
IN 201917029003
IN 201917029000
PATENTS
WO 2011013785
https://patents.google.com/patent/WO2011013785A1/en
[Production Example 6]: Synthesis of Compound 6
(1) Optically active substance of 2-benzylaminopropan-1-ol
To a solution of (S)-(+)-2-aminopropan-1-ol (50.0 g) and benzaldehyde (74 ml) in ethanol (500 ml) was added 5% palladium carbon (5.0 g) at room temperature and normal pressure. Hydrogenated for 8 hours. The reaction mixture was filtered through celite and concentrated under reduced pressure to give the title compound (111.2 g).
1 H-NMR (DMSO-D 6 ) δ: 7.34-7.27 (4H, m), 7.23-7.18 (1H, m), 4.53-4.47 (1H, m), 3.76 (1H, d, J = 13.5 Hz) , 3.66 (1H, d, J = 13.5 Hz), 3.29-3.24 (2H, m), 2.65-2.55 (1H, m), 1.99 (1H, br s), 0.93 (3H, d, J = 6.4 Hz) .
(2) Optically active substance of [benzyl- (2-hydroxy-1-methylethyl) -amino] acetic acid tert-butyl ester
To a mixture of optically active 2-benzylaminopropan-1-ol (111.2 g), potassium carbonate (111.6 g) and N, N-dimethylformamide (556 ml) cooled to 0 ° C., tert-butyl bromoacetate was added. Ester (109 ml) was added dropwise over 20 minutes and stirred at room temperature for 19.5 hours. The mixture was acidified to pH 2 by adding 2M aqueous hydrochloric acid and 6M aqueous hydrochloric acid, and washed with toluene (1000 ml). The separated organic layer was extracted with 0.1 M aqueous hydrochloric acid (300 ml). The combined aqueous layer was adjusted to pH 10 with 4M aqueous sodium hydroxide solution and extracted with ethyl acetate (700 ml). The organic layer was washed successively with water (900 ml) and saturated aqueous sodium chloride solution (500 ml). The separated aqueous layer was extracted again with ethyl acetate (400 ml). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the title compound (160.0 g).
1 H-NMR (DMSO-D 6 ) δ: 7.37-7.26 (4H, m), 7.24-7.19 (1H, m), 4.26 (1H, dd, J = 6.9, 3.9 Hz), 3.76 (1H, d, J = 14.1 Hz), 3.68 (1H, d, J = 13.9 Hz), 3.45-3.39 (1H, m), 3.29-3.20 (1H, m), 3.24 (1H, d, J = 17.2 Hz), 3.13 ( 1H, d, J = 17.0 Hz), 2.84-2.74 (1H, m), 1.37 (9H, s), 0.96 (3H, d, J = 6.8 Hz).
(3) Optically active substance of [benzyl- (2-chloropropyl) -amino] acetic acid tert-butyl ester
(3)-(1) Optically active form of [benzyl- (2-chloro-1-methylethyl) -amino] acetic acid tert-butyl ester
To a solution of [benzyl- (2-hydroxy-1-methylethyl) -amino] acetic acid tert-butyl ester optically active substance (160.0 g) cooled to 0 ° C. in chloroform (640 ml) was added thionyl chloride (50.0 ml). Was added dropwise and stirred at 60 ° C. for 2 hours. The reaction mixture was cooled to 0 ° C., saturated aqueous sodium hydrogen carbonate solution (1000 ml) and chloroform (100 ml) were added and stirred. The separated organic layer was washed with a saturated aqueous sodium chloride solution (500 ml), and the aqueous layer was extracted again with chloroform (450 ml). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the title compound (172.9 g).
1 H-NMR (CDCl 3 ) δ: 7.40-7.22 (5H, m), 4.05-3.97 (0.4H, m), 3.93-3.81 (2H, m), 3.70-3.65 (0.6H, m), 3.44- 3.38 (0.6H, m), 3.29 (0.8H, s), 3.27 (1.2H, d, J = 2.4 Hz), 3.24-3.15 (0.6H, m), 3.05-2.99 (0.4H, m), 2.94 -2.88 (0.4H, m), 1.50 (1.2H, d, J = 6.4 Hz), 1.48 (3.6H, s), 1.45 (5.4H, s), 1.23 (1.8H, d, J = 6.8 Hz) .
(3)-(2) Optically active form of [benzyl- (2-chloropropyl) -amino] acetic acid tert-butyl ester
[Benzyl- (2-chloro-1-methylethyl) -amino] acetic acid tert-butyl ester optically active substance (172.9 g) was dissolved in N, N-dimethylformamide (520 ml) and stirred at 80 ° C. for 140 minutes. did. The reaction mixture was cooled to 0 ° C., water (1200 ml) was added, and the mixture was extracted with n-hexane / ethyl acetate (2/1, 1000 ml). The organic layer was washed successively with water (700 ml) and saturated aqueous sodium chloride solution (400 ml), and the separated aqueous layer was extracted again with n-hexane / ethyl acetate (2/1, 600 ml). The combined organic layers were concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 50/1 to 40/1) to give the title compound (127.0 g )
1 H-NMR (CDCl 3 ) δ: 7.37-7.29 (4H, m), 7.28-7.23 (1H, m), 4.05-3.97 (1H, m), 3.91 (1H, d, J = 13.5 Hz), 3.86 (1H, d, J = 13.7 Hz), 3.29 (2H, s), 3.03 (1H, dd, J = 13.9, 6.6 Hz), 2.91 (1H, dd, J = 13.9, 6.8 Hz), 1.50 (3H, d, J = 6.4 Hz), 1.48 (9H, s).
(4) Optically active substance of 1-benzyl-3-methylazetidine-2-carboxylic acid tert-butyl ester
To a solution of [benzyl- (2-chloropropyl) -amino] acetic acid tert-butyl ester optically active substance (60.0 g) cooled to −72 ° C. and hexamethylphosphoramide (36.0 ml) in tetrahydrofuran (360 ml), Lithium hexamethyldisilazide (1.0 M tetrahydrofuran solution, 242 ml) was added dropwise over 18 minutes, and the temperature was raised to 0 ° C. over 80 minutes. A saturated aqueous ammonium chloride solution (300 ml) and water (400 ml) were sequentially added to the reaction mixture, and the mixture was extracted with ethyl acetate (500 ml). The organic layer was washed successively with water (700 ml) and saturated aqueous sodium chloride solution (500 ml), and the separated aqueous layer was extracted again with ethyl acetate (300 ml). The combined organic layers were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (developing solvent: n-hexane / ethyl acetate = 50/1 to 4/1). To give the title compound (50.9 g).
1 H-NMR (CDCl 3 ) δ: 7.34-7.21 (5H, m), 3.75 (1H, d, J = 12.6 Hz), 3.70-3.67 (1H, m), 3.58 (1H, d, J = 12.6 Hz ), 3.05-3.01 (1H, m), 2.99-2.95 (1H, m), 2.70-2.59 (1H, m), 1.41 (9H, s), 1.24 (3H, d, J = 7.1 Hz).
(5) Optically active substance of 3-methylazetidine-1,2-dicarboxylic acid di-tert-butyl ester
1-Benzyl-3-methylazetidine-2-carboxylic acid tert-butyl ester optically active substance (43.5 g) and di-tert-butyl dicarbonate (38.2 g) in tetrahydrofuran / methanol (130 ml / 130 ml) solution 20% Palladium hydroxide carbon (3.5 g) was added thereto, and hydrogenated at 4 atm for 2 hours. The mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to give the title compound (48.0 g).
1 H-NMR (DMSO-D 6 ) δ: 4.44 (1H, d, J = 8.8 Hz), 3.99-3.77 (1H, m), 3.45-3.37 (1H, m), 3.00-2.88 (1H, m) , 1.45 (9H, s), 1.40-1.30 (9H, m), 1.02 (3H, d, J = 7.2 Hz).
(6) Optically active substance of 3-methyl-2- (3-methyl-but-2-enyl) -azetidine-1,2-dicarboxylic acid di-tert-butyl ester
Optically active substance (48.0 g) of 3-methylazetidine-1,2-dicarboxylic acid di-tert-butyl ester cooled to -69 ° C. and 1-bromo-3-methyl-2-butene (25.4 ml) Lithium hexamethyldisilazide (1.0 M tetrahydrofuran solution, 200 ml) was added to a tetrahydrofuran solution (380 ml). The reaction mixture was warmed to −20 ° C. in 40 minutes and further stirred at the same temperature for 20 minutes. A saturated aqueous ammonium chloride solution (200 ml) and water (300 ml) were successively added to the reaction mixture, and the mixture was extracted with n-hexane / ethyl acetate (1 / 1,500 ml). The separated organic layer was washed successively with water (200 ml) and saturated aqueous sodium chloride solution (200 ml), dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 15/1 to 8/1) to give the titled compound (44.5 g).
1 H-NMR (CDCl 3 ) δ: 5.29-5.21 (1H, m), 3.77-3.72 (1H, m), 3.49-3.44 (1H, m), 2.73-2.52 (3H, m), 1.76-1.74 ( 3H, m), 1.66-1.65 (3H, m), 1.51 (9H, s), 1.43 (9H, s), 1.05 (3H, d, J = 7.3 Hz).
(7) Optically active substance of 3-methyl-2- (2-oxoethyl) azetidine-1,2-dicarboxylic acid di-tert-butyl ester
3-methyl-2- (3-methyl-but-2-enyl) -azetidine-1,2-dicarboxylic acid di-tert-butyl ester optically active substance (44.5 g) in chloroform / cooled to −70 ° C. An ozone stream was passed through the methanol solution (310 ml / 310 ml) for 1 hour. To this reaction mixture, a solution of triphenylphosphine (44.7 g) in chloroform (45 ml) was added little by little, and then the mixture was warmed to room temperature. To this mixture were added saturated aqueous sodium thiosulfate solution (200 ml) and water (300 ml), and the mixture was extracted with chloroform (500 ml). The separated organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain the title compound (95.0 g). This product was subjected to the next step without further purification.
1 H-NMR (DMSO-D 6 ) δ: 9.65 (1H, t, J = 2.6 Hz), 3.79-3.74 (1H, m), 3.45-3.40 (1H, m), 2.99-2.80 (3H, m) , 1.46 (9H, s), 1.34 (9H, s), 1.06 (3H, d, J = 7.2 Hz).
(8) Optically active substance of 2- (2-benzylaminoethyl) -3-methylazetidine-1,2-dicarboxylic acid di-tert-butyl ester
To a solution of the residue (95.0 g) obtained in (7) in tetrahydrofuran (300 ml) was added benzylamine (34 ml) at room temperature, and the mixture was stirred for 2 hours. The mixture was cooled to 0 ° C., sodium triacetoxyborohydride (83.3 g) was added, and the mixture was stirred at room temperature for 1.5 hours. Water (300 ml) was added to the reaction mixture, and the mixture was extracted with n-hexane / ethyl acetate (1/3, 600 ml). The separated organic layer was washed with water (300 ml) and saturated aqueous sodium chloride solution (200 ml), and then extracted twice with 5% aqueous citric acid solution (300 ml, 200 ml) and three times with 10% aqueous citric acid solution (250 ml × 3). . The combined aqueous layers were basified to pH 10 with 4M aqueous sodium hydroxide solution and extracted with chloroform (300 ml). The organic layer was washed with a saturated aqueous sodium chloride solution (200 ml), dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain the title compound (46.9 g).
1 H-NMR (DMSO-D 6 ) δ: 7.34-7.26 (4H, m), 7.22-7.17 (1H, m), 3.74-3.65 (2H, m), 3.61 (1H, t, J = 7.8 Hz) , 3.28 (1H, t, J = 7.5 Hz), 2.76-2.66 (2H, m), 2.57-2.45 (1H, m), 2.15 (1H, br s), 2.05-1.89 (2H, m), 1.42 ( 9H, s), 1.27 (9H, s), 0.96 (3H, d, J = 7.1 Hz).
(9) Optically active substance of 2- (2-benzylaminoethyl) -3-methylazetidine-2-dicarboxylic acid dihydrochloride
2- (2-Benzylaminoethyl) -3-methylazetidine-1,2-dicarboxylic acid di-tert-butyl ester optically active substance (46.5 g), 4M hydrochloric acid 1,4-dioxane (230 ml) and water (4.1 ml) was mixed and stirred at 80 ° C. for 2 hours. The mixture was concentrated under reduced pressure, azeotroped with toluene, and then slurry washed with n-hexane / ethyl acetate (1/1, 440 ml) to give the title compound (30.1 g).
1 H-NMR (DMSO-D 6 ) δ: 10.24 (1H, br s), 9.64 (2H, br s), 8.90 (1H, br s), 7.58-7.53 (2H, m), 7.47-7.41 (3H , m), 4.21-4.10 (2H, m), 4.02-3.94 (1H, m), 3.46-3.37 (1H, m), 3.20-3.10 (1H, m), 2.99-2.85 (2H, m), 2.69 -2.54 (2H, m), 1.10 (3H, d, J = 7.2 Hz).
(10) Optically active substance of 6-benzyl-3-methyl-1,6-diazaspiro [3.4] octan-5-one
To a solution of 2- (2-benzylaminoethyl) -3-methylazetidine-2-dicarboxylic acid dihydrochloride optically active substance (29.1 g) and N, N-diisopropylethylamine (65 ml) in chloroform (290 ml), At room temperature, O- (7-azabenzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate (41.3 g) was added and stirred for 4 hours. To this reaction mixture were added saturated aqueous sodium hydrogen carbonate solution (200 ml) and water (100 ml), and the mixture was extracted with chloroform (200 ml). The organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (developing solvent: chloroform / methanol = 20/1 to 10/1) to give the titled compound (21.3 g).
1 H-NMR (DMSO-D 6 ) δ: 7.38-7.31 (2H, m), 7.30-7.22 (3H, m), 4.52 (1H, d, J = 14.8 Hz), 4.29 (1H, d, J = 14.8 Hz), 3.35-3.27 (2H, m), 3.22-3.17 (1H, m), 3.05 (2H, dd, J = 9.5, 4.0 Hz), 2.77-2.66 (1H, m), 2.16-2.10 (1H , m), 1.96-1.87 (1H, m), 0.94 (3H, d, J = 7.1 Hz).
(11) Optically active substance of 6-benzyl-3-methyl-1,6-diazaspiro [3.4] octane-1-carboxylic acid tert-butyl ester
Concentrated sulfuric acid (4.8 ml) was slowly added dropwise to a suspension of lithium aluminum hydride (6.8 g) in tetrahydrofuran (300 ml) under ice cooling, and the mixture was stirred for 30 minutes. To this mixture was added dropwise a solution of 6-benzyl-3-methyl-1,6-diazaspiro [3.4] octan-5-one optically active substance (21.3 g) in tetrahydrofuran (100 ml) at the same temperature. Stir for 45 minutes. Water (7.0 ml), 4M aqueous sodium hydroxide solution (7.0 ml) and water (14.0 ml) were sequentially added to the reaction mixture, and the mixture was stirred as it was for 30 minutes. To this mixture was added anhydrous magnesium sulfate and ethyl acetate (100 ml), and the mixture was stirred and filtered through celite. Di-tert-butyl dicarbonate (23.4 g) was added to the filtrate at room temperature and stirred for 3 hours. The mixture was concentrated under reduced pressure to a half volume and washed twice with a saturated aqueous ammonium chloride solution (200 ml × 2). N-Hexane (200 ml) was added to the separated organic layer, and the mixture was extracted 5 times with a 10% aqueous citric acid solution. The separated aqueous layer was basified with 4M aqueous sodium hydroxide solution and extracted with chloroform. The organic layer was washed with a saturated aqueous sodium chloride solution (200 ml), dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (eluent: chloroform / methanol = 40/1 to 20/1) to give the titled compound (15.6 g).
1 H-NMR (DMSO-D 6 ) δ: 7.34-7.27 (4H, m), 7.26-7.21 (1H, m), 3.84-3.69 (1H, m), 3.62-3.47 (2H, m), 3.19- 3.05 (1H, m), 3.02-2.92 (1H, m), 2.76-2.69 (1H, m), 2.47-2.24 (4H, m), 1.95-1.77 (1H, m), 1.36 (9H, s), 1.03 (3H, d, J = 7.0 Hz).
(12) Optically active substance of 3-methyl-1,6-diazaspiro [3.4] octane-1-carboxylic acid tert-butyl ester
20% of optically active form of 6-benzyl-3-methyl-1,6-diazaspiro [3.4] octane-1-carboxylic acid tert-butyl ester (10.0 g) in tetrahydrofuran / methanol (50 ml / 50 ml) solution Palladium hydroxide on carbon (2.0 g) was added and hydrogenated at 4 atm for 24 hours. The mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to give the title compound (7.3 g).
1 H-NMR (DMSO-D 6 ) δ: 3.88-3.71 (1H, m), 3.44-3.06 (2H, m), 3.02-2.64 (4H, m), 2.55-2.38 (1H, m), 2.31- 2.15 (1H, m), 1.81-1.72 (1H, m), 1.37 (9H, s), 1.07 (3H, d, J = 7.0 Hz).
(13) Optical activity of 3-methyl-6- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1,6-diazaspiro [3.4] octane-1-carboxylic acid tert-butyl ester body
The optically active substance (6.9 g) of 3-methyl-1,6-diazaspiro [3.4] octane-1-carboxylic acid tert-butyl ester was converted into 4-chloro-7H-pyrrolo [2,3-d] pyrimidine ( 4.3 g), potassium carbonate (7.7 g) and water (65 ml) and stirred for 4 hours at reflux. The mixture was cooled to room temperature, water (60 ml) was added, and the mixture was extracted with chloroform / methanol (10/1, 120 ml). The organic layer was washed successively with water, saturated aqueous ammonium chloride solution and saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfate. To this mixture, silica gel (4 g) was added, stirred for 10 minutes, filtered through celite, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (developing solvent: chloroform / ethyl acetate = 1/1, then chloroform / methanol = 50/1 to 20/1) to give the title compound (10.0 g). Obtained.
1 H-NMR (DMSO-D 6 ) δ: 11.59 (1H, br s), 8.09 (1H, s), 7.12-7.09 (1H, m), 6.64-6.59 (1H, m), 4.09-3.66 (5H , m), 3.39-3.21 (1H, m), 2.64-2.44 (2H, m), 2.27-2.06 (1H, m), 1.36 (3H, s), 1.21 (6H, s), 1.11 (3H, d , J = 6.5 Hz).
(14) Optically active form of 4- (3-methyl-1,6-diazaspiro [3.4] oct-6-yl) -7H-pyrrolo [2,3-d] pyrimidine dihydrochloride
Optically active form of 3-methyl-6- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1,6-diazaspiro [3.4] octane-1-carboxylic acid tert-butyl ester (9 0.5 g), 4M hydrochloric acid 1,4-dioxane (50 ml), chloroform (50 ml) and methanol (100 ml) were mixed and stirred at 60 ° C. for 30 minutes. The mixture was concentrated under reduced pressure and azeotroped with toluene to give the title compound (9.3 g).
1 H-NMR (DMSO-D 6 ) δ: 12.91 (1H, br s), 9.97-9.64 (2H, m), 8.45-8.35 (1H, m), 7.58-7.47 (1H, m), 7.04-6.92 (1H, m), 4.99-4.65 (1H, m), 4.32-3.21 (7H, m), 3.04-2.90 (1H, m), 2.46-2.31 (1H, m), 1.27 (3H, d, J = 6.0 Hz).
(15) 3- [3-Methyl-6- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1,6-diazaspiro [3.4] oct-1-yl] -3-oxo Optically active form of propionitrile
4- (3-Methyl-1,6-diazaspiro [3.4] oct-6-yl) -7H-pyrrolo [2,3-d] pyrimidine dihydrochloride optically active substance (8.8 g) was converted to 1- The mixture was mixed with cyanoacetyl-3,5-dimethylpyrazole (6.8 g), N, N-diisopropylethylamine (20 ml) and 1,4-dioxane (100 ml) and stirred at 100 ° C. for 1 hour. The mixture was cooled to room temperature, saturated aqueous sodium hydrogen carbonate solution was added, and the mixture was extracted with chloroform / methanol (10/1). The separated organic layer was washed with a saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (developing solvent: chloroform / methanol = 30/1 to 9/1). The residue obtained by concentration under reduced pressure was slurry washed with n-heptane / ethanol (2/1, 90 ml) to obtain a solid (7.3 g). The solid was slurried again with n-heptane / ethanol (5/1, 90 ml) to give the title compound as crystals 1 (6.1 g).
1 H-NMR (DMSO-D 6 ) δ: 11.60 (1H, br s), 8.08 (1H, s), 7.11 (1H, dd, J = 3.5, 2.4 Hz), 6.58 (1H, dd, J = 3.4 , 1.9 Hz), 4.18-4.14 (1H, m), 4.09-3.93 (3H, m), 3.84-3.73 (1H, m), 3.71 (1H, d, J = 19.0 Hz), 3.66 (1H, d, J = 18.7 Hz), 3.58 (1H, dd, J = 8.2, 6.0 Hz), 2.70-2.58 (2H, m), 2.24-2.12 (1H, m), 1.12 (3H, d, J = 7.1 Hz).
[Α] D = + 47.09 ° (25 ° C., c = 0.55, methanol)
1-Butanol (39 ml) was added to the obtained crystal 1 (2.6 g), and the mixture was heated and stirred at 100 ° C. After complete dissolution, the solution was cooled to room temperature by 10 ° C. every 30 minutes and further stirred at room temperature overnight. The produced crystals were collected by filtration, washed with 1-butanol (6.2 ml), and dried under reduced pressure to give crystals 2 (2.1 g) of the title compound.
PATENTS
WO 2017006968
WO 2018117152
WO 2018117151
PATENT
WO 2018117153
https://patentscope.wipo.int/search/zh/detail.jsf?docId=WO2018117153&tab=FULLTEXT
Janus kinase (JAK) inhibitors are of current interest for the treatment of various diseases including autoimmune diseases, inflammatory diseases, and cancer. To date, two JAK inhibitors have been approved by the U.S. Food & Drug Administration (FDA). Ruxolitinib has been approved for the treatment of primary myelofibrosis and polycythemia vera (PV), and tofacitinib has been approved for the treatment of rheumatoid arthritis. Other JAK inhibitors are in the literature. The compound 3-((3S,4R)-3-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1,6-diazaspiro[3.4]octan-1-yl)-3-oxopropanenitrile (Compound A) (see structure below) is an example of a spirocyclic JAK inhibitor reported in U.S. Pat. Pub. Nos. 2011/0136778 and International Pat. Pub. No. PCT/JP2016/070046.
[Chem. 1]
Step A. Preparation of S-MABB-HC (Compound [5])
[Chem. 2]
Step 1
[Chem. 3]
S-BAPO [1] (35.0 g, 212 mmol) was added to water (175 mL) at room temperature under nitrogen atmosphere. To the resulting suspension were added toluene (53 mL) and potassium carbonate (32.2 g, 233 mmol) at room temperature. To the resulting solution was added dropwise TBBA (434.4 g, 223 mmol) at room temperature, and then the used dropping funnel was washed with toluene (17 mL) and the washings were added to the reaction mixture. The reaction mixture was stirred at 65°C for 21 hours, and then cooled to room temperature. After toluene (105 mL) was added to the reaction mixture and then the mixture was stirred, the organic layer was separated out. The organic layer was washed with water (175 mL), aqueous layer was removed, and then the solvent was removed out of the organic layer in vacuo. Toluene (105 mL) was added to the residue and the toluene solution was concentrated. The operation was repeated two more times to give a toluene solution of S-BBMO [2] (74.0 g, 212 mmol in theory). The given toluene solution of S-BBMO was used in the next step, assuming that the yield was 100 %.
A crude product of S-BBMO which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 7.36-7.13 (5H, m), 4.26 (1H, dd, J = 6.8, 3.9 Hz), 3.72 (2H, dd, J = 14.2, 6.8 Hz), 3.47-3.38 (1H, m), 3.30-3.08 (3H, m), 2.79 (1H, sext, J = 6.8 Hz), 1.35 (9H, s), 0.96 (3H, d, J = 6.8 Hz).
MS: m/z = 280 [M+H] +
[Chem. 2]
Step 1
[Chem. 3]
A crude product of S-BBMO which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 7.36-7.13 (5H, m), 4.26 (1H, dd, J = 6.8, 3.9 Hz), 3.72 (2H, dd, J = 14.2, 6.8 Hz), 3.47-3.38 (1H, m), 3.30-3.08 (3H, m), 2.79 (1H, sext, J = 6.8 Hz), 1.35 (9H, s), 0.96 (3H, d, J = 6.8 Hz).
MS: m/z = 280 [M+H] +
[0134]
Step 2
[Chem. 4]
To the toluene solution of S-BBMO [2] (74.0 g, 212 mmol) were added toluene (200 mL), tetrahydrofuran (35 mL), and then triethylamine (25.7 g, 254 mmol) at room temperature under nitrogen atmosphere. To the mixture was added dropwise methanesulfonyl chloride (26.7 g, 233 mmol) at 0°C, and then the used dropping funnel was washed with toluene (10 mL) and the washings were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours and further at 65°C for 22 hours, and then cooled to room temperature. After sodium bicarbonate water (105 mL) was added to the reaction mixture and then the mixture was stirred, the organic layer was separated out. The organic layer was washed with water (105 mL), aqueous layer was removed, and then the solvent was removed out of the organic layer in vacuo. Toluene (105 mL) was added to the residue, and the toluene solution was concentrated. The operation was repeated two more times to give a toluene solution of R-BCAB [3] (75.3 g, 212 mmol in theory). The given toluene solution of R-BCAB was used in the next step, assuming that the yield was 100 %.
A crude product of R-BCAB which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 7.28-7.11 (5H, m), 4.24-4.11 (1H, m), 3.80 (2H, d, J = 3.6 Hz), 3.24 (2H, d, J = 3.6 Hz), 2.98-2.78 (2H, m), 1.46-1.37 (12H, m).
MS: m/z = 298 [M+H] +
[Chem. 4]
A crude product of R-BCAB which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 7.28-7.11 (5H, m), 4.24-4.11 (1H, m), 3.80 (2H, d, J = 3.6 Hz), 3.24 (2H, d, J = 3.6 Hz), 2.98-2.78 (2H, m), 1.46-1.37 (12H, m).
MS: m/z = 298 [M+H] +
[0135]
Step 3
[Chem. 5]
To the toluene solution of R-BCAB [3] (75.3 g, 212 mmol) were added tetrahydrofuran (88.0 mL) and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (42.0 mL) at room temperature under nitrogen atmosphere. To the resulting solution was added dropwise a solution of lithium bis(trimethylsilyl)amide /tetrahydrofuran (195 mL, 233 mmol) at 0°C, and then the used dropping funnel was washed with tetrahydrofuran (17.0 mL) and the washings were added to the reaction mixture. The reaction mixture was stirred at 0°C for 1 hour, and then warmed to room temperature. After water (175 mL) and toluene (175 mL) were added to the reaction mixture and then the mixture was stirred, the organic layer was separated out. The resulting organic layer was washed with aqueous ammonium chloride (175 mL) and then water (175 mL), and the solvent was removed out of the organic layer in vacuo. Ethyl acetate (175 mL) was added to the residue and the ethyl acetate solution was concentrated. The operation was repeated two more times to give an ethyl acetate solution of S-MABB [4] (66.5 g, 212 mmol in theory). The given ethyl acetate solution of S-MABB was used in the next step, assuming that the yield was 100 %.
A crude product of S-MABB which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 7.28-7.25 (10H, m), 3.75 (1H, d, J = 12.7 Hz), 3.68 (1H, d, J = 1.4 Hz), 3.66 (1H, d, J = 6.7 Hz), 3.46 (2H, d, J = 12.7 Hz), 3.30-3.17 (2H, m), 2.95 (1H, dd, J = 6.2, 1.2 Hz), 2.77 (1H, dd, J = 6.1, 2.2 Hz), 2.65-2.55 (1H, m), 2.48-2.40 (2H, m), 1.35 (9H, s), 1.35 (9H, s), 1.12 (3H, d, J = 7.2 Hz), 1.09 (3H, d, J = 6.2 Hz).
MS: m/z = 262 [M+H] +
[Chem. 5]
A crude product of S-MABB which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 7.28-7.25 (10H, m), 3.75 (1H, d, J = 12.7 Hz), 3.68 (1H, d, J = 1.4 Hz), 3.66 (1H, d, J = 6.7 Hz), 3.46 (2H, d, J = 12.7 Hz), 3.30-3.17 (2H, m), 2.95 (1H, dd, J = 6.2, 1.2 Hz), 2.77 (1H, dd, J = 6.1, 2.2 Hz), 2.65-2.55 (1H, m), 2.48-2.40 (2H, m), 1.35 (9H, s), 1.35 (9H, s), 1.12 (3H, d, J = 7.2 Hz), 1.09 (3H, d, J = 6.2 Hz).
MS: m/z = 262 [M+H] +
[0136]
Step 4
[Chem. 6]
To the ethyl acetate solution of S-MABB [4] (66.5 g, 212 mmol in theory) were added ethyl acetate (175 mL) and active carbon (3.5 g) under nitrogen atmosphere, and then the mixture was stirred at room temperature for 2 hours. The active carbon was removed by filtration, and the residue on the filter was washed with ethyl acetate (175 mL). The washings were added to the filtrate. To the solution was added S-MABB-HC crystal (17.5 mg) that was prepared according to the method described herein at 0°C, and then 4 M hydrogen chloride/ethyl acetate (53.0 mL, 212 mmol) was dropped thereto at 0°C. The reaction mixture was stirred at 0°C for 17 hours, and then the precipitated solid was collected on a filter, and washed with ethyl acetate (70 mL). The resulting wet solid was dried in vacuo to give S-MABB-HC [5] (48.3 g, 162 mmol, yield: 76.4 %).
S-MABB-HC which was prepared by the same process was measured about NMR, MS, and Cl-content.
1H-NMR (DMSO-d 6) δ: 11.08 (1H, br s), 10.94 (1H, br s), 7.52-7.42 (10H, m), 5.34 (1H, t, J = 8.4 Hz), 4.90 (1H, br s), 4.45-4.10 (5H, m), 3.92-3.49 (3H, br m), 3.10-2.73 (2H, br m), 1.35 (9H, s), 1.29 (9H, s), 1.24 (3H, d, J = 6.7 Hz), 1.17 (3H, d, J = 7.4 Hz).
MS: m/z = 262 [M+H-HCl] +
Cl content (ion chromatography): 11.9 % (in theory: 11.9 %).
[Chem. 6]
S-MABB-HC which was prepared by the same process was measured about NMR, MS, and Cl-content.
1H-NMR (DMSO-d 6) δ: 11.08 (1H, br s), 10.94 (1H, br s), 7.52-7.42 (10H, m), 5.34 (1H, t, J = 8.4 Hz), 4.90 (1H, br s), 4.45-4.10 (5H, m), 3.92-3.49 (3H, br m), 3.10-2.73 (2H, br m), 1.35 (9H, s), 1.29 (9H, s), 1.24 (3H, d, J = 6.7 Hz), 1.17 (3H, d, J = 7.4 Hz).
MS: m/z = 262 [M+H-HCl] +
Cl content (ion chromatography): 11.9 % (in theory: 11.9 %).
[0137]
Step B. Preparation of S-MACB-HC (Compound [6])
[Chem. 7]
To a solution of S-MABB-HC [5] (5.0 g, 16.8 mmol) in methanol (15.0 mL) was added 5 % palladium carbon (made by Kawaken Fine Chemicals Co., Ltd., PH type, 54.1 % water-content 1.0 g) at room temperature under nitrogen atmosphere. The reaction vessel was filled with hydrogen, the reaction mixture was stirred at hydrogen pressure of 0.4 MPa at room temperature for 12 hours, the hydrogen in the reaction vessel was replaced with nitrogen, and then the 5 % palladium carbon was removed by filtration. The reaction vessel and the 5 % palladium carbon were washed with methanol (10 mL). The washings were added to the filtrate to give a methanol solution of S-MACB-HC [6] (24.8 g, 16.8 mmol in theory). The given methanol solution of S-MACB-HC was used in the next step, assuming that the yield was 100 %.
A crude product of S-MACB-HC which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 9.60 (br s, 1H), 4.97 (d, 1H, J = 9.2 Hz), 4.61 (d, 1H, J = 8.4 Hz), 4.01 (dd, 1H, J = 10.0, 8.4 Hz), 3.78-3.74 (m, 1H), 3.54 (dd, 1H, J = 9.6, 8.4 Hz), 3.35 (dd, 1H, J = 10.0, 6.0 Hz), 3.15-3.03 (m, 1H), 3.00-2.88 (m, 1H), 1.49 (s, 9H), 1.47 (s, 9H), 1.22 (d, 3H, J = 6.8 Hz), 1.14 (d, 3H, J = 7.2 Hz).
MS: m/z = 172 [M+H] + (free form)
[Chem. 7]
A crude product of S-MACB-HC which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 9.60 (br s, 1H), 4.97 (d, 1H, J = 9.2 Hz), 4.61 (d, 1H, J = 8.4 Hz), 4.01 (dd, 1H, J = 10.0, 8.4 Hz), 3.78-3.74 (m, 1H), 3.54 (dd, 1H, J = 9.6, 8.4 Hz), 3.35 (dd, 1H, J = 10.0, 6.0 Hz), 3.15-3.03 (m, 1H), 3.00-2.88 (m, 1H), 1.49 (s, 9H), 1.47 (s, 9H), 1.22 (d, 3H, J = 6.8 Hz), 1.14 (d, 3H, J = 7.2 Hz).
MS: m/z = 172 [M+H] + (free form)
[0138]
Step C. Preparation of S-ZMAB (Compound [7])
[Chem. 8]
To the methanol solution of S-MACB-HC [6] (24.8 g, 16.8 mmol in theory) was added dropwise N,N-diisopropylethylamine (4.8 g, 36.9 mmol) at room temperature under nitrogen atmosphere, and then the used dropping funnel was washed with tetrahydrofuran (2.5 mL) and the washings were added to the reaction mixture. To the resulting reaction mixture was added dropwise benzyl chloroformate (3.0 g, 17.6 mmol) at 0°C, and then the used dropping funnel was washed with tetrahydrofuran (2.5 mL) and the washings were added to the reaction mixture. The reaction mixture was stirred at 0°C for 1 hour, and then the solvent was removed in vacuo. After toluene (25.0 mL) and an aqueous solution of citric acid (25.0 mL) was added to the residue and then the mixture was stirred, the organic layer was separated out. The resulting organic layer was washed with sodium bicarbonate water (25.0 mL) and then water (25.0 mL), and the solvent in the organic layer was removed out of the organic layer in vacuo. Toluene (15.0 mL) was added to the residue and the toluene solution was concentrated. The operation was repeated one more time to give a toluene solution of S-ZMAB [7] (6.9 g, 16.8 mmol in theory). The given toluene solution of S-ZMAB was used in the next step, assuming that the yield was 100 %.
A crude product of S-ZMAB which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (CDCl 3) δ: 7.38-7.28 (m, 10H), 5.16-5.04 (m, 4H), 4.60 (d, 1H, J = 9.2 Hz), 4.18-4.12 (m, 2H), 4.04 (t, 1H, J = 8.6 Hz), 3.66 (dd, 1H, J = 7.6, 7.2 Hz), 3.50 (dd, 1H, J = 8.0, 5.2 Hz), 3.05-2.94 (m, 1H), 2.60-2.50 (m, 1H), 1.43 (br s, 18H), 1.33 (d, 3H, J = 6.5 Hz), 1.15 (d, 3H, J = 7.2 Hz).
MS: m/z = 328 [M+Na] +.
[Chem. 8]
A crude product of S-ZMAB which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (CDCl 3) δ: 7.38-7.28 (m, 10H), 5.16-5.04 (m, 4H), 4.60 (d, 1H, J = 9.2 Hz), 4.18-4.12 (m, 2H), 4.04 (t, 1H, J = 8.6 Hz), 3.66 (dd, 1H, J = 7.6, 7.2 Hz), 3.50 (dd, 1H, J = 8.0, 5.2 Hz), 3.05-2.94 (m, 1H), 2.60-2.50 (m, 1H), 1.43 (br s, 18H), 1.33 (d, 3H, J = 6.5 Hz), 1.15 (d, 3H, J = 7.2 Hz).
MS: m/z = 328 [M+Na] +.
[0139]
Step D. Preparation of RS-ZMBB (Compound [8])
[Chem. 9]
To the toluene solution of S-ZMAB [7] (6.9 g, 16.8 mmol) was added tetrahydrofuran (15.0 mL) at room temperature under nitrogen atmosphere. A solution of lithium bis(trimethylsilyl)amide/tetrahydrofuran (14.7 mL, 17.6 mmol) was added dropwise to the toluene solution at -70°C. The used dropping funnel was washed with tetrahydrofuran (2.5 mL) and the washings were added to the reaction mixture. The reaction mixture was stirred at -70°C for 6 hours, and then a solution of TBBA (3.4 g, 17.6 mmol) in tetrahydrofuran (2.5 mL) was added dropwise to the reaction mixture at -70°C. The used dropping funnel was washed with tetrahydrofuran (2.5 mL) and the washings were added to the reaction mixture. The reaction mixture was stirred at -70°C for 1 hour, and then warmed to room temperature. To the reaction mixture were added an aqueous ammonium chloride (25 mL) and toluene (25 mL) and then the mixture was stirred, the organic layer was separated out. The resulting organic layer was washed with an aqueous solution of citric acid (25 mL, x 2), sodium bicarbonate water (25 mL), and then water (25 mL), and then the solvent was removed out of the organic layer in vacuo. Acetonitrile (15 mL) was added to the residue and the acetonitrile solution was concentrated. The operation was repeated two more times. Acetonitrile (15 mL) and active carbon (0.25 g) were added to the residue, the mixture was stirred at room temperature for 2 hours. The active carbon was removed by filtration, and the reaction vessel and the residue on the filter was washed with acetonitrile (10 mL). The washings were added to the filtration, and then the filtration was concentrated in vacuo to give an acetonitrile solution of RS-ZMBB [8] (13.2 g, 16.8 mmol in theory). The given acetonitrile solution of RS-ZMBB was used in the next step, assuming that the yield was 100 %.
A crude product of RS-ZMBB which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 7.38-7.29 (m, 5H), 5.09-4.96 (m, 2H), 3.91 (t, 0.4H, J = 8.0 Hz), 3.79 (t, 0.6H, J = 8.0 Hz), 3.55 (t, 0.4H, J = 7.2 Hz), 3.46 (t, 0.6H, J = 7.5 Hz), 3.14-3.04 (m, 1H), 2.83-2.72 (m, 2H), 1.38 (br s, 9H), 1.37 (br s, 3.6H), 1.34 (br s, 5.4H), 1.12-1.09 (m, 3H).
MS: m/z = 420 [M+H] +.
[Chem. 9]
A crude product of RS-ZMBB which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 7.38-7.29 (m, 5H), 5.09-4.96 (m, 2H), 3.91 (t, 0.4H, J = 8.0 Hz), 3.79 (t, 0.6H, J = 8.0 Hz), 3.55 (t, 0.4H, J = 7.2 Hz), 3.46 (t, 0.6H, J = 7.5 Hz), 3.14-3.04 (m, 1H), 2.83-2.72 (m, 2H), 1.38 (br s, 9H), 1.37 (br s, 3.6H), 1.34 (br s, 5.4H), 1.12-1.09 (m, 3H).
MS: m/z = 420 [M+H] +.
[0140]
Step E. Preparation of RS-ZMAA-DN . 2H 2 O (Compound [9])
[Chem. 10]
To the acetonitrile solution of RS-ZMBB [8] (13.2 g, 16.8 mmol in theory) was added acetonitrile (15 mL) at room temperature under nitrogen atmosphere. p-Toluenesulfonic acid mono-hydrate (6.4 g, 33.6 mmol) was added to the solution at room temperature. The reaction mixture was stirred at 50°C for 12 hours, and then cooled to room temperature, and water (7.5 mL) was added dropwise to the reaction mixture. The reaction mixture was cooled to 0°C, and then 4 mol/L aqueous sodium hydroxide (17.6 mL, 70.5 mmol) was added dropwise thereto. After stirring the reaction mixture at room temperature for 1 hour, acetonitrile (75 mL) was added dropwise thereto at room temperature, and the reaction mixture was stirred for 3 hours. The precipitated solid was collected on a filter, and washed with a mixture of acetonitrile : water = 4 : 1 (10 mL) and then acetonitrile (10 mL). The resulting wet solid was dried in vacuo to give RS-ZMAA-DN .2H 2O [9] (5.2 g, 13.4 mmol, yield: 85.4 %).
RS-ZMAA-DN .2H 2O which was prepared by the same process was measured about NMR, MS, Na-content, and water-content.
1H-NMR (DMSO-d 6) δ: 7.32-7.22 (m, 5H), 4.97 (d, 1H, J = 12.7 Hz), 4.84 (d, 1H, J = 12.7 Hz), 3.79 (t, 1H, J = 8.0 Hz), 3.29 (d, 1H, J = 14.8 Hz), 3.16-3.12 (m, 1H), 2.17-2.09 (m, 2H), 1.07 (d, 3H, J = 6.9 Hz).
MS: m/z = 352 [M+H] + (anhydrate)
Na content (ion chromatography): 13.3 % (after correction of water content)(13.1 % in theory)
Water content (Karl Fischer’s method): 9.8 % (9.3 % in theory)
[Chem. 10]
RS-ZMAA-DN .2H 2O which was prepared by the same process was measured about NMR, MS, Na-content, and water-content.
1H-NMR (DMSO-d 6) δ: 7.32-7.22 (m, 5H), 4.97 (d, 1H, J = 12.7 Hz), 4.84 (d, 1H, J = 12.7 Hz), 3.79 (t, 1H, J = 8.0 Hz), 3.29 (d, 1H, J = 14.8 Hz), 3.16-3.12 (m, 1H), 2.17-2.09 (m, 2H), 1.07 (d, 3H, J = 6.9 Hz).
MS: m/z = 352 [M+H] + (anhydrate)
Na content (ion chromatography): 13.3 % (after correction of water content)(13.1 % in theory)
Water content (Karl Fischer’s method): 9.8 % (9.3 % in theory)
[0141]
Step F. Preparation of RS-ZMAA (Compound [10])
[Chem. 11]
To 1 mol/L hydrochloric acid (180 mL) were added RS-ZMAA-DN .2H 2O [9] (30 g, 77.5 mmol) and acetonitrile (60 mL), and the mixture was stirred at room temperature for about 15 minutes. After ethyl acetate (240 mL) was added to the reaction mixture and then the mixture was stirred, the organic layer was separated out. The organic layer was washed with 10 % brine (60 mL x 2). The organic layer was stirred with magnesium sulfate (6 g), the magnesium sulfate was removed by filtration, and the residue on the filter was washed with ethyl acetate (60 mL). The filtrate and the washings are combined, and the solvent was removed out in vacuo. Tetrahydrofuran (240 mL) was added to the residue and the tetrahydrofuran solution was concentrated. The operation was repeated two more times. Tetrahydrofuran (60 mL) was added to the residue to give a tetrahydrofuran solution of RS-ZMAA [10]. The given tetrahydrofuran solution of RS-ZMAA was used in the next step, assuming that the yield was 100 %.
RS-ZMAA which was prepared by the same process was measured about NMR and MS.
1H-NMR (DMSO-D 6) δ: 7.35-7.28 (m, 5H), 5.06-4.94 (m, 2H), 3.86 (dt, 1H, J = 48.4, 7.9 Hz), 3.50 (dt, 1H, J = 37.9, 7.4 Hz), 3.16-3.02 (br m, 1H), 2.91-2.77 (br m, 2H), 1.08 (d, 3H, J = 6.9 Hz)
MS: m/z = 308 [M+H] +.
[Chem. 11]
RS-ZMAA which was prepared by the same process was measured about NMR and MS.
1H-NMR (DMSO-D 6) δ: 7.35-7.28 (m, 5H), 5.06-4.94 (m, 2H), 3.86 (dt, 1H, J = 48.4, 7.9 Hz), 3.50 (dt, 1H, J = 37.9, 7.4 Hz), 3.16-3.02 (br m, 1H), 2.91-2.77 (br m, 2H), 1.08 (d, 3H, J = 6.9 Hz)
MS: m/z = 308 [M+H] +.
[0142]
Step G. Preparation of RS-ZMOO (Compound [11])
[Chem. 12]
To the tetrahydrofuran solution of RS-ZMAA [10] (25.8 mmol in theory) was added tetrahydrofuran (50 mL) under nitrogen atmosphere. Boron trifluoride etherate complex (4.40 g) was added dropwise thereto at 0°C to 5°C. The used dropping funnel was washed with tetrahydrofuran (5 mL) and the washings were added to the reaction mixture. To the reaction mixture was added dropwise 1.2 mol/L borane-tetrahydrofuran complex (43.0 mL) at 0°C to 5°C, and the reaction mixture was stirred at 0°C to 5°C for about 30 minutes, and then further stirred at room temperature overnight. To the reaction mixture was added dropwise 1.2 mol/L borane-tetrahydrofuran complex (21.1 mL) at 0°C to 5°C, and then the reaction mixture was stirred at room temperature overnight. After stirring, water (40 mL) was added dropwise to the reaction mixture at 0°C to 15°C. To the reaction mixture was added sodium bicarbonate (5.42 g) at 0°C to 15°C. The sodium bicarbonate left in the vessel was washed with water (10 mL), and the washings were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours, and then toluene (50 mL) was added thereto and the reaction mixture was further stirred. The organic layer was separated out. The resulting organic layer was washed with 10 % brine (20 mL x 1), a mixture (x 3) of 5 % sodium bicarbonate water (20 mL) and 10 % brine (20 mL), a mixture (x 1) of 5 % aqueous potassium hydrogensulfate (10 mL) and 10 % brine (10 mL), and then 10 % brine (20 mL x 2). The organic layer was stirred with magnesium sulfate (8.9 g), the magnesium sulfate was removed by filtration, and the residue on the filter was washed with toluene (20 mL). The washings were added to the filtration, and then the filtrate was concentrated in vacuo. To the concentrated residue was added toluene (80 mL). The solution was concentrated in vacuo, and toluene (15 mL) was added thereto to give a toluene solution of RS-ZMOO [11]. The given toluene solution of RS-ZMOO was used in the next step, assuming that the yield was 100 %.
RS-ZMOO which was prepared by the same process was measured about NMR and MS.
1H-NMR (CDCl 3) δ: 7.39-7.30 (m, 5H), 5.10 (s, 2H), 4.15-4.01 (br m, 2H), 3.83-3.73 (br m, 3H), 3.48 (dd, 1H, J = 8.3, 6.4 Hz), 2.59-2.50 (br m, 1H), 2.46-2.40 (br m, 1H), 2.07-1.99 (m, 1H), 1.14 (d, 3H, J = 7.2 Hz)
MS: m/z = 280 [M+H]+.
[Chem. 12]
RS-ZMOO which was prepared by the same process was measured about NMR and MS.
1H-NMR (CDCl 3) δ: 7.39-7.30 (m, 5H), 5.10 (s, 2H), 4.15-4.01 (br m, 2H), 3.83-3.73 (br m, 3H), 3.48 (dd, 1H, J = 8.3, 6.4 Hz), 2.59-2.50 (br m, 1H), 2.46-2.40 (br m, 1H), 2.07-1.99 (m, 1H), 1.14 (d, 3H, J = 7.2 Hz)
MS: m/z = 280 [M+H]+.
[0143]
Step H. Preparation of RS-ZMSS (Compound [12])
[Chem. 13]
To the toluene solution of RS-ZMOO [11] (23.7 mmol in theory) was added toluene (55 mL) under nitrogen atmosphere. And, triethylamine (5.27 g) was added dropwise thereto at -10°C to 10°C, and the used dropping funnel was washed with toluene (1.8 mL) and the washings were added to the reaction mixture. To this reaction mixture was added dropwise methanesulfonyl chloride (5.69 g) at -10°C to 10°C, and then the used dropping funnel was washed with toluene (1.8 mL) and the washings were added to the reaction mixture. The reaction mixture was stirred at 0°C to 10°C for about 2 hours, and then water (28 mL) was added dropwise thereto at 0°C to 20°C. The reaction mixture was stirred at 0°C to 20°C for about 30 minutes, and then, the organic layer was separated out. The resulting organic layer was washed twice with 10 % brine (18 mL). The organic layer was stirred with magnesium sulfate (2.75 g), the magnesium sulfate was removed by filtration, and the residue on the filter was washed with toluene (18 mL). The washings were added to the filtrate, and then the solvent was removed from the filtrate in vacuo. To the concentrated residue was added toluene up to 18 mL to give a toluene solution of RS-ZMSS [12]. The given toluene solution of RS-ZMSS was used in the next step, assuming that the yield was 100 %.
RS-ZMSS which was prepared by the same process was measured by NMR and MS.
1H-NMR (DMSO-D 6) δ: 7.37-7.27 (br m, 5H), 5.10-4.98 (m, 2H), 4.58-4.22 (br m, 4H), 3.84 (dt, 1H, J = 45.6, 8.1 Hz), 3.48-3.33 (br m, 1H), 3.17-3.10 (m, 6H), 2.81-2.74 (br m, 1H), 2.22-2.12 (m, 2H)
MS: m/z = 436 [M+H] +.
[Chem. 13]
RS-ZMSS which was prepared by the same process was measured by NMR and MS.
1H-NMR (DMSO-D 6) δ: 7.37-7.27 (br m, 5H), 5.10-4.98 (m, 2H), 4.58-4.22 (br m, 4H), 3.84 (dt, 1H, J = 45.6, 8.1 Hz), 3.48-3.33 (br m, 1H), 3.17-3.10 (m, 6H), 2.81-2.74 (br m, 1H), 2.22-2.12 (m, 2H)
MS: m/z = 436 [M+H] +.
[0144]
Step I. Preparation of SR-ZMDB (Compound [13])
[Chem. 14]
To a toluene solution of RS-ZMSS [12] (23.7 mmol in theory) was added toluene (55 mL) under nitrogen atmosphere. And, benzylamine (17.8 g) was added dropwise thereto at room temperature, and the used dropping funnel was washed with toluene (9.2 mL) and the washings were added to the reaction mixture. The reaction mixture was stirred at room temperature for about 1 hour, at 55°C to 65°C for about 3 hours, and then at 70°C to 80°C for 6 hours. After the reaction mixture was cooled to room temperature, 10 % NaCl (28 mL) was added dropwise thereto, and the reaction mixture was stirred at room temperature for about 30 minutes. After toluene (37 mL) was added to the reaction mixture and then the mixture was stirred, the organic layer was separated out. The resulting organic layer was washed with a mixture (x 2) of 10 % brine (18 mL) and acetic acid (2.84 g), and then 10 % brine (11 mL, x 1). The solvent of the organic layer was removed in vacuo to a half volume, and acetic anhydride (1.45 g) was added to the concentrated residue at room temperature. The mixture was stirred for about 3 hours. To the reaction mixture were added dropwise a solution of potassium hydrogensulfate (3.87 g) and water (92 mL) at room temperature. The reaction mixture was stirred, and then the aqueous layer was separated out. The resulting aqueous layer was washed with toluene (18 mL), and toluene (73 mL) and then sodium bicarbonate (6.56 g) were added to the aqueous layer at room temperature, and the mixture was stirred. The organic layer was separated out, and washed with 10 % brine (11 mL). The organic layer was stirred with magnesium sulfate (2.75 g), the magnesium sulfate was removed by filtration. The residue on the filter was washed with toluene (18 mL), and the washings were added to the filtrate, and then the filtrate was concentrated in vacuo. Toluene (44 mL) was added to the concentrated residue to give a toluene solution of SR-ZMDB [13]. The given toluene solution of SR-ZMDB was used in the next step, assuming that the yield was 100 %.
1H-NMR (CDCl 3) δ: 7.35-7.20 (m, 10H), 5.08 (d, 2H, J = 23.6 Hz), 3.94 (q, 1H, J = 7.9 Hz), 3.73-3.42 (br m, 2H), 3.30-3.23 (m, 1H), 3.05 (dd, 1H, J = 19.7, 9.5 Hz), 2.79 (dt, 1H, J = 69.6, 6.1 Hz), 2.57-2.32 (br m, 4H), 1.96-1.89 (m, 1H), 1.09 (d, 3H, J = 6.9 Hz)
MS: m/z = 351 [M+H] +.
[Chem. 14]
1H-NMR (CDCl 3) δ: 7.35-7.20 (m, 10H), 5.08 (d, 2H, J = 23.6 Hz), 3.94 (q, 1H, J = 7.9 Hz), 3.73-3.42 (br m, 2H), 3.30-3.23 (m, 1H), 3.05 (dd, 1H, J = 19.7, 9.5 Hz), 2.79 (dt, 1H, J = 69.6, 6.1 Hz), 2.57-2.32 (br m, 4H), 1.96-1.89 (m, 1H), 1.09 (d, 3H, J = 6.9 Hz)
MS: m/z = 351 [M+H] +.
[0145]
Step J. Preparation of SR-MDOZ (Compound [14])
[Chem. 15]
To a solution of 1-chloroethyl chloroformate (3.72 g) in toluene (28 mL) was added dropwise the toluene solution of SR-ZMDB [13] (23.7 mmol in theory) at 0°C to 10°C under nitrogen atmosphere, and then the used dropping funnel was washed with toluene (4.6 mL) and the washings were added to the reaction mixture. To the reaction mixture was added triethylamine (718 mg) at 0°C to 10°C, and the reaction mixture was stirred at 15°C to 25°C for about 2 hours. Then, methyl alcohol (46 mL) was added to the reaction mixture, and the mixture was stirred at 50°C to 60°C for additional about 2 hours. The solvent of the reaction mixture was removed in vacuo to a volume of about less than 37 mL. To the concentrated residue was added dropwise 2 mol/L hydrochloric acid (46 mL) at 15°C to 20°C, and the mixture was stirred, and the aqueous layer was separated out. The resulting aqueous layer was washed with toluene (28 mL, x 2). To the aqueous layer were added 20 % brine (46 mL) and tetrahydrofuran (92 mL), and then 8 mol/L aqueous sodium hydroxide (18 mL) was added dropwise thereto at 0°C to 10°C. The organic layer was separated out from the reaction mixture, washed with 20 % brine (18 mL, x 2), and then the solvent of the organic layer was removed in vacuo. To the concentrated residue was added tetrahydrofuran (92 mL), and the solution was concentrated in vacuo. The operation was repeated one more time. The concentrated residue was dissolved in tetrahydrofuran (92 mL). The solution was stirred with magnesium sulfate (2.75 g), and the magnesium sulfate was removed by filtration. The residue on the filter was washed with tetrahydrofuran (28 mL), the washings were added to the filtrate, and the filtrate was concentrated in vacuo. The volume of the concentrated residue was adjusted to about 20 mL with tetrahydrofuran to give a tetrahydrofuran solution of SR-MDOZ [14] (net weight: 4.01 g, 15.4 mol, yield: 65.0 %).
SR-MDOZ which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (CDCl 3) δ: 7.37-7.28 (m, 5H), 5.08 (dd, 2H, J = 16.8, 12.8 Hz), 4.00 (dd, 1H, J = 17.1, 8.3 Hz), 3.40-3.31 (m, 1H), 3.24 (d, 1H, J = 12.7 Hz), 3.00 (dd, 1H, J = 54.9, 12.4 Hz), 2.87-2.57 (m, 3H), 2.47-2.27 (m, 1H), 1.91-1.80 (m, 1H), 1.14 (d, 3H, J = 7.2 Hz)
MS: m/z = 261 [M+H] +.
[Chem. 15]
SR-MDOZ which was prepared by the same process was evaporated to dryness and then measured about NMR and MS.
1H-NMR (CDCl 3) δ: 7.37-7.28 (m, 5H), 5.08 (dd, 2H, J = 16.8, 12.8 Hz), 4.00 (dd, 1H, J = 17.1, 8.3 Hz), 3.40-3.31 (m, 1H), 3.24 (d, 1H, J = 12.7 Hz), 3.00 (dd, 1H, J = 54.9, 12.4 Hz), 2.87-2.57 (m, 3H), 2.47-2.27 (m, 1H), 1.91-1.80 (m, 1H), 1.14 (d, 3H, J = 7.2 Hz)
MS: m/z = 261 [M+H] +.
[0146]
Step K. Preparation of SR-MDOZ-OX (Compound [15])
[Chem. 16]
Under nitrogen atmosphere, oxalic acid (761 mg) was dissolved in tetrahydrofuran (40 mL), and the tetrahydrofuran solution of SR-MDOZ [14] (3.84 mmol in theory) was added dropwise to the solution of oxalic acid at room temperature. To the solution was added SR-MDOZ-OX crystal (1 mg) that was prepared according to the method described herein at room temperature, and the mixture was stirred at room temperature for about 3.5 hours to precipitate the crystal. To the slurry solution was added dropwise the tetrahydrofuran solution of SR-MDOZ (3.84 mmol) at room temperature, and the mixture was stirred at room temperature for about 1 hour. The slurry solution was heated, and stirred at 50°C to 60°C for about 2 hours, and then stirred at room temperature overnight. The slurry solution was filtrated, and the wet crystal on the filter was washed with tetrahydrofuran (10 mL), dried in vacuo to give SR-MDOZ-OX [15] (2.32 g, 6.62 mol, yield: 86.2 %).
SR-MDOZ-OX which was prepared by the same process was measured about NMR, MS, and elementary analysis.
1H-NMR (DMSO-D 6) δ: 7.37-7.30 (m, 5H), 5.15-5.01 (m, 2H), 3.92 (dt, 1H, J = 43.5, 8.4 Hz), 3.48-3.12 (br m, 5H), 2.67-2.56 (m, 1H), 2.46-2.35 (m, 1H), 2.12-2.05 (m, 1H), 1.13 (d, 3H, J = 6.9 Hz)
MS: m/z = 261 [M+H] +
elementary analysis: C 58.4wt % , H 6.4wt % , N 7.9 % wt % (theoretically, C 58.3wt % , H 6.3wt % , N 8.0wt % )
[Chem. 16]
SR-MDOZ-OX which was prepared by the same process was measured about NMR, MS, and elementary analysis.
1H-NMR (DMSO-D 6) δ: 7.37-7.30 (m, 5H), 5.15-5.01 (m, 2H), 3.92 (dt, 1H, J = 43.5, 8.4 Hz), 3.48-3.12 (br m, 5H), 2.67-2.56 (m, 1H), 2.46-2.35 (m, 1H), 2.12-2.05 (m, 1H), 1.13 (d, 3H, J = 6.9 Hz)
MS: m/z = 261 [M+H] +
elementary analysis: C 58.4wt % , H 6.4wt % , N 7.9 % wt % (theoretically, C 58.3wt % , H 6.3wt % , N 8.0wt % )
[0147]
Step L. Preparation of SR-MDPZ (Compound [16])
[Chem. 17]
To SR-MDOZ-OX [15] (12.0 g, 34.2 mmol) were added ethanol (36 mL), water (72 mL), CPPY [20] (5.36 g, 34.9 mmol), and then K 3PO 4 (21.8 g, 103 mmol) under nitrogen atmosphere. The reaction mixture was stirred at 80°C for 5 hours, and then cooled to 40°C. Toluene (120 mL) was added thereto at 40°C, and the organic layer was separated out. The resulting organic layer was washed with 20 % aqueous potassium carbonate (48 mL), followed by washing twice with water (48 mL). The solvent of the organic layer was then removed in vacuo. tert-butanol (60 mL) was added to the residue and the tert-butanol solution was concentrated. The operation was repeated two more times. tert-Butanol (36 mL) was added to the concentrated residue to give a solution of SR-MDPZ [16] in tert-butanol (61.1 g, 34.2 mmol in theory). The given tert-butanol solution of SR-MDPZ was used in the next step, assuming that the yield was 100 %.
SR-MDPZ which was prepared by the same process was isolated as a solid from a mixture of ethyl acetate and n-heptane, and then measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 11.59 (br s, 1H), 8.08 (s, 1H), 7.41-7.26 (br m, 3H), 7.22-7.08 (br m, 3H), 6.64-6.51 (br m, 1H), 5.07-4.91 (br m, 2H), 4.09-3.67 (br m, 5H), 3.47-3.32 (br m, 1H), 2.67-2.55 (br m, 2H), 2.21-2.15 (br m, 1H), 1.11 (d, 3H, J = 6.9 Hz).
MS: m/z = 378 [M+H] +
[Chem. 17]
SR-MDPZ which was prepared by the same process was isolated as a solid from a mixture of ethyl acetate and n-heptane, and then measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 11.59 (br s, 1H), 8.08 (s, 1H), 7.41-7.26 (br m, 3H), 7.22-7.08 (br m, 3H), 6.64-6.51 (br m, 1H), 5.07-4.91 (br m, 2H), 4.09-3.67 (br m, 5H), 3.47-3.32 (br m, 1H), 2.67-2.55 (br m, 2H), 2.21-2.15 (br m, 1H), 1.11 (d, 3H, J = 6.9 Hz).
MS: m/z = 378 [M+H] +
[0148]
Step M. Preparation of SR-MDOP (Compound [17])
[Chem. 18]
To the solution of SR-MDPZ [16] in tert-butanol (34.2 mmol in theory) were added ammonium formate (10.8 g, 171 mmol), water (60 mL), and 10 % palladium carbon (made by Kawaken Fine Chemicals Co., Ltd., M type, 52.6 % water-content, 1.20 g) under nitrogen atmosphere. The reaction mixture was stirred at 40°C for 13 hours, and then cooled to room temperature, and the resulting precipitate was removed by filtration. The reaction vessel and the residue on the filter were washed with tert-butanol (24 mL), the washings was added to the filtrate, and 8 M aqueous sodium hydroxide (25.7 mL, 205 mmol) and sodium chloride (13.2 g) were added to the filtrate. The reaction mixture was stirred at 50°C for 2 hours, and then toluene (84 mL) was added thereto at room temperature, and the organic layer was separated out. The resulting organic layer was washed with 20 % brine (60 mL), stirred with anhydrous sodium sulfate, and then the sodium sulfate was removed by filtration. The residue on the filter was washed with a mixture of toluene : tert-butanol = 1 : 1 (48 mL), the washings was added to the filtrate, and the filtrate was concentrated in vacuo. To the concentrated residue was added toluene (60 mL), and the solution was stirred at 50°C for 2 hours, and then the solvent was removed in vacuo. To the concentrated residue was added toluene (60 mL) again, and the solution was concentrated. To the concentrated residue was added toluene (48 mL), and the solution was stirred at room temperature for 1 hour, and then at ice temperature for 1 hour. The precipitated solid was collected on a filter, and washed with toluene (24 mL). The resulting wet solid was dried in vacuo to give SR-MDOP [17] (7.07 g, 29.1 mmol, yield: 84.8 %).
SR-MDOP which was prepared by the same process was measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 11.57 (br s, 1H), 8.07 (s, 1H), 7.10 (d, 1H, J = 3.2 Hz), 6.58 (d, 1H, J = 3.2 Hz), 3.92-3.59 (br m, 4H), 3.49 (dd, 1H, J = 8.3, 7.2 Hz), 2.93 (dd, 1H, J = 7.2, 6.1 Hz), 2.61-2.53 (m, 2H), 2.12-2.01 (br m, 2H), 1.10 (d, 3H, J = 6.9 Hz).
MS: m/z = 244 [M+H] +.
[Chem. 18]
SR-MDOP which was prepared by the same process was measured about NMR and MS.
1H-NMR (DMSO-d 6) δ: 11.57 (br s, 1H), 8.07 (s, 1H), 7.10 (d, 1H, J = 3.2 Hz), 6.58 (d, 1H, J = 3.2 Hz), 3.92-3.59 (br m, 4H), 3.49 (dd, 1H, J = 8.3, 7.2 Hz), 2.93 (dd, 1H, J = 7.2, 6.1 Hz), 2.61-2.53 (m, 2H), 2.12-2.01 (br m, 2H), 1.10 (d, 3H, J = 6.9 Hz).
MS: m/z = 244 [M+H] +.
[0149]
Step N. Preparation of Compound A mono-ethanolate (Compound [18])
[Chem. 19]
Under nitrogen atmosphere, acetonitrile (60 mL) and triethylamine (416 mg, 4.11 mmol) were added to SR-MDOP [17] (5.00 g, 20.5 mmol), and to the solution was added dropwise a solution of DPCN [21] (3.69 g, 22.6 mmol) in acetonitrile (35 mL) at 45°C, and then the used dropping funnel was washed with acetonitrile (5.0 mL) and the washings were added to the reaction mixture. The reaction mixture was stirred at 45°C for 3 hours, and then cooled to room temperature. After 5 % sodium bicarbonate water (25 mL), 10 % brine (25 mL), and ethyl acetate (50 mL) were added to the reaction mixture and then the mixture was stirred, the organic layer was separated out. The solvent of the organic layer was removed out in vacuo. Tetrahydrofuran (50 mL) was added to the residue and the tetrahydrofuran solution was concentrated. The operation was repeated three more times. To the concentrated residue was added tetrahydrofuran (50 mL), and water was added the solution to adjust the water content to 5.5 %. The resulting precipitate was removed by filtration. The reaction vessel and the residue on the filter were washed with tetrahydrofuran (15 mL), the washings were added to the filtrate, and the solvent was removed out of the filtrate in vacuo. To the concentrated residue were added ethanol (50 mL) and Compound A crystal (5.1 mg) that was prepared according to the method described in the following Example 15. The mixture was stirred at room temperature for 1 hour, and concentrated in vacuo. To the residue was ethanol (50 mL), and the solution was concentrated again. To the concentrated residue was added ethanol (15 mL), and the solution was stirred at room temperature for 1 hour. The precipitated solid was collected on the filter, and washed with ethanol (20 mL). The resulting wet solid was dried in vacuo to give Compound A mono-ethanolate [18] (6.26 g, 17.6 mmol, yield: 85.5 %).
Compound A mono-ethanolate which was prepared by the same process was measured by NMR and MS.
1H-NMR (DMSO-d 6) δ: 11.59 (br s, 1H), 8.08 (s, 1H), 7.11 (dd, 1H, J = 3.5, 2.3 Hz), 6.58 (dd, 1H, J = 3.5, 1.8 Hz), 4.34 (t, 1H, J = 5.1 Hz), 4.16 (t, 1H, J = 8.3 Hz), 4.09-3.92 (m, 3H), 3.84-3.73 (m, 1H), 3.71 (d, 1H, J = 19.0 Hz), 3.65 (d, 1H, J = 19.0 Hz), 3.58 (dd, 1H, J = 8.2, 5.9 Hz), 3.44 (dq, 2H, J = 6.7, 5.1 Hz), 2.69-2.60 (m, 2H), 2.23-2.13 (br m, 1H), 1.12 (d, 3H, J = 7.1 Hz), 1.06 (t, 3H, J = 6.7 Hz).
MS: m/z = 311 [M+H] +
[Chem. 19]
Compound A mono-ethanolate which was prepared by the same process was measured by NMR and MS.
1H-NMR (DMSO-d 6) δ: 11.59 (br s, 1H), 8.08 (s, 1H), 7.11 (dd, 1H, J = 3.5, 2.3 Hz), 6.58 (dd, 1H, J = 3.5, 1.8 Hz), 4.34 (t, 1H, J = 5.1 Hz), 4.16 (t, 1H, J = 8.3 Hz), 4.09-3.92 (m, 3H), 3.84-3.73 (m, 1H), 3.71 (d, 1H, J = 19.0 Hz), 3.65 (d, 1H, J = 19.0 Hz), 3.58 (dd, 1H, J = 8.2, 5.9 Hz), 3.44 (dq, 2H, J = 6.7, 5.1 Hz), 2.69-2.60 (m, 2H), 2.23-2.13 (br m, 1H), 1.12 (d, 3H, J = 7.1 Hz), 1.06 (t, 3H, J = 6.7 Hz).
MS: m/z = 311 [M+H] +
[0150]
Step O. Purification of Compound A (Compound [19])
[Chem. 20]
Compound A mono-ethanolate [18] (4.00 g, 11.2 mmol) and n-butanol (32 mL) were mixed under nitrogen atmosphere, and the mixture was dissolved at 110°C. The mixture was cooled to 85°C, and Compound A crystal (4.0 mg) that was prepared according to the method described herein was added thereto, and the mixture was stirred at 85°C for 2 hours, at 75°C for 1 hour, and then at room temperature for 16 hours. The precipitated solid was collected on a filter, and washed with n-butanol (8.0 mL) and then ethyl acetate (8.0 mL). The resulting wet solid was dried in vacuo to give Compound A [19] (3.18 g, 10.2 mmol, yield: 91.3 %).
Compound A which was prepared by the same process was measured by NMR and MS.
1H-NMR (DMSO-d 6) δ: 11.59 (br s, 1H), 8.08 (s, 1H), 7.11 (dd, 1H, J = 3.5, 2.5 Hz), 6.58 (dd, 1H, J = 3.5, 1.8 Hz), 4.16 (t, 1H, J = 8.3 Hz), 4.09-3.93 (m, 3H), 3.84-3.73 (m, 1H), 3.71 (d, 1H, J = 19.0 Hz), 3.65 (d, 1H, J = 19.0 Hz), 3.58 (dd, 1H, J = 8.2, 5.9 Hz), 2.69-2.59 (m, 2H), 2.23-2.13 (m, 1H), 1.12 (d, 3H, J = 7.2 Hz).
MS: m/z = 311 [M+H] +
[Chem. 20]
Compound A which was prepared by the same process was measured by NMR and MS.
1H-NMR (DMSO-d 6) δ: 11.59 (br s, 1H), 8.08 (s, 1H), 7.11 (dd, 1H, J = 3.5, 2.5 Hz), 6.58 (dd, 1H, J = 3.5, 1.8 Hz), 4.16 (t, 1H, J = 8.3 Hz), 4.09-3.93 (m, 3H), 3.84-3.73 (m, 1H), 3.71 (d, 1H, J = 19.0 Hz), 3.65 (d, 1H, J = 19.0 Hz), 3.58 (dd, 1H, J = 8.2, 5.9 Hz), 2.69-2.59 (m, 2H), 2.23-2.13 (m, 1H), 1.12 (d, 3H, J = 7.2 Hz).
MS: m/z = 311 [M+H] +
[0151]
Using Compound A, which was prepared by the same method, the single crystal X-ray analysis was carried out.
(1) Preparation of Single crystal
To 10 mg of Compound A in a LaPha ROBO Vial(R) 2.0 mL wide-mouthed vial was added 0.5 mL of chloroform. The vial was covered with a cap, in which Compound A was completely dissolved. In order to evaporate the solvent slowly, a hole was made on the septum attached in the cap with a needle of a TERUMO(R) syringe, and the vial was still stood at room temperature. The resulting single crystal was used in the structural analysis.
(2) Measuring instrument
Beam line: SPring-8 BL32B2
Detector: Rigaku R-AXIS V diffractometer
(3) Measuring method
The radiant light of 0.71068Å was irradiated to the single crystal to measure X-ray diffraction data.
(4) Assay method
Using the X-ray anomalous scattering effect of the chlorine atom in the resulting Compound A chloroform-solvate, the absolute configuration of Compound A was identified as (3S,4R). Based on the obtained absolute configuration of Compound A, the absolute configurations of each process intermediate were identified.
(1) Preparation of Single crystal
To 10 mg of Compound A in a LaPha ROBO Vial(R) 2.0 mL wide-mouthed vial was added 0.5 mL of chloroform. The vial was covered with a cap, in which Compound A was completely dissolved. In order to evaporate the solvent slowly, a hole was made on the septum attached in the cap with a needle of a TERUMO(R) syringe, and the vial was still stood at room temperature. The resulting single crystal was used in the structural analysis.
(2) Measuring instrument
Beam line: SPring-8 BL32B2
Detector: Rigaku R-AXIS V diffractometer
(3) Measuring method
The radiant light of 0.71068Å was irradiated to the single crystal to measure X-ray diffraction data.
(4) Assay method
Using the X-ray anomalous scattering effect of the chlorine atom in the resulting Compound A chloroform-solvate, the absolute configuration of Compound A was identified as (3S,4R). Based on the obtained absolute configuration of Compound A, the absolute configurations of each process intermediate were identified.
REFERENCES
1: Nakagawa H, Nemoto O, Yamada H, Nagata T, Ninomiya N. Phase 1 studies to assess the safety, tolerability and pharmacokinetics of JTE-052 (a novel Janus kinase inhibitor) ointment in Japanese healthy volunteers and patients with atopic dermatitis. J Dermatol. 2018 Jun;45(6):701-709. doi: 10.1111/1346-8138.14322. Epub 2018 Apr 17. PubMed PMID: 29665062; PubMed Central PMCID: PMC6001687.
2: Nakagawa H, Nemoto O, Igarashi A, Nagata T. Efficacy and safety of topical JTE-052, a Janus kinase inhibitor, in Japanese adult patients with moderate-to-severe atopic dermatitis: a phase II, multicentre, randomized, vehicle-controlled clinical study. Br J Dermatol. 2018 Feb;178(2):424-432. doi: 10.1111/bjd.16014. Epub 2018 Jan 15. PubMed PMID: 28960254.
3: Tanimoto A, Shinozaki Y, Yamamoto Y, Katsuda Y, Taniai-Riya E, Toyoda K, Kakimoto K, Kimoto Y, Amano W, Konishi N, Hayashi M. A novel JAK inhibitor JTE-052 reduces skin inflammation and ameliorates chronic dermatitis in rodent models: Comparison with conventional therapeutic agents. Exp Dermatol. 2018 Jan;27(1):22-29. doi: 10.1111/exd.13370. Epub 2017 Jul 3. PubMed PMID: 28423239.
4: Nomura T, Kabashima K. Advances in atopic dermatitis in 2015. J Allergy Clin Immunol. 2016 Dec;138(6):1548-1555. doi: 10.1016/j.jaci.2016.10.004. Review. PubMed PMID: 27931536.
5: Amano W, Nakajima S, Yamamoto Y, Tanimoto A, Matsushita M, Miyachi Y, Kabashima K. JAK inhibitor JTE-052 regulates contact hypersensitivity by downmodulating T cell activation and differentiation. J Dermatol Sci. 2016 Dec;84(3):258-265. doi: 10.1016/j.jdermsci.2016.09.007. Epub 2016 Sep 13. PubMed PMID: 27665390.
6: Tanimoto A, Shinozaki Y, Nozawa K, Kimoto Y, Amano W, Matsuo A, Yamaguchi T, Matsushita M. Improvement of spontaneous locomotor activity with JAK inhibition by JTE-052 in rat adjuvant-induced arthritis. BMC Musculoskelet Disord. 2015 Nov 6;16:339. doi: 10.1186/s12891-015-0802-0. PubMed PMID: 26546348; PubMed Central PMCID: PMC4636776.
7: Amano W, Nakajima S, Kunugi H, Numata Y, Kitoh A, Egawa G, Dainichi T, Honda T, Otsuka A, Kimoto Y, Yamamoto Y, Tanimoto A, Matsushita M, Miyachi Y, Kabashima K. The Janus kinase inhibitor JTE-052 improves skin barrier function through suppressing signal transducer and activator of transcription 3 signaling. J Allergy Clin Immunol. 2015 Sep;136(3):667-677.e7. doi: 10.1016/j.jaci.2015.03.051. Epub 2015 Jun 24. PubMed PMID: 26115905.
8: Tanimoto A, Ogawa Y, Oki C, Kimoto Y, Nozawa K, Amano W, Noji S, Shiozaki M, Matsuo A, Shinozaki Y, Matsushita M. Pharmacological properties of JTE-052: a novel potent JAK inhibitor that suppresses various inflammatory responses in vitro and in vivo. Inflamm Res. 2015 Jan;64(1):41-51. doi: 10.1007/s00011-014-0782-9. Epub 2014 Nov 12. PubMed PMID: 25387665; PubMed Central PMCID: PMC4286029.
References
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- “Anzupgo PI”. Union Register of medicinal products. 23 September 2024. Retrieved 27 September 2024.
- Dhillon S (April 2020). “Delgocitinib: First Approval”. Drugs. 80 (6): 609–615. doi:10.1007/s40265-020-01291-2. PMID 32166597. S2CID 212681247.
- Park B (5 August 2020). “Delgocitinib Cream Gets Fast Track Status for Chronic Hand Eczema”. empr.com.
- Szalus K, Trzeciak M, Nowicki RJ (November 2020). “JAK-STAT Inhibitors in Atopic Dermatitis from Pathogenesis to Clinical Trials Results”. Microorganisms. 8 (11): 1743. doi:10.3390/microorganisms8111743. PMC 7694787. PMID 33172122.
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/////////Delgocitinib, デルゴシチニブ , JAPAN 2020, 2020 APPROVALS, Corectim, UNII-9L0Q8KK220, JTE-052, 9L0Q8KK220, LEO 124249A, LEO 124249, HY-109053, CS-0031558, D11046, GTPL9619, JTE-052A, JTE052, LP-0133 , ROH-201, atopic dermatitis
CC1CN(C12CCN(C2)C3=NC=NC4=C3C=CN4)C(=O)CC#N
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| Clinical data | |
|---|---|
| Trade names | Corectim, others |
| Other names | JTE-052; JTE-052A |
| ATC code | |
| Legal status | |
| Legal status | |
| Identifiers | |
| CAS Number | |
| PubChem CID | |
| DrugBank | |
| ChemSpider | |
| UNII | |
| KEGG | |
| CompTox Dashboard (EPA) | |
| Chemical and physical data | |
| Formula | C16H18N6O |
| Molar mass | 310.361 g·mol−1 |
| 3D model (JSmol) | |
https://pubs.acs.org/doi/10.1021/acs.oprd.1c00031
https://www.chemicalbook.com/article/synthesis-of-delgocitinib.htm
Synthesis of Delgocitinib
Delgocitinib is synthesised using bromolactone as raw material by chemical reaction. The specific synthesis steps are as follows:
Synthesis of Delgocitinib
Dec 26，2023
Synthesis of Delgocitinib
Delgocitinib is synthesised using bromolactone as raw material by chemical reaction. The specific synthesis steps are as follows:
A stereocontrolled kilogram scale synthesis of delgocitinib has been disclosed, beginning with an SN2 reaction involving bromolactone 128 and benzyl amine to provide α-amino lactone 129, which was isolated as the HCl salt after precipitation from hydrochloric acid in ethyl acetate. Amine 129 was then acylated with enantiomerically pure acid chloride 131 (prepared by thionyl chloride treatment of commercial acid 130) to furnish lactone 132. In the crucial spirocyclic ring ringforming sequence of the synthesis, lactone 132 was treated with LHMDS to form an enolate that underwent SN2 displacement of the chloride, forming the spirolactone 133 and establishing both stereocenters with 98:2 dr and 96% ee.
The lactone ring of 133 was then opened by an attack of potassium phthalimide on the γ- carbon, and the resulting carboxylic acid was converted to the ethyl ester by treatment with ethyl iodide. Finally, treatment with diethylenetriamine released phthalimide, providing a free amine for subsequent cyclization to spirolactam 134 via the corresponding ethyl ester intermediate. This sequence took place in 80% yield over four steps and provided the spirolactam in >99% de after recrystallization.
The carbonyl groups within spirolactam 134 were then reduced with lithium aluminum hydride and aluminum chloride in THF, and the resulting diamine 135 was crystallized as a succinic acid salt in 86% yield. The SNAr reaction of 135 with chloropyrrolopyrimidine 136 followed by hydrogenative removal of the benzyl protecting group provided amine 137 in 92% yield over 2 steps. Finally, amine 137 was acylated with cyanoacetyl pyrazole 138 and recrystallized from n-butanol with 3 wt % BHT to provide delgocitinib in 86% yield, >99% ee, and >99% de.
Dotinurad ドチヌラド
Dotinurad
ドチヌラド
(3,5-dichloro-4-hydroxyphenyl)-(1,1-dioxo-2H-1,3-benzothiazol-3-yl)methanone
| Formula |
C14H9Cl2NO4S
|
|---|---|
| CAS |
1285572-51-1
|
| Mol weight |
358.1966
|
PMDA, Urece, APROVED JAPAN 2020/1/23, Antihyperuricemic
305EB53128UNII-305EB53128
1285572-51-1,
Dotinurad is a urate transporter inhibitor.
Patents
WO 2011040449
https://patents.google.com/patent/WO2011040449A1/en
Uric acid is produced by metabolizing a purine produced by the degradation of a nucleic acid in the body and adenosine triphosphate (ATP), which is an energy source of the living body, to xanthine, and further undergoes oxidation by xanthine oxidase or xanthine dehydrogenase. In humans, uric acid (dissociation constant pKa = 5.75) is the final metabolite of purines and exists in the body as free forms or salts.
Uric acid is normally excreted in the urine, but when uric acid production exceeds excretion and blood uric acid increases, hyperuricemia occurs. If a state in which the blood level of uric acid exceeds the upper limit of solubility (about 7 mg / dL) continues for a long period of time, crystals of urate (usually sodium salt) precipitate.
In the blood, the precipitated crystals deposit on cartilage tissue and joints, form precipitates and become gouty nodules, causing acute gouty arthritis, and then transition to chronic gouty arthritis.
When uric acid crystals are precipitated in urine, renal disorders such as interstitial nephritis (gouty kidney), urinary calculi, and the like are caused. After the seizures of acute gouty arthritis have subsided, drug therapy is given along with lifestyle improvement guidance to correct hyperuricemia.
Correcting hyperuricemia and appropriately managing uric acid levels are also important in preventing acute gouty arthritis, gouty kidneys, urinary tract stones, and the like.
Hyperuricemia is considered to be associated with a high rate of lifestyle-related diseases such as obesity, hyperlipidemia, impaired glucose tolerance, and hypertension (see Non-Patent Document 1 (pp7-9)). Increased serum uric acid levels are positively related to cardiovascular mortality, and higher serum uric acid levels increase mortality due to ischemic heart disease. It has been suggested that it is associated with the risk of death from disease (see Non-Patent Document 2).
Furthermore, serum uric acid levels have also been shown to be a powerful risk factor for myocardial infarction and stroke (see Non-Patent Document 3). To date, hyperuricemia is obesity, hyperlipidemia, dyslipidemia, impaired glucose tolerance, diabetes, metabolic syndrome, kidney disease (eg, renal failure, urine protein, end-stage renal disease (ESRD), etc.), heart It is known to be associated with vascular diseases (for example, hypertension, coronary artery disease, carotid artery disease, vascular endothelial disorder, arteriosclerosis, cardiac hypertrophy, cerebrovascular disease, etc.) or risk factors of these diseases (Non-Patent Documents 2 to 11) reference). In cerebrovascular dementia, it has also been reported that the concentration of uric acid in the cerebrospinal cord is increased (see Non-Patent Document 12).
Under such circumstances, it has been suggested that the treatment for lowering the blood uric acid level may delay the progression of kidney disease and reduce the risk of cardiovascular disease (Non-Patent Documents 5, 8, 13, 14), it has been reported that it should also be applied to asymptomatic hyperuricemia (see Non-Patent Document 14).
Therefore, reducing the blood uric acid level in the above-mentioned diseases is effective for the treatment or prevention of these diseases, and is considered to be important in terms of preventing recurrence of cardiovascular accidents and maintaining renal function.
The main factors that increase blood uric acid levels include excessive uric acid production and decreased uric acid excretion. Therefore, as a method for lowering blood uric acid level, it is conceivable to suppress the production of uric acid or promote the excretion of uric acid, and allopurinol is a drug having the former mechanism of action (uric acid production inhibitor). Benzbromarone, probenecid, JP-A 2006-176505 (Patent Document 1) and the like are known as drugs having the latter mechanism of action (uric acid excretion promoters).
According to the Japanese guidelines for treatment of hyperuricemia and gout, in principle, uric acid excretion-promoting agents are applied to hyperuricemia-reducing types and uric acid production-inhibiting agents are applied to excessive uric acid production types, respectively. (See Non-Patent Document 1 (pp31-32)).
In Japan, it is said that about 60% of hyperuricemia patients have a reduced uric acid excretion type, and about 25% are a mixed type of reduced uric acid excretion type and excessive uric acid production type (Non-patent Document 15). About 85% of the patients showed a decrease in uric acid excretion, and the average value of uric acid clearance was significantly lower than that of healthy individuals even in patients with excessive uric acid production, and the decrease in uric acid excretion was fundamental in all gout patients. Is also reported (Non-Patent Document 16).
Therefore, in hyperuricemia (especially gout), treatment for patients with reduced uric acid excretion is considered to be important, and the existence significance of uric acid excretion promoters is extremely large.
Among the major uric acid excretion promoters, probenecid is weakly used and is rarely used because of its gastrointestinal tract disorders and interactions with other drugs. On the other hand, severe liver damage has been reported for benzbromarone, which has a strong uric acid excretion promoting action and is widely used in Japan as a uric acid excretion promoting drug (see Non-Patent Document 17).
Benzbromarone or its analogs inhibit mitochondrial respiratory chain enzyme complex activity, uncoupling action, respiration inhibition, fatty acid β oxidation inhibition, mitochondrial membrane potential reduction, apoptosis, generation of reactive oxygen species, etc. Has been suggested to be involved in the development of liver damage (see Non-Patent Documents 18 and 19). Hexahydrate, which is the active body of benzbromarone, is also toxic to mitochondria.
Furthermore, benzbromarone has an inhibitory action on cytochrome P450 (CYP), which is a drug metabolizing enzyme. In particular, the inhibition against CYP2C9 is very strong, suggesting the possibility of causing a pharmacokinetic drug interaction (non-) (See Patent Documents 20 and 21).
Furthermore, although a nitrogen-containing fused ring compound having a URAT1 inhibitory action, which is a kind of uric acid transporter, and having a structure similar to that of the compound of the present invention is described in JP-A-2006-176505 (Patent Document 1), the effect is sufficient. In addition, no practical uric acid excretion promoter has been developed yet.
Recently, it has been found that the uric acid excretion promoting action depends on the urinary concentration of a drug having the same action, that is, the uric acid excretion promoting drug is excreted in the urine and exhibits a medicinal effect (Patent Document 2). Non-Patent Documents 22 and 23).
Therefore, a stronger pharmacological effect is expected if it is a uric acid excretion promoter that is excreted more in the urine, but the above existing uric acid excretion promoters have a very low concentration in urine, and a satisfactory activity can be obtained sufficiently. I can’t say that.
Regarding the urinary excretion of drugs, it is assumed that the administered drug is excreted as it is as an unchanged form or converted into an active metabolite and excreted. In the latter case, the active metabolite is produced. There is a risk that the individual difference in the amount becomes large, and in order to obtain stable drug efficacy and safety, a drug excreted as an unchanged substance is more desirable.
As described above, there is a demand for the development of a highly safe pharmaceutical having a high unchanged body urine concentration and a remarkable uric acid excretion promoting action as compared with existing uric acid excretion promoting drugs.
JP 2006-176505 A WO2005 / 121112
Treatment Guidelines for Hyperuricemia and Gout (1st Edition) pp7-9 and pp31-32, Gout and Nucleic Acid Metabolism, Volume 26, Supplement 1, 2002 Japan Gout and Nucleic Acid Metabolism Society JAMA 283: 2404-2410 (2000) Stroke 37: 1503-1507 (2006) Nephrology 9: 394-399 (2004) Semin. Nephrol. 25: 43-49 (2005)J. Clin. Hypertens. 8: 510-518 (2006) J. Hypertens. 17: 869-872 (1999) Curr. Med. Res. Opin. 20: 369-379 (2004) Curr. Pharm. Des. 11: 4139-4143 (2005)Hypertension 45: 991-996 (2005) Arch. Intern. Med. 169: 342-350 (2009) J. Neural. Transm. Park Dis. Dement. Sect. 6: 119-126 (1993) Am. J. Kidney Dis. 47: 51-59 (2006) Hyperuricemia and gout 9: 61-65 (2001) Japanese clinical trials 54: 3230-3236 (1996) Japanese clinical trial 54: 3248-3255 (1996) J. Hepatol. 20: 376-379 (1994) J. Hepatol. 35: 628-636 (2001) Hepatology 41: 925-935 (2005) Saitama Medical University Journal (J. Saitama. Med. School) 30: 187-194 (2004) Drug Metab. Dispos. 31: 967-971 (2003) 42nd Annual Meeting of the Japanese Gout and Nucleic Acid Metabolism General Assembly Program / Abstracts, p59 (2009) ACR 2008 Annual Scientific Meeting, No. 28
PATENT
JP 2011074017
PATENT
WO 2018199277
https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2018199277
//////////Dotinurad, Antihyperuricemic, JAPAN 2020, 2020 APPROVALS , ドチヌラド , VOFLAIHEELWYGO-UHFFFAOYSA-N, HY-109031, CS-0030545
C1N(C2=CC=CC=C2S1(=O)=O)C(=O)C3=CC(=C(C(=C3)Cl)O)Cl
IIIM-290
IIIM-290
4H-1-Benzopyran-4-one, 2-[2-(2,6-dichlorophenyl)ethenyl]-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-
| Molecular Weight |
462.32 |
|---|---|
| Formula |
C₂₃H₂₁Cl₂NO₅ |
| CAS No. |
2213468-64-3 |
CSIR-IIIM Jammu has filed an IND Application of “IIIM-290” to Drug Controller General of India for conducting Phase I/Phase II clinical trial of its capsule formulation in patients with locally advanced or metastatic pancreatic cancer. This IND candidate has emerged from the eight years of medicinal chemistry/ preclinical efforts of IIIM Jammu in the area of small molecule kinase inhibitors. IIIM-290 (NCE) is an orally bioavailable CDK inhibitor, obtained via semisynthetic modification of a natural product rohitukine. Institute has already secured a patent on this small molecule as well as on its oral capsule formulation.
IIIM-290 is a potent and oral CDK inhibitor with IC50s of 90 and 94 nM for CDK2/A and CDK9/T1.
PAPER
https://pubs.acs.org/doi/pdf/10.1021/acs.jmedchem.7b01765
Discovery and Preclinical Development of IIIM-290, an Orally Active Potent Cyclin-Dependent Kinase Inhibitor
- Sandip B. Bharate
- Vikas Kumar
- Shreyans K. Jain
- Mubashir J. Mintoo
- Santosh K. Guru
- Vijay K. Nuthakki
- Mohit Sharma
- Sonali S. Bharate
- Sumit G. Gandhi
- Dilip M. Mondhe
- Shashi Bhushan
- Ram A. Vishwa
View Author Information
Abstract
Rohitukine (1), a chromone alkaloid isolated from Indian medicinal plant Dysoxylum binectariferum, has inspired the discovery of flavopiridol and riviciclib, both of which are bioavailable only via intravenous route. With the objective to address the oral bioavailability issue of this scaffold, four series of rohitukine derivatives were prepared and screened for Cdk inhibition and cellular antiproliferative activity. The 2,6-dichloro-styryl derivative IIIM-290 (11d) showed strong inhibition of Cdk-9/T1 (IC50 1.9 nM) kinase and Molt-4/MIAPaCa-2 cell growth (GI50 < 1.0 μM) and was found to be highly selective for cancer cells over normal fibroblast cells. It inhibited the cell growth of MIAPaCa-2 cells via caspase-dependent apoptosis. It achieved 71% oral bioavailability with in vivo efficacy in pancreatic, colon, and leukemia xenografts at 50 mg/kg, po. It did not have CYP/efflux-pump liability, was not mutagenic/genotoxic or cardiotoxic, and was metabolically stable. The preclinical data presented herein indicates the potential of 11d for advancement in clinical studies.
Patent
IN201811026240
Patent
InventorRam A. VishwakarmaSandip B. BharateShashi BhushanDilip M. MondheShreyans K. JainSamdarshi MeenaSantosh K. GuruAnup S. PathaniaSuresh KumarAkanksha BehlMubashir J. MintooSonali S. BharatePrashant Joshi Current Assignee Council of Scientific and Industrial Research (CSIR)
https://patents.google.com/patent/US9932327B2/en
The disruption of any internal and external regulation of cellular growth leads to tumorogenesis by uncontrolled proliferation. This loss of control occurs at multiple levels in most of the cancer cases. Cyclin-dependent kinases (CDKs) have been recognized as key regulators of cell cycle progression. Alteration and deregulation of CDK activity have pathogenic link to the cancer. Number of cancers are associated with hyper-activation of CDKs as a result of mutation of the CDK genes or CDK inhibitor genes. Therefore, CDK inhibitors or modulators are of great interest to explore as novel therapeutic agents against cancer (Senderowicz, A. M. Leukemia 2001, 15, 1). Several classes of chemical inhibitors of CDK activity have been described (Zhang, J. et. al. Nat Rev Cancer. 2009, 9, 28) and some of them have reached to clinical pipeline for cancer.
Because CDK inhibitors are ATP competitive ligands; hence earlier they were typically described as purine class of compounds for example dimethylaminopurine, a first substance to be known as a CDK inhibitor (Neant, I. et al. Exp. Cell Res. 1988, 176, 68), olomoucine (Vesely, J. et al. Eur. J. Biochem. 1994, 224, 771) and roscovitine (Meijer, L. et al. Eur. J. Biochem. 1997, 243, 527). The IC50values of these purine class of compounds for CDK1/cyclin B are 120, 7 and 0.2-0.8 μM respectively (Gray, N. et al. Curr. Med. Chem. 1999, 6, 859). Some of the more potent members of this series have been prepared by the Schultz group using combinatorial approaches (Gray, N. S. et al. Science 1998, 281, 533). Number of synthetic flavoalkaloids having potent CDK inhibitory activity has been reviewed recently (Jain, S. K. et al. Mini–Rev. Med. Chem. 2012, 12, 632).
Specific CDKs operate in distinct phases of the cell cycle. CDK complexes with their respective type cyclin partners such as, complex of CDK2 and cyclin A is responsible for the cell’s progression from G1 phase to S phase (Sherr, C. J. Science 1996, 274, 1672). DNA synthesis (S phase) begins with the CDK mediated phosphorylation of Rb (retinoblastoma) protein. Phosphorylated Rb is released from its complex with E2F. The released E2F then promotes the transcription of numerous genes required for the cell to progress through S phase, including thymidylate synthase and dihydrofolate reductase which are required for cell progression (Hatakeyama, M. et. al, Cell Cycle Res. 1995, 1, 9; Zhang, H. S. et. al. Cell 1999, 97, 53). Majority of human cancers have abnormalities in some component of the Rb pathway because of hyper-activation of CDKs resulting from the over-expression of positive cofactors (cyclins/CDKs) or a decrease in negative factors (endogenous CDK inhibitors) or Rb gene mutations (Sausville, E. A. et. al, Pharmacol. Ther. 1999, 82, 285).
The CDK-9 is a member of the Cdc2-like family of kinases. Its cyclin partners are members of the family of cyclin T (T1, T2a and T2b) and cyclin K. The CDK-9/cyclin T complexes appear to be involved in regulating several physiological processes. CDK9/cyclin T1 belongs to the P-TEFb complex, and is responsible for the phosphorylation of carboxyl terminal domain of the RNA Polymerase II, thus promoting general elongation. CDK-9 has also been described as the kinase of the TAK complex, which is homologous to the P-TEFb complex and is involved in HIV replication. CDK9 also appears to be involved in the differentiation program of several cell types, such as muscle cells, monocytes and neurons, suggesting that it may have a function in controlling specific differentiative pathways. In addition, CDK-9 seems to have an anti-apoptotic function in monocytes, that may be related to its control over differentiation of monocytes. This suggests the involvement of CDK-9 in several physiological processes in the cell, the deregulation of which may be related to the genesis of transforming events that may in turn lead to the onset of cancer. In addition, since the complex CDK-9/cyclin T1 is able to bind to the HIV-1 product Tat, the study of the functions of CDK-9/cyclin T may be of interest in understanding the basal mechanisms that regulate HIV replication (Falco, G. D. and Giordano A. Cancer Biol. Therapy 2002, 1, 337).
Rohitukine belongs to a class of chromone alkaloids and it was isolated by chemists at Hoechst India Ltd. in the early 1990’s from Dysoxylum binectariferum Hook. which is phylogenetically related to the Ayurvedic plant, D. malabaricum Bedd., used for rheumatoid arthritis. Rohitukine was isolated as the constituent responsible for anti-inflammatory and immunomodulatory activity (Naik, R. G. et. al. Tetrahedron 1988, 44, 2081; U.S. Pat. No. 4,900,727, 1990). Medicinal chemistry efforts around this nature-derived flavone alkaloid led to discovery of two promising clinical candidates for treatment of cancer viz. flavopiridol of Sanofi-Aventis and P-276-00 of Piramal life sciences. Recently FDA has granted the orphan drug status to flavopiridol for treatment of chronic lymphocytic leukemia (CLL).
The molecular formula of rohitukine is C16H19NO5 and the structure has a molecular weight of 305.32 g/mol. The chemical structure of rohitukine (1) is shown below. The present invention reports new semi-synthetic analogs of rohitukine as promising inhibitors of cyclin-dependent kinases such as CDK-2 and CDK-9.
Synthesis of styryl analog 2-(2,6-dichlorostyryl)-5,7-dihydroxy-8-(3-hydroxy-1-methylpiperidin-4-yl)-4H-chromen-4-one (33)
This compound was synthesized using the procedure as described in example 4. Yellow solid; 1H NMR (DMSO-d6, 400 MHz): δ 7.68 (m, 2H), 7.61 (d, J=16 Hz, 1H), 7.49 (t, J=8 Hz, 1H), 7.14 (d, J=16 Hz, 1H), 6.41 (s, 1H), 5.85 (s, 1H), 4.53 (brs, 1H), 3.10-2.50 (m, 6H of piperidine), 2.65 (s, 3H), 1.62 (m, 1H); 13C NMR (DMSO-d6, 125 MHz): δ 179.68. 171.27, 159.20, 158.02, 154.03, 133.12, 131.49, 129.75, 128.35 (2C), 128.20, 127.90, 108.81, 106.79, 100.88, 100.52, 66.35, 59.82, 54.45, 43.15, 35.79, 22.01, 20.33, ESI-MS: m/z 462.01 [M+H]+; IR (CHCl3): νmax 3400, 2921, 1652, 1577, 1550, 1417, 1380, 1191, 1085 cm−1.
///////////IIIM-290, nda, india, phase 1, dcgi, CSIR, ROHITUKINE
|
OC1=C2C(OC(/C=C/C3=C(Cl)C=CC=C3Cl)=CC2=O)=C([C@]4([H])[C@H](O)CN(C)CC4)C(O)=C1 |
Zanubrutinib, ザヌブルチニブ , занубрутиниб , زانوبروتينيب ,
Zanubrutinib, BGB-3111
| Formula |
C27H29N5O3
|
|---|---|
| CAS |
1691249-45-2
|
| Mol weight |
471.5509
|
FDA , 2019/11/14, Brukinsa
ザヌブルチニブ ,
занубрутиниб [Russian]
زانوبروتينيب [Arabic]
(7S)-7-(1-Acryloyl-4-piperidinyl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide
Pyrazolo[1,5-a]pyrimidine-3-carboxamide, 4,5,6,7-tetrahydro-7-[1-(1-oxo-2-propen-1-yl)-4-piperidinyl]-2-(4-phenoxyphenyl)-, (7S)-
Antineoplastic, Bruton’s tyrosine kinase inhibitor, Mantle cell lymphoma
NEW PA
https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2023062504&_gid=202316
Zanubrutinib, sold under the brand name Brukinsa, is for the treatment of adult patients with mantle cell lymphoma (MCL) who have received at least one prior therapy.[3]
It was approved for medical use in the United States in November 2019.[4][3][5][6]
Zanubrutinib is classified as a Bruton’s tyrosine kinase (BTK) inhibitor. It is administered orally.
History
Efficacy was evaluated in BGB-3111-206 (NCT03206970), a phase II open-label, multicenter, single-arm trial of 86 patients with mantle cell lymphoma (MCL) who received at least one prior therapy.[5] Zanubrutinib was given orally at 160 mg twice daily until disease progression or unacceptable toxicity.[5] Efficacy was also assessed in BGB-3111-AU-003 (NCT 02343120), a phase I/II, open-label, dose-escalation, global, multicenter, single-arm trial of B‑cell malignancies, including 32 previously treated MCL patients treated with zanubrutinib administered orally at 160 mg twice daily or 320 mg once daily.[5][6]
The primary efficacy outcome measure in both trials was overall response rate (ORR), as assessed by an independent review committee.[5] In trial BGB-3111-206, FDG-PET scans were required and the ORR was 84% (95% CI: 74, 91), with a complete response rate of 59% (95% CI 48, 70) and a median response duration of 19.5 months (95% CI: 16.6, not estimable).[5] In trial BGB-3111-AU-003, FDG-PET scans were not required and the ORR was 84% (95% CI: 67, 95), with a complete response rate of 22% (95% CI: 9, 40) and a median response duration of 18.5 months (95% CI: 12.6, not estimable).[5] Trial 1 was conducted at 13 sites in China, and Trial 2 was conducted at 25 sites in the United States, United Kingdom, Australia, New Zealand, Italy, and South Korea.[6]
The U.S. Food and Drug Administration (FDA) granted zanubrutinib priority review, accelerated approval, breakthrough therapydesignation, and orphan drug designation.[3][5][7]
The FDA approved zanubrutinib in November 2019, and granted the application for Brukinsa to BeiGene USA Inc.[3][5][8]
PAPER
https://www.x-mol.com/paper/5799457
Discovery of Zanubrutinib (BGB-3111), a Novel, Potent, and Selective Covalent Inhibitor of Bruton’s Tyrosine Kinase Journal of Medicinal Chemistry ( IF 6.054 ) Pub Date: 2019-08-19 , DOI: 10.1021 / acs.jmedchem.9b00687
Yunhang Guo, Ye Liu, Nan Hu, Desheng Yu, Changyou Zhou, Gongyin Shi, Bo Zhang, Min Wei, Junhua Liu, Lusong Luo, Zhiyu Tang, Huipeng Song, Yin Guo, Xuesong Liu, Dan Su, Shuo Zhang, Xiaomin Song , Xing Zhou, Yuan Hong, Shuaishuai Chen, Zhenzhen Cheng, Steve Young, Qiang Wei, Haisheng Wang, Qiuwen Wang, Lei Lv, Fan Wang, Haipeng Xu, Hanzi Sun, Haimei Xing, Na Li, Wei Zhang, Zhongbo Wang, Guodong Liu, Zhijian Sun, Dongping Zhou, Wei Li, Libin Liu, Lai Wang, Zhiwei Wang
 |
Aberrant activation of Bruton’s tyrosine kinase (BTK) plays an important role in pathogenesis of B-cell lymphomas, suggesting that inhibition of BTK is useful in the treatment of hematological malignancies. The discovery of a more selective on-target covalent BTK inhibitor is of high value. Herein, we disclose the discovery and preclinical characterization of a potent, selective, and irreversible BTK inhibitor as our clinical candidate by using in vitro potency, selectivity, pharmacokinetics (PK), and in vivo pharmacodynamic for prioritizing compounds. Compound BGB-3111 (31a, Zanubrutinib) demonstrates (i) potent activity against BTK and excellent selectivity over other TEC, EGFR and Src family kinases, (ii) desirable ADME, excellent in vivo pharmacodynamic in mice and efficacy in OCI-LY10 xenograft models.
PATENT
WO 2014173289
WO 2018033135
PATENT
WO 2018033853
Bruton’s tyrosine kinase (Btk) belongs to the Tec tyrosine kinase family (Vetrie et al., Nature 361: 226-233, 1993; Bradshaw, Cell Signal. 22: 1175-84, 2010). Btk is primarily expressed in most hematopoietic cells such as B cells, mast cells and macrophages (Smith et al., J. Immunol. 152: 557-565, 1994) and is localized in bone marrow, spleen and lymph node tissue. Btk plays important roles in B-cell receptor (BCR) and FcR signaling pathways, which involve in B-cell development, differentiation (Khan, Immunol. Res. 23: 147, 2001). Btk is activated by upstream Src-family kinases. Once activated, Btk in turn phosphorylates PLC gamma, leading to effects on B-cell function and survival (Humphries et al., J. Biol.Chem. 279: 37651, 2004).
[0003] These signaling pathways must be precisely regulated. Mutations in the gene encoding Btk cause an inherited B-cell specific immunodeficiency disease in humans, known as X-linked agammaglobulinemia (XLA) (Conley et al., Annu. Rev. Immunol. 27: 199-227, 2009). Aberrant BCR-mediated signaling may result in dysregulated B-cell activation leading to a number of autoimmune and inflammatory diseases. Preclinical studies show that Btk deficient mice are resistant to developing collagen- induced arthritis. Moreover, clinical studies of Rituxan, a CD20 antibody to deplete mature B-cells, reveal the key role of B-cells in a number of inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis (Gurcan et al, Int. Immunopharmacol. 9: 10-25, 2009). Therefore, Btk inhibitors can be used to treat autoimmune and/or inflammatory diseases.
[0004] In addition, aberrant activation of Btk plays an important role in pathogenesis of B-cell lymphomas indicating that inhibition of Btk is useful in the treatment of hematological malignancies (Davis et al, Nature 463: 88-92, 2010). Preliminary clinical trial results showed that the Btk inhibitor PCI-32765 was effective in treatment of several types of B-cell lymphoma (for example, 54thAmerican Society of Hematology (ASH) annual meeting abstract, Dec. 2012: 686 The Bruton’s Tyrosine Kinase (Btk) Inhibitor, Ibrutinib (PCI- 32765), Has Preferential Activity in the ABC Subtype of Relapsed/Refractory De Novo Diffuse Large B-Cell Lymphoma (DLBCL): Interim Results of a Multic enter, Open-Label, Phase I Study). Because Btk plays a central role as a mediator in multiple signal transduction pathways, inhibitors of Btk are of great interest as anti-inflammatory and/or anti-cancer agents {Mohamed et al., Immunol. Rev. 228: 58-73, 2009; Pan, Drug News perspect 21: 357-362, 200%; Rokosz et al., Expert Opin. Ther. Targets 12: 883-903, 2008; Uckun et al., Anti-cancer Agents Med. Chem. 7: 624-632, 2007; Lou et al, J. Med. Chem. 55(10): 4539-4550, 2012).
[0005] International application WO2014173289A disclosed a series of fused heterocyclic compounds as Btk inhibitors. In particular, WO2014173289A disclosed
(S)-7-(l-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetra-hydropyrazolo[l,5-a]pyrimi dine-3-carboxamide (hereinafter C
Compound 1
[0006] Compound 1 is a potent, specific and irreversible BTK kinase inhibitor. The data generated in preclinical studies using biochemical, cell based and animal studies suggested that Compound 1 could offer significant benefit in inhibiting tumor growth in B-cell malignancies. As Compound 1 was shown to be more selective than ibrutinib for inhibition of BTK vs. EGFR, FGR, FRK, HER2, HER4, ITK, JAK3, LCK, and TEC, it is expected to give rise to less side-effects than ibrutinib in clinic. In addition, Compound 1 showed significantly less inhibition of rituximab-induced antigen-dependent cell-mediated cytotoxicity (ADCC) than ibrutinib due to weaker ITK inhibition, and therefore may provide better efficacy when combined with rituximab or other ADCC-dependent antibody in treating B-cell malignancies.
[0007] Preclinical safety evaluation has demonstrated that Compound 1 was safer than ibrutinib in terms of the overall tolerance and severe toxicities in both rat and dog single and repeat dose toxicity studies up to 28 days. Additionally, Compound 1 had better bioavailability without accumulation issues observed for ibrutinib. These unique characteristics warrant further evaluation of Compound 1 in clinical studies.
[0008] However, Compound 1 was found to be an amorphous form according to the preparation method for Compound 27 in WO 2014173289A, which was further confirmed by the X-Ray Powder Diffraction pattern of FIG. 7A. The amorphous form was shown to have a low glass transition temperature as shown in FIG. 7B, indicating some difficulties in the drug formulation with the amorphous form, such as low stability and hard to purify. Therefore, it’s necessary to develop a new form of Compound 1 which possesses characteristics such as high melting point and better stability, suitable for drug formulation.
Scheme 1: Preparation of Compound 1 and deuterium-labeled Compound 1
Deuterium-Labeled Compound 1
Step 15: Synthesis of
(S)-7-(l-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)-4,5,6,7-tetrahydropyrazolori,5-a1pyrimi dine-3-carboxamide (Compound 1
[0105] Under N2 atmosphere, ACN (12.0 v), water (12.5 v), BG-13 (8.0 Kg, 1.0 eq), and NaHC03 (2.5 eq.) were added to a reactor. The mixture was then cooled to -5-0 °C. To the mixture, the solution of acryloyl chloride (1.1 eq.) in MeCN (0.5 v) was added dropwise and
stirred until the reaction was completed. EA (6.0 v) was then added to the reactor, and stirred. The organic phase was collected. The aqueous layer was further extracted with EA (3.0 v). The organic phases were combined and washed with brine. The organic layer was collected and concentrated.
[0106] The residue was purified by silica gel (2 wt) column, eluted with 3% w/w methanol in DCM (21.0 v). The Compound 1 solution was collected and concentrated under vacuum. The residue was precipitated from EA/MTBE (2.0 v). The cake was collected by centrifugation as the product.
Step 15: Synthesis of (S)-7-(l-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl)
-4,5,6,7-tetrahydropyrazolori,5-a1pyrimidine-3-carboxamide (Compound 1, alternative method)
[0107] A mixture of CHsCN (10.0 v), purified water (5.0 v), NaOH (1.5 eq.) and BG-13 (1.0 eq.) was stirred to get a clear solution. EtOAc (6.0 v) was then charged to the reaction and separated. The organic phase was collected and washed with 15% brine (3.0 v) twice. The organic phase prepared above was concentrated and the solvent was swapped to CH3CN (residue volume: NMT 5.0 v). CH3CN (7.5 v) and purified water (12.5 v) were charged and cooled to 15-20°C. L-(+)-tartaric acid (0.5 eq) and NaHCCb (2.5 eq.) were charged to the reaction mixture. A solution of acryloyl chloride (1.1 eq.) in CH3CN (0.5 v) was charged drop-wise to the reaction mixture. After the reaction was completed, EtOAc (6.0 v) was charged to the reaction mixture and organic layer was collected. Aqueous phase was further extracted with EA (3.0 v). The organic layers were combined, washed with 15% brine (5.0 v) and concentrated. The solvent was swapped to DCM (volume of residue: 1.5-2.0 v) and purified by silica gel column (silica gel: 100-200 mush, 2.0 w/ w; eluent: 3%> w/ w MeOH in DCM (about 50 v). The collected solution was concentrated and swapped to EtOAc (4.0 v). MTBE (6.4 v) was charged drop-wise to residue at 50°C. The mixture was then cooled to 5°C and the cake was collected centrifugation.
Step 16: Preparation of Crystalline Form A of Compound 1
[0108] The above cake of Compound 1 was dissolved in 7.0 volumes of DCM, and then swapped to solvent EA. After recrystallization from EA/MTBE, the cakes was collected by centrifugation, and was dried under vacuum. This gave 4.44 Kg product (Yield: 70.2%).
[0109] The product was then characterized by X-ray powder diffraction (XRPD) pattern method, which was generated on a PANalytical Empyrean X-ray powder diffractometer with the XRPD parameters as follows: X-Ray wavelength (Cu, ka, Kal (A): 1.540598, Ka2(A): 1.544426; Ka2/Kal intensity ratio: 0.50); X-Ray tube setting (45 Kv, 40mA); divergence slit (automatic); scan mode (Continuous); scan range (°2TH) (3°-40); step size (°2TH) (0.0131); scan speed (°/min) (about 10). The XRPD result found the resultant product as a crystalline shown in FIG. 1.
[0110] The differential scanning calorimetry (DSC) curves shown as in FIG. 2 was generated on a TA Q2000 DSC from TA Instruments. The DSC parameters used includes: temperature (25°C-desired temperature); heating rate (10°C/min) ; method (ramp); sample pan (aluminum, crimped); purge gas (N2). DSC result showed a sharp melting point at 139.4°C (onset temperature).
[0111] The thermo-gravimetric analysis (TGA) curves shown as in FIG. 3 was generated on a TA Q5000 TGA from TA Instruments. The TGA parameters used includes: temperature
(RT-desired temperature); heating rate (10°C/min); method (ramp); sample pan (platinum, open); purge gas (N2). TGA result showed is anhydrous with no weight loss even up to 110 °C.
[0112] The proton nuclear magnetic resonance ^H-NMR) shown as in FIG. 4 was collected on a Bruker 400M NMR Spectrometer in DMSO-de. ¾-NMR (DMSO-de) δ 7.50 (d, J= 8.6 Hz, 2H), 7.46-7.38 (m, 2H), 7.17 (t, J = 7.6 Hz, 1H), 7.08 (d, J= 7.6 Hz, 2H), 7.05 (d, J= 8.8 Hz, 2H), 6.85-6.72 (m, 1H), 6.67 (s, 1H), 6.07 (dd, J= 16.8, 2.2 Hz, 1H), 5.64 (dd, J= 10.4 Hz, 2.2 Hz, 1H), 4.55-4.38 (m, 1H), 4.17-3.94 (m, 2H), 3.33-3.22 (m, 2H), 3.08-2.88 (m, 1H), 2.67-2.51 (m, 1H), 2.36-2.15 (m, 1H), 2.12-1.82 (m, 2H), 1.79-1.65 (m, 1H), 1.63-1.49 (m, 1H), 1.38-1.08 (m, 2H).
[0113] The carbon nuclear magnetic resonance (13C-NMR) shown as in FIG. 5 was collected on a Bruker 400M NMR Spectrometer in DMSO-de. 13C-NMR spectra for Crystalline Form A of Compound 1.
PATENT
WO 2019108795
Step 15: Synthesis of (S)-7-(1-acrvlovlpiperidin-4-vl)-2-(4-phenoxvphenyl)-4.5.6.7-tetrahvdropvrazolo[1.5-a1pvrimidine-3-carboxamide (Compound 1)
[0119] Under N2 atmosphere, ACN (12.0 v), water (12.5 v), BG-13 (8.0 Kg, 1.0 eq), and NaHCO3 (2.5 eq.) were added to a reactor. The mixture was then cooled to -5-0 °C. To the mixture, the solution of acryloyl chloride (1.1 eq.) in MeCN (0.5 v) was added dropwise and stirred until the reaction was completed. EA (6.0 v) was then added to the reactor, and stirred. The organic phase was collected. The aqueous layer was further extracted with EA (3.0 v). The organic phases were combined and washed with brine. The organic layer was collected and concentrated.
[0120] The residue was purified by silica gel (2 wt) column, eluted with 3% w/w methanol in DCM (21.0 v). The Compound 1 solution was collected and concentrated under vacuum. The residue was precipitated from EA/MTBE (2.0 v). The cake was collected by centrifugation as the product.
Step 15: Synthesis of (S)-7-(l-acryloylpiperidin-4-yl)-2-(4-phenoxyphenyl) -4.5.6.7-tetrahvdropvrazolori.5-a1pvrimidine-3-carboxamide (Compound 1. alternative method)
[0121] A mixture of CH3CN (10.0 v), purified water (5.0 v), NaOH (1.5 eq.) and BG-13 (1.0 eq.) was stirred to get a clear solution. EtOAc (6.0 v) was then charged to the reaction and separated. The organic phase was collected and washed with 15% brine (3.0 v) twice. The organic phase prepared above was concentrated and the solvent was swapped to CH3CN (residue volume: NMT 5.0 v). CH3CN (7.5 v) and purified water (12.5 v) were charged and cooled to 15-20°C. L-(+)-tartaric acid (0.5 eq) and NaHCO3 (2.5 eq.) were charged to the reaction mixture. A solution of acryloyl chloride (1.1 eq.) in CH3CN (0.5 v) was charged drop-wise to the reaction mixture. After the reaction was completed, EtOAc (6.0 v) was charged to the reaction mixture and organic layer was collected. Aqueous phase was further extracted with EA (3.0 v). The organic layers were combined, washed with 15% brine (5.0 v) and concentrated. The solvent was swapped to DCM (volume of residue: 1.5-2.0 v) and purified by silica gel column (silica gel: 100-200 mush, 2.0 w/ w; eluent: 3% w/ w MeOH in DCM (about 50 v). The collected solution was concentrated and swapped to EtOAc (4.0 v). MTBE (6.4 v) was charged drop-wise to residue at 50°C. The mixture was then cooled to 5°C and the cake was collected centrifugation.
References
- ^ “Zanubrutinib (Brukinsa) Use During Pregnancy”. Drugs.com. 3 January 2020. Retrieved 26 January 2020.
- ^ “Zanubrutinib”. DrugBank. Retrieved 15 November 2019.
- ^ Jump up to:a b c d “FDA approves therapy to treat patients with relapsed and refractory mantle cell lymphoma supported by clinical trial results showing high response rate of tumor shrinkage”. U.S. Food and Drug Administration (FDA) (Press release). 14 November 2019. Retrieved 15 November 2019.
This article incorporates text from this source, which is in the public domain. - ^ “Brukinsa (zanubrutinib) FDA Approval History”. Drugs.com. 14 November 2019. Archived from the original on 15 November 2019. Retrieved 15 November 2019.
- ^ Jump up to:a b c d e f g h i “FDA grants accelerated approval to zanubrutinib for mantle cell lymphoma”. U.S. Food and Drug Administration (FDA)(Press release). 15 November 2019. Archived from the original on 28 November 2019. Retrieved 27 November 2019. This article incorporates text from this source, which is in the public domain.
- ^ Jump up to:a b c “Drug Trials Snapshots Brukinsa”. U.S. Food and Drug Administration (FDA). 14 November 2019. Retrieved 26 January 2020. This article incorporates text from this source, which is in the public domain.
- ^ “Zanubrutinib Orphan Drug Designation and Approval”. U.S. Food and Drug Administration (FDA). 28 November 2019. Archived from the original on 28 November 2019. Retrieved 27 November 2019. This article incorporates text from this source, which is in the public domain.
- ^ “Drug Approval Package: Brukinsa”. U.S. Food and Drug Administration (FDA). 27 November 2019. Archived from the original on 28 November 2019. Retrieved 27 November 2019. This article incorporates text from this source, which is in the public domain.
External links
- “Zanubrutinib”. Drug Information Portal. U.S. National Library of Medicine.
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| Clinical data | |
|---|---|
| Trade names | Brukinsa |
| Other names | BGB-3111 |
| AHFS/Drugs.com | Monograph |
| License data |
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| Pregnancy category |
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| Routes of administration |
By mouth |
| Drug class | Bruton’s tyrosine kinase(BTK) inhibitor |
| Legal status | |
| Legal status |
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| Identifiers | |
| CAS Number | |
| PubChem CID | |
| PubChem SID | |
| DrugBank | |
| ChemSpider | |
| UNII | |
| KEGG | |
| ChEMBL | |
| Chemical and physical data | |
| Formula | C27H29N5O3 |
| Molar mass | 471.5509 g·mol−1 |
| 3D model (JSmol) | |
/////////////////Zanubrutinib, FDA 2019, ザヌブルチニブ , занубрутиниб , زانوبروتينيب , BGB-3111
Teprotumumab-trbw
Tepezza (teprotumumab-trbw)
Company: Horizon Therapeutics plc
Date of Approval: January 21, 2020
Treatment for: Thyroid Eye Disease
UNIIY64GQ0KC0A
CAS number1036734-93-6
R-1507 / R1507 / RG-1507 / RG1507 / RO-4858696 / RO-4858696-000 / RO-4858696000 / RO4858696 / RO4858696-000 / RV-001 / RV001
Tepezza (teprotumumab-trbw) is a fully human monoclonal antibody (mAb) and a targeted inhibitor of the insulin-like growth factor 1 receptor (IGF-1R) for the treatment of active thyroid eye disease (TED).
FDA Approves Tepezza (teprotumumab-trbw) for the Treatment of Thyroid Eye Disease (TED) – January 21, 2020
Today, the U.S. Food and Drug Administration (FDA) approved Tepezza (teprotumumab-trbw) for the treatment of adults with thyroid eye disease, a rare condition where the muscles and fatty tissues behind the eye become inflamed, causing the eyes to be pushed forward and bulge outwards (proptosis). Today’s approval represents the first drug approved for the treatment of thyroid eye disease.
“Today’s approval marks an important milestone for the treatment of thyroid eye disease. Currently, there are very limited treatment options for this potentially debilitating disease. This treatment has the potential to alter the course of the disease, potentially sparing patients from needing multiple invasive surgeries by providing an alternative, non surgical treatment option,” said Wiley Chambers, M.D., deputy director of the Division of Transplant and Ophthalmology Products in the FDA’s Center for Drug Evaluation and Research. “Additionally, thyroid eye disease is a rare disease that impacts a small percentage of the population, and for a variety of reasons, treatments for rare diseases are often unavailable. This approval represents important progress in the approval of effective treatments for rare diseases, such as thyroid eye disease.”
Thyroid eye disease is associated with the outward bulging of the eye that can cause a variety of symptoms such as eye pain, double vision, light sensitivity or difficulty closing the eye. This disease impacts a relatively small number of Americans, with more women than men affected. Although this condition impacts relatively few individuals, thyroid eye disease can be incapacitating. For example, the troubling ocular symptoms can lead to the progressive inability of people with thyroid eye disease to perform important daily activities, such as driving or working.
Tepezza was approved based on the results of two studies (Study 1 and 2) consisting of a total of 170 patients with active thyroid eye disease who were randomized to either receive Tepezza or a placebo. Of the patients who were administered Tepezza, 71% in Study 1 and 83% in Study 2 demonstrated a greater than 2 millimeter reduction in proptosis (eye protrusion) as compared to 20% and 10% of subjects who received placebo, respectively.
The most common adverse reactions observed in patients treated with Tepezza are muscle spasm, nausea, alopecia (hair loss), diarrhea, fatigue, hyperglycemia (high blood sugar), hearing loss, dry skin, dysgeusia (altered sense of taste) and headache. Tepezza should not be used if pregnant, and women of child-bearing potential should have their pregnancy status verified prior to beginning treatment and should be counseled on pregnancy prevention during treatment and for 6 months following the last dose of Tepezza.
The FDA granted this application Priority Review, in addition to Fast Track and Breakthrough Therapy Designation. Additionally, Tepezza received Orphan Drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases or conditions. Development of this product was also in part supported by the FDA Orphan Products Grants Program, which provides grants for clinical studies on safety and efficacy of products for use in rare diseases or conditions.
The FDA granted the approval of Tepezza to Horizon Therapeutics Ireland DAC.
Teprotumumab (RG-1507), sold under the brand name Tepezza, is a medication used for the treatment of adults with thyroid eye disease, a rare condition where the muscles and fatty tissues behind the eye become inflamed, causing the eyes to be pushed forward and bulge outwards (proptosis).[1]
The most common adverse reactions observed in people treated with teprotumumab-trbw are muscle spasm, nausea, alopecia (hair loss), diarrhea, fatigue, hyperglycemia (high blood sugar), hearing loss, dry skin, dysgeusia (altered sense of taste) and headache.[1] Teprotumumab-trbw should not be used if pregnant, and women of child-bearing potential should have their pregnancy status verified prior to beginning treatment and should be counseled on pregnancy prevention during treatment and for six months following the last dose of teprotumumab-trbw.[1]
It is a human monoclonal antibody developed by Genmab and Roche. It binds to IGF-1R.
Teprotumumab was first investigated for the treatment of solid and hematologic tumors, including breast cancer, Hodgkin’s and non-Hodgkin’s lymphoma, non-small cell lung cancer and sarcoma.[2][3] Although results of phase I and early phase II trials showed promise, research for these indications were discontinued in 2009 by Roche. Phase II trials still in progress were allowed to complete, as the development was halted due to business prioritization rather than safety concerns.
Teprotumumab was subsequently licensed to River Vision Development Corporation in 2012 for research in the treatment of ophthalmic conditions. Horizon Pharma (now Horizon Therapeutics, from hereon Horizon) acquired RVDC in 2017, and will continue clinical trials.[4] It is in phase III trials for Graves’ ophthalmopathy (also known as thyroid eye disease (TED)) and phase I for diabetic macular edema.[5] It was granted Breakthrough Therapy, Orphan Drug Status and Fast Track designations by the FDA for Graves’ ophthalmopathy.[6]
In a multicenter randomized trial in patients with active Graves’ ophthalmopathy Teprotumumab was more effective than placebo in reducing the clinical activity score and proptosis.[7] In February 2019 Horizon announced results from a phase 3 confirmatory trial evaluating teprotumumab for the treatment of active thyroid eye disease (TED). The study met its primary endpoint, showing more patients treated with teprotumumab compared with placebo had a meaningful improvement in proptosis, or bulging of the eye: 82.9 percent of teprotumumab patients compared to 9.5 percent of placebo patients achieved the primary endpoint of a 2 mm or more reduction in proptosis (p<0.001). Proptosis is the main cause of morbidity in TED. All secondary endpoints were also met and the safety profile was consistent with the phase 2 study of teprotumumab in TED.[8] On 10th of July 2019 Horizon submitted a Biologics License Application (BLA) to the FDA for teprotumumab for the Treatment of Active Thyroid Eye Disease (TED). Horizon requested priority review for the application – if so granted (FDA has a 60-day review period to decide) it would result in a max. 6 month review process.[9]
History[edit]
Teprotumumab-trbw was approved for use in the United States in January 2020, for the treatment of adults with thyroid eye disease.[1]
Teprotumumab-trbw was approved based on the results of two studies (Study 1 and 2) consisting of a total of 170 patients with active thyroid eye disease who were randomized to either receive teprotumumab-trbw or a placebo.[1] Of the subjects who were administered Tepezza, 71% in Study 1 and 83% in Study 2 demonstrated a greater than two millimeter reduction in proptosis (eye protrusion) as compared to 20% and 10% of subjects who received placebo, respectively.[1]
The U.S. Food and Drug Administration (FDA) granted the application for teprotumumab-trbw fast track designation, breakthrough therapy designation, priority review designation, and orphan drug designation.[1] The FDA granted the approval of Tepezza to Horizon Therapeutics Ireland DAC.[1]
References
- ^ Jump up to:a b c d e f g h “FDA approves first treatment for thyroid eye disease”. U.S. Food and Drug Administration (FDA) (Press release). 21 January 2020. Retrieved 21 January 2020. This article incorporates text from this source, which is in the public domain.
- ^ https://clinicaltrials.gov/ct2/show/NCT01868997
- ^ http://adisinsight.springer.com/drugs/800015801
- ^ http://www.genmab.com/product-pipeline/products-in-development/teprotumumab
- ^ http://adisinsight.springer.com/drugs/800015801
- ^ http://www.genmab.com/product-pipeline/products-in-development/teprotumumab
- ^ Smith, TJ; Kahaly, GJ; Ezra, DG; Fleming, JC; Dailey, RA; Tang, RA; Harris, GJ; Antonelli, A; Salvi, M; Goldberg, RA; Gigantelli, JW; Couch, SM; Shriver, EM; Hayek, BR; Hink, EM; Woodward, RM; Gabriel, K; Magni, G; Douglas, RS (4 May 2017). “Teprotumumab for Thyroid-Associated Ophthalmopathy”. The New England Journal of Medicine. 376 (18): 1748–1761. doi:10.1056/NEJMoa1614949. PMC 5718164. PMID 28467880.
- ^ “Horizon Pharma plc Announces Phase 3 Confirmatory Trial Evaluating Teprotumumab (OPTIC) for the Treatment of Active Thyroid Eye Disease (TED) Met Primary and All Secondary Endpoints”. Horizon Pharma plc. Retrieved 22 March 2019.
- ^ “Horizon Therapeutics plc Submits Teprotumumab Biologics License Application (BLA) for the Treatment of Active Thyroid Eye Disease (TED)”. Horizon Therapeutics plc. Retrieved 27 August 2019.
External links
- “Teprotumumab”. Drug Information Portal. U.S. National Library of Medicine.
| Monoclonal antibody | |
|---|---|
| Type | Whole antibody |
| Source | Human |
| Target | IGF-1R |
| Clinical data | |
| Other names | teprotumumab-trbw, RG-1507 |
| ATC code |
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| Legal status | |
| Legal status |
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| Identifiers | |
| CAS Number | |
| DrugBank | |
| ChemSpider |
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| UNII | |
| KEGG | |
| ChEMBL | |
| ECHA InfoCard | 100.081.384  |
| Chemical and physical data | |
| Formula | C6476H10012N1748O2000S40 |
| Molar mass | 145.6 kg/mol g·mol−1 |
/////////Teprotumumab-trbw, APPROVALS 2020, FDA 2020, ORPHAN, BLA, fast track designation, breakthrough therapy designation, priority review designation, and orphan drug designation, Tepezza, Horizon Therapeutics, MONOCLONAL ANTIBODY, 2020 APPROVALS, active thyroid eye disease, Teprotumumab
https://www.fda.gov/news-events/press-announcements/fda-approves-first-treatment-thyroid-eye-disease
ENCORAFENIB, エンコラフェニブ
ENCORAFENIB, エンコラフェニブ
UNII:8L7891MRB6
Formula:C22H27ClFN7O4S, Average: 540.01
1269440-17-6
- BRAFTOVI
- NVP-LGX818
- NVP-LGX-818-NXA
- NVP-LGX818-NXA
- ENCORAFENIB [USAN]
- ENCORAFENIB [WHO-DD]
- ENCORAFENIB
- ENCORAFENIB [INN]
- METHYL N-((2S)-1-((4-(3-(5-CHLORO-2-FLUORO-3-(METHANESULFONAMIDO)PHENYL)(-1-(PROPAN-2-YL)-1H-PYRAZOL-4-YL(PYRIMIDIN-2-YL)AMINO)PROPAN-2-YL)CARBAMATE
- CARBAMIC ACID, N-((1S)-2-((4-(3-(5-CHLORO-2-FLUORO-3-((METHYLSULFONYL)AMINO)PHENYL)-1-(1-METHYLETHYL)-1H-PYRAZOL-4-YL)-2-PYRIMIDINYL)AMINO)-1-METHYLETHYL)-, METHYL ESTER
- LGX818
- LGX-818
Encorafenib, also known as BRAFTOVI, is a kinase inhibitor. Encorafenib inhibits BRAF gene, which encodes for B-raf protein, which is a proto-oncogene involved in various genetic mutations Label. This protein plays a role in regulating the MAP kinase/ERK signaling pathway, which impacts cell division, differentiation, and secretion. Mutations in this gene, most frequently the V600E mutation, are the most commonly identified cancer-causing mutations in melanoma, and have been isolated in various other cancers as well, including non-Hodgkin lymphoma, colorectal cancer, thyroid carcinoma, non-small cell lung carcinoma, hairy cell leukemia and adenocarcinoma of the lung 6.
On June 27, 2018, the Food and Drug Administration approved encorafenib and Binimetinib(BRAFTOVI and MEKTOVI, Array BioPharma Inc.) in combination for patients with unresectable or metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test Label.
Array Biopharma (a wholly owned subsidiary of Pfizer ), under license from Novartis , and licensees Pierre Fabre and Ono Pharmaceutical have developed and launched the B-Raf kinase inhibitor encorafenib . In January 2020, the US FDA’s Orange Book was seen to list encorafenib patents such as US8946250 , US8501758 , US9314464 and US9763941 , expiring in the range of 2029-2032. At that time Orange Book also reported that encorafenib as having NCE exclusivity expiring on July 27, 2023.
Encorafenib (trade name Braftovi) is a drug for the treatment of certain melanomas. It is a small molecule BRAF inhibitor [1] that targets key enzymes in the MAPK signaling pathway. This pathway occurs in many different cancers including melanoma and colorectal cancers.[2] The substance was being developed by Novartis and then by Array BioPharma. In June 2018, it was approved by the FDA in combination with binimetinib for the treatment of patients with unresectable or metastatic BRAF V600E or V600K mutation-positive melanoma.[3][4]
The most common (≥25%) adverse reactions in patients receiving the drug combination were fatigue, nausea, diarrhea, vomiting, abdominal pain, and arthralgia.[3]
Indication
Used in combination with Binimetinib in metastatic melanoma with a BRAF V600E or V600K mutation, as detected by an FDA-approved test 5.
Associated Conditions
Pharmacodynamics
Encorafenib has shown improved efficacy in the treatment of metastatic melanoma 3.
Encorafenib, a selective BRAF inhibitor (BRAFi), has a pharmacologic profile that is distinct from that of other clinically active BRAFis 7.
Once-daily dosing of single-agent encorafenib has a distinct tolerability profile and shows varying antitumor activity across BRAFi-pretreated and BRAFi-naïve patients with advanced/metastatic stage melanoma 7.
Mechanism of action
Encorafenib is a kinase inhibitor that specifically targets BRAF V600E, as well as wild-type BRAF and CRAF while tested with in vitro cell-free assays with IC50 values of 0.35, 0.47, and 0.3 nM, respectively. Mutations in the BRAF gene, including BRAF V600E, result in activated BRAF kinases that mahy stimulate tumor cell growth. Encorafenib is able to bind to other kinases in vitro including JNK1, JNK2, JNK3, LIMK1, LIMK2, MEK4, and STK36 and significantly reduce ligand binding to these kinases at clinically achievable concentrations (≤ 0.9 μM) Label.
In efficacy studies, encorafenib inhibited the in vitro cell growth of tumor cell lines that express BRAF V600 E, D, and K mutations. In mice implanted with tumor cells expressing the BRAF V600E mutation, encorafenib induced tumor regressions associated with RAF/MEK/ERK pathway suppression Label.
Encorafenib and binimetinib target two different kinases in the RAS/RAF/MEK/ERK pathway. Compared with either drug alone, co-administration of encorafenib and binimetinib result in greater anti-proliferative activity in vitro in BRAF mutation-positive cell lines and greater anti-tumor activity with respect to tumor growth inhibition in BRAF V600E mutant human melanoma xenograft studies in mice. In addition to the above, the combination of encorafenib and binimetinib acted to delay the emergence of resistance in BRAF V600E mutant human melanoma xenografts in mice compared with the administration of either drug alone Label.
Pharmacology
Encorafenib acts as an ATP-competitive RAF kinase inhibitor, decreasing ERK phosphorylation and down-regulation of CyclinD1.[5]This arrests the cell cycle in G1 phase, inducing senescence without apoptosis.[5] Therefore it is only effective in melanomas with a BRAF mutation, which make up 50% of all melanomas.[6] The plasma elimination half-life of encorafenib is approximately 6 hours, occurring mainly through metabolism via cytochrome P450 enzymes.[7]
Clinical trials
Several clinical trials of LGX818, either alone or in combinations with the MEK inhibitor MEK162,[8] are being run. As a result of a successful Phase Ib/II trials, Phase III trials are currently being initiated.[9]
History
Approval of encorafenib in the United States was based on a randomized, active-controlled, open-label, multicenter trial (COLUMBUS; NCT01909453) in 577 patients with BRAF V600E or V600K mutation-positive unresectable or metastatic melanoma.[3] Patients were randomized (1:1:1) to receive binimetinib 45 mg twice daily plus encorafenib 450 mg once daily, encorafenib 300 mg once daily, or vemurafenib 960 mg twice daily.[3] Treatment continued until disease progression or unacceptable toxicity.[3]
The major efficacy measure was progression-free survival (PFS) using RECIST 1.1 response criteria and assessed by blinded independent central review.[3] The median PFS was 14.9 months for patients receiving binimetinib plus encorafenib, and 7.3 months for the vemurafenib monotherapy arm (hazard ratio 0.54, 95% CI: 0.41, 0.71, p<0.0001).[3] The trial was conducted at 162 sites in Europe, North America and various countries around the world.[4]
SYN
PATENT
WO2010010154 , expiry , EU states, 2029, US in 2030 with US154 extension.
WO 2011025927
WO 2016089208
Patent
WO-2020011141
Novel deuterated analogs of diarylpyrazole compounds, particularly encorafenib are B-RAF and C-RAF kinase inhibitors, useful for treating proliferative diseases such as melanoma and colorectal cancer. Family members of the product case, WO2010010154 , expire in most of the EU states until 2029 and will expire in the US in 2030 with US154 extension. In January 2020, the US FDA’s Orange Book was seen to list encorafenib patents such as US8946250 , US8501758 , US9314464 and US9763941 , expiring in the range of 2029-2032. At that time Orange Book also reported that encorafenib as having NCE exclusivity expiring on July 27, 2023.
The mitogen-activated protein kinase (MAPK) pathway mediates the activity of many effector molecules that coordinately control cell proliferation, survival, differentiation, and migration. Cells are bound by plasma factors such as growth factors, cytokines, or hormones to plasma membrane-associated Ras and GTP and thereby activated to recruit Raf. This interaction induces Raf’s kinase activity, resulting in direct phosphorylation of MAPK / ERK (MEK), which in turn phosphorylates extracellular signal-related kinase (ERK). Activated ERK phosphorylates a range of effector molecules, such as kinases, phosphatases, transcription factors, and cytoskeleton proteins. Therefore, the Ras-Raf-MEK-ERK signaling pathway transmits signals from cell surface receptors to the nucleus and is essential for cell proliferation and survival.
[0003]
According to Raf’s ability to interact with upstream regulator Ras, Raf has three different isoforms, namely A-Raf, B-Raf, and C-Raf. An activating mutation of one of the Ras genes can be observed in about 20% of all tumors, and the Ras-Raf-MEK-ERK pathway is activated in about 30% of all tumors. Activation mutations in the B-Raf kinase domain occur in approximately 70% of melanoma, 40% of papillary cancer, 30% of low-grade ovarian cancer, and 10% of colorectal cancer. Most B-Raf mutations are found in the kinase domain, with a single substitution (V600E) accounting for 80%. The mutated B-Raf protein activates the Raf-MEK-ERK pathway by increasing kinase activity against MEK or by activating C-Raf. B-Raf inhibitors inhibit cells involved in B-Raf kinase by blocking the signal cascade in these cancer cells and eventually inducing cell arrest and / or death.
[0004]
Encorafenib (aka LGX-818, chemical name is (S)-(1-((4- (3- (5-chloro-2-fluoro-3- (methylsulfonylamino) phenyl) -1-iso Propyl-1H-pyrazol-4-yl) pyrimidin-2-yl) amino) prop-2-yl) methyl carbamate, which has the following structural formula) is a new oral BRAF jointly developed by Novartis and Array Pharmaceuticals Inhibitors can inhibit the activation of the MAPK pathway caused by B-Raf kinase mutations (such as V600 mutations, that is, glutamate mutations at amino acid 600). Encorafenib alone or in combination with MEK inhibitor Binimetinib is used to treat patients with advanced BRAF v600 mutant melanoma. On June 27, 2018, the FDA approved Encorafenib (commercial name BRAFTOVI) capsules in combination with Binimetinib (commercial name: MEKTOVI) tablets for treating melanoma patients with BRAF V600E or BRAFV 600K mutations.
It is known that poor absorption, distribution, metabolism, and / or excretion (ADME) properties are the main cause of the failure of many candidate drug clinical trials. Many drugs currently on the market also limit their scope of application due to poor ADME properties. The rapid metabolism of drugs will cause many drugs that could be highly effective in treating diseases to be difficult to make because they are metabolized from the body too quickly. Although frequent or high-dose medication may solve the problem of rapid drug removal, this method will bring problems such as poor patient compliance, side effects caused by high-dose medication, and rising treatment costs. In addition, rapidly metabolizing drugs may also expose patients to adverse toxic or reactive metabolites.
[0007]
Although Encoratenib as a BRAF inhibitor can effectively treat BRAF V600 mutant melanoma, there are still serious clinical unmet needs in this field, and the Encoratenib compound is a class II BCS with poor water solubility at weakly acidic and neutral pH Compounds have poor oral availability, so finding new compounds that have a therapeutic effect on BRAF kinase mutations, have good oral bioavailability, and have pharmaceutical properties is still a challenging task. Therefore, there remains a need in the art to develop compounds that have selective inhibitory activity and / or better pharmacodynamics / pharmacokinetics for use as BRAF inhibitors, and the present invention provides such compounds.
PAPER
European journal of cancer (Oxford, England : 1990) (2018), 88, 67-76.
References
- ^ Koelblinger P, Thuerigen O, Dummer R (March 2018). “Development of encorafenib for BRAF-mutated advanced melanoma”. Current Opinion in Oncology. 30 (2): 125–133. doi:10.1097/CCO.0000000000000426. PMC 5815646. PMID 29356698.
- ^ Burotto M, Chiou VL, Lee JM, Kohn EC (November 2014). “The MAPK pathway across different malignancies: a new perspective”. Cancer. 120 (22): 3446–56. doi:10.1002/cncr.28864. PMC 4221543. PMID 24948110.
- ^ Jump up to:a b c d e f g “FDA approves encorafenib and binimetinib in combination for unresectable or metastatic melanoma with BRAF mutations”. U.S. Food and Drug Administration (FDA)(Press release). 27 June 2018. Archived from the original on 18 December 2019. Retrieved 28 June 2018. This article incorporates text from this source, which is in the public domain.
- ^ Jump up to:a b “Drug Trial Snapshot: Braftovi”. U.S. Food and Drug Administration (FDA). 16 July 2018. Archived from the original on 19 December 2019. Retrieved 18 December 2019. This article incorporates text from this source, which is in the public domain.
- ^ Jump up to:a b Li Z, Jiang K, Zhu X, Lin G, Song F, Zhao Y, Piao Y, Liu J, Cheng W, Bi X, Gong P, Song Z, Meng S (January 2016). “Encorafenib (LGX818), a potent BRAF inhibitor, induces senescence accompanied by autophagy in BRAFV600E melanoma cells”. Cancer Letters. 370 (2): 332–44. doi:10.1016/j.canlet.2015.11.015. PMID 26586345.
- ^ Hodis E, Watson IR, Kryukov GV, Arold ST, Imielinski M, Theurillat JP, et al. (July 2012). “A landscape of driver mutations in melanoma”. Cell. 150 (2): 251–63. doi:10.1016/j.cell.2012.06.024. PMC 3600117. PMID 22817889.
- ^ Koelblinger P, Thuerigen O, Dummer R (March 2018). “Development of encorafenib for BRAF-mutated advanced melanoma”. Current Opinion in Oncology. 30 (2): 125–133. doi:10.1097/CCO.0000000000000426. PMC 5815646. PMID 29356698.
- ^ “18 Studies found for: LGX818”. Clinicaltrials.gove.
- ^ Clinical trial number NCT01909453 for “Study Comparing Combination of LGX818 Plus MEK162 and LGX818 Monotherapy Versus Vemurafenib in BRAF Mutant Melanoma (COLUMBUS)” at ClinicalTrials.gov
External links
- “Encorafenib”. Drug Information Portal. U.S. National Library of Medicine.
- Li Z, Jiang K, Zhu X, Lin G, Song F, Zhao Y, Piao Y, Liu J, Cheng W, Bi X, Gong P, Song Z, Meng S: Encorafenib (LGX818), a potent BRAF inhibitor, induces senescence accompanied by autophagy in BRAFV600E melanoma cells. Cancer Lett. 2016 Jan 28;370(2):332-44. doi: 10.1016/j.canlet.2015.11.015. Epub 2015 Nov 14. [PubMed:26586345]
- Koelblinger P, Thuerigen O, Dummer R: Development of encorafenib for BRAF-mutated advanced melanoma. Curr Opin Oncol. 2018 Mar;30(2):125-133. doi: 10.1097/CCO.0000000000000426. [PubMed:29356698]
- Moschos SJ, Pinnamaneni R: Targeted therapies in melanoma. Surg Oncol Clin N Am. 2015 Apr;24(2):347-58. doi: 10.1016/j.soc.2014.12.011. Epub 2015 Jan 24. [PubMed:25769717]
- Dummer R, Ascierto PA, Gogas HJ, Arance A, Mandala M, Liszkay G, Garbe C, Schadendorf D, Krajsova I, Gutzmer R, Chiarion-Sileni V, Dutriaux C, de Groot JWB, Yamazaki N, Loquai C, Moutouh-de Parseval LA, Pickard MD, Sandor V, Robert C, Flaherty KT: Encorafenib plus binimetinib versus vemurafenib or encorafenib in patients with BRAF-mutant melanoma (COLUMBUS): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol. 2018 May;19(5):603-615. doi: 10.1016/S1470-2045(18)30142-6. Epub 2018 Mar 21. [PubMed:29573941]
- FDA approves encorafenib and binimetinib in combination for unresectable or metastatic melanoma with BRAF mutations [Link]
- BRAF B-Raf proto-oncogene, serine/threonine kinase [ Homo sapiens (human) ] [Link]
- Phase I Dose-Escalation and -Expansion Study of the BRAF Inhibitor Encorafenib (LGX818) in Metastatic BRAF-Mutant Melanoma [Link]
- Encorafenib FDA label [File]
- Encorafenib review [File]
 |
|
| Clinical data | |
|---|---|
| Trade names | Braftovi |
| Other names | LGX818 |
| AHFS/Drugs.com | Monograph |
| MedlinePlus | a618040 |
| License data |
|
| Routes of administration |
Oral |
| Drug class | Antineoplastic Agents |
| ATC code | |
| Legal status | |
| Legal status |
|
| Identifiers | |
| CAS Number | |
| PubChem CID | |
| DrugBank | |
| ChemSpider | |
| UNII | |
| KEGG | |
| ChEMBL | |
| CompTox Dashboard (EPA) | |
| Chemical and physical data | |
| Formula | C22H27ClFN7O4S |
| Molar mass | 540.011 g/mol g·mol−1 |
| 3D model (JSmol) | |
///////////ENCORAFENIB, 1269440-17-6, BRAFTOVI, NVP-LGX818, LGX818, LGX 818, エンコラフェニブ ,
COC(=O)N[C@@H](C)CNc1nccc(n1)c2cn(nc2c3cc(Cl)cc(NS(=O)(=O)C)c3F)C(C)C
patent
Method for preparing compounds of the invention
[0165]
The compounds of the invention, including their salts, can be prepared using known organic synthesis techniques, and can be synthesized according to any of a number of possible synthetic routes, such as those in the schemes below. The reaction for preparing the compound of the present invention can be performed in a suitable solvent, and a person skilled in the art of organic synthesis can easily select a solvent. Suitable solvents may be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is performed (e.g., a temperature ranging from the solvent freezing temperature to the solvent boiling point temperature). A given reaction may be performed in one solvent or a mixture of more than one solvent. The skilled person can select a solvent for a specific reaction step depending on the specific reaction step.
[0166]
The preparation of the compounds of the invention may involve the protection and removal of different chemical groups. Those skilled in the art can easily determine whether protection and removal of protection are needed and the choice of an appropriate protecting group. The chemical nature of the protecting group can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Edition, John Wiley & Sons: New Jersey, (2006), which is incorporated herein by reference in its entirety.
[0167]
The compound of the present invention can be prepared into a single stereo by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereomeric compounds, separating the diastereomers, and recovering the optically pure enantiomer isomer. Enantiomeric resolution can be performed using diastereomeric derivatives of the compounds of the present invention, with preferentially dissociable complexes (e.g., crystalline diastereomeric salts). Diastereomers have significantly different physical properties (eg, melting points, boiling points, solubility, reactivity, etc.) and can be easily separated by the advantages of these dissimilarities. Diastereomers can be separated by chromatography, preferably by separation / resolution techniques based on differences in solubility. The optically pure enantiomer is then recovered, along with the resolving reagent, by any practical means that does not allow racemization. A more detailed description of techniques suitable for resolution of stereoisomers of compounds starting from racemic mixtures can be found in Jean Jacques, Andre Collet, Samue1H. Wilen, “Enantiomers, Racemates and Resolution” (“Enantiomers, Racemates and Resolutions “), John Wiley And Sons, Inc., 1981.
[0168]
The reaction can be monitored according to any suitable method known in the art. For example, it may be by spectroscopic means such as nuclear magnetic resonance (NMR) spectroscopy (e.g. 1 H or 13 C), infrared (IR) spectroscopy, spectrophotometry (e.g. UV-visible light), mass spectrometry (MS)) or by chromatography Methods such as high performance liquid chromatography (HPLC) or thin layer chromatography (TLC) to monitor product formation.
[0169]
The compound of formula (I) of the present invention can be prepared by the following reaction scheme 1:
[0170]
Reaction Flowchart 1
[0171]
[0172]
Wherein Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , R 1 , R 2 , R 3 , R 4 , X 1 , X 2 , X 3 , X 4 and X 5 are as defined in the present invention. The compound of formula (I) can be obtained by using a compound of formula (I-1) and a sulfonating agent X 5 SO 2 Cl at a suitable base (for example, pyridine, triethylamine, 4- (N, N-dimethylamino) pyridine, etc.) Reaction with a suitable solvent (e.g., dichloromethane, THF, etc.). The reaction is performed at a temperature ranging from about 0 ° C to about 1000 ° C, and may take up to about 20 hours to complete. The reaction mixture is optionally further reacted to remove any protecting groups.
[0173]
The compound of formula (I-1) can be prepared by the following reaction scheme 2:
[0174]
Reaction Flowchart 2
[0175]
[0176]
Wherein Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , R 1 , R 2 , R 3 , R 4 , X 1 , X 2 , X 3 , X 4 and X 5 are as defined in the present invention, M is a leaving group (for example, iodine, bromine, chlorine, trifluoromethanesulfonyloxy, etc.), each Z may be, for example, hydrogen, methyl, etc., or two Z groups may be connected to form a boric acid ester. Both P groups can be H, or two P groups taken together represent a suitable nitrogen protecting group (eg, one P can be hydrogen and the other can be Boc). The compound of formula (I-2) can be obtained by using a compound of formula (I-4) and a compound of formula (I-3) in a suitable transition metal catalyst (for example, Pd (PPh 3 ) 4 or PdCl 2(dppf)), a suitable solvent (for example, DME, dioxane, toluene, ethanol, etc.) and a suitable base (for example, anhydrous potassium carbonate or sodium carbonate, etc.) are reacted. The reaction is carried out at a temperature ranging from about 20 ° C to 120 ° C, and may take about 2 hours to complete. Compounds of formula (I) can be synthesized by leaving the protecting group P from compounds of formula (I-2) (eg, by treatment with a strong acid such as hydrogen chloride in the presence of DME and dioxane).
[0177]
Compounds of formula (I-4) can be prepared by the following reaction scheme 3:
[0178]
Reaction Flowchart 3
[0179]
[0180]
Wherein Y 1 , Y 2 , R 1 , R 2 , R 3 , R 4 , X 1 , X 2 , X 3 and X 4 are as defined in the present invention, and M is a leaving group (for example, iodine, Bromine, chlorine, trifluoromethanesulfonyloxy, etc.), and V is a leaving group (eg, iodine, bromine, chlorine, trifluoromethanesulfonyloxy, etc.). Compounds of formula (I-4) can be prepared by reacting an amine compound of formula (I-5) and a compound of formula (I-6). The reaction is performed in a suitable solvent (for example, DMSO, NMP, dioxane, or isopropanol) in the presence of a suitable base (for example, sodium carbonate or potassium carbonate, etc.) at a temperature ranging from about 25 ° C to about 120 ° C.
[0181]
Examples
[0182]
The present invention will be further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally based on conventional conditions or conditions recommended by the manufacturer. Unless stated otherwise, parts and percentages are parts by weight and percent by weight.
[0183]
The abbreviations used in this article have the following meanings:
[0184]
[TABLE 0001]
| APCI | Atmospheric pressure chemical dissociation |
| HPLC | High performance liquid chromatography |
| TLC | TLC |
| h | hour |
| DMF | N, N-dimethylformamide |
| K 2 CO 3 | Potassium carbonate |
| DCM | Dichloromethane |
| THF | Tetrahydrofuran |
| CH 3 MgBr | Methyl magnesium bromide |
| PTSA | p-Toluenesulfonic acid |
| TFA | Trifluoroacetate |
| NMP | N-methylpyrrolidone |
| Diguanidinium carbonate | Guanidine carbonate |
| MTBE | Methyl tert-butyl ether |
| POCl 3 | Phosphorus oxychloride |
| DMSO | Dimethyl sulfoxide |
| Pd (dppf) Cl 2 | [1,1′-Bis (diphenylphosphino) ferrocene] Palladium dichloride |
| Dioxane | Dioxane |
| TsCl | 4-toluenesulfonyl chloride |
| Boc | Tert-butoxy carbon |
| DIPEA | N, N-diisopropylethylamine |
| CDCl 3 | Deuterated chloroform |
| TEA | Triethylamine |
| DMAP | 4-dimethylaminopyridine |
| Na 2 CO 3 | Sodium carbonate |
| HCl | hydrochloric acid |
[0185]
[表 0002]
| MsCl | Methanesulfonyl chloride |
| Tol | Toluene |
[0186]
Preparation of intermediate A 2-chloro-4- (3-iodo-1- (prop-2-yl-d 7) -1H-pyrazol-4-yl) pyrimidine.
[0187]
[0188]
Use the following route for synthesis:
[0189]
[0190]
Step 1 Synthesis of Compound A-2
[0191]
Compound 1 (5.0 g, 73.4 mmol) was added to a 47% solution of hydrobromic acid (20 ml). The reaction solution was reacted at 80 ° C for 3 hours, and distilled under normal pressure. The 60-70 ° C fraction was collected to obtain a colorless liquid. 6.2g, yield 65%.
[0192]
Step 2 Synthesis of Compound A-4
[0193]
Compound A-3 (3.0 g, 27.8 mmol) was added to a DMF (20 ml) solution, the solution was lowered to 0 ° C, K 2 CO 3 (4.6 g, 33.3 mmol, 10 ml) was added, and the mixture was stirred at low temperature for 0.5 h. Then compound A-2 (4.3g, 33.3mmol) was slowly added dropwise. After the dropwise addition, the temperature was raised to 90 ° C for 10 hours. The reaction solution was extracted with DCM (50ml × 3). The organic phases were combined and dried over anhydrous sodium sulfate. The concentrated solution was subjected to column separation (eluent: petroleum ether / ethyl acetate (v / v) = 1: 1) to obtain 3.1 g of a white solid in a yield of 72%. LC-MS (APCI): m / z = 158.21.06 (M + 1) + .
[0194]
Step 3 Synthesis of Compound A-5
[0195]
Under nitrogen protection, compound A-4 (3.0 g, 19.1 mmol) was added to a solution of anhydrous THF (40 ml), and the temperature was lowered to -5 ° C, and CH 3 MgBr (19.1 ml, 57.3 mmol, 3 ml / L) was added dropwise . Anhydrous THF solution. After the dropwise addition was completed, the temperature was gradually raised to reflux for 4 h. The reaction was quenched with saturated ammonium chloride, then the pH was adjusted to neutral with dilute hydrochloric acid, and the mixture was extracted with ethyl acetate (50 ml × 3). The phases were dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: petroleum ether / ethyl acetate (v / v) = 2: 3) to obtain 2.0 g of a yellow solid with a yield of 61%. LC-MS (APCI): m / z = 175.21.06 (M + 1) + .
[0196]
Step 4 Synthesis of Compound A-6
[0197]
Compound A-5 (2.0g, 11.5mmol), PTSA (4.2g, 23.0mmol) were added to the acetonitrile (15ml) solution, and after dropping to 0 ° C, sodium nitrite (1.43g, 20.7mmol) and Aqueous solution (5 ml) of potassium iodide (3.82 g, 23.0 mmol). The reaction solution was reacted at room temperature for 3 hours, and extracted with ethyl acetate (30 ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and spin-dried to obtain 2.5 g of an orange solid with a yield of 75%.
[0198]
Step 5 Synthesis of Compound A-8
[0199]
Under nitrogen protection, compound A-6 (2.0 g, 7.01 mmol) was added to a DMF (15 ml) solution, and the temperature was raised to 120 ° C. Then compound A-7 (1.9 g, 10.5 mmol, 10 ml) was added at 120 ° C. The reaction was stirred for 0.5h. Dichloromethane (30ml × 3) was extracted. The organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was separated by column. ) To obtain 1.9 g of the product in a yield of 80%. LC-MS (APCI): m / z = 341.06 (M + 1) + .
[0200]
Step 6 Synthesis of Compound A-9
[0201]
Under nitrogen protection, compound A-8 (1.9 g, 5.6 mmol) and guanidine carbonate (1.6 g, 12.8 mmol) were sequentially added to the NMP (20 ml) solution. At the same time, a water separation device was set up to raise the solution to 130 ° C. The reaction was stirred at 130 ° C for 10 hours. After the reaction was completed, dichloromethane (30ml × 3) was extracted, the organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was separated by column (eluent: petroleum ether / ethyl acetate (v / v) = 2: 3), 1.5 g of product was obtained with a yield of 81%. LC-MS (APCI): m / z = 336.86 (M + 1) + .
[0202]
Step 7 Synthesis of Compound A-10
[0203]
Compound A-9 (1.5 g, 4.5 mmol) was added to the TFA (15 ml) solution. After reducing to 0 ° C, sodium nitrite (0.93 g, 13.4 mmol) was added as a solid. The reaction solution was reacted at room temperature for 1 h. Extract with ethyl acetate (30ml × 3), combine the organic phases, dry over anhydrous sodium sulfate, spin dry the oil with MTBE (10ml), and filter to obtain 1.3g of white solid, 87% yield, LC-MS (APCI) : m / z = 338.15 (M + 1) + .
[0204]
Step 8 Synthesis of intermediate compound A
[0205]
Compound A-10 (1.3 g, 3.86 mmol) was added to a solution of POCl 3 (15 ml), and the temperature was raised to 110 ° C., and the reaction was refluxed at this temperature for 10 h. After the reaction was completed, the reaction solution was spin-dried and dichloromethane (30 ml × 2) Extraction, combined organic phases, dried over anhydrous sodium sulfate, and column separation of the concentrated solution (eluent: petroleum ether / ethyl acetate (v / v) = 4: 1), 1.0 g of product was obtained, yield 73% . LC-MS (APCI): m / z = 356.32 (M + 1) + .
[0206]
Preparation of intermediate B (S)-(methyl-d 3) (1-aminoprop-2-yl) aminocarbonate.
[0207]
[0208]
Use the following route for synthesis:
[0209]
[0210]
Step 1 Synthesis of Compound B-2
[0211]
Compound B-1 (1.3 g, 4.5 mmol) was dissolved in a toluene (15 ml) solution, the temperature was lowered to 0 ° C, and CD 3 OD (0.5 g, 15 mmol) and triethylamine (1.7 g, 17 mmol) in toluene were added dropwise . (10ml) solution, reacted at room temperature for 2h after the dropwise addition, washed three times with ice water, dried over anhydrous sodium sulfate, filtered to obtain a toluene solution of compound B-2, and directly used in the next step.
[0212]
Step 2 Synthesis of Compound B-4
[0213]
At 0 ° C, the hydrochloride (0.5 g, 2.4 mmol) and triethylamine (0.73 g, 7.2 mmol) of compound B-3 were added to a solution of dichloromethane (10 ml) in this order, and one step of compound B was added dropwise. -2 toluene solution, reacted for 5 hours at room temperature after the addition, quenched by adding water (10ml), extracted with dichloromethane (20ml × 3), combined organic phases, dried over anhydrous sodium sulfate, and concentrated the column for separation (elution Agent: petroleum ether / ethyl acetate (v / v) = 4: 1), 0.45 g of white solid product was obtained with a yield of 80%.
[0214]
Step 3 Synthesis of intermediate compound B
[0215]
At 0 ° C, a solution of 4M hydrochloric acid in dioxane (4ml) was slowly added to a solution of compound B-4 (0.45g, 1.9mmol) in dichloromethane (10ml), and the reaction was continued at room temperature for 6h. After the reaction was completed, the solution was spin-dried, petroleum ether (10 ml) was slurried, and 0.2 g of the product was obtained by suction filtration with a yield of 77%.
[0216]
Preparation of intermediate C (1-aminoprop-2-yl-1,1,3,3,3-d 5) carbamate.
[0217]
[0218]
Use the following route for synthesis:
[0219]
[0220]
Step 1 Synthesis of compound C-3
[0221]
A mixture of compound C-2 (4.6 g, 61.8 mmol), compound C-1 (11.5 g, 67.6 mmol) and sodium hydroxide (7.16 g, 67.7 mmol) in water (60 ml) was stirred and reacted at 0 ° C for 3 h. After the reaction was completed, water (60 ml) was added, and the mixture was extracted with ethyl acetate (60 ml x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: petroleum ether / ethyl acetate (v / v) = 10: 1), 11.2 g of an oily substance was obtained, and the yield was 88%.
[0222]
Step 2 Synthesis of Compound C-4
[0223]
Under a nitrogen atmosphere, DMSO (4.8 g, 61.5 mmol) was slowly added to a solution of oxalyl chloride (6.0 g, 47.2 mmol) in DCM (60 ml) at -78 ° C, and the mixture was stirred at -78 ° C for half an hour. Then, a solution of compound C-3 (8.0 g, 38.2 mmol) in DCM (20 ml) was added to the mixture, and the mixture was further stirred at -78 ° C for 1 h, and then triethylamine (16 ml) was added to the mixture, and the mixture was raised to At room temperature, it was washed with 1N hydrochloric acid (50ml) and sodium bicarbonate aqueous solution (50ml) successively. The organic phase was dried over anhydrous sodium sulfate, heat-shrinked, and then slurried with a volume ratio of PE: EA = 8: 1 to obtain 5.3g of a white solid product. The rate is 87%.
[0224]
Step 3 Synthesis of Compound C-5
[0225]
1,5,7-Triazabicyclo [4.4.0] dec-5-ene (0.27 g, 1.9 mmol) was added to a solution of compound C-4 (4.0 g, 19.3 mmol) in deuterated chloroform (30 ml) After the reaction solution was stirred at room temperature for 30 hours, water (10 ml) was added to quench the reaction, and the organic phase was separated and washed with saturated sodium chloride. The organic phase was dried and spin-dried to obtain 3.9 g of an oil with a yield of 98%.
[0226]
Step 4 Synthesis of compound C-6
[0227]
Under a nitrogen atmosphere, compound C-5 (4.0 g, 18.9 mmol) and tert-butylsulfinamide (2.7 g, 22.6 mmol) were added to the THF (60 ml) solution, and tetraisopropyl titanate was added at room temperature. Ester (11.8 g, 41.5 mmol), and then heated to 60 ° C for 3 h. The reaction solution was cooled to room temperature, quenched by adding water, and extracted with ethyl acetate (60 ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: petroleum ether / ethyl acetate ( v / v) = 4: 1), 3.5 g of product was obtained with a yield of 58%. LC-MS (APCI): m / z = 315.80 (M + 1) + .
[0228]
Step 5 Synthesis of compound C-7
[0229]
At -50 ° C, NaBH 4 (0.73 g, 19.1 mmol) was added to a solution of compound C-6 (2.0 g, 6.3 mmol) in methanol (20 ml), and then the reaction was continued at low temperature for 1 h. 1M hydrochloric acid was added to quench the reaction, and the mixture was extracted with dichloromethane (30 ml × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and spin-dried to obtain 2.1 g of an oily product.
[0230]
Step 6 Synthesis of compound C-8
[0231]
A solution of 4M hydrochloric acid in dioxane (10 ml) was slowly added to a solution of compound C-7 (2.0 g, 6.3 mmol) in dichloromethane (20 ml) at 0 ° C, and the reaction was continued at 0 ° C for 6 h. After the reaction is completed, the solvent is spin-dried and directly used in the next step without further processing.
[0232]
Step 7 Synthesis of compound C-9
[0233]
Triethylamine (1.43 g, 14.1 mmol) was added to a solution of compound C-8 (1.5 g, 7.0 mmol) in dichloromethane (20 ml) at 0 ° C, and methyl chloroformate was added dropwise to the mixture. (0.8g, 8.5mmol), and react at room temperature for 5 hours after the addition. After the reaction is complete, water (10ml) is added to quench the reaction. The reaction solution is extracted with dichloromethane (20ml × 2). Sodium was dried, and the concentrated solution was subjected to column separation (eluent: petroleum ether / ethyl acetate (v / v) = 4: 1) to obtain 1.1 g of a white solid product with a yield of 58%.
[0234]
Step 8 Synthesis of intermediate compound C
[0235]
Under a hydrogen atmosphere, Pd-C (0.2g, 10%) was added to the compound C-9 (1.0g, 3.7mmol) in ethanol (5ml) and a 1N hydrochloric acid solution (5ml), and the reaction was stirred for 5h. After the reaction was completed, It was filtered and the filtrate was directly concentrated to obtain 0.4 g of the product.
[0236]
Example 1 (S) -methyl- (1-((4- (3- (5-chloro-2-fluoro-3- (methylsulfonylamino) phenyl) -1- (propan-2-yl -d 7) Preparation of 1H-pyrazol-4-yl) pyrimidin-2-yl) amino) propan-2-yl) carbamate (compound L-1).
[0237]
[0238]
Use the following route for synthesis:
[0239]
[0240]
Step 1 Synthesis of Compound 2
[0241]
Under nitrogen protection, intermediate compound A (1.0 g, 2.8 mmol), compound 1 (0.52 g, 3.1 mmol), and sodium carbonate (1.2 g, 11.2 mmol) were sequentially added to the DMSO (20 ml) solution, and the temperature was raised to 90 ° C. The reaction was stirred at this temperature for 16h. After the reaction was completed, DCM (30ml × 3) was extracted, the organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was separated by column (eluent: petroleum ether / ethyl acetate (v / v) = 1: 2), 0.8 g of product was obtained with a yield of 63%. LC-MS (APCI): m / z = 452.33 (M + 1) + .
[0242]
Step 2 Synthesis of Compound 4
[0243]
Under nitrogen protection, compound 2 (0.5 g, 1.11 mmol), compound 3 (0.5 g, 1.33 mmol), sodium carbonate (0.47 g, 4.43 mmol), and Pd (dppf) Cl2 (0.09 g, 0.11 mmol) were added in this order. Into a mixed solution of toluene (20 ml) and water (4 ml), heated to 80 ° C. for 2 h. The reaction solution was cooled to room temperature, extracted with ethyl acetate (30 ml × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: petroleum ether / ethyl acetate (v / v) = 1: 1) 0.2 g of product was obtained with a yield of 31%. LC-MS (APCI): m / z = 569.09 (M + 1) + .
[0244]
Step 3 Synthesis of Compound 5
[0245]
At 0 ° C, a solution of 4M hydrochloric acid in dioxane (4ml) was slowly added to a solution of compound 4 (0.2g, 0.35mmol) in DCM (10ml), and the reaction mixture was warmed to room temperature for 6h. After the reaction is complete, the solution is spin-dried and directly sent to the next step without further processing. LC-MS (APCI): m / z = 469.27 (M + 1) + .
[0246]
Step 4 Synthesis of Compound L-1
[0247]
Compound 5 (0.15 g, 0.32 mmol) and triethylamine (0.16 g, 1.6 mmol) were sequentially added to the DCM (10 ml) solution. After the temperature was lowered to 0 ° C, MsCl (0.11 g, 1.0 mmol) was slowly added dropwise. After the addition was completed, the reaction temperature was raised to room temperature for 5 hours. After the reaction was completed, the reaction solution was spin-dried to obtain a residue. Toluene (9 ml), methanol (1 ml), water (10 ml), and sodium carbonate (2 g) were sequentially added to the residue. The reaction temperature was raised to 85 ° C for 10 hours, cooled to room temperature, and extracted with ethyl acetate (20ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 20: 1), 50 mg of product was obtained with a yield of 35%. LC-MS (APCI): m / z = 547.31 (M + 1) + . 1 H NMR (400MHz, CDCl 3 ) δ 8.08 (d, J = 11.4 Hz, 2H), 7.61 (d, J = 6.3 Hz, 1H), 7.42 (d, J = 5.6 Hz, 1H), 6.48 (d , J = 5.1 Hz, 1H), 5.32 (d, J = 18.8 Hz, 1H), 5.17 (s, 1H), 4.59 (d, J = 13.2 Hz, 1H), 3.79 (s, 1H), 3.61 (s , 3H), 3.24 (s, 1H), 2.98 (d, J = 16.6Hz, 3H), 2.01 (s, 1H), 1.31 (s, 3H).
[0248]
Example 2 (S)-(methyl-d 3)-(1-((4- (3- (5-chloro-2-fluoro-3- (methylsulfonylamino) phenyl) -1-iso Preparation of propyl-1H-pyrazol-4-yl) pyrimidin-2-yl) amino) propan-2-yl) carbamate (compound L-2).
[0249]
[0250]
Use the following route for synthesis:
[0251]
[0252]
Step 1 Synthesis of compound 7
[0253]
Under nitrogen protection, compound 6 (0.5 g, 1.5 mmol), intermediate compound B (0.2 g, 1.5 mmol), and sodium carbonate (0.63 g, 6.0 mmol) were sequentially added to the DMSO (20 ml) solution, and the temperature was raised to 90 ° C. The reaction was stirred at this temperature for 16h. After the reaction was completed, DCM (30ml × 3) was extracted, the organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was separated by column (eluent: petroleum ether / ethyl acetate (v / v) = 1: 2), 0.42 g of product was obtained in a yield of 65%. LC-MS (APCI): m / z = 447.80 (M + 1) + .
[0254]
Step 2 Synthesis of Compound 8
[0255]
Under nitrogen protection, compound 7 (0.4 g, 0.90 mmol), compound 3 (0.4 g, 1.07 mmol), sodium carbonate (0.38 g, 3.6 mmol), and Pd (dppf) Cl2 (0.07 g, 0.09 mmol) were added in this order. Into a mixed solution of toluene (20 ml) and water (4 ml), the mixture was heated to 80 ° C. and reacted for 2 h. The reaction solution was cooled to room temperature, extracted with ethyl acetate (30 ml × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: petroleum ether / ethyl acetate (v / v) = 1: 1) 0.2 g of product was obtained with a yield of 40%. LC-MS (APCI): m / z = 565.03 (M + 1) + .
[0256]
Step 3 Synthesis of Compound 9
[0257]
At 0 ° C, a solution of 4M hydrochloric acid in dioxane (4 ml) was slowly added to a solution of compound 8 (0.2 g, 0.35 mmol) in DCM (10 ml), and the reaction mixture was warmed to room temperature and continued to react for 6 h. After the reaction is complete, the solution is spin-dried and directly sent to the next step without further processing. LC-MS (APCI): m / z = 465.27 (M + 1) + .
[0258]
Step 4 Synthesis of Compound L-2
[0259]
Compound 9 (0.2 g, 0.43 mmol) and triethylamine (0.22 g, 2.1 mmol) were sequentially added to the DCM (10 ml) solution. After lowering to 0 ° C, MsCl (0.15 g, 1.3 mmol) was slowly added dropwise. After the addition was completed, the reaction temperature was raised to room temperature for 5 hours. After the reaction was completed, the reaction solution was spin-dried to obtain a residue. Toluene (9 ml), methanol (1 ml), water (10 ml), and sodium carbonate (2 g) were sequentially added to the residue. The reaction temperature was raised to 85 ° C for 10 hours, cooled to room temperature, and extracted with ethyl acetate (20ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 20: 1), 70 mg of product was obtained with a yield of 30%. LC-MS (APCI): m / z = 543.21 (M + 1) + . 1 H NMR (400MHz, CDCl 3 ) δ 8.01 (d, J = 11.4 Hz, 2H), 7.63 (d, J = 6.3 Hz, 1H), 7.40 (d, J = 5.8 Hz, 1H), 6.58 (d , J = 6.1 Hz, 1H), 5.47 (d, J = 18.8 Hz, 1H), 5.17 (s, 1H), 4.59 (d, J = 12.2, Hz, 1H), 3.80 (s, 1H), 3.61 ( s, 1H) 3.24 (s, 1H), 3.10 (d, J = 16.6 Hz, 3H), 2.21 (s, 1H), 1.35 (s, 3H), 1.27 (d, 6H).
[0260]
Example 3 (S)-(methyl-d 3)-(1-((4- (3- (5-chloro-2-fluoro-3- (methylsulfonylamino) phenyl) -1- ( Preparation of prop-2-yl-d 7) -1H-pyrazol-4 -yl) pyrimidin-2-yl) amino) prop-2-yl) carbamate (compound L-3).
[0261]
[0262]
Take the following synthetic route:
[0263]
[0264]
Step 1 Synthesis of compound 10
[0265]
Under nitrogen protection, intermediate compound A (0.6 g, 1.7 mmol), intermediate compound B (0.23 g, 1.7 mmol), and sodium carbonate (0.71 g, 6.8 mmol) were added to the DMSO (20 ml) solution in this order, and the temperature was raised to The reaction was stirred at 90 ° C for 16h at this temperature. After the reaction was completed, DCM (30ml × 3) was extracted, the organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: petroleum ether / ethyl acetate ( v / v) = 1: 2), 0.65 g of product was obtained with a yield of 84%. LC-MS (APCI): m / z = 454.92 (M + 1) + .
[0266]
Step 2 Synthesis of Compound 11
[0267]
Under nitrogen protection, compound 10 (0.6 g, 1.3 mmol), compound 3 (0.59 g, 1.6 mmol), sodium carbonate (0.56 g, 5.3 mmol), and Pd (dppf) Cl2 (0.10 g, 0.13 mmol) were added in this order. Into a mixed solution of toluene (20 ml) and water (4 ml), heated to 80 ° C. for 2 h. The reaction solution was cooled to room temperature, extracted with ethyl acetate (30 ml × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: petroleum ether / ethyl acetate (v / v) = 1: 1) 0.32 g of the product was obtained with a yield of 43%. LC-MS (APCI): m / z = 572.10 (M + 1) + .
[0268]
Step 3 Synthesis of Compound 12
[0269]
At 0 ° C, a solution of 4M hydrochloric acid in dioxane (4 ml) was slowly added to a solution of compound 11 (0.3 g, 0.52 mmol) in DCM (10 ml), and the reaction mixture was warmed to room temperature and continued to react for 6 h. After the reaction is complete, the solution is spin-dried and directly sent to the next step without further processing. LC-MS (APCI): m / z = 472.09 (M + 1) + .
[0270]
Step 4 Synthesis of Compound L-3
[0271]
Compound 12 (0.25 g, 0.53 mmol) and triethylamine (0.27 g, 2.6 mmol) were sequentially added to the DCM (10 ml) solution. After dropping to 0 ° C, MsCl (0.18 g, 1.6 mmol) was slowly added dropwise. After the addition was completed, the reaction temperature was raised to room temperature for 5 hours. After the reaction was completed, the reaction solution was spin-dried to obtain a residue. Toluene (9 ml), methanol (1 ml), water (10 ml), and sodium carbonate (2 g) were sequentially added to the residue. The reaction temperature was raised to 85 ° C for 10 hours, cooled to room temperature, and extracted with ethyl acetate (20ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: dichloromethane / methanol (v / v) = 20: 1), 75 mg of product was obtained with a yield of 26%. LC-MS (APCI): m / z = 550.29 (M + 1) + . 1 H NMR (400 MHz, CDCl 3 ) δ 8.13 (d, J = 11.4 Hz, 2 H), 7.63 (d, J = 6.3 Hz, 1 H), 7.40 (d, J = 5.8 Hz, 1 H), 6.65 (d , J = 6.1 Hz, 1H), 5.47 (d, J = 18.8 Hz, 1H), 5.17 (s, 1H), 4.63 (d, J = 12.2, Hz, 1H), 3.70 (s, 1H), 3.54 ( s, 1H), 3.16 (d, J = 16.6 Hz, 3H), 2.11 (s, 1H), 1.38 (s, 3H).
[0272]
Example 4 (1-((4- (3- (5-chloro-2-fluoro-3- (methylsulfonylamino) phenyl) -1- (prop-2-yl) -1H-pyrazole- 4- yl) pyrimidin-2-yl) amino ) propan-2-yl -1,1,3,3,3-d 5) carbamate (compound L-4),
[0273]
(S)-(1-((4- (3- (5-chloro-2-fluoro-3- (methylsulfonylamino) phenyl) -1- (prop-2-yl) -1H-pyrazole 4-yl) pyrimidin-2-yl) amino) propan-2- yl-1,1,3,3,3-d 5) methyl carbamate (compound L-4-S) and
[0274]
(R)-(1-((4- (3- (5-chloro-2-fluoro-3- (methylsulfonylamino) phenyl) -1- (prop-2-yl) -1H-pyrazole 4-yl) pyrimidin-2-yl) amino) propan-2- yl-1,1,3,3,3-d 5) Preparation of methyl carbamate (compound L-4-R).
[0275]
[0276]
Take the following synthetic route:
[0277]
[0278]
Step 1 Synthesis of compound 13
[0279]
Under nitrogen protection, compound 6 (0.4 g, 1.1 mmol), intermediate compound C (0.16 g, 1.1 mmol), and sodium carbonate (0.50 g, 4.6 mmol) were sequentially added to the DMSO (15 ml) solution, and the solution was raised to The reaction was stirred at 90 ° C at this temperature for 16 hours. After the reaction was completed, the reaction solution was extracted with dichloromethane (30 ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: petroleum ether). / Ethyl acetate (v / v) = 1: 2) to obtain 0.40 g of the product in a yield of 75%. LC-MS (APCI): m / z = 449.53 (M + 1) + .
[0280]
Step 2 Synthesis of Compound 14
[0281]
Under a nitrogen atmosphere, compound 13 (0.4 g, 0.9 mmol), compound 3 (0.5 g, 1.4 mmol), sodium carbonate (0.40 g, 3.56 mmol), and Pd (dppf) Cl 2 (0.08 g, 0.1 mmol) were added in this order. Into a mixed solution of toluene (20 ml) and water (4 ml), heated to 80 ° C. for 2 h. The reaction solution was cooled to room temperature, and then extracted with ethyl acetate (30 ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: petroleum ether / ethyl acetate (v / v) = 1: 1), 0.28 g of product was obtained with a yield of 55%. LC-MS (APCI): m / z = 567.12 (M + 1) + .
[0282]
Step 3 Synthesis of Compound 15
[0283]
At 0 ° C, a solution of 4M hydrochloric acid in dioxane (2ml) was slowly added to a solution of compound 14 (0.28g, 0.50mmol) in dichloromethane (10ml), and the reaction was continued at room temperature for 6h. After the reaction is completed, the solution is directly spin-dried and directly sent to the next step without further processing. LC-MS (APCI): m / z = 467.29 (M + 1) + .
[0284]
Step 4 Synthesis of compound L-4
[0285]
Triethylamine (0.13 g, 1.28 mmol) was added to a solution of compound 15 (0.2 g, 0.43 mmol) in dichloromethane (10 ml). After the solution was lowered to 0 ° C, methanesulfonyl chloride (0.15 g, 1.3 mmol) was slowly added dropwise to the upper solution. The reaction solution was reacted at room temperature for 5 hours. After the reaction was completed, the reaction solution was spin-dried. To the residue were added toluene (9 ml), methanol (1 ml), and water (10 ml). Sodium carbonate (2g), the solution was reacted at 85 ° C for 10h, the reaction solution was cooled to room temperature, and then extracted with ethyl acetate (20ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation ( Eluent: dichloromethane / methanol (v / v) = 20: 1) to obtain 65 mg of the product with a yield of 27%. LC-MS (APCI): m / z = 545.08 (M + 1) + . 1 H NMR (400MHz, CDCl 3 ) δ8.05 (d, 2H), 7.61 (d, 1H), 7.45 (d, 1H), 6.40 (d, 1H), 5.29 (d, 1H), 5.18 (s, 1H), 4.62 (d, 1H), 3.89 (d, 1H), 3.58 (s, 3H), 3.10 (d, 3H), 2.05 (s, 1H), 1.29 (d, 6H).
[0286]
Step 5 Synthesis of compounds L-4-S and L-4-R
[0287]
The racemic compound L-4 was separated using a chiral preparative column to obtain compounds L-4-S and L-4-R.
[0288]
Example 5 (1-((4- (3- (5-chloro-2-fluoro-3- (methylsulfonylamino) phenyl) -1- (prop-2-yl-d 7))-1H -Pyrazol-4-yl) pyrimidin-2-yl) amino) propan-2-yl-1,1,3,3,3-d 5) methyl carbamate (compound L-5),
[0289]
(S)-(1-((4- (3- (5-chloro-2-fluoro-3- (methylsulfonylamino) phenyl) -1- (prop-2-yl-d 7))- 1H-pyrazol-4-yl) pyrimidin-2-yl) amino) propan-2-yl-1,1,3,3,3-d 5) methyl carbamate (compound L-5-S) and
[0290]
(R)-(1-((4- (3- (5-chloro-2-fluoro-3- (methylsulfonylamino) phenyl) -1- (prop-2-yl-d 7))- 1H-pyrazol-4-yl) pyrimidin-2-yl) amino) propan-2-yl-1,1,3,3,3-d 5) Preparation of methyl carbamate (compound L-5-R) .
[0291]
[0292]
[0293]
Take the following synthetic route:
[0294]
[0295]
Step 1 Synthesis of compound 16
[0296]
Under nitrogen protection, intermediate compound A (0.5 g, 1.5 mmol), intermediate compound C (0.2 g, 1.5 mmol), and sodium carbonate (0.63 g, 6.0 mmol) were added to the DMSO (20 ml) solution in this order. The temperature was raised to 90 ° C, and the reaction was stirred at this temperature for 16 hours. After the reaction was completed, the reaction solution was extracted with dichloromethane (30ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: Petroleum ether / ethyl acetate (v / v) = 1: 2) to obtain 0.45 g of the product with a yield of 68%. LC-MS (APCI): m / z = 456.68 (M + 1) + .
[0297]
Step 2 Synthesis of Compound 17
[0298]
Under a nitrogen atmosphere, compound 16 (0.45 g, 0.98 mmol), compound 3 (0.55 g, 1.54 mmol), sodium carbonate (0.42 g, 3.95 mmol), and Pd (dppf) Cl 2 (0.08 g, 0.1 mmol) were sequentially added Into a mixed solution of toluene (20 ml) and water (4 ml), heated to 80 ° C. for 2 h. The reaction solution was cooled to room temperature, and then extracted with ethyl acetate (30 ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation (eluent: petroleum ether / ethyl acetate (v / v) = 1: 1), 0.40 g of the product was obtained in a yield of 70%. LC-MS (APCI): m / z = 574.16 (M + 1) + .
[0299]
Step 3 Synthesis of Compound 18
[0300]
A solution of 4M hydrochloric acid in dioxane (2 ml) was slowly added to a solution of compound 17 (0.40 g, 0.70 mmol) in dichloromethane (10 ml) at 0 ° C, and the reaction was allowed to proceed to room temperature for 6 h. After the reaction is completed, the solution is directly spin-dried and directly sent to the next step without further processing. LC-MS (APCI): m / z = 474.21 (M + 1) + .
[0301]
Step 4 Synthesis of Compound L-5
[0302]
Triethylamine (0.23 g, 2.21 mmol) was added to a solution of compound 18 (0.35 g, 0.74 mmol) in dichloromethane (10 ml). After the solution was lowered to 0 ° C, methanesulfonyl chloride (0.25 g, 2.2 mmol) was slowly added dropwise to the upper solution. The reaction solution was reacted at room temperature for 5 hours. After the reaction was completed, the reaction solution was spin-dried. To the residue were added toluene (9 ml), methanol (1 ml), and water (10 ml). Sodium carbonate (2g), the solution was reacted at 85 ° C for 10h, the reaction solution was cooled to room temperature, and then extracted with ethyl acetate (20ml x 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the concentrated solution was subjected to column separation Eluent: dichloromethane / methanol (v / v) = 20: 1) to obtain 120 mg of the product in a yield of 30%. LC-MS (APCI): m / z = 552.33 (M + 1) + . 1 H NMR (400MHz, CDCl 3 ) δ 8.02 (d, 2H), 7.61 (d, 1H), 7.45 (d, 1H), 6.40 (d, 1H), 5.22 (d, 1H), 5.18 (s, 1H), 4.59 (d, 1H), 3.58 (s, 3H), 2.98 (d, 3H), 2.05 (s, 1H).
[0303]
Step 5 Synthesis of compounds L-5-S and L-5-R
[0304]
The racemic compound L-4 was separated using a chiral preparative column to obtain compounds L-5-S and L-5-R.
GRAPIPRANT
GRAPIPRANT
- Molecular FormulaC26H29N5O3S
- Average mass491.605 Da
CAS 415903-37-6
UNII-J9F5ZPH7NB, CJ 023423, CJ-023423,
Phase II, Arrys Therapeutics, CANCER,
PAIN, AskAt Phase II,
N-{2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)phenyl]ethyl}-N’-[(4-methylphenyl)sulfonyl]urea
RQ-00000007, MR10A7
9763
AAT-007
Benzenesulfonamide, N-[[[2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)phenyl]ethyl]amino]carbonyl]-4-methyl-
CJ-023,423
- N-[[[2-[4-(2-Ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)phenyl]ethyl]amino]carbonyl]-4-methylbenzenesulfonamide
- 1-[2-[4-(2-Ethyl-4,6-dimethylimidazo[4,5-c]pyridin-1-yl)phenyl]ethyl]-3-(4-methylphenyl)sulfonylurea
- 2-Ethyl-4,6-dimethyl-1-[4-[2-[[[[(4-methylphenyl)sulfonyl]amino]carbonyl]amino]ethyl]phenyl]-1H-imidazo[4,5-c]pyridine
- AAT 007
- CJ 023423
- Grapiprant
- MR 10A7
- RQ 00000007
- RQ 7
Synonyms and Mappings
- 415903-37-6
- GRAPIPRANT [GREEN BOOK]
- CJ-023
- GRAPIPRANT [INN]
- GRAPIPRANT [WHO-DD]
- MR-10A7
- AAT-007
- MR10A7
- RQ-00000007
- RQ-7
- GRAPIPRANT [USAN]
- GRAPIPRANT
- 2-ETHYL-4,6-DIMETHYL-1-(4-(2-(((((4-METHYLPHENYL)SULFONYL)AMINO)CARBONYL)AMINO)ETHYL)PHENYL)-1H-IMIDAZO(4,5-C)PYRIDINE
- N-(((2-(4-(2-ETHYL-4,6-DIMETHYL-1H-IMIDAZO(4,5-C)PYRIDIN-1-YL)PHENYL)ETHYL)AMINO)CARBONYL)-4-METHYLBENZENESULFONAMIDE
- CJ 023423
- BENZENESULFONAMIDE, N-(((2-(4-(2-ETHYL-4,6-DIMETHYL-1H-IMIDAZO(4,5-C)PYRIDIN-1-YL)PHENYL)ETHYL)AMINO)CARBONYL)-4-METHYL-
- CJ-023,423
- N-(((2-(4-(2-ETHYL-4,6-DIMETHYL-1H-IMIDAZO(4,5-C)PYRIDIN-1-YL)PHENYL)ETHYL)AMINO)CARBONYL)-4-METHYL-BENZENESULFONAMIDE
- CJ-023423
SYN
Arrys Therapeutics (under license from AskAt ) and affiliate Ikena Oncology (formerly known as Kyn Therapeutics ) are developing ARY-007 , an oral formulation of grapiprant, for treating cancers; in December 2019, preliminary data were expected in 2020
Grapiprant (trade name Galliprant) is a small molecule drug that belongs in the piprant class. This analgesic and anti-inflammatory drug is primarily used as a pain relief for mild to moderate inflammation related to osteoarthritis in dogs. Grapiprant has been approved by the FDA’s Center for Veterinary Medicine and was categorized as a non-cyclooxygenase inhibiting non-steroidal anti-inflammatory drug (NSAID) in March 2016.[1]
Preclinical studies also indicate that grapiprant is not only efficacious as a acute pain but also in chronic pain relief and inflammation drug. The effect of the drug is directly proportional to the dosage and its effects were comparable to human medication such as rofecoxib and piroxicam.[2]
Grapiprant, a prostanoid EP4 receptor antagonist, is in phase II clinical trials at AskAt for the treatment of chronic pain. Phase I/II clinical trials are ongoing at Arrys Therapeutics in combination with pembrolizumab for the treatment of patients with microsatellite stable colorectal cancer and in patients with advanced or metastatic PD-1/L1 refractory non-small cell lung cancer (NSCLC).
Grapiprant is also used in humans, and was researched to be used as a pain control and inflammation associated with osteoarthritis. The effect of grapiprant could be explained through the function of prostaglandin E2, in which acts as a pro-inflammatory mediator of redness of the skin, edema and pain which are the typical signs of inflammation. The effect of PGE2 stems from its action through the four prostaglandin receptor subgroups EP1, EP2, EP3 and EP4, in which the prostaglandin EP4 receptor acts as the main intermediary of the prostaglandin-E2-driven inflammation. Grapiprant is widely accepted in veterinary medicine due to its specific and targeted approach to pain management in dogs. The serum concentration of grapiprant is increased when used in conjunction with other drugs such as acetaminophen, albendazole, and alitretinoin.
Common side effects are intestinal related effects such as mild diarrhea, appetite loss, and vomiting.[3] Additionally, it is found that it might lead to reduced tear production due to it being a sulfa-based medication and also reduced albumin levels.
Grapiprant, a prostanoid EP4 receptor antagonist, is in phase II clinical trials at AskAt for the treatment of chronic pain. Phase I/II clinical trials are ongoing at Arrys Therapeutics in combination with pembrolizumab for the treatment of patients with microsatellite stable colorectal cancer and in patients with advanced or metastatic PD-1/L1 refractory non-small cell lung cancer (NSCLC).
Medical uses
Grapiprant is used once a day as an oral pain relief for dogs with inflammation-related osteoarthritis. It is a non-steroidal anti-inflammatory (NSAID) that functions as a targeted action to treat osteoarthritis pain and inflammation in dogs.
Mechanism of action
Grapiprant acts as a specific antagonist that binds and blocks the prostaglandin EP4 receptor, one out of the four prostaglandin E2 (PGE2) receptor subgroups. The EP4 receptor then mediates the prostaglandin-E2-elicited response to pain, and hence grapiprant was proven to be effective in the decrease of pain in several inflammatory pain models of rats. It was also proven to be effective in reducing osteoarthritis-related pain in humans, which serves as a proof for its mechanism of action. The approximate calculation for canine efficacy dose is between the range of 1.3 and 1.7 mg/kg, in conjunction with a methylcellulose suspending agent. Based on the calculations from the comparisons of binding affinity of grapiprant to the EP4 receptors of dogs, rats, and humans, the study of plasma and serum protein binding determinations, the effective doses determined in inflammation pain models of rats, and human-related clinical studies, it is evaluated that Grapiprant should be administered just once a day. The approved dose of the commercial Grapiprant tablet by the FDA for the pain relief and inflammation associated with osteoarthritis to dogs is reported to be 2 mg/kg a day.[4]
Absorption
Studies in animals such as horses have shown the presence of Grapiprant in serum 72 hours with a concentration >0.005 ng/ml after the initial administration of a dose of 2 mg/kg. Grapiprant is expeditiously absorbed and the reported serum concentration was reported to be 31.9 ng/ml in an amount of time of 1.5 hours. The actual body exposure to grapiprant after administration of one dose was shown to be 2000 ng.hr/ml. The degree and rate at which grapiprant is absorbed into the body, presents a mean bioavailability of 39%. A significant reduction in the bioavailability, concentration time and maximal concentration were reported to have occurred after food intake.[1] And thus, grapiprant is usually not administered with food as it will not be as efficient.[5]
Distribution
The volume of distribution in cat studies was reported to be 918 ml/kg.[1]
Route of elimination
Following an oral administration, the majority of the dose was metabolized within the first 72 hours. Equine studies have shown that grapiprant is present in urine 96 hours after the first administration of a dose of 2 mg/kg and has a concentration >0.005 ng/ml. From the excreted dose conducted in horses, it is found that 55%, 15% and 19% of the orally-administered dose was excreted in bile, urine, and faeces respectively.[1]
Toxicity
Safety studies conducted on grapiprant have demonstrated that it generally possesses an exceptional safety profile and a wide safety margin in veterinary studies.[6] In animal studies, a research on 2.5-12 times overdose was conducted for grapiprant and the study resulted in soft-blobs and mucous-filled faeces, occasional bloody stools and emesis.
PATENT
WO-2020014465
Novel crystalline forms of grapiprant and their salts eg HCl (designated as Form A), useful for inhibiting prostaglandin EP4 receptor activity and treating cancers.
Prostaglandins are mediators of pain, fever and other symptoms associated with inflammation. Prostaglandin E2 (PGE2) is the predominant eicosanoid detected in inflammation conditions. In addition, it is also involved in various physiological and/or pathological conditions such as hyperalgesia, uterine contraction, digestive peristalsis, awakeness, suppression of gastric acid secretion, blood pressure, platelet function, bone metabolism, angiogenesis or the like.
[0003] Four PGE2 receptor subtypes (EP1, EP2, EP3 and EP4) displaying different pharmacological properties exist. The EP4 subtype, a Gs-coupled receptor, stimulates cAMP production as well as PI3K and GSK3P signaling, and is distributed in a wide variety of tissue suggesting a major role in PGE2-mediated biological events. Various EP4 inhibitors have been described previously, for example, in WO 2002/032900, WO 2005/021508, EiS 6,710,054, and US 7,238,714, the contents of which are incorporated herein by reference in their entireties.
[0004] Accordingly, there is a need for treating, preventing, and/or reducing severity of a proliferative disorder associated with prostaglandin EP4 receptor activity. The present invention addresses such a need.
It has now been found that compounds of the present invention, and compositions thereof, are useful for treating, preventing, and/or reducing severity of a proliferative disorder associated with prostaglandin EP4 receptor activity. In general, salt forms and co-crystal forms, and pharmaceutically acceptable compositions thereof, are useful for treating or lessening the severity of proliferative disorders associated with prostaglandin EP4 receptor activity, as described in detail herein. Such compounds are represented by the chemical structure below, denoted as compound A (also known as grapiprant):
A
or a pharmaceutically acceptable salt thereof.
United States Patent 7,960,407, filed March 1, 2006 and issued June 14, 2011 (“the ‘407 patent,” the entirety of which is hereby incorporated herein by reference), describes certain EP4 inhibitor compounds. Such compounds include compound A:
or a pharmaceutically acceptable salt thereof.
[0037] Compound A, N-[({2-[4-(2-Ethyl-4,6-dimethyl-lH-imidazo[4,5-c]pyridin-l-yl) phenyl]ethyl}amino)carbonyl]-4-methylbenzenesulfonamide, is described in detail in the ‘407
patent, including its synthetic route. The ‘407 patent also discloses a variety of physical forms of compound A.
[0038] It would be desirable to provide a solid form of compound A (e.g., as a co-crystal thereof or salt thereof) that imparts characteristics such as improved aqueous solubility, stability and ease of formulation. Accordingly, the present invention provides both co-crystal forms and salt forms of compound A:
A.
PATENT
WO 2002032900
PATENT
WO 2002032422
Family members of the product case ( WO0232422 ) of grapiprant have protection in most of the EU states until October 2021 and expire in the US in October 15, 2021.
PATENT
WO 2003086371
PATENT
WO2020014445 covering combinations of grapiprant and an immuno-oncology agent.
WO 2005102389
WO 2006095268
US 7960407
US 20190314390
References
- ^ Jump up to:a b c d “Grapiprant”. http://www.drugbank.ca. Retrieved 2019-05-15.
- ^ PubChem. “Grapiprant”. pubchem.ncbi.nlm.nih.gov. Retrieved 2019-05-15.
- ^ Paul Pion, D. V. M.; Spadafori, Gina (2017-08-08). “Veterinary Partner”. VIN.com.
- ^ Nagahisa, A.; Okumura, T. (2017). “Pharmacology of grapiprant, a novel EP4 antagonist: receptor binding, efficacy in a rodent postoperative pain model, and a dose estimation for controlling pain in dogs”. Journal of Veterinary Pharmacology and Therapeutics. 40 (3): 285–292. doi:10.1111/jvp.12349. ISSN 1365-2885. PMID 27597397.
- ^ Paul Pion, D. V. M.; Spadafori, Gina (2017-08-08). “Veterinary Partner”. VIN.com.
- ^ Kirkby Shaw, Kristin; Rausch-Derra, Lesley C.; Rhodes, Linda (February 2016). “Grapiprant: an EP4 prostaglandin receptor antagonist and novel therapy for pain and inflammation”. Veterinary Medicine and Science. 2 (1): 3–9. doi:10.1002/vms3.13. ISSN 2053-1095. PMC 5645826. PMID 29067176.
 |
|
| Clinical data | |
|---|---|
| Trade names | Galliprant |
| Routes of administration |
Oral |
| ATCvet code | |
| Pharmacokinetic data | |
| Bioavailability | 6.6 L/kg, high volume of distribution |
| Elimination half-life | 5.86 hours in horses |
| Excretion | Urine |
| Identifiers | |
| CAS Number | |
| PubChem CID | |
| DrugBank | |
| ChemSpider | |
| UNII | |
| CompTox Dashboard (EPA) | |
| Chemical and physical data | |
| Formula | C26H29N5O3S |
| Molar mass | 491.61 g·mol−1 |
| 3D model (JSmol) | |
//////////////GRAPIPRANT, 415903-37-6, UNII-J9F5ZPH7NB, CJ 023423, CJ-023423, RQ-00000007, MR10A7, Galliprant, Phase II, Arrys Therapeutics, CANCER, PAIN, AskAt
CCC1=NC2=C(N1C3=CC=C(C=C3)CCNC(=O)NS(=O)(=O)C4=CC=C(C=C4)C)C=C(N=C2C)C
DR ANTHONY MELVIN CRASTO
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