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DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with AFRICURE PHARMA, ROW2TECH, NIPER-G, Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India as ADVISOR, earlier assignment was
with GLENMARK LIFE SCIENCES LTD, as CONSUlTANT, Retired from GLENMARK in Jan2022 Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 32 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri, etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him Open superstar worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international,
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and implementation them on commercial scale over a 32 PLUS year tenure till date Feb 2023, Around 35 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 100 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 100 Lakh plus views on dozen plus blogs, 227 countries, 7 continents, He makes himself available to all, contact him on +91 9323115463, email amcrasto@gmail.com, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 38 lakh plus views on New Drug Approvals Blog in 227 countries......https://newdrugapprovals.wordpress.com/ , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc
He has total of 32 International and Indian awards
It also shows broad-spectrum antiviral effectiveness against a range of other RNA virus families, including bunyaviruses, arenaviruses, paramyxoviruses, coronaviruses, flaviviruses and phleboviruses.[3] BCX4430 has been demonstrated to protect against both Ebola and Marburg viruses in both rodents and monkeys, even when administered up to 48 hours after infection,[1] and development for use in humans was then being fast-tracked due to concerns about the lack of treatment options for the 2013-2016 Ebola virus epidemic in West Africa.[4]
BCX4430 later showed efficacy against Zika virus in a mouse model, though there are no plans for human trials at this stage.[5]
When any new virus emerges, drug and vaccine developers spring into action, searching for products to stop it in its tracks. Drug discovery campaigns launch, vaccine development efforts ramp up, and everyone mobilizes to get it all into the clinic as quickly as possible.
The current pandemic, driven by a coronavirus known as SARS-CoV-2, is no different. Already, a Phase I study of an mRNA-based vaccine developed by Moderna has begun, and major pharma companies and small biotechs are working on other types of vaccines. But even if they work, the most optimistic timelines put a vaccine a year to 18 months away.
The more immediate approach to an outbreak is to scour the medicine cabinet for existing molecules that could be repurposed against a new virus. The most advanced potential treatment is Gilead Sciences’ remdesivir, an antiviral discovered during the 2014 Ebola epidemic. The compound is already being tested in four, Phase III trials—two in China and two in the US—against the respiratory disease COVID-19. Gilead expects the first dataset from those studies to come out in April.
A new paper from CAS explored remdesivir and other possible options the cabinet might contain (ACS Cent. Sci. 2020, DOI: 10.1021/acscentsci.0c00272). CAS, a division of the American Chemical Society, which publishes C&EN, looked at the landscape of patent and journal articles covering small molecules, antibodies, and other therapeutic classes to identify therapies with potential activity against COVID-19.
SARS-CoV-2, belongs to the same family as two coronaviruses responsible for earlier outbreaks, Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). Because all three feature structurally similar proteins that allow entry into and replication inside host cells, CAS searched for patent data related to those more well-studied coronaviruses.
C&EN has assembled the relevant small molecules identified by CAS, which can be explored by the stage in the viral life cycle they aim to disrupt.
Hydroxychloroquine (HCQ), sold under the brand name Plaquenil among others, is a medication used for the prevention and treatment of certain types of malaria.[2] Specifically it is used for chloroquine-sensitive malaria.[3] Other uses include treatment of rheumatoid arthritis, lupus, and porphyria cutanea tarda.[2] It is taken by mouth.[2] It is also being used as an experimental treatment for coronavirus disease 2019 (COVID-19).[4]
Hydroxychloroquine was approved for medical use in the United States in 1955.[2] It is on the World Health Organization’s List of Essential Medicines, the safest and most effective medicines needed in a health system.[6] The wholesale cost in the developing world is about US$4.65 per month as of 2015, when used for rheumatoid arthritis or lupus.[7] In the United States the wholesale cost of a month of treatment is about US$25 as of 2020.[8] In the United Kingdom this dose costs the NHS about £ 5.15.[9] In 2017, it was the 128th most prescribed medication in the United States with more than five million prescriptions.[10]
In 2014, its efficacy to treat Sjögren syndrome was questioned in a double-blind study involving 120 patients over a 48-week period.[11]
Hydroxychloroquine is widely used in the treatment of post-Lymearthritis. It may have both an anti-spirochaete activity and an anti-inflammatory activity, similar to the treatment of rheumatoid arthritis.[12]
Contraindications
The drug label advises that hydroxychloroquine should not be prescribed to individuals with known hypersensitivity to 4-Aminoquinoline compounds.[13] There are a range of other contraindications[14][15] and caution is required if patients have certain heart conditions, diabetes, psoriasis etc.
Side effects[
The most common adverse effects are a mild nausea and occasional stomach cramps with mild diarrhea. The most serious adverse effects affect the eye, with dose-related retinopathy as a concern even after hydroxychloroquine use is discontinued.[2] For short-term treatment of acute malaria, adverse effects can include abdominal cramps, diarrhea, heart problems, reduced appetite, headache, nausea and vomiting.[2]
For prolonged treatment of lupus or rheumatoid arthritis, adverse effects include the acute symptoms, plus altered eye pigmentation, acne, anemia, bleaching of hair, blisters in mouth and eyes, blood disorders, convulsions, vision difficulties, diminished reflexes, emotional changes, excessive coloring of the skin, hearing loss, hives, itching, liver problems or liver failure, loss of hair, muscle paralysis, weakness or atrophy, nightmares, psoriasis, reading difficulties, tinnitus, skin inflammation and scaling, skin rash, vertigo, weight loss, and occasionally urinary incontinence.[2] Hydroxychloroquine can worsen existing cases of both psoriasis and porphyria.[2]
Children may be especially vulnerable to developing adverse effects from hydroxychloroquine.[2]
One of the most serious side effects is retinopathy (generally with chronic use).[2][16] People taking 400 mg of hydroxychloroquine or less per day generally have a negligible risk of macular toxicity, whereas the risk begins to go up when a person takes the medication over 5 years or has a cumulative dose of more than 1000 grams. The daily safe maximum dose for eye toxicity can be computed from one’s height and weight using this calculator. Cumulative doses can also be calculated from this calculator. Macular toxicity is related to the total cumulative dose rather than the daily dose. Regular eye screening, even in the absence of visual symptoms, is recommended to begin when either of these risk factors occurs.[17]
Toxicity from hydroxychloroquine may be seen in two distinct areas of the eye: the cornea and the macula. The cornea may become affected (relatively commonly) by an innocuous cornea verticillata or vortex keratopathy and is characterized by whorl-like corneal epithelial deposits. These changes bear no relationship to dosage and are usually reversible on cessation of hydroxychloroquine.
The macular changes are potentially serious. Advanced retinopathy is characterized by reduction of visual acuity and a “bull’s eye” macular lesion which is absent in early involvement.
Overdose
Due to rapid absorption, symptoms of overdose can occur within a half an hour after ingestion. Overdose symptoms include convulsions, drowsiness, headache, heart problems or heart failure, difficulty breathing and vision problems.
Hydroxychloroquine overdoses are rarely reported, with 7 previous cases found in the English medical literature. In one such case, a 16-year-old girl who had ingested a handful of hydroxychloroquine 200mg presented with tachycardia (heart rate 110 beats/min), hypotension (systolic blood pressure 63 mm Hg), central nervous system depression, conduction defects (ORS = 0.14 msec), and hypokalemia (K = 2.1 meq/L). Treatment consisted of fluid boluses and dopamine, oxygen, and potassium supplementation. The presence of hydroxychloroquine was confirmed through toxicologic tests. The patient’s hypotension resolved within 4.5 hours, serum potassium stabilized in 24 hours, and tachycardia gradually decreased over 3 days.[18]
Specifically, the FDA drug label for hydroxychloroquine lists the following drug interactions [13]:
Digoxin (wherein it may result in increased serum digoxin levels)
Insulin or antidiabetic drugs (wherein it may enhance the effects of a hypoglycemic treatment)
Drugs that prolong QT interval and other arrhythmogenic drugs (as Hydroxychloroquine prolongs the QT interval and may increase the risk of inducing ventricular arrhythmias if used concurrently)
Mefloquine and other drugs known to lower the convulsive threshold (co-administration with other antimalarials known to lower the convulsion threshold may increase risk of convulsions)
Antiepileptics (concurrent use may impair the antiepileptic activity)
Methotrexate (combined use is unstudied and may increase the frequency of side effects)
Cyclosporin (wherein an increased plasma cylcosporin level was reported when used together).
Pharmacology[
Pharmacokinetics
Hydroxychloroquine has similar pharmacokinetics to chloroquine, with rapid gastrointestinal absorption and elimination by the kidneys. Cytochrome P450 enzymes (CYP2D6, 2C8, 3A4 and 3A5) metabolize hydroxychloroquine to N-desethylhydroxychloroquine.[21]
Pharmacodynamics
Antimalarials are lipophilic weak bases and easily pass plasma membranes. The free base form accumulates in lysosomes (acidic cytoplasmic vesicles) and is then protonated,[22] resulting in concentrations within lysosomes up to 1000 times higher than in culture media. This increases the pH of the lysosome from 4 to 6.[23] Alteration in pH causes inhibition of lysosomal acidic proteases causing a diminished proteolysis effect.[24] Higher pH within lysosomes causes decreased intracellular processing, glycosylation and secretion of proteins with many immunologic and nonimmunologic consequences.[25] These effects are believed to be the cause of a decreased immune cell functioning such as chemotaxis, phagocytosis and superoxide production by neutrophils.[26] HCQ is a weak diprotic base that can pass through the lipid cell membrane and preferentially concentrate in acidic cytoplasmic vesicles. The higher pH of these vesicles in macrophages or other antigen-presenting cells limits the association of autoantigenic (any) peptides with class II MHC molecules in the compartment for peptide loading and/or the subsequent processing and transport of the peptide-MHC complex to the cell membrane.[27]
In 2003, a novel mechanism was described wherein hydroxychloroquine inhibits stimulation of the toll-like receptor (TLR) 9 family receptors. TLRs are cellular receptors for microbial products that induce inflammatory responses through activation of the innate immune system.[29]
As with other quinoline antimalarial drugs, the mechanism of action of quinine has not been fully resolved. The most accepted model is based on hydrochloroquinine and involves the inhibition of hemozoinbiocrystallization, which facilitates the aggregation of cytotoxic heme. Free cytotoxic heme accumulates in the parasites, causing their deaths.[citation needed]
Brand names
It is frequently sold as a sulfate salt known as hydroxychloroquine sulfate.[2] 200 mg of the sulfate salt is equal to 155 mg of the base.[2]
Brand names of hydroxychloroquine include Plaquenil, Hydroquin, Axemal (in India), Dolquine, Quensyl, Quinoric.[30]
Hydroxychloroquine and chloroquine have been recommended by Chinese and South Korean health authorities for the experimental treatment of COVID-19.[31][32]In vitro studies in cell cultures demonstrated that hydroxychloroquine was more potent than chloroquine against SARS-CoV-2.[33]
On 17 March 2020, the AIFA Scientific Technical Commission of the Italian Medicines Agency expressed a favorable opinion on including the off-label use of chloroquine and hydroxychloroquine for the treatment of SARS-CoV-2 infection.[34]
^World Health Organization (2019). World Health Organization model list of essential medicines: 21st list. Geneva: World Health Organization. hdl:10665/325771. WHO/MVP/EMP/IAU/2019.06. License: CC BY-NC-SA 3.0 IGO.
^“NADAC as of 2019-08-07”. Centers for Medicare and Medicaid Services. Retrieved 19 March 2020. Typical dose is 600mg per day. Costs 0.28157 per dose. Month has about 30 days.
^British national formulary: BNF 69 (69 ed.). British Medical Association. 2015. p. 730. ISBN9780857111562.
^Effects of Hydroxychloroquine on Symptomatic Improvement in Primary Sjögren Syndrome, Gottenberg, et al. (2014) “Archived copy”. Archived from the original on 11 July 2015. Retrieved 10 July 2015.
^Steere, AC; Angelis, SM (October 2006). “Therapy for Lyme Arthritis: Strategies for the Treatment of Antibiotic-refractory Arthritis”. Arthritis and Rheumatism. 54 (10): 3079–86. doi:10.1002/art.22131. PMID17009226.
^Mohammad, Samya; Clowse, Megan E. B.; Eudy, Amanda M.; Criscione-Schreiber, Lisa G. (March 2018). “Examination of Hydroxychloroquine Use and Hemolytic Anemia in G6PDH-Deficient Patients”. Arthritis Care & Research. 70 (3): 481–485. doi:10.1002/acr.23296. ISSN2151-4658. PMID28556555.
^Hurst, NP; French, JK; Gorjatschko, L; Betts, WH (1988). “Chloroquine and Hydroxychloroquine Inhibit Multiple Sites in Metabolic Pathways Leading to Neutrophil Superoxide Release”. The Journal of Rheumatology. 15 (1): 23–27. PMID2832600.
^Fox, R (1996). “Anti-malarial Drugs: Possible Mechanisms of Action in Autoimmune Disease and Prospects for Drug Development”. Lupus. 5: S4–10. doi:10.1177/096120339600500103. PMID8803903.
^Waller; et al. Medical Pharmacology and Therapeutics (2nd ed.). p. 370.
Umifenovir[2] (trade names ArbidolRussian: Арбидол, Chinese: 阿比朵尔) is an antiviral treatment for influenza infection used in Russia[3] and China. The drug is manufactured by Pharmstandard (Russian: Фармстандарт). Although some Russian studies have shown it to be effective, it is not approved for use in other countries. It is not approved by FDA for the treatment or prevention of influenza.[4] Chemically, umifenovir features an indole core, functionalized at all but one positions with different substituents. The drug is claimed to inhibit viral entry into target cells and stimulate the immune response. Interest in the drug has been renewed as a result of the SARS-CoV-2 outbreak.
Umifenovir is manufactured and made available as tablets, capsules and syrup.
Testing of umifenovir’s efficacy has mainly occurred in China and Russia,[5][6] and it is well known in these two countries.[7] Some of the Russian tests showed the drug to be effective[5] and a direct comparison with Tamiflu showed similar efficiency in vitro and in a clinical setting.[8] In 2007, Arbidol (umifenovir) had the highest sales in Russia among all over-the-counter drugs.
Mode of action
Biochemistry
Umifenovir inhibits membrane fusion.[3] Umifenovir prevents contact between the virus and target host cells. Fusion between the viral envelope (surrounding the viral capsid) and the cell membrane of the target cell is inhibited. This prevents viral entry to the target cell, and therefore protects it from infection.[9]
Some evidence suggests that the drug’s actions are more effective at preventing infections from RNA viruses than infections from DNA viruses.[10]
More recent studies indicate that umifenovir also has in vitro effectiveness at preventing entry of Ebolavirus Zaïre Kikwit, Tacaribe arenavirus and human herpes virus 8 in mammalian cell cultures, while confirming umifenovir’s suppressive effect in vitro on Hepatitis B and poliovirus infection of mammalian cells when introduced either in advance of viral infection or during infection.[13][14]
Research
In February 2020, Li Lanjuan, an expert of the National Health Commission of China, proposed using Arbidol (umifenovir) together with darunavir as a potential treatment during the 2019–20 coronavirus pandemic.[15] Chinese experts claim that preliminary tests had shown that arbidol and darunavir could inhibit replication of the virus.[16][17] So far without additional effect if added on top of recombinant human interferon α2b spray.[18]
In 2007, the Russian Academy of Medical Sciences stated that the effects of Arbidol (umifenovir) are not scientifically proven.[20]
Russian media criticized lobbying attempts by Tatyana Golikova (then-Minister of Healthcare) to promote umifenovir,[21] and the unproven claim that Arbidol can speed up recovery from flu or cold by 1.3-2.3 days.[22] They also debunked claims that the efficacy of umifenovir is supported by peer-reviewed studies.[23][24]
Arbidol hydrochloride, chemical name: 6-bromo-4-(dimethylaminomethyl)-5-hydroxy-1-methyl-2-(phenylthiomethyl)-1H- Indole-3-carboxylic acid ethyl ester hydrochloride, the structural formula is as follows:
Arbidol hydrochloride is an antiviral drug developed by the Soviet Medicinal Chemistry Research Center. It was first listed in Russia in 1993. It is used as a monohydrate for medicinal purposes. This product not only has immunomodulatory and interferon-inducing effects, but also has good anti-influenza virus activity, and is clinically used for the prevention and treatment of influenza and other acute viral respiratory tract infections.
The preparation of Arbidol hydrochloride has multiple synthetic routes, Chinese patent CN1687033A and Wang Dun, Wu Xiujing, Gong Ping’s “Synthesis of Arbidol Hydrochloride” bibliographical report in Chinese Pharmaceutical Industry Magazine 2004,35(8) are Taking p-benzoquinone and 3-aminocrotonic acid ethyl ester as starting materials, through Neitzescu reaction, O-acylation, N-alkylation, bromination, thiophenol reaction, Mannich amine methylation reaction, hydrochloric acid acidification to obtain hydrochloric acid Arbidol, the total reaction yield was 22.9%.
The synthetic route is as follows:
The Nenitzescu reaction used in the synthesis of indole rings in this method, the reaction yield of this step is about 60%, resulting in a total yield of 22.9%.
U.S. Patent US5198552 and World Patent WO9008135 reported that 5-hydroxy-1,2-dimethylindole-3-ethyl carboxylate was used as raw material, and arbidol hydrochloride was prepared through bromination, condensation, Mannich reaction and salt-forming reaction you.
Although the synthesis steps of this method are short, the raw material 5-hydroxy-1,2-dimethylindole-3-carboxylic acid ethyl ester is not easy to obtain, and the large-scale application is difficult.
Song Yanling, Zhao Yanfang, Gong Pingren reported in the 3rd National Symposium on Pharmaceutical Engineering Technology and Education “Synthesis Research on Arbidol Hydrochloride” in the literature report using thiophenol as the starting material, and chloroacetoacetic acid. After the substitution reaction of the ethyl ester, the thiophenyl fragment in the molecule is introduced, which is then condensed with methylamine, followed by the Neitzescu reaction with p-benzoquinone, and the dimethylamine methyl group is introduced through the Mannich reaction. Reaction, then carry out deprotection reaction, and finally obtain the final product Arbidol hydrochloride through salification reaction
Its synthetic route is as follows:
Since the Nenitzescu reaction yield in this method is only 33.7%, the total yield is only 11.2%.
There are also bibliographical reports (Wen Yanzhen, Gao Zhiwei, Wei Wenlong, Zhi Cuimei, Wang Qi etc. in China Pharmaceutical Industry Journal 2006, “The Synthetic Route Diagram of Arbidol Hydrochloride” reported in 2006,37(12)) is based on ethyl acetoacetate. Ester and methylamine are used as starting materials, and Arbidol hydrochloride is obtained by Neitzescu reaction, acylation to protect hydroxyl group, bromination, thiophenol reaction, Mannich reaction, and acidification.
The synthetic route is as follows:
The method has relatively mild reaction conditions and relatively easy-to-obtain raw materials, but the total yield is still low, about 20%.
The above synthesis methods of Arbidol hydrochloride all use the Nenitzescu indole ring synthesis method to synthesize the indole ring of Arbidol hydrochloride, resulting in a low total reaction yield of about 10% to 20%.
SUMMARY OF THE INVENTION
In view of the above-mentioned problems, the object of the present invention is to provide a preparation method of Arbidol hydrochloride, the raw materials are easy to obtain, the reaction technical conditions are relatively simple, the reaction conditions are mild, and the total reaction yield is relatively high, reaching more than 30%. The cost is low, and it is suitable for industrial production. The method of the invention is based on the starting material of 3-iodo-4-nitrophenol, which is protected by a hydroxyl group, synthesized by indole ring, N-methylated, brominated, thiophenolated, and Mannich amine. Methylation reaction, acidification with hydrochloric acid, and purification to obtain Arbidol hydrochloride.
The reaction formula of the inventive method is as follows:
Fe stands for iron powder
CH 3 COOH stands for acetic acid
H 2 O is for water
(CH 3 ) 2 SO 4 Represents dimethyl sulfate
K 2 CO 3 stands for potassium carbonate
Example 1:
A preparation method of Arbidol hydrochloride, its steps are (preparation of compound 1):
A. Preparation of compound 1: 53 g of 3-iodo-4-nitrophenol was added to 160 g of acetone (drying over anhydrous potassium carbonate), 30.3 g of triethylamine was added, and 37.7 g of triethylamine was added dropwise at room temperature (20-25° C., the same below). g acetyl chloride, dripped in 1 hour, the reaction solution was automatically raised to reflux temperature T=56°C, reacted for 0.5h, cooled to room temperature T=25°C naturally, the reaction solution was poured into 1000g ice water, stirred, filtered, and the filter cake was washed with water , and vacuum-dried to obtain 57.4 g of compound 1 crude product with a yield of 93.6%. The next reaction was carried out directly without further purification.
B. Preparation of compound 2: 48.6 g of ethyl acetoacetate and 180 ml of freshly distilled tetrahydrofuran were added to a dry flask. Over 2 hours, 41.9 g of potassium tert-butoxide was added in portions with stirring. The temperature was raised to T=70°C (reflux), and the solution of 57.4 g of compound 1 obtained in the step and 75 mL of freshly distilled tetrahydrofuran was added dropwise, and the drop was completed in 2 hours. TLC plates monitor the reaction endpoint. After the reaction mixture was cooled to room temperature T=25°C, 93.5 ml of a 4 mol/L hydrochloric acid solution was added dropwise. The precipitated potassium chloride was removed by filtration, the solvent was evaporated under reduced pressure, and the obtained solid was washed with 45 mL of water and 60 mL of petroleum ether in turn to obtain 56.6 g of a crude product of compound 2 with a yield of 98%. The crude product can be recrystallized from the mixed solution of petroleum ether and ethyl acetate to obtain pure product.
C. Preparation of compound 3: add 56.6 g of compound 2, 160 mL of acetic acid and 160 mL of water to the flask, stir under nitrogen protection, add 30.8 g of iron powder in batches, stir vigorously, and heat the reaction mixture to T=80 °C for 4 h. End (TLC plate detection). Iron and its oxides were removed by filtration, water and acetic acid were distilled off under reduced pressure, neutralized with saturated sodium carbonate solution to weakly alkaline, extracted with ethyl acetate, dried over anhydrous magnesium sulfate, and concentrated to obtain 44.1 g of compound 3 crude product in a yield of 44.1 g. 92.3%.
D. Preparation of compound 3: add 10.0 g of compound 2, 28 mL of acetic acid and 28 mL of water to the flask, stir under nitrogen protection, add 7.2 g of iron powder in batches, stir vigorously, and simultaneously heat the reaction mixture to T=80 ° C, 4h reaction End (TLC plate detection). Iron and its oxides were removed by filtration, water and acetic acid were evaporated under reduced pressure, neutralized with saturated sodium carbonate solution to weakly alkaline, extracted with ethyl acetate, dried over anhydrous magnesium sulfate, and concentrated to obtain 7.5 g of compound 3 crude product, yield 89.4%.
E. Preparation of compound 4: 44.1 g of compound 3 prepared in step C was added to 230 ml of DMF, and after adding 35.0 g of anhydrous potassium carbonate, 31.9 g of dimethyl sulfate was slowly added dropwise at 100° C. under stirring, and the same temperature T= The reaction was carried out at 100 °C for 4 h. The reaction solution was cooled to room temperature of T=25°C, 280 ml of water was added under stirring, left to stand for crystallization, suction filtered, the filter cake was washed with water and dried to obtain 44.6 g of a crude compound 4, which was recrystallized with methanol to obtain 36.8 g of a refined compound of compound 4, Yield 79.3%
F. Preparation of compound 5: 36.8g of compound 4 was added to 200ml of carbon tetrachloride, 0.1g of benzoyl peroxide was added, heated to T=76°C and refluxed, 45.0g of bromine was added dropwise, and the reaction was completed within 2h. For 4 h, the reaction solution was cooled in an ice-water bath, filtered, and the filter cake was washed with a small amount of carbon tetrachloride, and dried to obtain 47.5 g of compound 5 crude product, with a yield of 82%.
G. Preparation of compound 6: dissolve 15.4 g of potassium hydroxide in 360 ml of methanol, stir, cool to 0-10° C. in an ice-water bath, add 12.7 g of thiophenol, react for 10 min, add 47.5 g of compound 5, and warm to room temperature, The reaction was carried out for 3 to 3.5 h, the reaction solution was poured into 1500 ml of ice water, adjusted to pH 2 with hydrochloric acid under stirring, filtered, the filter cake was washed with water, and dried in vacuo to obtain 42.9 g of crude compound 6 with a yield of 93.1%. The crude product was recrystallized with ethyl acetate, 10 g of activated carbon was decolorized, and 36.0 g of the dried compound 6 was purified, with a purification yield of 84%. The mother liquor of recrystallization is concentrated and recovered. Or to prepare compound 6, dissolve 3.3 g of potassium hydroxide in 75 ml of methanol, stir, cool to 0-10° C. in an ice-water bath, add 2.7 g of thiophenol, react for 10 min, add 10.0 g of compound 5, warm to room temperature, and react For 3-3.5 h, the reaction solution was poured into 300 ml of ice water, adjusted to pH 2 with hydrochloric acid under stirring, filtered, the filter cake was washed with water, and dried in vacuo to obtain 9.0 g of crude compound 6 with a yield of 93.1%. The crude product was recrystallized with isopropanol, 2 g of activated carbon was decolorized, and 6.3 g of the dried refined product of compound 6 was obtained, with a purification yield of 70%. The mother liquor of recrystallization is concentrated and recovered.
H. Preparation of compound 7:
In 320ml of ethanol, add, (33%) dimethylamine aqueous solution 29.2g, (37-40%) formaldehyde aqueous solution 23.8g, stir for 10min, add 36.0g compound 6, react at 60°C for 5h, the reaction is completed, 5.0g activated carbon decolorization, Filtration while hot, tetrahydrofuran was distilled off from the filtrate under reduced pressure, and dried to obtain 40.4 g of compound 7 crude product, with a yield of 99.0%. Or to prepare compound 7, under stirring and cooling conditions, 8.1 g of (33%) dimethylamine aqueous solution, 6.6 g of (37-40%) formaldehyde solution and 10 g of compound 6 were sequentially added to 100 ml of glacial acetic acid, and placed in 70 The reaction was carried out at °C for 6 hours. After the completion of the reaction, the reaction solution was concentrated under reduced pressure, 100 ml of water was added, and the pH was adjusted to 12 with trimethylamine solution. The aqueous phase was extracted three times with dichloromethane (20 ml×3), and the organic phase was dried over anhydrous sodium sulfate. Concentrate under reduced pressure and dry to obtain 9.6 g of crude compound 7, with a yield of 85.0%.
J. Preparation of compound 8:
40.4 g of the crude product of compound 7 obtained in the above step H was heated and dissolved in 150 ml of acetone, adjusted to pH=2 with hydrochloric acid while hot, a solid was precipitated, cooled to about 0°C in an ice-water bath, filtered, and the filter cake was washed with frozen acetone and dried in vacuo to obtain compound 8 Crude product 40.5g, yield 89.8%.
The above crude compound J was recrystallized from acetone-ethanol-water (3:1:1). 36.5 g of product were obtained, and the yield was 90.0%.
Research and develop a kind of method for efficient green synthesis of arbidol hydrochloride intermediate, the structural formula of arbidol hydrochloride is as follows:
Example 1: Ethyl 5-acetoxy-1,2-dimethylindole-3-carboxylate
After the device was installed, 150 mL of acetic anhydride solvent was added to the three-necked flask, and then solid ethyl 5-hydroxy-1,2-dimethylindole-3-carboxylate (23.3 g, 0.1 mol) was added while stirring. After all dissolved, heated to reflux for 4 h, after the reaction was completed, the reaction solution was cooled, and the solid was obtained by suction filtration. Wash the solid with water for 4 times (150 mL-200 mL of water each time), and slowly add 0.15 mol/L ammonia water to the solution in the third time to control the pH of the mixed system by adding water to the solid to be 8 to 9. Finally, suction filtration A solid was obtained, which was dried in an oven at 70° C. for 5 h to obtain a crude product. Recrystallization from methanol gave 18.8 g of ethyl 5-acetoxy-1,2-dimethylindole-3-carboxylate as brown crystals. Yield 65%.
Example 2: Ethyl 5-acetoxy-6-bromo-2-bromomethyl-1-methylindole-3-carboxylate
After installing the device, in a three-necked flask, ethyl 5-acetoxy-1,2-dimethylindole-3-carboxylate (17.9g, 0.065mol), catalyst (p-cymene)- Ruthenium dichloride dimer (4.0g, 0.0065moL), N-bromosuccinimide NBS (46.28g, 0.26moL) and 200mL dimethylacetamide DMA, slowly warmed to 90°C in oil bath under nitrogen protection , maintain the reaction temperature for 24h, after the reaction is over; cool the reaction solution to room temperature, add an appropriate amount of water to the reaction solution, extract 5 times with ethyl acetate, combine the organic phases, dry, spin dry the solvent to obtain a solid, use acetone After recrystallization, a white powdery solid was precipitated, which was dried in vacuo to obtain 23.3 g of ethyl 5-acetoxy-6-bromo-2-bromomethyl-1-methylindole-3-carboxylate. Yield 80%.
Example 3: Ethyl 6-bromo-5-hydroxy-1-methyl-2-phenylthiomethylindole-3-carboxylate
Install the device, add 150 mL of methanol solvent to the three-necked flask, slowly add 8.6 g of solid potassium hydroxide under stirring, cool to room temperature after all dissolved, then add thiophenol (6.2 g, 0.05 mL) under stirring, After about 15 min, ethyl 5-acetoxy-6-bromo-2-bromomethyl-1-methylindole-3-carboxylate (23.3 g, 0.05 moL) was finally added, and the reaction was stirred at room temperature for 4 h. After the reaction is completed. 10% acetic acid was added dropwise to the reaction solution until the pH of the reaction solution was 3-4. After a large amount of yellow solid was precipitated, the solid was obtained by suction filtration, washed once with water, filtered with suction, and dried at 70 °C for 5 h in a drying box. get crude products. Recrystallization from ethyl acetate gave 12.6 g of ethyl 6-bromo-5-hydroxy-1-methyl-2-phenylthiomethylindole-3-carboxylate as yellow-white crystals. Yield 60%.
Example 4: Arbidol
After installing the device, add 100 mL of glacial acetic acid solution to the three-necked flask, cool it to 0 °C, slowly add 40 mL of 40% methylamine aqueous solution, and then add 10 mL of 37% formaldehyde aqueous solution, and after the reaction is stirred for 15 min, add 6- Ethyl bromo-5-hydroxy-1-methyl-2-phenylthiomethylindole-3-carboxylate (12.6g, 0.03moL), stirred uniformly for 10 min, then began to heat up to 80°C, maintaining the reaction temperature , and react for 4 h after complete dissolution. After the reaction is over, pour the reaction solution into water, add an appropriate amount of 20% potassium hydroxide solution to neutralize it with stirring, adjust the pH of the solution to 7.0, precipitate solids, filter with suction, and wash with water once. The solid was obtained by suction filtration, and dried in an oven at 70 °C for 5 h to obtain a crude product. Recrystallize with acetonitrile, after complete dissolution, add 1 g of activated carbon to reflux for 30 min, filter hot, cool, and precipitate 8.5 g of brown solid Arbidol. Yield 60%.
Example 5: Arbidol hydrochloride
Install the device, add an appropriate amount of acetone solvent to the three-necked flask, add Arbidol (8.5g, 0.018moL) under stirring, heat to reflux, add 10mL of concentrated hydrochloric acid dropwise, reflux for 30 min, and after the reaction is over, cool the reaction The liquid was brought to room temperature, and filtered with suction to obtain crude Arbidol hydrochloride, which was dried in an oven at 50 °C for 3 h. Recrystallize with acetone:ethanol (3:2) solvent, cool at room temperature for 10 h, freeze in refrigerator for 10 h, suction filtration, wash the solid with a small amount of acetone, and obtain 7.0 g of refined Arbidol hydrochloride in a yield of 75%. MS (EI): m/z: 513.8754 ([M]+).
1,2-Dimethyl-5-hydroxyindole-3-acetic acid ethyl ester (I) is acetylated with acetic anhydride affording the O-acyl derivative (II) , which is brominated to the corresponding dibromide compound (III) . The reaction of (III) with thiophenol in KOH yields (IV) , which is then submitted to a conventional Mannich condensation with formaldehyde and dimethylamine in acetic acid, giving the free base of arbidol (V), which is treated with aqueous hydrochloric acid .
Umifenovir (Arbidol®) is an indole derivative first marketed in 1993 for the prophylactic treatment of infections caused by influenza A and B viruses [74]. Produced by Pharmstandard, it is still currently used in Russia and China to treat influenza infections [75]. Umifenovir is marketed in 50 and 100 mg capsules, being administered orally. The pharmacokinetics is limited, presenting rapid absorption and reaching the maximum concentration in 1.6–1.8 h. It is a slow elimination drug, with a half-life of 16 to 21 h, and may be administered twice a day [76].
The drug’s anti-influenza mechanism of action is related to arbidol’s ability to bind to the haemagglutinin (HA) protein [77]. The haemagglutinin (HA) protein is a homotrimeric glycoprotein found on the surface of the influenza virus, and it is essential for its infectivity. This protein is responsible for allowing the influenza virus binding to the sialic acid present on the surface of the target cells (respiratory tract cells or erythrocytes). As a result of this interaction, the virus is internalized in the host cell. Once umifenovir binds to the HA protein, this glycoprotein is prevented from binding to sialic acid, so the virus is no longer able to penetrate the host cell [78].
The structural similarity between the SARS-CoV-2 peak and the influenza virus (H3N2) HA glycoproteins justifies the fact that drugs that are capable of binding to HA can also do so to the SARS-CoV-2 spike protein. This fact was evidenced by molecular modeling studies, wherein was demonstrated that umifenovir is able to bind to the protein peak, preventing its trimerization, which would be a determining factor for the mechanism of cell adhesion (Fig. 8) [78].
Fig. 8. Umifenovir (in orange) binding region in SARS-CoV-2 spike glycoprotein. Reprinted from International Journal of Antimicrobial Agents, 56, N. Vankadari, “Arbidol: A potential antiviral drug for the treatment of SARS-CoV-2 by blocking trimerization of the spike glycoprotein”, Page 2, with permission of Elsevier. Copyright 2020. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Recently in 2020, in vitro studies performed with Vero cells confirmed that arbidol efficiently inhibits SARS-CoV-2 infection with an EC50 of 4.11 μM. The author also determined that arbidol was able to efficiently block both viral entry and post-entry stages, and also concluded that the drug prevented the viral attachment and release of SARS-CoV-2 from the intracellular vesicles. Importantly, the EC50 of arbidol against SARS-CoV-2 led the authors to suggest that the dose of arbidol currently recommended by the Chinese Guidelines (200 mg, 3 times/day) should be elevated in order to achieve ideal therapeutic efficacy to inhibit the SARS-CoV-2 infection [79].
A clinical trial was conducted at Wuhan Jinyintan Hospital, in 2020, from February 2 to March 20 conducted to evaluate the effectiveness and safety of umifenovir in the treatment of COVID-19 patients. In this study, 81 patients were evaluated: 45 received 200 mg of umifenovir three times a day, and 36 were in the control group. The authors concluded that baseline clinical and laboratory characteristics were similar in the two groups, and patients in the umifenovir group had a longer hospital stay than those in the control [80]. Although such results may seem discouraging, further clinical trials with higher doses of umifenovir may be required in order to verify its clinical efficiency against the SARS-CoV-2 infection.
The synthesis of umifenovir was described in 2006 starting from the reaction between ethyl acetoacetate 63 and methylamine, giving enaminone 64, which next undergoes a Nentizescu condensation reaction with 1,4-benzoquinone to produce indole derivative 65 (Scheme 9). Then, an acetylation reaction is carried out to protect the hydroxyl group in 65, producing 66, which is converted to 67 after a bromination step. The reaction of intermediate 67 with thiophenol in basic medium leads to the formation of 68, which finally affords umifenovir after a Mannich reaction [81].
Drug-repurposing studies are testing a range of compounds to treat COVID-19, but manufacturers may struggle to meet demand if any of these candidates prove effective against SARS-CoV-2. The pandemic has already strained global supply chains and limited the availability of a number of products, including hand sanitizer and diagnostic test reagents. The raw materials needed to make a new antiviral drug would most likely face similar pressures. But a team led by Tim Cernak of the University of Michigan has used an AI-based retrosynthesis program called Synthia to devise alternative routes to 12 leading drug candidates under investigation. The work appears on a preprint server and has not been peer reviewed (ChemRxiv 2020, DOI: 10.26434/chemrxiv.12765410.v1). “If the world runs out of one of the drugs currently in the clinic, we are providing a backup recipe,” Cernak says. Using alternative starting materials that are readily available, the researchers aimed to find routes of similar length and cost to those of existing syntheses. For each compound, the researchers whittled down a long list of options offered by Synthia to identify the most suitable synthetic strategies. Then the team tested some of these syntheses in the lab, including four new routes to the antiviral umifenovir, currently being investigated in eight clinical trials against COVID-19. Cernak says this approach could be used more generally to rapidly identify alternative synthetic routes whenever crises cause supply chain disruptions in drug manufacturing.
Artificial intelligence finds alternative routes to COVID-19 drug candidates
If drug-repurposing studies hit pay dirt, backup recipes could help antiviral manufacturers avoid supply chain problems
The research on the disease COVID-19 is an ongoing process since its outbreak as a pandemic. The repurposing of existing approved drugs has received priority attention due to some promising results obtained regarding COVID-19. In this article, some of the important chemical methodologies adopted for the synthesis of umifenovir, (s)-cidofovir, ribavirin, and ruxolitinib have been discussed. The repurposing of these approved drugs has received priority attention due to some promising results obtained regarding COVID-19 and some drugs are under more therapeutic trials. This manuscript has highlighted the synthetic strategies of four heterocyclic-based approved drugs, umifenovir, (s)-cidofovir, ribavirin, and ruxolitinib, repurposed for the treatment of COVID-19.
Ethyl 5-acetoxy-2-methyl-1H-indole-3-carboxylate 1a: Acetic anhydride (25.9 ml, 274 mmol 20 eq.) was added to a stirred solution of ethyl 5-hydroxy-2-methyl-1H-indole- 3-carboxylate 1 (3.00 g, 13.6 mmol, 1.0 eq.) in pyridine (3.32 mL, 41.1 mmol, 3.0 eq.) and the reaction heated to reflux. After 1 h, the reaction was allowed to cool back to rt before pouring the mixture into a solution of aqueous saturated sodium bicarbonate (40 mL). The product was extracted with ethyl acetate (3 x 40 ml) and the combined organic layers were washed with water (40 mL), dried (Na 2 SO 4 ) and concentrated In vacua to yield the product as a white solid which was used without further purification (3.4g, 96%). NMR: δH ( 400 MHz, CDCl 3) 8.34 (1H, s; NH), 7.75 (1H, s, H 4 ), 7.21 (1H, d, J 8.5, H 6 ), 6.89 (1H, d, J 8.5, H 7 ), 4.38 (2H , q, J 7.1 , CO 2 CH 2 CH 3 ), 2.71 (3H, s, C 1 CH 3 ), 2.34 (3H, s, CO 2 CH 3 ), 1.43 (3H, t, J 7.1, CO 2 CH 2 CH 3 ). δ c (100 MHz, CDCl 3 ) 170.8<a name=”
0.31 (40% ethyl acetate in hexane), HRMS. (ESI-TOF): C 14 H 15 O 4 N ([M+H] + ) requires 262.1074, found 262.1074.
Ethyl 5-acetoxy-1,2-dimethyl-1H-indole-3-carboxylate 1b: Protected indole 1b (1.35 g, 5.17 mmol, 1 eq.) was dissolved in DMF (15 mL). To this solution, methyl iodide (0.965 ml, 15.5 mmol, 3.0 eq.) was added and the resulting mixture was cooled on ice. Sodium hydride (0.186 g, 7.75 mmol, 1.5 eq.) was added and the reaction was left to stir on ice for 1.5 h. After this time, a small amount of water (5.0 mL) was added to the reaction and the solvents removed in vacuo. The resultant brown oil was then purified directly by column chromatography (30% ethyl acetate in petrol) to yield the title compound as a pale yellow solid (1.50 g, 95%). NMR: δ Η (500 MHz, CDCl 3 ) 7.79 (s, 1H, H 4 ), 7.26 (m, 1H, H 6 ), 6.96 (ddd, J = 8.8, 2.4, 0.8 Hz, 1H, H7 ), 4.38 (f, J = 7.1 Hz, 2H, CO 2 CH 2 CH 3 ), 3.69 (s, 3H, NCH 3 ), 2.77 (d, J = 1.3 Hz, 3H, ΑrCΗ 3 ), 2.33 (s , 3H, CO 2 CH 3 ), 1.43 (t, J= 7.1 Hz, 3H, CO 2 CH 2 CH 3 ). δ C (150 MHz, CDCl 3 ) 170.5 (CO 2 CH 3 ), 166.0 (CO 2 Et), 146.5 (C 5 ), 146.0 (C 2 ), 134.5 (C 8 ), 127.2 (C 3 ), 116.2 ( C6 ), 114.0 (C4 ) , 109.6 (C7 ), 104.4 (C 1 ), 59.6 (CO 2 CH 2 CH 3 ), 29.9 (NCH 3 ), 21.3 (C 1 CH 3 ), 14.8 (CO 2 CH 2 CH 3 ), 12.1 (CO 2 CH 3 ) . R f : 0.4 (30% ethyl acetate in hexane). HRMS (ESI-TOF): C 15 H 17 O 4 N ,([M+H] + ) requires 276.1230, found 276.1229.
Ethyl 6-bromo-2-(bromomethyl)-5-hydroxy-1-methyl-1H-indole-3-carboxylate 2: Bromine (558 μL, 10.9 mmol, 3.0 eq.) was added to a stirred solution of protected indole ( 1b, 1.00 g, 3.63 mmol, 1.0 eq.) in carbon tetrachloride (100 mL). After refluxing for 16 h, the reaction was cooled and aqueous sodium thlosulphate (10%. w/v, 100 mL) was added and left to stir for 20 min until the orange color disappeared. After this time, the organic layer was separated, washed with water (2 x 100 mL), dried (Na 2 SO 4 and concentrated in vacuo to yield a pale yellow solid, which was used without further purification (1.40 g, 99%) NMR: δ Η (400 MHz, PDCl 3 ) 7.86 (1H, s, H 4 ), 7.54 (1H, s, H 7), 5.05 (2H, s, CH 2 Br), 4.41 (2H, q, J 7.1, CO 2 CH 2 CH 3 ), 3.69 (3H, s, NCH 3 ), 2.39 (3H, s, CO 2 CH 3 ), 1.45 (3H, t, J 7.1, CO 2 CH 2 CH 3 ), δ C (100 MHz, CDCl 3 )
1.4.5 (CO 2 CH 2 CH 3 ), R f : 0.75 (CH 2 Cl 2 ). HRMS (ESI-TOF): C 15 Η 15 O 3 ΝΒr ([M+H] + ) requires 431.9441, found 431.9441.<a name=”
Ethyl 6-bromo-5-hydroxy-1-methyl-2-((phenylthio)methyl)-1H-indole-3-carboxylate 3: Thiophenol (99.8 μL, 0.972 mmol, 1.0 eq.) was added to a solution of potassium hydroxide (164 mg, 2.92 mmol, 3.0 eq.) in methanol (2 ml) and left to stir at room temperature for 15 min. After this time, the solution was cooled on ice and bromo indole 2 (880 mg, 0.972 mmol, 10 eq.) in CH 2 Cl 2 (5 mL) was added. The reaction was left to stir for 3 h before neutralization with acetic acid. The solvent was removed in vacuo and columned directly (20% EtOAc in petrol) to yield the title product as a pale yellow solid (362 mg, 86%). NMR: δ Η (600 MHz, CDCl 3 ) 7.74 (s, 1 H, Hr), 7.43 (s, 1H, H 4 ), 7.36 (dq, J = 5.2, 3.4, 2.4 Hz, 2H, H10 ), 7.25 (dd, J = 5.2, 1.9 Hz, 3H, H 11 and 5.33 (s, 2H, SCH 2 ), 4.29 (q, J = 7.3 Hz, 2H, CO 2 CH 2 CH 3 ), 3.60 (d, J = 18.1 Hz, 3H, NCH 3 ), 1.38 (t, J = 7.3 Hz, 3H, CO 2 CH 2 CH 3 ), δ c (150 MHz, CDCl 3 ) 165.1, 147.7, 144.2, 134.1 R f : 0.35 (20% EtOAc in petrol) HRMS (ESI-TOF)-: C 19 H 18 BrNO 3 S ([M+H] + ) requires 420.0263, found 420.0260.
Arbidol [Ethyl 6-bromo-4((dimethylamino)methyl)-5-hydroxy-1-methyl-2-((phenylthio)methyl)-1H-indole-3-carboxylate] 4: 1 Indole 3 (200 mg, 0.476 mmol, 1.0 eq.) and N, N, N’, N’-tetramethylaminomethane (1-95 μL, 1.43 mmol, 3.0 eq.) were dissolved in 1,4-dioxane (2 mL). The reaction was. heated to reflux for 3.5 h before removing the solvent in vacuo. The reaction was then re-dissolved in ethyl acetate and 1 M HCl was added to the solution causing the title product to crash out as a pale yellow solid (117 mg, 51%). NMR-: δ B (500 MHz, MeOD) 7.87 (s, 1 H, H 7 ), 7.39 (dd, J = 7.4, 2.2 Hz, 2H, H 10 ), 7.35 – 7.31 (m, 3H, H 11 and H12 ) . 4.87 (s, 2H, SCH 2), 4.71 (s, 2H, CH 2 NMe 2 ), 4.33 (q, J = 7.1 Hz, 2H, CO 2 CH 2 CB 3 ), 3.63 (s, 3H, NCH 3 ), 2.97 (s, 6H, N (CH 3 ) 2 ), 1.39 (t, J = 7.1 Hz, 3H, CO 2 CH 2 CH 3 ). δ c (150 MHz, MeOD) 169.7 (CO 2 Et), 152.7 (C s ), 149.0 (C), 137.7 (C 10 ), 136.4 (C 3 ), 136.0 (C 8 ), 132.3 (C 11 ), 131.3 (CH), 129.3 (C12 ) , 119.8 (C7 ) , 113.1 (C2), 111.3 ( C6), 108.3 (C4 ) , 64.2. (CO 2 CH 2 CH 3 ), 57.4 (CH 2 NMe 2 ), 45.4 (CH 2 N(CR 3 ) 2 ); 33.7 (CH 2 SPh), 32.9 (NCH 3 ), 16.6 (CO 2 CH 2 CH 3 ). R f : 0.25 (EtOAc). HRMS (ESI-TOF): G 22 H 25 BrN 2 O 3 S ([M+H] + ) requires 477.0842, found 477.0844.
Synthesis of Arbidol Analogues
Ethyl 5-acetoxy-6-bromo-2-(((3-hydroxyhpenyl)thio)methyl)-1-methyl-1H-indole-3-carboxylate 8a: 3-hydroxythiophenol. (117 μL, 1.15 mmol, 1.0 eq.) was added to a solution of sodium carbonate (367 mg, 3.46 mmol, 3.0: eq.) and bromo indole 2 (500 mg, 1.15 mmol, 1.0 eq.) in dry ethyl acetate (10mL). The reaction was heated to 100°C and stirred for 5 h before<a name=”cooling, filtering and removing the solvent in vacuo. The compound was purified by column chromatography (40% EtOAc in Hexanes) to produce the title product as a pale yellow solid (240 mg, 44%). NMR: δ H (500 MHz,. CDCl 3 ) 7.85 (s, 1H, H 7 ), 7.56 (s, 1 H, H 4 ), 7.12 (t, J = 7.9 Ηz, 1Η, H 13 ), 6.95 – 6.90.(m, 1H, Η 14 ), 6.78 (s, 1H, H 10 ), 6.75-6.71 (m, 1H,.H 12 ), 4.69 (s, 2H, SCH 2 ), 4.30 (q, J = 7.4 Hz, 3H, CO 2 CH 2 CH 3 ), -3.66 (s, 3H, NGH 3 ), 2.4Q (s, 3H, COCH 3), 1.38 (t, J = 7.4 Hz, 3H, CO 2 CH 2 CH 3 ). Δ C (150 MHz, CDCL 3 ) 169.8, 165.0, 156.1, 144.6, 143.3, 135.6, 135.1, 130.1, 126.1, 124.8, 119.3, 113.9, 111.1, 105.8, 60.1, 30.4, 29.9, 21.0, 14. . R f : OAS (30% EtOAc in Hexane). HRMS (ESS-TOF): C 21 H 20 SrNO 5 S ([M+H] + ) requires 473.0318, found 478.0317.
Ethyl 6-bromo-5-hydroxy-24(((3-hydroxyphenyl)thio)methyl)-1-methyl-1H-indole-3-carboxylate 8: Sodium carbonate (106 mg. 1.00 mmol, 2.0 eq.) was added to a stirred solution of meta-hydroxy indole 8a (240 mg, 0.502 mmol, 1.0 eq.) in methanol (40 ml) and left to stir for 2h, The solution was then filtered and the solvent removed in vacuo, The product was re -dissolved in ethyl acetate (10 mL) and washed once with water (40 mL) before drying (Na 2 SO 4 and concentrating in vacuo to give the title product as a white solid, which could be used without further purification (160 mg, 67%), NMR-: δ H (600 MHz, MeOD) 7.60 (s, 1H, H 7 ), 7.58 (s, 1 H, H 4 ), 7.07 (dd, J = 8.2, 7.7 Hz, 1H, H 13), 6.83 – 6.81 (m, 1Η, Η 14 ), 6.79 (ddd, J = 7.7, 1.8, 0.9, 1H, H 10 ), 6.7.0 (dd, J = 8.2, 1.8, 0.9 Hz, 1H, H 12 ), 4.70 (s, 2H, SCH 2 ), 4.26 (q, J = 7.1 Hz, 2H, CO 2 CH 2 CH 3 ), 3.64 (s, 3H, NCH 3 ), 1.39 (t, J = 7.1 Hz , 3H, CO 2 CH 2 CH 3 ). δ c (150 MHz, MeOD) 166.9, 158.9, 150.6, 145.5, 136.4, 133.6, 131.0,. 130.7, 128.0, 1.24.9, 120.5, 116.0, 114.8, 107.9, 104.8, 60.8, 30.5, 30.4, 14.8. R f : 0.45 (1% MeOH in CΗ 2 Cl 2 ). HRMS (ESI-TOF): C 7 PM18 BrNO 4 S ([M+H] + ) requires 436.0213, found 436.0215.
Ethyl 2-(((3-aminophenyl)thio)methyl)-6-bromo-5-hydroxy-1-methyl-1H-indole-3-carboxyiate 9: 3-aminothiophenol (54.3 μL, 0.511 mmol, 1.0 eq.) was added to a solution of potassium hydroxide (86 mg, 1.53 mmol, 3.0 eq.) in methanol (2 ml) and left to stir at room temperature for 15 min. After this time, the solution was cooled on ice and bromo indole 2 (200 mg, 0.511 mmol, 1.0 eq.) in CH 2 Cl 2 (5 ml) was added. The reaction was left to stir for 3 h before neutralization with acetic-acid. The solvent was removed in vacuo and purified directly by preparative TLC (1% MeOH in CH 2 Cl 2 ) to yield the title product as a pale yellow solid (138 mg, 62%), NMR: δ H (500 MHz, CDCl 3) 7.74 (d, J = 1.8 Hz, 1H, H 7 ), .7.42 (d, J = 1.8 Hz., 1H, H 4 ), 7.03 (t, J = 8.1 Hz, 1 H, H 13 ), 6.75 (d, J= 7.7 Hz, 1H, H 14 ), 6.68 (s, 1H, H 10 ), 6.55 (d, J = 6.1 Hz, 1H, H 12 ), 4.68 (d, J = 1.9 Hz, 2H, SCH 2 ), 4.35 ™ 4.30 (m, 2H, COCH 2 GH 3 ),. 3.60 (d, J = 1.9 Hz, 3H, NCH 3 ), 1.40 (td, J = 7.1, 1.8 Hz, 3H, COCH 2 CH 3 ). δ c (150 MHz, CDCl 3 ) 1-66.9, 150.6, 149.6,<a name=”
146.0, 136.0, 133.5, 1 . 30.5, 128.1, 123.1, 120.2, 117.6, 115.8, 114.8, 107.9, 104.6, 68.1, 60.8, 30.4, 14.8. R f : 0.85 (1% MeOH in CH 2 Cl 2 ), HRMS (ESI-TOF): C 19 H 19 BrN 2 O 3 S ([M+H] + ) requires 435.0372, found 435.0370.
Ethyl 2-(((3-aminophenyl)thio)methyl)-6-bromo-5-hydroxy-1-methyl-1H-indole-3-carboxylate 10: 2-napthalenethiol (82.0 mg, 0.511 mmot, 1.0 eq.) was added to a solution of potassium hydroxide (86 mg, 1.53 mmol, 3.0 eq.) in methanol (2 mL) and left to stir at room temperature for 15 min. After this time, the solution was cooled on ice and bromo indole 2 (200 mg, 0.511 mmol, 1.0 eq.) in CH 2 Cl 2 (5 mL) was added. The reaction was left to stir for 3 h before neutralization with acetic acid. The solvent was removed in vacuo and purified directly by preparative TLC (1% MeOH in CH 2 Cl 2 ) to yield the title product as a pale yellow solid (118 mg, 50%). NMR: δR(600 MHz, DMSO) 9.77 (s, 1H, OH), 7.83 (d, J = 1.8 Hz, 1H, Ar), 7.81 -7.79 (m, 1H, Ar), 7.75 (d, J = 8.6 Hz, 1H , Ar), 7.72 – 7.70 (m, 1H, Ar), 7.66 (s, 1H, Hz), 7.46 (s, 1H, H 4 ), 7.45 – 7.40 (m, 2H, Ar), 7.34 (dd, J = 8.5, 1.9 Hz, 1H, Ar), 4.82 (s, 2H, CH 2 SPh), 4.04 (q, J = 7.1 Hz, 2H, CO 2 CH 2 CH 3 ), 3.63 (s, 3H, NC. %), 1.14 (t, J = 7.1 Hz, 3H, CO 2 CH 2 CH 3 ). δ c (150 MHz, DMSO) 164.3, 149.3, 143.4, 133.2, 132.0, 131.7, 131.6, 128.9, 128.4, 128.4, 127.7, 127.2, 126.8, 126.3, 1:26.0, 116.3, 116.3, 116.3 06.3, 103.3, 59.2, 30.3, 28.1, 14.3. R f : 0.75 (1% MeOH in CH2 Cl 2 ). HRMS (ESI-TOF): C 23 H 20 BrNO 3 S (fM+Hf) requires 470.0420, found 470.0420.
Ethyl 6-bromo-4-((dimethylammino)methyl)-5-hydroxy-2-(((3-hydroxyohenyl)thio)methyl)-1-methyl-1H-indole-3-carboxylate 11; Meta-hydroxy indole 8 (30.0 mg, 0.069 mmol, 1.0 eq,) and N, N,N’,N’-tetramethyldiaminomethane (47.0 μL, 0.344 mmol, 5.0 eq.) were dissolved in CH 2 Cl 2 (30 mL) . The reaction was heated to reflux for 3.5 h before removing the solvent in vacuo to, yield the title product as a pale yellow solid (34 mg. 99%). NMR; δH (500 MHz, CDCl 3 ) 7.47 (s, 1H, H 7 ), 7.12 (t, J = 7.9 Hz, 1H, H 13 ), 6.90 (d, J = 7.9 Hz, 1H, H 14 ), 6.90 ( d, J = 7.9 Hz, 1H, H 12 ), 6.66 (s, 1H, H 10 ), 4.41 (s, 2H, CH 2 NMe2 ), 4.34 (s, 2H, CH 2 SPh), 4.15 (q, J = 7.1 Hz, 2H, CO 2 CH 2 CH 3 ), 3.60 (s, 3H, NCH 3 ), 2.55 (s, 6H, CH 2N (CH 3 ) 2 ), . 1.33 – 1.21 (m, 3H, CO 2 CH 2 CH 3 ). δ c ( . 150 MHz, CDCl 3 ) 165.9, 156.7, 150.9, 142.6, 135.1, 132.2, 131.0, 130.0, 128.9, 124.6, 124.3, 119.3, 115.5, 113.4, 106.8, 106.8, 106.8, 106.8, 165.5 58.7, 44.0, 30.4, 29.9, 14.3, R f : 0.15 (10% MeOH in CH 2 Cl 2 ). HRMS (ESS-TOF): C22 H 25 BrN 2 O 4 S ([M+H] + ) requires 493.0791, found 493.0792,
0.0419 mmol, 1.0 eq.) and 1-(2-((trimethylsilyl)oxy)ethyl)piperazine (25 mg, 0.126 mmol, 3.0 eq.) were dissolved in 1,4-dioxane (2 mL) and refluxed overnight. The solvent was then removed in vacuo and the reaction columned directly to yield the title product as a yellow solid (12 mg, 51%). NMR: δ Η (400 MHz, MeOD) 7.56 (s, 1.H, H 7 ), 7.22 – 7.30 (m, 5H, SPh), 4.57 (s, 2H, CH 2 SPh), 4.14 – 4.19 (m, 4H, CH 2 NR 2 and CΟ 2 CH 2 CΗ 3 ), 3.68 (t, J = 5.9 Hz, 2H, . CH 2 OH), 3.60 (s, 3H, NCH 3 ), 2.53 – 2.70 (m, 10H, piperazine ring and CH 2 CH2 CH 2 OH), . 134 – 1.30 (rn, 3H, CO 2 CH 2 CH 3 ), δ c (150 MHz, MeOD) 167.2, 151.7, 143.9, 135.4, 134.2, 133.4, 130.1, 129.0, 125.5, 114.1, 113.6, 107.0, 108.9, 108.9, 108.9 61.5, 61.1, 59.8, 59.1, 54.3, 52.9, 30.9, 30.5, 14.7. R f : 0.15 (5% MeOH in CH 2 Cl 2 ), HRMS (ESI-TOF): C 26 H 32 BrN 3 O 4 S ([M+H] + ) requires 562.1370, found 562.1368,
Ethyl 6-bromo-5-hydroxy-2-(((2-hydroxyphenyl)thio)methyl)-1-methyl-1H-indole-3-carboxylate 16: 2-hydroxythiophenol (26.0 μL, 0.256 mmol, 1.0 eq.) was added to a solution of sodium carbonate (81.0 mg, 0.767 mmol, 3.0 eq.) and promo indole 2 (100 mg, 0.256 mmol, 1.0 eq.) in ethyl acetate (2 mL). The reaction was heated to 50°C and stirred for 2 h before cooling and removing the solvent in vacuo. The product was then re-dissolved in methanol (2 mL) and potassium hydroxide (21.5 mg, 0.384 mmol, 1.5 eq.) was added. The reaction was stirred at room temperature for 3 h before direct purification by preparative TLC (2% MeOH in CH 2 Cl 2 ) to yield the title product as. a white solid (20.5 mg, 1.8%), NMR: δ H (500 MHz, MeOD) 7.59 (s, 1H, H 7), 7.54 (s, 1H, Η 4 ), 7.17 (t, J = 7.7 Hz, 1H, H 12 ), 7.08 (d, J = 7.7 Hz, 1H, H 14 ), 6.85 (d, J =7.7 Hz , 1H, H 13 ), 6.66 (t, J = 7.7 Hz, 1H, H 15 ), 4.58 (s, 2H, SCH 2 ), 4.24 (q, J = 7.1 Hz, 2H, CO 2 CH 2 CH 3 ) , 3.59 (s, 3H, NCH 3 ), 1.40 (t, J = 7.1 Hz, 3H, CO 2 CH 2 CH 3 ). δ c (150 MHz, MeOD) 161.3, 146.4, 143.3, 134.8, 132.0, 130.4, 129.4, 123.7, 123.0, 121.8, 120.5, 114.4, 113.6, 110.0, 102.7, 60.04, 390.2, 390.2, 390.04 R f : 0.6 (2% MeOH in CH 2 Cl2 ). HRMS (ESI-TOF): C 19 H 18 BrNO 4 S ([M+H] + ) requires 436.0213, found 436.0212.
Ethyl 6-bromo-5-hydroxy-2-(((4-hydroxyphenyl)thmetihoyl)-)1-methyl-1H-indole-3-carboxylate 17: 4-hydroxythiophenol (26.0 μL, 0.256 mmol, 1.0 eq.) was added to a solution of sodium carbonate (81.0 mg, 0.767 mmol, 3.0 eq.) and bromo indole 2 (100 mg, 0.256 mmol, 1.0 eq.) in ethyl acetate (2 mL). The reaction was heated to 50°C. and stirred for 2 hours before cooling<a name=”and removing the solvent in vacuo. The product was then re-dissolved in methanol (2 mL) and potassium hydroxide (21.5 mg, 0.384 mmol, 1.5 eq.) was added. The reaction was stirred at room temperature for 3 h before direct purification by preparative TLC (2% MeOH in CH 2 Cl 2 ) to yield the title product as a white solid (2.5 mg, 2%). NMR: δ Η (600 MHz, DMSO) 7.65 (s, 1H, H 7 ), 7.49 (s, 1 H, H 4 ), 7.03 (d, J = 8.7 Hz, 2H , H 12 ), 6.58 (d, J = 8.7 Hz, 2. HH 13 ) , 4.52 (s, 2H, SCH 2 ), 4.08 (q, J = 7.2 Hz, 2H, CO 2 CH 2 CH 3 ), 3.51 (s, 3H, NCH3 ), 1.23 (t, J = 7.1 Hz, 3H, CO 2 CH 2 CH 3 ). δ c (150 MHz, DMSO) 164.2, 157.9, 149.2, 144.3, 135.7, 131.4, 126.1, 1214, 115.9, 114.1, 106.3, 102.9, 79.2, 59.0, 55.4, 30.0, R 14.3, f ; 0.5 (2% MeOH in CH 2 Cl 2 ). HRMS (ESI-TOF): C 19 H 18 BrNO 4 S ([Μ+H] + ) requires 436.0213, found 436.0213.
Ethyl 5-acetoxy-6-bromo-2-(((3-methoxyphenyl) thio)methyl)-1-methyl-1H-indole-3-carboxylate 18a: 3-methpxythiophenol (14.6 μL, 0.118 mmol, 1.0 eq.) was added to a solution of sodium carbonate (37.4 mg, 0.353 mmol, 3.0 eq.) and bromo indoles 2 (46.0 mg, 0.118 mmol, 1.0 eq.) in dry ethyl acetate (20 ml). The reaction was heated to 50°C and stirred for 2 h before addition of water. The organic layer was separated, dried (Na 2 SO 4 ) and concentrated in vacuo. The compound was purified by column chromatography (20% EtOAc in Hexanes) to produce the title product as a white solid (34 mg, 59%). UMR; δ Η (600 MHz, DMSO) 7.92 (s, 1 H, H 7 ), 7.6.6 (s, 1 H, H 4 ), 7.13 (t, J = 7.9 Hz, 1H, H 13), 6.8.7 – 6.84 (m, 1 H, H 14 , 6.79 – 6.74 (m, 2H, H 10 and H 1 2 ), 4.77 (s, 2H, SCH 2 ), 4.13 (q, J = 7.1 Hz , 2H, CO 2 CH 2 CH 3 ), 3.70 (s, 3H, NCH 3 ), 3.58 (s, 3H, SPhOCH 3 ), 2.27 (s, 3H, COCH 3 ), 1.20 (t, J = 7.1 Hz, 3H, CO 2 CH 2 CW 3 ).δ c (150 MHz, DMSO) 169.1, 163.9, 159.4, 144.9, 142.6, 135.1, 135.0, 129.9, 125.2, 123.3, 116.2, 115.1, 114.73, 110.73, 110.2 104.3, 59.5, 55.1, 30.6, 28.1, 20.7, 14.3 R f : 0.4 (20% EtOAc in Hexane) HRMS (ESI-TOF): C 22H 22 BrNO s S ([M+H] + ) requires 492.0475, found 492.0472.
Ethyl 6-bromo-5-hydroxy-2-((3(-methoxyphenyl)thio)methyl)-1-methyl-1H-indole-3-carboxylate 18: Sodium carbonate (41.3 mg, 0.390 mmol, 2.0 eq.) was added to a stirred solution of meta-methoxy indole 18a (96.0 mg, 0.195 mmot, 1.0 eq.) in methanol (10 mL) and left to stir for 2h, the solution was then filtered and the solvent removed in vacuo. The product-was re-dissolved in ethyl acetate (10 mL) and washed once with water (10 mL) before drying (Na 2 SO 4 ) and concentrating in vacuo to give the title product as a white solid, which could he used without further purification (80 mg, 91%), NMR: δ Η (600 MHz, CDCl 3 ) 7.73 (s, 1H, Η 7 ), 7.41 (s, 1H, H 4 ), 7.17 – 7.13 (m, H 13), 7.07 (m, 1H, H 14 ), 6.96 (dt, J = 7.7, 1.3, 1H, H 10 ), 6.85 (m, 1H, H 12 ),4.71 (s, 2H, SCH 2 ), 4.30 ( q, J = 7.1 Hz, 2H, CO 2 CH 2 CH 3 ), 3.63 (s, 3H, NCH 3 ), 1.41 (t, J = 7.1 Hz, 3H, CO 2 CH 2 CH 3 ). Δ C (150 MHz, CDCL 3 ) 165.2, 159.8, 147.7, 135.4, 132.6, 129.8, 12.7.2, 124.6, 119.7, 117.3, 114.1, 112.5, 107.5, 107.9, 55.4, 29.9, 29.6, 14.7 Rf :. _<a name=”0.55 (1% MeOH in CH 2 Cl 2 ). HRMS (ESl-TOF): C 20 H 20 BrNO 4 S ([M+H] + ) requires 450.0369, found 450.0367.
Ethyl 6-bromo-4-((dimethylamino)methy-5-hydroxy-2-(((2-hydroxyphenyl)thio)methyl)-1-methyl-1H-indole-3-carboxylatete 19: Ortho-hydroxy indole: 16 (13.5 mg, 0.0309 mmol, 1.0 eq.) and N, N, N’,N’-tetramethyldiaminomethane (12.7 μL, 0.0928 mmol, 3.0 eq.) were dissolved in 1,4-dioxane (2.0 mL). reaction was heated io reflux for 3.5 h before removing the solvent in vacuo to yield the title product as a white solid (13 mg, 85 %).HMR: δ H (500 MHz, MeOD) 7.53 (s, 1H, H 7 ) , 7.19 – 7.11 (m, 1H, H 4 ), 7.03 (dd, J = 7.6, 1.7 Hz, 1 H, H 12 ), 6.86 – 6.78 (m, TH, H 14 ), 6.63 (dt, J = 13.7 , 7.6 Hz, 1H, H 15 ), 4.48 (s, 2H, CH 2 Sph), 4.34 (s, 2H, CH 2NMe 2 ), 4.22 (dq, J = 10.8, 7.1, 6.3 Hz, 2H, CO 2 CH 2 CH 3 ), 3.56 (s, 3H, NCH 3 ), 2.49 (d, J = 11.4 Hz, 6H, CH 2 N(CH 3 ) 2 ), 1.42 – 1.37 (m, 3H, CO 2 CH 2 CH 3 ). Δ C (150 MHz, MEOD) 167.5, 160.4, 137.0, 136.6, 132.5, 131.6, 130.6, 126.1, 114.5, 112.6, 110.6, 106.0, 68.1, 61.4, 60.1, 43.5, 29.9, 29.9 14.6. R f : 0.4 (5% MeOH in CH 2 Cl 2 ), HRMS (ESI-TOF): C 22 H 25 BrN 2 O4 S ([M+H] + ) requires 493.0791, found 493.0793.
Ethyl 6-bromo-4-((dimethylamino)methyl-5-hydroxy-2-(((3-methoxyphenyl)thio)methyl)-1-methyl-1H-indole-3-carboxylate 21: Sodium carbonate (17.5 mg, 0165 mmol, 3.0 eq.) was added to a stirred solution of meta-methoxy indole 18 (27.0 mg, 0.055 mmol, 1 .0 eq.) in ethyl acetate (8 mL) and methanol (1 mL). to stir for 3h before filtering and removing the solvent in vacuo.The compound was then re-dissolved in 1,4-dioxane (5 mL) and N, N, N’,N’-tetramethyldiaminomethane (5.5 μL, 0.04 mmol, 3.0 eq.) as added. The reaction was heated to reflux overnight before removing the solvent in vacuo. Purification by preparative TLC (5% MeOH In CH 2 Cl 2 ) yielded the title product as a pale yellow solid (7 mg, 24%) .NMR: δ Μ (600 MHz, CDCl 3) 7.44 (s, 1H, H 7 ), 7.19 (t, J = 7.9 Hz, 1 H, H 15 ), 6.96 (rn, 1H, H 14 ), 6.82 (m, 1H, W 10 ), 6.77 (m , 1H, H 12 ), 4.52 (s, 2H, CH 2 SPh), 4.21 (qd, J = 7.2, 0.8 Hz, 2H, CO 2 CH 2 CH 3 ), 4.17 (s, 2H, CH 2 NMe 2 ), 3.66 (s, 3H, NCH 3 ), 3.58 (s, 3H, OCH 3 ), 2.38 (s, 6H, CH 2 H(CH 3 ) 2 ), 1.34 (m, 3H, CO 2 CH 2 CH 3 ). δ c (150 MHz, CDCl 3) 165.6, 159.9, 151.7, 141.7, 135.5, 131.9, 129.9, 124.7, 117.5, 114.2, 113.1, 112.6, 108.6. 106.2, 60.5, 59.9, 55.3, 44.2, 30.5, 29.9, 14.5. R f : 0.35 (5% MeOH in CH 2 Cl 2 ). HUMS (ESI-TOF): C 23 H 27 BrN 2 O 5 S ((M+H] + ) requires 523.0897, found
^ Jump up to:abLeneva IA, Russell RJ, Boriskin YS, Hay AJ (February 2009). “Characteristics of arbidol-resistant mutants of influenza virus: implications for the mechanism of anti-influenza action of arbidol”. Antiviral Research. 81 (2): 132–40. doi:10.1016/j.antiviral.2008.10.009. PMID19028526.
^Wang MZ, Cai BQ, Li LY, Lin JT, Su N, Yu HX, Gao H, Zhao JZ, Liu L (June 2004). “[Efficacy and safety of arbidol in treatment of naturally acquired influenza]”. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. Acta Academiae Medicinae Sinicae. 26 (3): 289–93. PMID15266832.
^Boriskin YS, Leneva IA, Pécheur EI, Polyak SJ (2008). “Arbidol: a broad-spectrum antiviral compound that blocks viral fusion”. Current Medicinal Chemistry. 15 (10): 997–1005. doi:10.2174/092986708784049658. PMID18393857.
^Leneva IA, Burtseva EI, Yatsyshina SB, Fedyakina IT, Kirillova ES, Selkova EP, Osipova E, Maleev VV (February 2016). “Virus susceptibility and clinical effectiveness of anti-influenza drugs during the 2010-2011 influenza season in Russia”. International Journal of Infectious Diseases. 43: 77–84. doi:10.1016/j.ijid.2016.01.001. PMID26775570.
^Shi L, Xiong H, He J, Deng H, Li Q, Zhong Q, Hou W, Cheng L, Xiao H, Yang Z (2007). “Antiviral activity of arbidol against influenza A virus, respiratory syncytial virus, rhinovirus, coxsackie virus and adenovirus in vitro and in vivo”. Archives of Virology. 152 (8): 1447–55. doi:10.1007/s00705-007-0974-5. PMID17497238.
^Glushkov RG, Gus’kova TA, Krylova LIu, Nikolaeva IS (1999). “[Mechanisms of arbidole’s immunomodulating action]”. Vestnik Rossiiskoi Akademii Meditsinskikh Nauk (in Russian) (3): 36–40. PMID10222830.
^Hulseberg CE, Fénéant L, Szymańska-de Wijs KM, Kessler NP, Nelson EA, Shoemaker CJ, Schmaljohn CS, Polyak SJ, White JM. Arbidol and Other Low-Molecular-Weight Drugs That Inhibit Lassa and Ebola Viruses. J Virol. 2019 Apr 3;93(8). pii: e02185-18. doi:10.1128/JVI.02185-18PMID30700611
Umifenovir is an indole-based, hydrophobic, dual-acting direct antiviral/host-targeting agent used for the treatment and prophylaxis of influenza and other respiratory infections.13 It has been in use in Russia for approximately 25 years and in China since 2006. Its invention is credited to a collaboration between Russian scientists from several research institutes 40-50 years ago, and reports of its chemical synthesis date back to 1993.13 Umifenovir’s ability to exert antiviral effects through multiple pathways has resulted in considerable investigation into its use for a variety of enveloped and non-enveloped RNA and DNA viruses, including Flavivirus,2 Zika virus,3 foot-and-mouth disease,4 Lassa virus,6 Ebola virus,6 herpes simplex,8, hepatitis B and C viruses, chikungunya virus, reovirus, Hantaan virus, and coxsackie virus B5.13,9 This dual activity may also confer additional protection against viral resistance, as the development of resistance to umifenovir does not appear to be significant.13
Umifenovir is currently being investigated as a potential treatment and prophylactic agent for COVID-19 caused by SARS-CoV2 infections in combination with both currently available and investigational HIV therapies.1,16,17
Indication
Umifenovir is currently licensed in China and Russia for the prophylaxis and treatment of influenza and other respiratory viral infections.13 It has demonstrated activity against a number of viruses and has been investigated in the treatment of Flavivirus,2 Zika virus,3 foot-and-mouth disease,4 Lassa virus,6 Ebola virus,6 and herpes simplex.8 In addition, it has shown in vitro activity against hepatitis B and C viruses, chikungunya virus, reovirus, Hantaan virus, and coxsackie virus B5.13,9
Umifenovir is currently being investigated as a potential treatment and prophylactic agent for the prevention of COVID-19 caused by SARS-CoV-2 infections.1,16
Pharmacodynamics
Umifenovir exerts its antiviral effects via both direct-acting virucidal activity and by inhibiting one (or several) stage(s) of the viral life cycle.13 Its broad-spectrum of activity covers both enveloped and non-enveloped RNA and DNA viruses. It is relatively well-tolerated and possesses a large therapeutic window – weight-based doses up to 100-fold greater than those used in humans failed to produce any pathological changes in test animals.13
Umifenovir does not appear to result in significant viral resistance. Instances of umifenovir-resistant influenza virus demonstrated a single mutation in the HA2 subunit of influenza hemagglutinin, suggesting resistance is conferred by prevention of umifenovir’s activity related to membrane fusion. The mechanism through which other viruses may become resistant to umifenovir requires further study.13
Mechanism of action
Umifenovir is considered both a direct-acting antiviral (DAA) due to direct virucidal effects and a host-targeting agent (HTA) due to effects on one or multiple stages of viral life cycle (e.g. attachment, internalization), and its broad-spectrum antiviral activity is thought to be due to this dual activity.13 It is a hydrophobic molecule capable of forming aromatic stacking interactions with certain amino acid residues (e.g. tyrosine, tryptophan), which contributes to its ability to directly act against viruses. Antiviral activity may also be due to interactions with aromatic residues within the viral glycoproteins involved in fusion and cellular recognition,5,7 with the plasma membrane to interfere with clathrin-mediated exocytosis and intracellular trafficking,10 or directly with the viral lipid envelope itself (in enveloped viruses).13,12 Interactions at the plasma membrane may also serve to stabilize it and prevent viral entry (e.g. stabilizing influenza hemagglutinin inhibits the fusion step necessary for viral entry).13
Due to umifenovir’s ability to interact with both viral proteins and lipids, it may also interfere with later stages of the viral life cycle. Some virus families, such as Flaviviridae, replicate in a subcellular compartment called the membranous web – this web requires lipid-protein interactions that may be hindered by umifenovir. Similarly, viral assembly of hepatitis C viruses is contingent upon the assembly of lipoproteins, presenting another potential target.13
Absorption
Umifenovir is rapidly absorbed following oral administration, with an estimated Tmax between 0.65-1.8 hours.14,15,13 The Cmax has been estimated as 415 – 467 ng/mL and appears to increase linearly with dose,14,15 and the AUC0-inf following oral administration has been estimated to be approximately 2200 ng/mL/h.14,15
Volume of distribution
Data regarding the volume of distribution of umifenovir are currently unavailable.
Protein binding
Data regarding protein-binding of umifenovir are currently unavailable.
Metabolism
Umifenovir is highly metabolized in the body, primarily in hepatic and intestinal microsomess, with approximately 33 metabolites having been observed in human plasma, urine, and feces.14 The principal phase I metabolic pathways include sulfoxidation, N-demethylation, and hydroxylation, followed by phase II sulfate and glucuronide conjugation. In the urine, the major metabolites were sulfate and glucuronide conjugates, while the major species in the feces was unchanged parent drug (~40%) and the M10 metabolite (~3.0%). In the plasma, the principal metabolites are M6-1, M5, and M8 – of these, M6-1 appears of most importance given its high plasma exposure and long elimination half-life (~25h), making it a potentially important player in the safety and efficacy of umifenovir.14
Enzymes involved in the metabolism of umifenovir include members of the cytochrome P450 family (primarily CYP3A4), flavin-containing monooxygenase (FMO) family, and UDP-glucuronosyltransferase (UGT) family (specifically UGT1A9 and UGT2B7).14,11
Lu H: Drug treatment options for the 2019-new coronavirus (2019-nCoV). Biosci Trends. 2020 Jan 28. doi: 10.5582/bst.2020.01020. [PubMed:31996494]
Haviernik J, Stefanik M, Fojtikova M, Kali S, Tordo N, Rudolf I, Hubalek Z, Eyer L, Ruzek D: Arbidol (Umifenovir): A Broad-Spectrum Antiviral Drug That Inhibits Medically Important Arthropod-Borne Flaviviruses. Viruses. 2018 Apr 10;10(4). pii: v10040184. doi: 10.3390/v10040184. [PubMed:29642580]
Fink SL, Vojtech L, Wagoner J, Slivinski NSJ, Jackson KJ, Wang R, Khadka S, Luthra P, Basler CF, Polyak SJ: The Antiviral Drug Arbidol Inhibits Zika Virus. Sci Rep. 2018 Jun 12;8(1):8989. doi: 10.1038/s41598-018-27224-4. [PubMed:29895962]
Herod MR, Adeyemi OO, Ward J, Bentley K, Harris M, Stonehouse NJ, Polyak SJ: The broad-spectrum antiviral drug arbidol inhibits foot-and-mouth disease virus genome replication. J Gen Virol. 2019 Sep;100(9):1293-1302. doi: 10.1099/jgv.0.001283. Epub 2019 Jun 4. [PubMed:31162013]
Kadam RU, Wilson IA: Structural basis of influenza virus fusion inhibition by the antiviral drug Arbidol. Proc Natl Acad Sci U S A. 2017 Jan 10;114(2):206-214. doi: 10.1073/pnas.1617020114. Epub 2016 Dec 21. [PubMed:28003465]
Hulseberg CE, Feneant L, Szymanska-de Wijs KM, Kessler NP, Nelson EA, Shoemaker CJ, Schmaljohn CS, Polyak SJ, White JM: Arbidol and Other Low-Molecular-Weight Drugs That Inhibit Lassa and Ebola Viruses. J Virol. 2019 Apr 3;93(8). pii: JVI.02185-18. doi: 10.1128/JVI.02185-18. Print 2019 Apr 15. [PubMed:30700611]
Zeng LY, Yang J, Liu S: Investigational hemagglutinin-targeted influenza virus inhibitors. Expert Opin Investig Drugs. 2017 Jan;26(1):63-73. doi: 10.1080/13543784.2017.1269170. Epub 2016 Dec 14. [PubMed:27918208]
Li MK, Liu YY, Wei F, Shen MX, Zhong Y, Li S, Chen LJ, Ma N, Liu BY, Mao YD, Li N, Hou W, Xiong HR, Yang ZQ: Antiviral activity of arbidol hydrochloride against herpes simplex virus I in vitro and in vivo. Int J Antimicrob Agents. 2018 Jan;51(1):98-106. doi: 10.1016/j.ijantimicag.2017.09.001. Epub 2017 Sep 7. [PubMed:28890393]
Pecheur EI, Borisevich V, Halfmann P, Morrey JD, Smee DF, Prichard M, Mire CE, Kawaoka Y, Geisbert TW, Polyak SJ: The Synthetic Antiviral Drug Arbidol Inhibits Globally Prevalent Pathogenic Viruses. J Virol. 2016 Jan 6;90(6):3086-92. doi: 10.1128/JVI.02077-15. [PubMed:26739045]
Blaising J, Levy PL, Polyak SJ, Stanifer M, Boulant S, Pecheur EI: Arbidol inhibits viral entry by interfering with clathrin-dependent trafficking. Antiviral Res. 2013 Oct;100(1):215-9. doi: 10.1016/j.antiviral.2013.08.008. Epub 2013 Aug 25. [PubMed:23981392]
Song JH, Fang ZZ, Zhu LL, Cao YF, Hu CM, Ge GB, Zhao DW: Glucuronidation of the broad-spectrum antiviral drug arbidol by UGT isoforms. J Pharm Pharmacol. 2013 Apr;65(4):521-7. doi: 10.1111/jphp.12014. Epub 2012 Dec 24. [PubMed:23488780]
Teissier E, Zandomeneghi G, Loquet A, Lavillette D, Lavergne JP, Montserret R, Cosset FL, Bockmann A, Meier BH, Penin F, Pecheur EI: Mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol. PLoS One. 2011 Jan 25;6(1):e15874. doi: 10.1371/journal.pone.0015874. [PubMed:21283579]
Blaising J, Polyak SJ, Pecheur EI: Arbidol as a broad-spectrum antiviral: an update. Antiviral Res. 2014 Jul;107:84-94. doi: 10.1016/j.antiviral.2014.04.006. Epub 2014 Apr 24. [PubMed:24769245]
Deng P, Zhong D, Yu K, Zhang Y, Wang T, Chen X: Pharmacokinetics, metabolism, and excretion of the antiviral drug arbidol in humans. Antimicrob Agents Chemother. 2013 Apr;57(4):1743-55. doi: 10.1128/AAC.02282-12. Epub 2013 Jan 28. [PubMed:23357765]
Liu MY, Wang S, Yao WF, Wu HZ, Meng SN, Wei MJ: Pharmacokinetic properties and bioequivalence of two formulations of arbidol: an open-label, single-dose, randomized-sequence, two-period crossover study in healthy Chinese male volunteers. Clin Ther. 2009 Apr;31(4):784-92. doi: 10.1016/j.clinthera.2009.04.016. [PubMed:19446151]
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Nature Biotechnology: Coronavirus puts drug repurposing on the fast track [Link]
The drug substance is a white to light yellow powder. It is sparingly soluble in acetonitrile and in methanol, and slightly soluble in water and in ethanol (99.5). It is slightly soluble at pH 2.0 to 5.5 and sparingly soluble at pH 5.5 to 6.1. The drug substance is not hygroscopic at 25°C/51% to 93%RH. The melting point is 187°C to 193°C, and the dissociation constant (pKa) is 5.1 due to the hydroxyl group of favipiravir. Measurement results on the partition ratio of favipiravir in water/octanol at 25°C indicate that favipiravir tends to be distributed in the 1-octanol phase at pH 2 to 4 and in the water phase at pH 5 to 13.
Any batch manufactured by the current manufacturing process is in Form A. The stability study does not show any change in crystal form over time; and a change from Form A to Form B is unlikely.
T-705 is an RNA-directed RNA polymerase (NS5B) inhibitor which has been filed for approval in Japan for the oral treatment of influenza A (including avian and H1N1 infections) and for the treatment of influenza B infection.
The compound is a unique viral RNA polymerase inhibitor, acting on viral genetic copying to prevent its reproduction, discovered by Toyama Chemical. In 2005, Utah State University carried out various studies under its contract with the National Institute of Allergy and Infectious Diseases (NIAID) and demonstrated that T-705 has exceptionally potent activity in mouse infection models of H5N1 avian influenza.
T-705 (Favipiravir) is an antiviral pyrazinecarboxamide-based, inhibitor of of the influenza virus with an EC90 of 1.3 to 7.7 uM (influenza A, H5N1). EC90 ranges for other influenza A subtypes are 0.19-1.3 uM, 0.063-1.9 uM, and 0.5-3.1 uM for H1N1, H2N2, and H3N2, respectively. T-705 also exhibits activity against type B and C viruses, with EC90s of 0.25-0.57 uM and 0.19-0.36 uM, respectively. (1) Additionally, T-705 has broad activity against arenavirus, bunyavirus, foot-and-mouth disease virus, and West Nile virus with EC50s ranging from 5 to 300 uM.
Studies show that T-705 ribofuranosyl triphosphate is the active form of T-705 and acts like purines or purine nucleosides in cells and does not inhibit DNA synthesis
In 2012, MediVector was awarded a contract from the U.S. Department of Defense’s (DOD) Joint Project Manager Transformational Medical Technologies (JPM-TMT) to further develop T-705 (favipiravir), a broad-spectrum therapeutic against multiple influenza viruses.
Several novel anti-influenza compounds are in various phases of clinical development. One of these, T-705 (favipiravir), has a mechanism of action that is not fully understood but is suggested to target influenza virus RNA-dependent RNA polymerase. We investigated the mechanism of T-705 activity against influenza A (H1N1) viruses by applying selective drug pressure over multiple sequential passages in MDCK cells. We found that T-705 treatment did not select specific mutations in potential target proteins, including PB1, PB2, PA, and NP. Phenotypic assays based on cell viability confirmed that no T-705-resistant variants were selected. In the presence of T-705, titers of infectious virus decreased significantly (P < 0.0001) during serial passage in MDCK cells inoculated with seasonal influenza A (H1N1) viruses at a low multiplicity of infection (MOI; 0.0001 PFU/cell) or with 2009 pandemic H1N1 viruses at a high MOI (10 PFU/cell). There was no corresponding decrease in the number of viral RNA copies; therefore, specific virus infectivity (the ratio of infectious virus yield to viral RNA copy number) was reduced. Sequence analysis showed enrichment of G→A and C→T transversion mutations, increased mutation frequency, and a shift of the nucleotide profiles of individual NP gene clones under drug selection pressure. Our results demonstrate that T-705 induces a high rate of mutation that generates a nonviable viral phenotype and that lethal mutagenesis is a key antiviral mechanism of T-705. Our findings also explain the broad spectrum of activity of T-705 against viruses of multiple families.
In February 2020 Favipiravir was being studied in China for experimental treatment of the emergent COVID-19 (novel coronavirus)disease.[7][8] On March 17 Chinese officials suggested the drug had been effective in treating COVID in Wuhan and Shenzhen.[9][10]
Discovered by Toyama Chemical Co., Ltd. in Japan, favipiravir is a modified pyrazine analog that was initially approved for therapeutic use in resistant cases of influenza.7,9 The antiviral targets RNA-dependent RNA polymerase (RdRp) enzymes, which are necessary for the transcription and replication of viral genomes.7,12,13
Not only does favipiravir inhibit replication of influenza A and B, but the drug shows promise in the treatment of influenza strains that are resistant to neuramidase inhibitors, as well as avian influenza.9,19 Favipiravir has been investigated for the treatment of life-threatening pathogens such as Ebola virus, Lassa virus, and now COVID-19.10,14,15
Mechanism of action
The mechanism of its actions is thought to be related to the selective inhibition of viral RNA-dependent RNA polymerase.[11] Other research suggests that favipiravir induces lethal RNA transversion mutations, producing a nonviable viral phenotype.[12] Favipiravir is a prodrug that is metabolized to its active form, favipiravir-ribofuranosyl-5′-triphosphate (favipiravir-RTP), available in both oral and intravenous formulations.[13][14] Human hypoxanthine guanine phosphoribosyltransferase (HGPRT) is believed to play a key role in this activation process.[15] Favipiravir does not inhibit RNA or DNA synthesis in mammalian cells and is not toxic to them.[1] In 2014, favipiravir was approved in Japan for stockpiling against influenza pandemics.[16] However, favipiravir has not been shown to be effective in primary human airway cells, casting doubt on its efficacy in influenza treatment.[17]
Approval status
In 2014, Japan approved Favipiravir for treating viral strains unresponsive to current antivirals.[18]
Some research has been done suggesting that in mouse models Favipiravir may have efficacy against Ebola. Its efficacy against Ebola in humans is unproven.[20][21][22] During the 2014 West Africa Ebola virus outbreak, it was reported that a French nurse who contracted Ebola while volunteering for MSF in Liberia recovered after receiving a course of favipiravir.[23] A clinical trial investigating the use of favipiravir against Ebola virus disease was started in Guéckédou, Guinea, during December 2014.[24] Preliminary results showed a decrease in mortality rate in patients with low-to-moderate levels of Ebola virus in the blood, but no effect on patients with high levels of the virus, a group at a higher risk of death.[25] The trial design has been criticised by Scott Hammer and others for using only historical controls.[26] The results of this clinical trial were presented in February 2016 at the annual Conference on Retroviruses and Opportunistic Infections (CROI) by Daouda Sissoko[27] and published on March 1, 2016 in PLOS Medicine.[28]
SARS-CoV-2 virus disease
In March 2020, Chinese officials suggested Favipiravir may be effective in treating COVID-19.[29]
Drug Discoveries & Therapeutics. 2014; 8(3):117-120.
As a RNA polymerase inhibitor, 6-fluoro-3-hydroxypyrazine-2-carboxamide commercially named favipiravir has been proved to have potent inhibitory activity against RNA viruses in vitro and in vivo. A four-step synthesis of the compound is described in this article, amidation, nitrification, reduction and fluorination with an overall yield of about 8%. In addition, we reported the crystal structure of the title compound. The molecule is almost planar and the intramolecular O−H•••O hydrogen bond makes a 6-member ring. In the crystal, molecules are packing governed by both hydrogen bonds and stacking interactions.
2.2.1. Preparation of 3-hydroxypyrazine-2-carboxamide To a suspension of 3-hydroxypyrazine-2-carboxylic acid (1.4 g, 10 mmol) in 150 mL MeOH, SOCl2 was added dropwise at 40°C with magnetic stirring for 6 h resulting in a bright yellow solution. The reaction was then concentrated to dryness. The residue was dissolved in 50 mL 25% aqueous ammonia and stirred overnight to get a suspension. The precipitate was collected and dried. The solid yellow-brown crude product was recrystallization with 50 mL water to get the product as pale yellow crystals (1.1 g, 78%). mp = 263-265°C. 1 H-NMR (300 MHz, DMSO): δ 13.34 (brs, 1H, OH), 8.69 (s, 1H, pyrazine H), 7.93-8.11 (m, 3H, pyrazine H, CONH2). HRMS (ESI): m/z [M + H]+ calcd for C5H6N3O2 + : 140.0460; found: 140.0457.
2.2.2. Preparation of 3-hydroxy-6-nitropyrazine-2- carboxamide In the solution of 3-hydroxypyrazine-2-carboxamide (1.0 g, 7 mmol) in 6 mL concentrate sulfuric acid under ice-cooling, potassium nitrate (1.4 g, 14 mmol) was added. After stirring at 40°C for 4 h, the reaction mixture was poured into 60 mL water. The product was collected by fi ltration as yellow solid (0.62 g, 48%). mp = 250-252°C. 1 H-NMR (600 MHz, DMSO): δ 12.00- 15.00 (br, 1H, OH), 8.97 (s, 1H, pyrazine H), 8.32 (s, 1H, CONH2), 8.06 (s, 1H, CONH2). 13C-NMR (75 MHz, DMSO): δ 163.12, 156.49, 142.47, 138.20, 133.81. HRMS (ESI): m/z [M + H]+ calcd for C5H5N4O4 + : 185.0311; found: 185.0304.
2.2.3. Preparation of 6-amino-3-hydroxypyrazine-2- carboxamide 3-Hydroxy-6-nitropyrazine-2-carboxamide (0.6 g, 3.3 mmol) and a catalytic amount of raney nickel were suspended in MeOH, then hydrazine hydrate was added dropwise. The resulting solution was refl uxed 2 h, cooled, filtered with diatomite, and then MeOH is evaporated in vacuo to get the crude product as dark brown solid without further purification (0.4 g, 77%). HRMS (ESI): m/z [M + H]+ calcd for C5H7N4O2 + : 155.0569; found:155.0509.
2.2.4. Preparation of 6-fluoro-3-hydroxypyrazine-2- carboxamide To a solution of 6-amino-3-hydroxypyrazine-2- carboxamide (0.4 g, 2.6 mmol) in 3 mL 70% HFpyridine aqueous at -20°C under nitrogen atmosphere, sodium nitrate (0.35 g, 5.2 mmol) was added. After stirring 20 min, the solution was warmed to room temperature for another one hour. Then 20 mL ethyl acetate/water (1:1) were added, after separation of the upper layer, the aqueous phase is extracted with four 20 mL portions of ethyl acetate. The combined extracts are dried with anhydrous magnesium sulfate and concentrated to dryness to get crude product as oil. The crude product was purified by chromatography column as white solid (0.12 g, 30%). mp = 178-180°C. 1 H-NMR (600 MHz, DMSO): δ 12.34 (brs, 1H, OH), 8.31 (d, 1H, pyrazine H, J = 8.0 Hz), 7.44 (s, 1H, CONH2), 5.92 (s, 1H, CONH2). 13C-NMR (75 MHz, DMSO): δ 168.66, 159.69, 153.98, 150.76, 135.68. HRMS (ESI): m/z [M + H]+ calcd for C5H5FN3O2 + : 158.0366; found: 158.0360.
SEE
Chemical Papers (2019), 73(5), 1043-1051.
PAPER
Medicinal chemistry (Shariqah (United Arab Emirates)) (2018), 14(6), 595-603
Furuta, Y.; Takahashi, K.; Shiraki, K.; Sakamoto, K.; Smee, D. F.; Barnard, D. L.; Gowen, B. B.; Julander, J. G.; Morrey, J. D. (2009). “T-705 (favipiravir) and related compounds: Novel broad-spectrum inhibitors of RNA viral infections”. Antiviral Research82 (3): 95–102. doi:10.1016/j.antiviral.2009.02.198. PMID19428599. edit
WO 2000010569
WO 2008099874
WO 201009504
WO 2010104170
WO 2012063931
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Influenza virus is a central virus of the cold syndrome, which has attacked human being periodically to cause many deaths amounting to tens millions. Although the number of deaths shows a tendency of decrease in the recent years owing to the improvement in hygienic and nutritive conditions, the prevalence of influenza is repeated every year, and it is apprehended that a new virus may appear to cause a wider prevalence.
For prevention of influenza virus, vaccine is used widely, in addition to which low molecular weight substances such as Amantadine and Ribavirin are also used
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Synthesis of Favipiravir
ZHANG Tao1, KONG Lingjin1, LI Zongtao1,YUAN Hongyu1, XU Wenfang2*
(1. Shandong Qidu PharmaceuticalCo., Ltd., Linzi 255400; 2. School of Pharmacy, Shandong University, Jinan250012) ABSTRACT: Favipiravir was synthesized from3-amino-2-pyrazinecarboxylic acid by esterification, bromination with NBS,diazotization and amination to give 6-bromo-3-hydroxypyrazine-2-carboxamide,which was subjected to chlorination with POCl3, fluorination with KF, andhydrolysis with an overall yield of about 22％.
To a 17.5 ml N,N-dimethylformamide solution of 5.0 g of 3,6-difluoro-2-pyrazinecarbonitrile, a 3.8 ml water solution of 7.83 g of potassium acetate was added dropwise at 25 to 35° C., and the solution was stirred at the same temperature for 2 hours. 0.38 ml of ammonia water was added to the reaction mixture, and then 15 ml of water and 0.38 g of active carbon were added. The insolubles were filtered off and the filter cake was washed with 11 ml of water. The filtrate and the washing were joined, the pH of this solution was adjusted to 9.4 with ammonia water, and 15 ml of acetone and 7.5 ml of toluene were added. Then 7.71 g of dicyclohexylamine was added dropwise and the solution was stirred at 20 to 30° C. for 45 minutes. Then 15 ml of water was added dropwise, the solution was cooled to 10° C., and the precipitate was filtered and collected to give 9.44 g of dicyclohexylamine salt of 6-fluoro-3-hydroxy-2-pyradinecarbonitrile as a lightly yellowish white solid product. 1H-NMR (DMSO-d6) δ values: 1.00-1.36 (10H, m), 1.56-1.67 (2H, m), 1.67-1.81 (4H, m), 1.91-2.07 (4H, m), 3.01-3.18 (2H, m), 8.03-8.06 (1H, m), 8.18-8.89 (1H, broad)
Example 1-2
4.11 ml of acetic acid was added at 5 to 15° C. to a 17.5 ml N,N-dimethylformamide solution of 5.0 g of 3,6-difluoro-2-pyrazinecarbonitrile. Then 7.27 g of triethylamine was added dropwise and the solution was stirred for 2 hours. 3.8 ml of water and 0.38 ml of ammonia water were added to the reaction mixture, and then 15 ml of water and 0.38 g of active carbon were added. The insolubles were filtered off and the filter cake was washed with 11 ml of water. The filtrate and the washing were joined, the pH of the joined solution was adjusted to 9.2 with ammonia water, and 15 ml of acetone and 7.5 ml of toluene were added to the solution, followed by dropwise addition of 7.71 g of dicyclohexylamine. Then 15 ml of water was added dropwise, the solution was cooled to 5° C., and the precipitate was filtered and collected to give 9.68 g of dicyclohexylamine salt of 6-fluoro-3-hydroxy-2-pyrazinecarbonitrile as a slightly yellowish white solid product.
Examples 2 to 5
The compounds shown in Table 1 were obtained in the same way as in Example 1-1.
TABLE 1
Example No.
Organic amine
Example No.
Organic amine
2
Dipropylamine
4
Dibenzylamine
3
Dibutylamine
5
N-benzylmethylamine
Dipropylamine salt of 6-fluoro-3-hydroxy-2-pyrazinecarbonitrile 1H-NMR (DMSO-d6) 6 values: 0.39 (6H, t, J=7.5 Hz), 1.10 (4H, sex, J=7.5 Hz), 2.30-2.38 (4H, m), 7.54 (1H, d, J=8.3 Hz)
Dibutylamine salt of 6-fluoro-3-hydroxy-2-pyrazinecarbonitrile 1H-NMR (DMSO-d6) 6 values: 0.36 (6H, t, J=7.3 Hz), 0.81 (4H, sex, J=7.3 Hz), 0.99-1.10 (4H, m), 2.32-2.41 (4H, m), 7.53 (1H, d, J=8.3 Hz)
Dibenzylamine salt of 6-fluoro-3-hydroxy-2-pyrazinecarbonitrile 1H-NMR (DMSO-d6) δ values: 4.17 (4H, s), 7.34-7.56 (10H, m), 8.07 (1H, d, J=8.3 Hz)
N-benzylmethylamine salt of 6-fluoro-3-hydroxy-2-pyrazinecarbonitrile 1H-NMR (DMSO-d6) δ values: 2.57 (3H, s), 4.14 (2H, s), 7.37-7.53 (5H, m), 8.02-8.08 (1H, m)
Preparation Example 1
300 ml of toluene was added to a 600 ml water solution of 37.5 g of sodium hydroxide. Then 150 g of dicyclohexylamine salt of 6-fluoro-3-hydroxy-2-pyrazinecarbonitrile was added at 15 to 25° C. and the solution was stirred at the same temperature for 30 minutes. The water layer was separated and washed with toluene, and then 150 ml of water was added, followed by dropwise addition of 106 g of a 30% hydrogen peroxide solution at 15 to 30° C. and one-hour stirring at 20 to 30° C. Then 39 ml of hydrochloric acid was added, the seed crystals were added at 40 to 50° C., and 39 ml of hydrochloric acid was further added dropwise at the same temperature. The solution was cooled to 10° C. the precipitate was filtered and collected to give 65.6 g of 6-fluoro-3-hydroxy-2-pyrazinecarboxamide as a slightly yellowish white solid. 1H-NMR (DMSO-d6) δ values: 8.50 (1H, s), 8.51 (1H, d, J=7.8 Hz), 8.75 (1H, s), 13.41 (1H, s)
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jan 2014
Investigational flu treatment drug has broad-spectrum potential to fight multiple viruses
First patient enrolled in the North American Phase 3 clinical trials for investigational flu treatment drug
BioDefense Therapeutics (BD Tx)—a Joint Product Management office within the U.S. Department of Defense (DoD)—announced the first patient enrolled in the North American Phase 3 clinical trials for favipiravir (T-705a). The drug is an investigational flu treatment candidate with broad-spectrum potential being developed by BD Tx through a contract with Boston-based MediVector, Inc.
Favipiravir is a novel, antiviral compound that works differently than anti-flu drugs currently on the market. The novelty lies in the drug’s selective disruption of the viralRNA replication and transcription process within the infected cell to stop the infection cycle.
“Favipiravir has proven safe and well tolerated in previous studies,” said LTC Eric G. Midboe, Joint Product Manager for BD Tx. “This first patient signifies the start of an important phase in favipiravir’s path to U.S. Food and Drug Administration (FDA) approval for flu and lays the groundwork for future testing against other viruses of interest to the DoD.”
In providing therapeutic solutions to counter traditional, emerging, and engineered biological threats, BD Tx chose favipiravir not only because of its potential effectiveness against flu viruses, but also because of its demonstrated broad-spectrum potential against multiple viruses. In addition to testing favipiravir in the ongoing influenzaprogram, BD Tx is testing the drug’s efficacy against the Ebola virus and other viruses considered threats to service members. In laboratory testing, favipiravir was found to be effective against a wide variety of RNA viruses in infected cells and animals.
“FDA-approved, broad-spectrum therapeutics offer the fastest way to respond to dangerous and potentially lethal viruses,” said Dr. Tyler Bennett, Assistant Product Manager for BD Tx.
MediVector is overseeing the clinical trials required by the FDA to obtain drug licensure. The process requires safety data from at least 1,500 patients treated for flu at the dose and duration proposed for marketing of the drug. Currently, 150 trial sites are planned throughout the U.S.
Malpani Y, Achary R, Kim SY, Jeong HC, Kim P, Han SB, Kim M, Lee CK, Kim JN, Jung YS.
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^Li G, De Clercq E. Therapeutic options for the 2019 novel coronavirus (2019-nCoV). Nature Reviews Drug Discovery 2020 Feb doi:10.1038/d41573-020-00016-0
^Gatherer D (August 2014). “The 2014 Ebola virus disease outbreak in West Africa”. The Journal of General Virology. 95 (Pt 8): 1619–24. doi:10.1099/vir.0.067199-0. PMID24795448.
^Oestereich L, Lüdtke A, Wurr S, Rieger T, Muñoz-Fontela C, Günther S (May 2014). “Successful treatment of advanced Ebola virus infection with T-705 (favipiravir) in a small animal model”. Antiviral Research. 105: 17–21. doi:10.1016/j.antiviral.2014.02.014. PMID24583123.
^Smither SJ, Eastaugh LS, Steward JA, Nelson M, Lenk RP, Lever MS (April 2014). “Post-exposure efficacy of oral T-705 (Favipiravir) against inhalational Ebola virus infection in a mouse model”. Antiviral Research. 104: 153–5. doi:10.1016/j.antiviral.2014.01.012. PMID24462697.
Beigel J, Bray M: Current and future antiviral therapy of severe seasonal and avian influenza. Antiviral Res. 2008 Apr;78(1):91-102. doi: 10.1016/j.antiviral.2008.01.003. Epub 2008 Feb 4. [PubMed:18328578]
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Gowen BB, Wong MH, Jung KH, Sanders AB, Mendenhall M, Bailey KW, Furuta Y, Sidwell RW: In vitro and in vivo activities of T-705 against arenavirus and bunyavirus infections. Antimicrob Agents Chemother. 2007 Sep;51(9):3168-76. Epub 2007 Jul 2. [PubMed:17606691]
Sidwell RW, Barnard DL, Day CW, Smee DF, Bailey KW, Wong MH, Morrey JD, Furuta Y: Efficacy of orally administered T-705 on lethal avian influenza A (H5N1) virus infections in mice. Antimicrob Agents Chemother. 2007 Mar;51(3):845-51. Epub 2006 Dec 28. [PubMed:17194832]
Furuta Y, Takahashi K, Kuno-Maekawa M, Sangawa H, Uehara S, Kozaki K, Nomura N, Egawa H, Shiraki K: Mechanism of action of T-705 against influenza virus. Antimicrob Agents Chemother. 2005 Mar;49(3):981-6. [PubMed:15728892]
Furuta Y, Takahashi K, Fukuda Y, Kuno M, Kamiyama T, Kozaki K, Nomura N, Egawa H, Minami S, Watanabe Y, Narita H, Shiraki K: In vitro and in vivo activities of anti-influenza virus compound T-705. Antimicrob Agents Chemother. 2002 Apr;46(4):977-81. [PubMed:11897578]
Furuta Y, Komeno T, Nakamura T: Favipiravir (T-705), a broad spectrum inhibitor of viral RNA polymerase. Proc Jpn Acad Ser B Phys Biol Sci. 2017;93(7):449-463. doi: 10.2183/pjab.93.027. [PubMed:28769016]
Venkataraman S, Prasad BVLS, Selvarajan R: RNA Dependent RNA Polymerases: Insights from Structure, Function and Evolution. Viruses. 2018 Feb 10;10(2). pii: v10020076. doi: 10.3390/v10020076. [PubMed:29439438]
Madelain V, Nguyen TH, Olivo A, de Lamballerie X, Guedj J, Taburet AM, Mentre F: Ebola Virus Infection: Review of the Pharmacokinetic and Pharmacodynamic Properties of Drugs Considered for Testing in Human Efficacy Trials. Clin Pharmacokinet. 2016 Aug;55(8):907-23. doi: 10.1007/s40262-015-0364-1. [PubMed:26798032]
Nguyen TH, Guedj J, Anglaret X, Laouenan C, Madelain V, Taburet AM, Baize S, Sissoko D, Pastorino B, Rodallec A, Piorkowski G, Carazo S, Conde MN, Gala JL, Bore JA, Carbonnelle C, Jacquot F, Raoul H, Malvy D, de Lamballerie X, Mentre F: Favipiravir pharmacokinetics in Ebola-Infected patients of the JIKI trial reveals concentrations lower than targeted. PLoS Negl Trop Dis. 2017 Feb 23;11(2):e0005389. doi: 10.1371/journal.pntd.0005389. eCollection 2017 Feb. [PubMed:28231247]
de Farias ST, Dos Santos Junior AP, Rego TG, Jose MV: Origin and Evolution of RNA-Dependent RNA Polymerase. Front Genet. 2017 Sep 20;8:125. doi: 10.3389/fgene.2017.00125. eCollection 2017. [PubMed:28979293]
Shu B, Gong P: Structural basis of viral RNA-dependent RNA polymerase catalysis and translocation. Proc Natl Acad Sci U S A. 2016 Jul 12;113(28):E4005-14. doi: 10.1073/pnas.1602591113. Epub 2016 Jun 23. [PubMed:27339134]
Nagata T, Lefor AK, Hasegawa M, Ishii M: Favipiravir: a new medication for the Ebola virus disease pandemic. Disaster Med Public Health Prep. 2015 Feb;9(1):79-81. doi: 10.1017/dmp.2014.151. Epub 2014 Dec 29. [PubMed:25544306]
Rosenke K, Feldmann H, Westover JB, Hanley PW, Martellaro C, Feldmann F, Saturday G, Lovaglio J, Scott DP, Furuta Y, Komeno T, Gowen BB, Safronetz D: Use of Favipiravir to Treat Lassa Virus Infection in Macaques. Emerg Infect Dis. 2018 Sep;24(9):1696-1699. doi: 10.3201/eid2409.180233. Epub 2018 Sep 17. [PubMed:29882740]
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Launched – 1985, in Japan by Ono for the oral treatment of postoperative reflux esophagitis and chronic pancreatitis.
Camostat mesilate is a synthetic serine protease inhibitor that has been launched in Japan by Ono for the oral treatment of postoperative reflux esophagitis and chronic pancreatitis. It has been demonstrated that the drug has the ability to inhibit proteases such as trypsin, kallikrein, thrombin, plasmin, and C1 esterase, and that it decreases inflammation by directly suppressing the activity of monocytes and pancreatic stellate cells (PSCs).
In 2011, orphan drug designation was received in the U.S. by Stason Pharmaceuticals for the treatment of chronic pancreatitis. In 2017, Kangen Pharmaceuticals acquired KC Specialty Therapeutics (formerly a wholly-owned subsidiary of Stason Pharmaceuticals).
Camostat (INN; development code FOY-305) is a serine protease inhibitor. Serine protease enzymes have a variety of functions in the body, and so camostat has a diverse range of uses. It is used in the treatment of some forms of cancer and is also effective against some viral infections, as well as inhibiting fibrosis in liver or kidney disease or pancreatitis.[1][2][3][4][5] It is approved in Japan for the treatment of pancreatitis.[6][7]
An in vitro study shows that Camostat reduces significantly the infection of Calu-3 lung cells by SARS-CoV-2, the virus responsible for COVID-19.[8]
SYN
DE 2548886; FR 2289181; GB 1472700; JP 76054530; US 4021472
The reaction of p-hydrophenylacetic acid (I) with N,N-dimethylbromoacetamide (II) by means of triethylamine in reftuxing acetonitrile gives N,N-dimethylcarbamoylmethyl-p-hydroxyphenylacetate (III), which is then condensed with p-guanidinobenzoyl chloride (IV) [obtained from the corresponding acid p-guanidinobenzoic acid (V) and thionyl chloride] in pyridine.
By reaction of N,N-dimethylcarbamoylmethyl-p-(p-aminobenzoyloxy)phenylacetate (VI) with cyanamide (VII).
Camostat mesilate, chemical name is 4-(4-guanidine radicals benzoyloxy group) toluylic acid-N, N-dimethyl carbamoyl methyl esters mesylate, be the non-peptide proteinoid enzyme inhibitors of Japanese little Ye medicine Co., Ltd. exploitation, first in January, 1985 go on the market with trade(brand)name Foipan in Japan.Pharmacological evaluation shows: camostat mesilate has very strong restraining effect to trypsinase, kallikrein, Tryptase, zymoplasm, C1 esterase, oral rear kassinin kinin generation system, fibrinolytic system, blood coagulation system and the complement system acting on rapidly body, suppress the exception of the enzymic activity of these systems hyperfunction, thus control the symptom of chronic pancreatitis, alleviating pain, reduce amylase value, the clinical alleviation for chronic pancreatitis acute symptom.In addition, this product is also used for the treatment of diffusivity blood vessel blood coagulation disease.Pharmacological evaluation also finds, camostat mesilate also has the effects such as anticancer, antiviral, and effectively can reduce proteinuria, and play the effect of preliminary conditioning, further research is still underway.Current this product not yet in Discussion on Chinese Listed, also without the report succeeded in developing.
A preparation method for camostat mesilate, comprises the steps:
(1), by 160g methylene dichloride DCM join stirring in reaction vessel, cooling, be cooled to start when 0–10 DEG C to drip 51g 50% dimethylamine agueous solution, drip 30g chloroacetyl chloride simultaneously; Drip process control temp 5–10 DEG C, system pH controls 4-7, at 5–10 DEG C, react 1h after dripping off, reaction process pH controls 5-7, and reaction terminates rear standing 20min, separatory, water layer is with 54g dichloromethane extraction, and organic layer is concentrating under reduced pressure below 80 DEG C, obtains 3-pyrrolidone hydrochloride, crude, 3-pyrrolidone hydrochloride, crude carries out underpressure distillation within 130 DEG C, obtains 3-pyrrolidone hydrochloride distillation product; Output is 31g;
(2), the 3-pyrrolidone hydrochloride of 30.6g, 9g triethylamine TEA, 0.4g sodium bisulfite and 40g p-hydroxyphenylaceticacid p-hydroxyphenylaceticacid drop in order in reaction vessel and carry out stirring at low speed, and then drip the triethylamine of 17.6g, dropping temperature 40-95 DEG C, drip off rear maintenance 80-95 DEG C reaction 3h, after reaction terminates, add aqueous solution of sodium bisulfite (0.05gNaHSO3+90gH2O), add and start more than temperature 70 C, add finishing temperature more than 48 DEG C, after adding, cool, crystal seed is added when 40 DEG C, keep cooling temperature 0-5 DEG C, crystallization 2h, filter after crystallization, filter cake 100g purified water is washed, camostat mesilate crude product is obtained after draining, camostat mesilate crude product, 50mL ethyl acetate are joined heating for dissolving in aqueous solution of sodium bisulfite (0.2g NaHSO3+20g H2O), after having dissolved, cooling crystallization, keep recrystallization temperature 0-5 DEG C, crystallization time 1h, suction filtration after crystallization, filter cake, with 10mL water washing, washs with 20mL ethyl acetate after draining again, again at 60 ± 3 DEG C of drying under reduced pressure 2h after draining, obtain camostat mesilate refined silk, output is about 47g,
(3), the camostat mesilate refined silk of 47g is joined heating for dissolving in 30mL acetonitrile, after dissolving terminates, cooling temperature is to 0-5 DEG C, crystallization 1h, after crystallization terminates, suction filtration, filter cake with 17mL acetonitrile wash, drain, drying under reduced pressure 2h at 60 ± 3 DEG C, obtain camostat mesilate product, output is about 45g.
German researchers identify potential drug for Covid-19
Scientists at the German Primate Center – Leibniz Institute for Primate Research have found that an existing drug may help treat Covid-19.
As well as Charité – Universitätsmedizin Berlin, the scientists worked with researchers at the University of Veterinary Medicine Hannover Foundation, the BG-Unfallklinik Murnau, the LMU Munich, the Robert Koch Institute and the German Center for Infection Research.
The research aimed to understand the entry of the novel coronavirus, SARS-CoV-2, into host cells, as well as determine approaches to block the process.
Research findings showed that SARS-CoV-2 requires cellular protein TMPRSS2 to enter hosts’ lung cells.
German Primate Center Infection Biology Unit head Stefan Pöhlmann said: “Our results show that SARS-CoV-2 requires the protease TMPRSS2, which is present in the human body, to enter cells. This protease is a potential target for therapeutic intervention.”
Summary: A clinically proven drug known to block an enzyme essential for the viral entry of Coronavirus into the lungs blocks the COVID 19 (SARS-CoV-2) infection. The drug, Camostat mesilate, is a drug approved in Japan to treat pancreatic inflammation. Results suggest this drug may also protect against COVID 19. Researchers call for further clinical trials.
Viruses must enter cells of the human body to cause disease. For this, they attach to suitable cells and inject their genetic information into these cells. Infection biologists from the German Primate Center – Leibniz Institute for Primate Research in Göttingen, together with colleagues at Charité – Universitätsmedizin Berlin, have investigated how the novel coronavirus SARS-CoV-2 penetrates cells. They have identified a cellular enzyme that is essential for viral entry into lung cells: the protease TMPRSS2. A clinically proven drug known to be active against TMPRSS2 was found to block SARS-CoV-2 infection and might constitute a novel treatment option.
The findings have been published in Cell.
Several coronaviruses circulate worldwide and constantly infect humans, which normally caused only mild respiratory disease. Currently, however, we are witnessing a worldwide spread of a new coronavirus with more than 101,000 confirmed cases and almost 3,500 deaths. The new virus has been named SARS coronavirus-2 and has been transmitted from animals to humans. It causes a respiratory disease called COVID-19 that may take a severe course. The SARS coronavirus-2 has been spreading since December 2019 and is closely related to the SARS coronavirus that caused the SARS pandemic in 2002/2003. No vaccines or drugs are currently available to combat these viruses.
Stopping virus spread
A team of scientists led by infection biologists from the German Primate Centre and including researchers from Charité, the University of Veterinary Medicine Hannover Foundation, the BG-Unfallklinik Murnau, the LMU Munich, the Robert Koch Institute and the German Center for Infection Research, wanted to find out how the new coronavirus SARS-CoV-2 enters host cells and how this process can be blocked. The researchers identified a cellular protein that is important for the entry of SARS-CoV-2 into lung cells. “Our results show that SARS-CoV-2 requires the protease TMPRSS2, which is present in the human body, to enter cells,” says Stefan Pöhlmann, head of the Infection Biology Unit at the German Primate Center. “This protease is a potential target for therapeutic intervention.”
The SARS coronavirus-2 has been spreading since December 2019 and is closely related to the SARS coronavirus that caused the SARS pandemic in 2002/2003. No vaccines or drugs are currently available to combat these viruses. The image is credited to CDC.
Promising drug
Since it is known that the drug camostat mesilate inhibits the protease TMPRSS2, the researchers have investigated whether it can also prevent infection with SARS-CoV-2. “We have tested SARS-CoV-2 isolated from a patient and found that camostat mesilate blocks entry of the virus into lung cells,” says Markus Hoffmann, the lead author of the study. Camostat mesilate is a drug approved in Japan for use in pancreatic inflammation. “Our results suggest that camostat mesilate might also protect against COVID-19,” says Markus Hoffmann. “This should be investigated in clinical trials.”
References
^Okuno, M.; Kojima, S.; Akita, K.; Matsushima-Nishiwaki, R.; Adachi, S.; Sano, T.; Takano, Y.; Takai, K.; Obora, A.; Yasuda, I.; Shiratori, Y.; Okano, Y.; Shimada, J.; Suzuki, Y.; Muto, Y.; Moriwaki, Y. (2002). “Retinoids in liver fibrosis and cancer”. Frontiers in Bioscience. 7 (4): d204-18. doi:10.2741/A775. PMID11779708.
^Hsieh, H. P.; Hsu, J. T. (2007). “Strategies of development of antiviral agents directed against influenza virus replication”. Current Pharmaceutical Design. 13 (34): 3531–42. doi:10.2174/138161207782794248. PMID18220789.
^Kitamura, K.; Tomita, K. (2012). “Proteolytic activation of the epithelial sodium channel and therapeutic application of a serine protease inhibitor for the treatment of salt-sensitive hypertension”. Clinical and Experimental Nephrology. 16 (1): 44–8. doi:10.1007/s10157-011-0506-1. PMID22038264.
Kunze H, Bohn E (May 1983). “Effects of the serine protease inhibitors FOY and FOY 305 on phospholipase A1 (EC 3.1.1.32) activity in rat – liver lysosomes”. Pharmacol Res Commun. 15 (5): 451–9. doi:10.1016/S0031-6989(83)80065-4. PMID6412250.
Göke B, Stöckmann F, Müller R, Lankisch PG, Creutzfeldt W (1984). “Effect of a specific serine protease inhibitor on the rat pancreas: systemic administration of camostate and exocrine pancreatic secretion”. Digestion. 30 (3): 171–8. doi:10.1159/000199102. PMID6209186.
L-Alanine, N-[(S)-hydroxyphenoxyphosphinyl)-, 2-ethylbutyl ester,6-ester with 2-C-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-2,5-anhydro-D-altrononitrile
GS-5734
Treatment of viral infections
Phase III, clinical trials for the treatment of hospitalized patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19). National Institute of Allergy and Infectious Diseases (NIAID) is evaluating remdesivir in phase II/III clinical trials for the treatment of Ebola virus infection.
The compound has been evaluated in preclinical studies for the potential treatment of Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV) infections.
Remdesivir is a nucleoside analogue, with effective antiviral activity, with EC50s of 74 nM for ARS-CoV and MERS-CoV in HAE cells, and 30 nM for murine hepatitis virus in delayed brain tumor cells.
Laboratory tests suggest remdesivir is effective against a wide range of viruses, including SARS-CoV and MERS-CoV. The medication was pushed to treat the West African Ebola virus epidemic of 2013–2016. Although the drug turned out to be safe, it was not particularly effective against filoviruses such as the Ebola virus.
Ebola virus
Remdesivir was rapidly pushed through clinical trials due to the West African Ebola virus epidemic of 2013–2016, eventually being used in at least one human patient despite its early development stage at the time. Preliminary results were promising and it was used in the emergency setting during the Kivu Ebola epidemic that started in 2018 along with further clinical trials, until August 2019, when Congolese health officials announced that it was significantly less effective than monoclonal antibody treatments such as mAb114 and REGN-EB3. The trials, however, established its safety profile.[9][10][11][12][13][14][15][16]
SARS-CoV-2
In response to the 2019–20 coronavirus outbreak induced by coronavirus SARS-CoV-2, Gilead provided remdesivir for a “small number of patients” in collaboration with Chinese medical authorities for studying its effects.[17]
Gilead also started laboratory testing of remdesivir against SARS-CoV-2. Gilead stated that remdesivir was “shown to be active” against SARS and MERS in animals.[3][18]
In late January 2020, remdesivir was administered to the first US patient to be confirmed to be infected by SARS-CoV-2, in Snohomish County, Washington, for “compassionate use” after he progressed to pneumonia. While no broad conclusions were made based on the single treatment, the patient’s condition improved dramatically the next day,[7] and he was eventually discharged.[19]
Also in late January 2020, Chinese medical researchers stated to the media that in exploratory research considering a selection of 30 drug candidates. Remdesivir and two other drugs, chloroquine and lopinavir/ritonavir, seemed to have “fairly good inhibitory effects” on SARS-CoV-2 at the cellular level. Requests to start clinical testing were submitted,[20][21]. On February 6, 2020, a clinical trial of remdesivir began in China.[22]
Other viruses
The active form of remdesivir, GS-441524, shows promise for treating feline coronavirus.[23]
Mechanism of action and resistance
Remdesivir is a prodrug that metabolizes into its active form GS-441524. GS-441524 is an adenosine nucleotide analog that confuses viral RNA polymerase and evades proofreading by viral exoribonuclease (ExoN), causing a decrease in viral RNA production. It was unknown whether it terminates RNA chains or causes mutations in them.[24]However, it has been learned that the RNA dependent RNA polymerase of ebolavirus is inhibited for the most part by delayed chain termination.[25]
Mutations in the mouse hepatitis virusRNA replicase that cause partial resistance were identified in 2018. These mutations make the viruses less effective in nature, and the researchers believe they will likely not persist where the drug is not being used.[24]
MORE SYNTHESIS COMING, WATCH THIS SPACE…………………..
SYNTHESIS
Remdesivir can be synthesized in multiple steps from ribose derivatives. The figure below is one of the synthesis route of remdesivir invented by Chun et al. from Gilead Sciences.[26]In this method, intermediate a is firstly prepared from L-alanine and phenyl phosphorodichloridate in presence of triethylamine and dichloromethane; triple benzyl-protected ribose is oxidized by dimethyl sulfoxide with acetic anhydride and give the lactone intermediate b; pyrrolo[2,1-f][1,2,4]triazin-4-amine is brominated, and the amine group is protected by excess trimethylsilyl chloride. n-Butyllithium undergoes a halogen-lithium exchange reaction with the bromide at -78 °C to yield the intermediate c. The intermediate b is then added to a solution containing intermediate c dropwise. After quenching the reaction in a weakly acidic aqueous solution, a mixture of 1: 1 anomers was obtained. It was then reacted with an excess of trimethylsilyl cyanide in dichloromethane at -78 °C for 10 minutes. Trimethylsilyl triflate was added and reacts for an additional 1 hour, and the mixture was quenched in an aqueous sodium hydrogen carbonate. A nitrile intermediate was obtained. The protective group, benzyl, was then removed with boron trichloride in dichloromethane at -20 °C. The excess of boron trichloride was quenched in a mixture of potassium carbonate and methanol. A benzyl-free intermediate was obtained. The isomers were then separated via reversed-phase HPLC. The optically pure compound and intermediate a are reacted with trimethyl phosphate and methylimidazole to obtain a diastereomer mixture of remdesivir. In the end, optically pure remdesivir can be obtained through methods such as chiral resolution.
The synthesis of Remdesivir was invented by Byoung Kwon Chun et al. from Gilead Sciences, Inc. and claimed in the patent, WO2016069826A1.
Prevention and treatment methods for some Arenaviridae , Coronaviridae , Filoviridae, Flaviviridae, and Paramyxoviridae viruses present challenges due to a lack of vaccine or post-exposure treatment modality for preventing or managing these infections. In some cases, patients only receive supportive and resource intensive therapy such as electrolyte and fluid balancing, oxygen, blood pressure maintenance, or treatment for secondary infections. Thus, there is a need for antiviral therapies having a potential for broad antiviral activity.
[0004] The compound (S)-2-ethylbutyl 2-(((S)-(((2R,3 S,4R,5R)-5-(4-aminopyrrolo[2, 1-f][l,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy) phosphoryl)amino)propanoate, referred herein as Compound 1 or Formula I, is known to exhibit antiviral properties against Arenaviridae, Coronaviridae, Filoviridae, and
Paramyxoviridae viruses as described in Warren, T. et al., Nature (2016) 531 :381-385 and antiviral activities against Flaviviridae viruses as described in co-pending United States provisional patent application no. 62/325,419 filed April 20, 2016.
propanoate or 2-ethylbutyl ((S)-(((2R,3 S,4R,5R)-5-(4-aminopyrrolo[2, l-f][l,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate, (Formula I), has the following structure:
Example 1. (2S)-ethyl 2-(chloro(phenoxy)phosphorylamino)pro anoate (Chloridate A)
[0246] Ethyl alanine ester hydrochloride salt (1.69 g, 11 mmol) was dissolved in anhydrous CH2CI2 (10 mL) and the mixture stirred with cooling to 0 °C under N2(g). Phenyl dichlorophosphate (1.49 mL, 10 mmol) was added followed by dropwise addition of Et3N over 10 min. The reaction mixture was then slowly warmed to RT and stirred for 12 h. Anhydrous Et20 (50 mL) was added and the mixture stirred for 30 min. The solid that formed was removed by filtration, and the filtrate concentrated under reduced pressure. The residue was subjected to silica gel chromatography eluting with 0-50% EtOAc in hexanes to provide intermediate A (1.13 g, 39%). H NMR (300 MHz, CDC13) δ 7.39-7.27 (m, 5H), 4.27 (m, 3H), 1.52 (m, 3H), 1.32 (m, 3H). 31P NMR (121.4 MHz, CDC13) δ 8.2, 7.8.
Example 2. (2S)-2-ethylbutyl 2-(chloro(phenoxy)phosphorylamino)propanoate
(Chloridate B
[0247] The 2-ethylbutyl alanine chlorophosphoramidate ester B was prepared using the same procedure as chloridate A except substituting 2-ethylbutyl alanine ester for ethyl alanine ester. The material is used crude in the next reaction. Treatment with methanol or ethanol forms the displaced product with the requisite LCMS signal.
Example 3. (2S)-isopropyl 2-(chloro(phenoxy)phosphorylamino)propanoate
(Chloridate C)
C
[0248] The isopropyl alanine chlorophosphoramidate ester C was prepared using the same procedure as chloridate A except substituting isopropyl alanine ester for the ethyl alanine ester. The material is used crude in the next reaction. Treatment with methanol or ethanol forms the displaced product with the requisite LCMS signal.
Example 4. (2S)-2-ethylbutyl 2-((((2R,3S,4R,5R)-5-(4-aminopyrrolo[l,2-firi,2,41triazin- 7-yl)-5-cvano-3,4-dihvdroxytetrahydrofuran-2- yl)methoxy)(phenoxy)phosphorylamino)propanoate (Compound 9)
[0249] Compound 9 can be prepared by several methods described below. Procedure 1
[0250] Prepared from Compound 1 and chloridate B according to the same method as for the preparation of compound 8 as described in PCT Publication no. WO 2012/012776. 1H NMR (300 MHz, CD3OD) δ 7.87 (m, 1H), 7.31-7.16 (m, 5H), 6.92-6.89 (m, 2H), 4.78 (m, 1H), 4.50-3.80 (m, 7H), 1.45-1.24 (m, 8H), 0.95-0.84 (m, 6H). 31P NMR (121.4 MHz, CD3OD) δ 3.7. LCMS m/z 603.1 [M+H], 601.0 [M-H].
Procedure 2
9
[0251] (2S)-2-ethylbutyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7- yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino) propanoate. (2S)-2-ethylbutyl 2-(((4-nitrophenoxy)(phenoxy)phosphoryl)amino)propanoate (1.08 g, 2.4 mmol) was dissolved in anhydrous DMF (9 mL) and stirred under a nitrogen atmosphere at RT. (2R,3R,4S,5R)-2-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)-3,4- dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (350 mg, 1.2 mmol) was added to the reaction mixture in one portion. A solution of i-butylmagnesium chloride in THF (1M, 1.8 mL, 1.8 mmol) was then added to the reaction drop wise over 10 minutes. The reaction was stirred for 2 h, at which point the reaction mixture was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium bicarbonate solution (3 x 15 mL) followed by saturated aqueous sodium chloride solution (15 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting oil was purified with silica gel column chromatography (0-10% MeOH in DCM) to afford (2S)-2- ethylbutyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2, l-f][l,2,4]triazin-7-yl)-5-cyano-3,4- dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino) propanoate (311 mg, 43%, 1 :0.4 diastereomeric mixture at phosphorus) as a white solid. H NMR (400 MHz, CD3OD) δ 7.85 (m, 1H), 7.34 – 7.23 (m, 2H), 7.21 – 7.09 (m, 3H), 6.94 – 6.84 (m, 2H), 4.78 (d, / = 5.4 Hz, 1H), 4.46 – 4.33 (m, 2H), 4.33 – 4.24 (m, 1H), 4.18 (m, 1H), 4.05 – 3.80 (m, 3H), 1.52 – 1.39 (m, 1H), 1.38 – 1.20 (m, 7H), 0.85 (m, 6H). 31P NMR (162 MHz, CD3OD) δ 3.71, 3.65. LCMS m/z 603.1 [M+H], 600.9 [M-H]. HPLC (2-98% MeCN-H20 gradient with 0.1% TFA modifier over 8.5 min, 1.5mL/min, Column: Phenomenex Kinetex C18, 2.6 um 100 A, 4.6 x 100 mm ) tR = 5.544 min, 5.601 min
Separation of the (S) and (R) Diastereomers
[0252] (2S)-2-ethylbutyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,l-f][l,2,4]triazin-7-yl)- 5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)amino) propanoate was dissolved in acetonitrile. The resulting solution was loaded onto Lux Cellulose-2 chiral column, equilibrated in acetonitrile, and eluted with isocratic
acetonitrile/methanol (95 :5 vol/vol). The first eluting diastereomer had a retention time of 17.4 min, and the second eluting diastereomer had a retention time of 25.0 min.
[0253] First Eluting Diastereomer is (S)-2-ethylbutyl 2-(((R)-(((2R,3S,4R,5R)-5-(4- aminopyrrolo[2, 1 -f] [ 1 ,2,4]triazin-7-yl)-5-cyano-3 ,4-dihydroxytetrahydrofuran-2- yl)methoxy)(phenoxy)phos horyl)amino)propanoate:
Example 5. (S)-2-ethylbutyl 2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolor2J- f|[l,2,41triazin-7-yl)-5-cvano-3,4-dihvdroxytetrahvdrofuran-2- yl)methoxy)(phenoxy)phosphoryl)amino)propanoate (32)
[0255] The preparation of (S)-2-ethylbutyl 2-(((S)-(((2R,3S,4R,5R)-5-(4-aminopyrrolo[2,l f][l,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2- yl)methoxy)(phenoxy)phosphoryl)amino)propanoate is described below.
Preparation of (3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)dihydrofuran-2(3H)- one.
[0256] (3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol (15.0g) was combined with MTBE (60.0 mL), KBr (424.5 mg), aqueous K2HP04solution (2.5M, 14.3 mL), and TEMPO (56 mg). This mixture was cooled to about 1 °C. Aqueous bleach solution (7.9%wt.) was slowly charged in portions until complete consumption of starting material as indicated through a starch/iodide test. The layers were separated, and the aqueous layer was extracted with MTBE. The combined organic phase was dried over MgS04 and concentrated under reduced pressure to yield the product as a solid.
[0257] To a cold solution of 4-aminopyrrolo[2, l-f][l,2,4]-triazine (10.03 g; 74.8 mmol) in N,N-dimethylformamide (70.27 g), N-iodosuccinimide (17.01g; 75.6 mmol) was charged in portions, while keeping the contents at about 0 °C. Upon reaction completion (about 3 h at about 0 °C), the reaction mixture was transferred into a 1 M sodium hydroxide aqueous solution (11 g NaOH and 276 mL water) while keeping the contents at about 20-30 °C. The resulting slurry was agitated at about 22 °C for 1.5 h and then filtered. The solids are rinsed with water (50 mL) and dried at about 50 °C under vacuum to yield 4-amino-7- iodopyrrolo[2,l-f] [l,2,4]triazine as a solid. !H NMR (400 MHz, DMSO-d6) δ 7.90 (s, 1H), 7.78 (br s, 2H), 6.98 (d, J = 4.4 Hz, 1H), 6.82 (d, J = 4.4 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 155.7, 149.1, 118.8, 118.1, 104.4, 71.9. MS m/z = 260.97 [M+H].
Preparation (3R,4R,5R)-2-(4-aminopyrrolor2, l-firi,2,41triazin-7-yl)-3,4-bis(benzyloxy)-5- ((benzyloxy)methyl)tetrahvdrofuran-2-ol via (4-amino-7-iodopyrrolor2,l-fl ri,2,41triazine)
[0258] To a reactor under a nitrogen atmosphere was charged iodobase 2 (81 g) and THF (1.6 LV). The resulting solution was cooled to about 5 °C, and TMSC1 (68 g) was charged. PhMgCl (345mL, 1.8 M in THF) was then charged slowly while maintaining an internal temperature at about < 5°C. The reaction mixture was stirred at about 0°C for 30 min, and then cooled to about -15 °C. z‘PrMgCl-LiCl (311 mL, 1.1 M in THF) was charged slowly while maintaining an internal temperature below about -12 °C. After about 10 minutes of stirring at about -15 °C, the reaction mixture was cooled to about -20 °C, and a solution of lactone 1 (130 g) in THF (400 mL) was charged. The reaction mixture was then agitated at about -20 °C for about 1 h and quenched with AcOH (57 mL). The reaction mixture was warmed to about 0 °C and adjusted to pH 7-8 with aqueous NaHCC>3 (5 wt%, 1300 mL). The reaction mixture was then diluted with EtOAc (1300 mL), and the organic and aqueous layers were separated. The organic layer was washed with IN HC1 (1300 mL), aqueous NaHCC>3 (5 wt%, 1300 mL), and brine (1300 mL), and then dried over anhydrous Na2S04 and concentrated to dryness. Purification by silica gel column chromatography using a gradient consisting of a mixture of MeOH and EtOAc afforded the product.
Preparation ((2S)-2-ethylbutyl 2- (((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate) (mixture of Sp and Rp):
1 ) phenyl dichlorophosphate
CH2CI2, -78 °C to ambient
2) pentafluorophenol
Et3N, 0 °C to ambient
[0259] L- Alanine 2-ethylbutyl ester hydrochloride (5.0 g, 23.84 mmol) was combined with methylene chloride (40 mL), cooled to about -78 °C, and phenyl dichlorophosphate (3.65 mL, 23.84 mmol) was added. Triethylamine (6.6 mL, 47.68 mmol) was added over about 60 min at about -78 °C and the resulting mixture was stirred at ambient temperature for 3h. The reaction mixture was cooled to about 0 °C and pentafluorophenol (4.4 g, 23.84 mmol) was added. Triethylamine (3.3 mL, 23.84 mmol) was added over about 60 min. The mixture was stirred for about 3h at ambient temperature and concentrated under reduced pressure. The residue was dissolved in EtOAc, washed with an aqueous sodium carbonate solution several times, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of EtOAc and hexanes (0 to 30%). Product containing fractions were concentrated under reduced pressure to give (2S)-2-ethylbutyl 2-(((perfluorophenoxy)(phenoxy)phosphoryl)amino)propanoate as a solid. H NMR (400 MHz, Chloroform-d) δ 7.41 – 7.32 (m, 4H), 7.30 – 7.17 (m, 6H), 4.24 – 4.16 (m, 1H), 4.13 – 4.03 (m, 4H), 4.01 – 3.89 (m, 1H), 1.59 – 1.42 (m, 8H), 1.40 – 1.31 (m, 8H), 0.88 (t, J = 7.5 Hz, 12H). 31P NMR (162 MHz, Chloroform-d) δ – 1.52. 19F NMR (377 MHz, Chloroform-d) δ – 153.63, – 153.93 (m), – 160.05 (td, J = 21.9, 3.6 Hz), – 162.65 (qd, J = 22.4, 20.5, 4.5 Hz). MS m/z = 496 [M+H]. Preparation of Title Compound (mixture of Sp and Rp):
[0260] The nucleoside (29 mg, 0.1 mmol) and the phosphonamide (60 mg, 0.12 mmol) and N,N-dimethylformamide (2 mL) were combined at ambient temperature. 7¾ri-Butyl magnesiumchloride (1M in THF, 0.15 mL) was slowly added. After about lh, the reaction was diluted with ethyl acetate, washed with aqueous citric acid solution (5%wt.), aqueous saturated NaHC03 solution and saturated brine solution. The organic phase was dried over Na2S04 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of methanol and CH2CI2 (0 to 5%). Product containing fractions were concentrated under reduced pressure to provide the product.
Preparation of (3aR,4R,6R,6aR)-4-(4-aminopyrrolor2, l-firi,2,41triazin-7-yl)-6- (hvdroxymethyl)-2,2-dimethyltetrahydrofuror3,4-diri,31dioxole-4-carbonitrile:
[0261] To a mixture of (2R,3R,4S,5R)-2-(4-aminopyrrolo[2, l-f] [l,2,4]triazin-7-yl)-3,4- dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (5.8g, 0.02 mol), 2,2- dimethoxypropane (11.59 mL, 0.09 mol) and acetone (145 mL) at ambient temperature was added sulfuric acid (18M, 1.44 mL). The mixture was warmed to about 45 °C. After about 30 min, the mixture was cooled to ambient temperature and sodium bicarbonate (5.8 g) and water 5.8 mL) were added. After 15 min, the mixture was concentrated under reduced pressure. The residue was taken up in ethyl acetate (150 mL) and water (50 mL). The aqueous layer was extracted with ethyl acetate (2 x 50 mL). The combined organic phase was dried over sodium sulfate and concentrated under reduced pressure to give crude (2R,3R,4S,5R)-2-(4-aminopyrrolo[2, l-f] [l,2,4]triazin-7-yl)-3,4-dihydroxy-5- (hydroxymethyl)tetrahydrofuran-2-carbonitrile. !H NMR (400 MHz, CD3OD) δ 7.84 (s, 1H), 6.93 (d, / = 4.6 Hz, 1H), 6.89 (d, / = 4.6 Hz, 1H), 5.40 (d, / = 6.7 Hz, 1H), 5.00 (dd, / = 6.7, 3.3 Hz, 1H), 4.48 – 4.40 (m, 1H), 3.81 – 3.72 (m, 2H), 1.71 (s, 3H), 1.40 (s, 3H). MS m/z = 332.23 [M+l].
Preparation of (2S)-2-ethylbutyl 2-(((((2R,3S,4R,5R)-5-(4-aminopyrrolor2,l-firi,2,41triazin- 7-yl)-5-cvano-3,4-dihvdroxytetrahydrofuran-2- yl)methoxy)(phenoxy)phosphoryl)amino)propanoate:
[0262] Acetonitrile (100 mL) was combined with (2S)-2-ethylbutyl 2-(((4- nitrophenoxy)(phenoxy)phosphoryl)-amino)propanoate (9.6 g, 21.31 mmol), the substrate alcohol (6.6 g, 0.02 mol), magnesium chloride (1.9 g, 19.91 mmol) at ambient temperature. The mixture was agitated for about 15 min and N,N-diisopropylethylamine (8.67 mL, 49.78 mmol) was added. After about 4h, the reaction was diluted with ethyl acetate (100 mL), cooled to about 0 °C and combined with aqueous citric acid solution (5%wt., 100 mL). The organic phase was washed with aqueous citric acid solution (5%wt., 100 mL) and aqueous saturated ammonium chloride solution (40 mL), aqueous potassium carbonate solution
[0263] The crude acetonide (12.85 g) was combined with tetrahydrofuran (50 mL) and concentrated under reduced pressure. The residue was taken up in tetrahydrofuran (100 mL), cooled to about 0 °C and concentrated HC1 (20 mL) was slowly added. The mixture was allowed to warm to ambient temperature. After consumption of the starting acetonide as indicated by HPLC analysis, water (100 mL) was added followed by aqueous saturated sodium bicarbonate solution (200 mL). The mixture was extracted with ethyl acetate (100 mL), the organic phase washed with aqueous saturated brine solution (50 mL), dried over sodium sulfated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of methanol and ethyl acetate (0 to 20%).
Product containing fractions were concentrated under reduced pressure to provide the product.
PATENT
US 20170071964
US 20160122374
PAPER
Journal of Medicinal Chemistry (2017), 60(5), 1648-1661.
The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1′-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3–14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [ Nature 2016, 531, 381−385]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.
The drug substance is a white to light yellow powder. It is sparingly soluble in acetonitrile and in methanol, and slightly soluble in water and in ethanol (99.5). It is slightly soluble at pH 2.0 to 5.5 and sparingly soluble at pH 5.5 to 6.1. The drug substance is not hygroscopic at 25°C/51% to 93%RH. The melting point is 187°C to 193°C, and the dissociation constant (pKa) is 5.1 due to the hydroxyl group of favipiravir. Measurement results on the partition ratio of favipiravir in water/octanol at 25°C indicate that favipiravir tends to be distributed in the 1-octanol phase at pH 2 to 4 and in the water phase at pH 5 to 13.
Any batch manufactured by the current manufacturing process is in Form A. The stability study does not show any change in crystal form over time; and a change from Form A to Form B is unlikely.
T-705 is an RNA-directed RNA polymerase (NS5B) inhibitor which has been filed for approval in Japan for the oral treatment of influenza A (including avian and H1N1 infections) and for the treatment of influenza B infection.
The compound is a unique viral RNA polymerase inhibitor, acting on viral genetic copying to prevent its reproduction, discovered by Toyama Chemical. In 2005, Utah State University carried out various studies under its contract with the National Institute of Allergy and Infectious Diseases (NIAID) and demonstrated that T-705 has exceptionally potent activity in mouse infection models of H5N1 avian influenza.
T-705 (Favipiravir) is an antiviral pyrazinecarboxamide-based, inhibitor of of the influenza virus with an EC90 of 1.3 to 7.7 uM (influenza A, H5N1). EC90 ranges for other influenza A subtypes are 0.19-1.3 uM, 0.063-1.9 uM, and 0.5-3.1 uM for H1N1, H2N2, and H3N2, respectively. T-705 also exhibits activity against type B and C viruses, with EC90s of 0.25-0.57 uM and 0.19-0.36 uM, respectively. (1) Additionally, T-705 has broad activity against arenavirus, bunyavirus, foot-and-mouth disease virus, and West Nile virus with EC50s ranging from 5 to 300 uM.
Studies show that T-705 ribofuranosyl triphosphate is the active form of T-705 and acts like purines or purine nucleosides in cells and does not inhibit DNA synthesis
In 2012, MediVector was awarded a contract from the U.S. Department of Defense’s (DOD) Joint Project Manager Transformational Medical Technologies (JPM-TMT) to further develop T-705 (favipiravir), a broad-spectrum therapeutic against multiple influenza viruses.
Several novel anti-influenza compounds are in various phases of clinical development. One of these, T-705 (favipiravir), has a mechanism of action that is not fully understood but is suggested to target influenza virus RNA-dependent RNA polymerase. We investigated the mechanism of T-705 activity against influenza A (H1N1) viruses by applying selective drug pressure over multiple sequential passages in MDCK cells. We found that T-705 treatment did not select specific mutations in potential target proteins, including PB1, PB2, PA, and NP. Phenotypic assays based on cell viability confirmed that no T-705-resistant variants were selected. In the presence of T-705, titers of infectious virus decreased significantly (P < 0.0001) during serial passage in MDCK cells inoculated with seasonal influenza A (H1N1) viruses at a low multiplicity of infection (MOI; 0.0001 PFU/cell) or with 2009 pandemic H1N1 viruses at a high MOI (10 PFU/cell). There was no corresponding decrease in the number of viral RNA copies; therefore, specific virus infectivity (the ratio of infectious virus yield to viral RNA copy number) was reduced. Sequence analysis showed enrichment of G→A and C→T transversion mutations, increased mutation frequency, and a shift of the nucleotide profiles of individual NP gene clones under drug selection pressure. Our results demonstrate that T-705 induces a high rate of mutation that generates a nonviable viral phenotype and that lethal mutagenesis is a key antiviral mechanism of T-705. Our findings also explain the broad spectrum of activity of T-705 against viruses of multiple families.
In February 2020 Favipiravir was being studied in China for experimental treatment of the emergent COVID-19 (novel coronavirus)disease.[7][8] On March 17 Chinese officials suggested the drug had been effective in treating COVID in Wuhan and Shenzhen.[9][10]
Discovered by Toyama Chemical Co., Ltd. in Japan, favipiravir is a modified pyrazine analog that was initially approved for therapeutic use in resistant cases of influenza.7,9 The antiviral targets RNA-dependent RNA polymerase (RdRp) enzymes, which are necessary for the transcription and replication of viral genomes.7,12,13
Not only does favipiravir inhibit replication of influenza A and B, but the drug shows promise in the treatment of influenza strains that are resistant to neuramidase inhibitors, as well as avian influenza.9,19 Favipiravir has been investigated for the treatment of life-threatening pathogens such as Ebola virus, Lassa virus, and now COVID-19.10,14,15
Mechanism of action
The mechanism of its actions is thought to be related to the selective inhibition of viral RNA-dependent RNA polymerase.[11] Other research suggests that favipiravir induces lethal RNA transversion mutations, producing a nonviable viral phenotype.[12] Favipiravir is a prodrug that is metabolized to its active form, favipiravir-ribofuranosyl-5′-triphosphate (favipiravir-RTP), available in both oral and intravenous formulations.[13][14] Human hypoxanthine guanine phosphoribosyltransferase (HGPRT) is believed to play a key role in this activation process.[15] Favipiravir does not inhibit RNA or DNA synthesis in mammalian cells and is not toxic to them.[1] In 2014, favipiravir was approved in Japan for stockpiling against influenza pandemics.[16] However, favipiravir has not been shown to be effective in primary human airway cells, casting doubt on its efficacy in influenza treatment.[17]
Approval status
In 2014, Japan approved Favipiravir for treating viral strains unresponsive to current antivirals.[18]
Some research has been done suggesting that in mouse models Favipiravir may have efficacy against Ebola. Its efficacy against Ebola in humans is unproven.[20][21][22] During the 2014 West Africa Ebola virus outbreak, it was reported that a French nurse who contracted Ebola while volunteering for MSF in Liberia recovered after receiving a course of favipiravir.[23] A clinical trial investigating the use of favipiravir against Ebola virus disease was started in Guéckédou, Guinea, during December 2014.[24] Preliminary results showed a decrease in mortality rate in patients with low-to-moderate levels of Ebola virus in the blood, but no effect on patients with high levels of the virus, a group at a higher risk of death.[25] The trial design has been criticised by Scott Hammer and others for using only historical controls.[26] The results of this clinical trial were presented in February 2016 at the annual Conference on Retroviruses and Opportunistic Infections (CROI) by Daouda Sissoko[27] and published on March 1, 2016 in PLOS Medicine.[28]
SARS-CoV-2 virus disease
In March 2020, Chinese officials suggested Favipiravir may be effective in treating COVID-19.[29]
Drug Discoveries & Therapeutics. 2014; 8(3):117-120.
As a RNA polymerase inhibitor, 6-fluoro-3-hydroxypyrazine-2-carboxamide commercially named favipiravir has been proved to have potent inhibitory activity against RNA viruses in vitro and in vivo. A four-step synthesis of the compound is described in this article, amidation, nitrification, reduction and fluorination with an overall yield of about 8%. In addition, we reported the crystal structure of the title compound. The molecule is almost planar and the intramolecular O−H•••O hydrogen bond makes a 6-member ring. In the crystal, molecules are packing governed by both hydrogen bonds and stacking interactions.
2.2.1. Preparation of 3-hydroxypyrazine-2-carboxamide To a suspension of 3-hydroxypyrazine-2-carboxylic acid (1.4 g, 10 mmol) in 150 mL MeOH, SOCl2 was added dropwise at 40°C with magnetic stirring for 6 h resulting in a bright yellow solution. The reaction was then concentrated to dryness. The residue was dissolved in 50 mL 25% aqueous ammonia and stirred overnight to get a suspension. The precipitate was collected and dried. The solid yellow-brown crude product was recrystallization with 50 mL water to get the product as pale yellow crystals (1.1 g, 78%). mp = 263-265°C. 1 H-NMR (300 MHz, DMSO): δ 13.34 (brs, 1H, OH), 8.69 (s, 1H, pyrazine H), 7.93-8.11 (m, 3H, pyrazine H, CONH2). HRMS (ESI): m/z [M + H]+ calcd for C5H6N3O2 + : 140.0460; found: 140.0457.
2.2.2. Preparation of 3-hydroxy-6-nitropyrazine-2- carboxamide In the solution of 3-hydroxypyrazine-2-carboxamide (1.0 g, 7 mmol) in 6 mL concentrate sulfuric acid under ice-cooling, potassium nitrate (1.4 g, 14 mmol) was added. After stirring at 40°C for 4 h, the reaction mixture was poured into 60 mL water. The product was collected by fi ltration as yellow solid (0.62 g, 48%). mp = 250-252°C. 1 H-NMR (600 MHz, DMSO): δ 12.00- 15.00 (br, 1H, OH), 8.97 (s, 1H, pyrazine H), 8.32 (s, 1H, CONH2), 8.06 (s, 1H, CONH2). 13C-NMR (75 MHz, DMSO): δ 163.12, 156.49, 142.47, 138.20, 133.81. HRMS (ESI): m/z [M + H]+ calcd for C5H5N4O4 + : 185.0311; found: 185.0304.
2.2.3. Preparation of 6-amino-3-hydroxypyrazine-2- carboxamide 3-Hydroxy-6-nitropyrazine-2-carboxamide (0.6 g, 3.3 mmol) and a catalytic amount of raney nickel were suspended in MeOH, then hydrazine hydrate was added dropwise. The resulting solution was refl uxed 2 h, cooled, filtered with diatomite, and then MeOH is evaporated in vacuo to get the crude product as dark brown solid without further purification (0.4 g, 77%). HRMS (ESI): m/z [M + H]+ calcd for C5H7N4O2 + : 155.0569; found:155.0509.
2.2.4. Preparation of 6-fluoro-3-hydroxypyrazine-2- carboxamide To a solution of 6-amino-3-hydroxypyrazine-2- carboxamide (0.4 g, 2.6 mmol) in 3 mL 70% HFpyridine aqueous at -20°C under nitrogen atmosphere, sodium nitrate (0.35 g, 5.2 mmol) was added. After stirring 20 min, the solution was warmed to room temperature for another one hour. Then 20 mL ethyl acetate/water (1:1) were added, after separation of the upper layer, the aqueous phase is extracted with four 20 mL portions of ethyl acetate. The combined extracts are dried with anhydrous magnesium sulfate and concentrated to dryness to get crude product as oil. The crude product was purified by chromatography column as white solid (0.12 g, 30%). mp = 178-180°C. 1 H-NMR (600 MHz, DMSO): δ 12.34 (brs, 1H, OH), 8.31 (d, 1H, pyrazine H, J = 8.0 Hz), 7.44 (s, 1H, CONH2), 5.92 (s, 1H, CONH2). 13C-NMR (75 MHz, DMSO): δ 168.66, 159.69, 153.98, 150.76, 135.68. HRMS (ESI): m/z [M + H]+ calcd for C5H5FN3O2 + : 158.0366; found: 158.0360.
SEE
Chemical Papers (2019), 73(5), 1043-1051.
PAPER
Medicinal chemistry (Shariqah (United Arab Emirates)) (2018), 14(6), 595-603
Furuta, Y.; Takahashi, K.; Shiraki, K.; Sakamoto, K.; Smee, D. F.; Barnard, D. L.; Gowen, B. B.; Julander, J. G.; Morrey, J. D. (2009). “T-705 (favipiravir) and related compounds: Novel broad-spectrum inhibitors of RNA viral infections”. Antiviral Research82 (3): 95–102. doi:10.1016/j.antiviral.2009.02.198. PMID19428599. edit
WO 2000010569
WO 2008099874
WO 201009504
WO 2010104170
WO 2012063931
CLIP Influenza virus is a central virus of the cold syndrome, which has attacked human being periodically to cause many deaths amounting to tens millions. Although the number of deaths shows a tendency of decrease in the recent years owing to the improvement in hygienic and nutritive conditions, the prevalence of influenza is repeated every year, and it is apprehended that a new virus may appear to cause a wider prevalence. For prevention of influenza virus, vaccine is used widely, in addition to which low molecular weight substances such as Amantadine and Ribavirin are also used
CLIP Synthesis of Favipiravir ZHANG Tao1, KONG Lingjin1, LI Zongtao1,YUAN Hongyu1, XU Wenfang2* (1. Shandong Qidu PharmaceuticalCo., Ltd., Linzi 255400; 2. School of Pharmacy, Shandong University, Jinan250012) ABSTRACT: Favipiravir was synthesized from3-amino-2-pyrazinecarboxylic acid by esterification, bromination with NBS,diazotization and amination to give 6-bromo-3-hydroxypyrazine-2-carboxamide,which was subjected to chlorination with POCl3, fluorination with KF, andhydrolysis with an overall yield of about 22％.
To a 17.5 ml N,N-dimethylformamide solution of 5.0 g of 3,6-difluoro-2-pyrazinecarbonitrile, a 3.8 ml water solution of 7.83 g of potassium acetate was added dropwise at 25 to 35° C., and the solution was stirred at the same temperature for 2 hours. 0.38 ml of ammonia water was added to the reaction mixture, and then 15 ml of water and 0.38 g of active carbon were added. The insolubles were filtered off and the filter cake was washed with 11 ml of water. The filtrate and the washing were joined, the pH of this solution was adjusted to 9.4 with ammonia water, and 15 ml of acetone and 7.5 ml of toluene were added. Then 7.71 g of dicyclohexylamine was added dropwise and the solution was stirred at 20 to 30° C. for 45 minutes. Then 15 ml of water was added dropwise, the solution was cooled to 10° C., and the precipitate was filtered and collected to give 9.44 g of dicyclohexylamine salt of 6-fluoro-3-hydroxy-2-pyradinecarbonitrile as a lightly yellowish white solid product. 1H-NMR (DMSO-d6) δ values: 1.00-1.36 (10H, m), 1.56-1.67 (2H, m), 1.67-1.81 (4H, m), 1.91-2.07 (4H, m), 3.01-3.18 (2H, m), 8.03-8.06 (1H, m), 8.18-8.89 (1H, broad) Example 1-2 4.11 ml of acetic acid was added at 5 to 15° C. to a 17.5 ml N,N-dimethylformamide solution of 5.0 g of 3,6-difluoro-2-pyrazinecarbonitrile. Then 7.27 g of triethylamine was added dropwise and the solution was stirred for 2 hours. 3.8 ml of water and 0.38 ml of ammonia water were added to the reaction mixture, and then 15 ml of water and 0.38 g of active carbon were added. The insolubles were filtered off and the filter cake was washed with 11 ml of water. The filtrate and the washing were joined, the pH of the joined solution was adjusted to 9.2 with ammonia water, and 15 ml of acetone and 7.5 ml of toluene were added to the solution, followed by dropwise addition of 7.71 g of dicyclohexylamine. Then 15 ml of water was added dropwise, the solution was cooled to 5° C., and the precipitate was filtered and collected to give 9.68 g of dicyclohexylamine salt of 6-fluoro-3-hydroxy-2-pyrazinecarbonitrile as a slightly yellowish white solid product. Examples 2 to 5 The compounds shown in Table 1 were obtained in the same way as in Example 1-1.
TABLE 1
Example No.
Organic amine
Example No.
Organic amine
2
Dipropylamine
4
Dibenzylamine
3
Dibutylamine
5
N-benzylmethylamine
Dipropylamine salt of 6-fluoro-3-hydroxy-2-pyrazinecarbonitrile 1H-NMR (DMSO-d6) 6 values: 0.39 (6H, t, J=7.5 Hz), 1.10 (4H, sex, J=7.5 Hz), 2.30-2.38 (4H, m), 7.54 (1H, d, J=8.3 Hz) Dibutylamine salt of 6-fluoro-3-hydroxy-2-pyrazinecarbonitrile 1H-NMR (DMSO-d6) 6 values: 0.36 (6H, t, J=7.3 Hz), 0.81 (4H, sex, J=7.3 Hz), 0.99-1.10 (4H, m), 2.32-2.41 (4H, m), 7.53 (1H, d, J=8.3 Hz) Dibenzylamine salt of 6-fluoro-3-hydroxy-2-pyrazinecarbonitrile 1H-NMR (DMSO-d6) δ values: 4.17 (4H, s), 7.34-7.56 (10H, m), 8.07 (1H, d, J=8.3 Hz) N-benzylmethylamine salt of 6-fluoro-3-hydroxy-2-pyrazinecarbonitrile 1H-NMR (DMSO-d6) δ values: 2.57 (3H, s), 4.14 (2H, s), 7.37-7.53 (5H, m), 8.02-8.08 (1H, m) Preparation Example 1
300 ml of toluene was added to a 600 ml water solution of 37.5 g of sodium hydroxide. Then 150 g of dicyclohexylamine salt of 6-fluoro-3-hydroxy-2-pyrazinecarbonitrile was added at 15 to 25° C. and the solution was stirred at the same temperature for 30 minutes. The water layer was separated and washed with toluene, and then 150 ml of water was added, followed by dropwise addition of 106 g of a 30% hydrogen peroxide solution at 15 to 30° C. and one-hour stirring at 20 to 30° C. Then 39 ml of hydrochloric acid was added, the seed crystals were added at 40 to 50° C., and 39 ml of hydrochloric acid was further added dropwise at the same temperature. The solution was cooled to 10° C. the precipitate was filtered and collected to give 65.6 g of 6-fluoro-3-hydroxy-2-pyrazinecarboxamide as a slightly yellowish white solid. 1H-NMR (DMSO-d6) δ values: 8.50 (1H, s), 8.51 (1H, d, J=7.8 Hz), 8.75 (1H, s), 13.41 (1H, s)
CLIP jan 2014
Investigational flu treatment drug has broad-spectrum potential to fight multiple viruses First patient enrolled in the North American Phase 3 clinical trials for investigational flu treatment drug
BioDefense Therapeutics (BD Tx)—a Joint Product Management office within the U.S. Department of Defense (DoD)—announced the first patient enrolled in the North American Phase 3 clinical trials for favipiravir (T-705a). The drug is an investigational flu treatment candidate with broad-spectrum potential being developed by BD Tx through a contract with Boston-based MediVector, Inc.
Favipiravir is a novel, antiviral compound that works differently than anti-flu drugs currently on the market. The novelty lies in the drug’s selective disruption of the viralRNA replication and transcription process within the infected cell to stop the infection cycle.
“Favipiravir has proven safe and well tolerated in previous studies,” said LTC Eric G. Midboe, Joint Product Manager for BD Tx. “This first patient signifies the start of an important phase in favipiravir’s path to U.S. Food and Drug Administration (FDA) approval for flu and lays the groundwork for future testing against other viruses of interest to the DoD.”
In providing therapeutic solutions to counter traditional, emerging, and engineered biological threats, BD Tx chose favipiravir not only because of its potential effectiveness against flu viruses, but also because of its demonstrated broad-spectrum potential against multiple viruses. In addition to testing favipiravir in the ongoing influenzaprogram, BD Tx is testing the drug’s efficacy against the Ebola virus and other viruses considered threats to service members. In laboratory testing, favipiravir was found to be effective against a wide variety of RNA viruses in infected cells and animals.
“FDA-approved, broad-spectrum therapeutics offer the fastest way to respond to dangerous and potentially lethal viruses,” said Dr. Tyler Bennett, Assistant Product Manager for BD Tx.
MediVector is overseeing the clinical trials required by the FDA to obtain drug licensure. The process requires safety data from at least 1,500 patients treated for flu at the dose and duration proposed for marketing of the drug. Currently, 150 trial sites are planned throughout the U.S.
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^Oestereich L, Lüdtke A, Wurr S, Rieger T, Muñoz-Fontela C, Günther S (May 2014). “Successful treatment of advanced Ebola virus infection with T-705 (favipiravir) in a small animal model”. Antiviral Research. 105: 17–21. doi:10.1016/j.antiviral.2014.02.014. PMID24583123.
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It was developed by the German pharmaceutical company Bayer AG and is co-marketed in Japan by Bayer and Nippon Shinyaku Co. Ltd. under the trade name Baynas.
SYN
Science 1976,193163-5
Proc Natl Acad Sci USA 1975,72(8),2994-8
The synthesis of Bay u 3405 was carried out as follows: Reductive amination of 3-oxo-1,2,3,4-tetrahydrocarbazole (I) with S-phenethylamine (II) afforded a mixture of diastereomeric amines, of which the desired isomer (III) crystallized in high diastereomeric purity as the hydrogensulfate. Cleavage of the phenethyl group by transfer hydrogenolysis with amminium formate and palladium on charcoal yielded the enantiomerically pure (3R)-3-amino-1,2,3,4-tetrahydrocarbazole (IV). Sulfonylation of (IV) with 4-fluorobenzenesulfonyl chloride (V) to the sulfonamide (VI) followed by addition of acrylonitrile and subsequent hydrolysis gave Bay u 3405.
SYN
J Label Compd Radiopharm 1994,34(12),1207
The synthesis of [14C]-labeled Bay-u-3405 by two closely related ways has been described: 1) [14C]-Labeled aniline (I) is diazotized and reduced with sodium sulfite, yielding the labeled hydrazine (II), which is condensed with the monoketal of cyclohexane-1,4-dione (III) under Fisher’s indole synthesis (ZnCl2) to afford the tetrahydrocarbazole (IV). The hydrolysis of (IV) with HCl in THF/water yields 1,2,3,4-tetrahydrocarbazol-3-one (V), which is submitted to a reductive condensation with (S)-1-phenylethylamine (VI) by means of tetrabutylammonium borohydride, yielding preferentially the secondary amine (VII), which, after purification, is dealkylated with ammonium formate and Pd/C to afford 1,2,3,4-tetrahydrocarbazole-3(R)-amine (VIII). The acylation of (VIII) with 4-fluorophenylsulfonyl chloride (IX) gives the corresponding sulfonamide (X), which is condensed with acrylonitrile by means of NaH, yielding 3-[3(R)-(4-fluorophenylsulfonamido)-1,2,3,4-tetrahydrocarbazol-9-yl]pro pionitrile (XI). Finally, this compound is hydrolyzed in the usual way. 2) The condensation of the sulfonamide (X) with methyl acrylate by means of NaH as before gives 3-[3(R)-(4-fluorophenylsulfonamido)-1,2,3,4-tetrahydrocarbazol-9-yl]propionic acid methyl ester (XII), which is finally hydrolyzed in the usual way.
This invention relates to 2-amino- tetrahydrocarbazole-propanoic acid and a new process for its synthesis .
2-Amino-tetrahydrocarbazole-propanoic acid is a key intermediate for the synthesis of Ramatroban, a thromboxaneA2 receptor (TP) antagonist with clinical efficacy in asthma and allergic rhinitis.
US Patent 4988820 discloses the synthesis of this compound stating from compound 1, which is condensed with phenylhydrazine and ring-closed to give indole 2. Deprotection of 2 using acid provides ketone 3. Reductive amination of ketone with s-phenylethylamine in the presence of tetrabutylammonium borohydride provides compound 4, which undergoes palladium catalyzed hydrogenation to give key intermediate 5.
Ramatroban
The process, however, has disadvantages: the starting material 1 is relatively expensive, and the yield of the amination step is only 40% and needs expensive tetrabutylammonium borohydride as the reducing agent. And also the subsequent hydrogenation provides only 70% of the desired compound 5. [0006] US Patent 4988820 also describes an alternative synthesis of compound 5 starting from compound 6, which is oxidized by chromium trioxide to afford ketone 7. Condensation of compound 7 with phenylhydrazine and ring closure give indole 8. The subsequent hydrolysis using HCl provides indole 9. The intermediate 5 is obtained by resolution of racemic 9 using ( + ) -mandelic acid as the resolving agent.
9 5
However, this process has crucial disadvantages: the first step oxidation reaction needs the heavy metal reagent chromium trioxide, which is toxic and expensive, and the resolution of indole 9 using (+) -mandelic acid affords only -10 % of compound 5.
US Patent 5684158 discloses the synthesis of 2- amino-tetrahydrocarbazole-propanoic acid ethyl ester 10 by the alkylation of compound 5 in the presence of about 1 mol of alkali metal hydroxides and phase-transfer catalysts such as potassium hydroxide and benzyltriethylammonium chloride.
The problem with this reaction is that the insoluble material in the reaction mixture becomes very sticky during the reaction. The reaction mixture must be filtered in hot solvent in order to remove insoluble material during work up and the sticky material tents to block the filtration. [0010] Therefore, there is a great need for a new process for the synthesis of 2-amino-tetrahydrocarbazole- propanoic acid.
0.91 g 9-(2-Cyanoethyl)-4-[N-(4-fluorphenylsulfonyl)-N-(2-cyanoethyl)aminomethyl]-1,2,3,4-tetrahydrocarbazol be hydrolyzed analogously to Example 7.One obtains 0.77 g (89% of theory) of crystalline product as the sodium salt.
5.8 g (0.0128 mol) of Example 67 are dissolved in 60 ml isopropanol, treated with 130 ml of 10% potassium hydroxide solution, after 16 hours heating under reflux, is cooled, diluted with water and extracted with ethyl acetate.The aqueous phase is concentrated in vacuo and then treated dropwise with vigorous stirring with conc.Hydrochloric acid.The case precipitated acid is filtered off, washed with water and dried thoroughly in vacuo.Obtained 4.4 g (86.6% of theory) of the product..: Mp 85-95 ° C rotation [α] 20 = 42.55 ° (CHCl 3) D
The preparation of Example 70 from Example 68 is carried out analogously to the preparation of Example 69 from Example 67. m.p .: 85-95 ° C optical rotation: [α] 20 = -37.83 ° (CHCl 3) D
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Synthesis pathway
Synthesis of a)
Trade names
Country
Trade name
Manufacturer
Japan
Baynas
Bayer
Ukraine
no
no
Formulations
50 mg tablet 75 mg
Reference
DE 3631824 (Bayer AG; appl. 19.9.1986; prior. 21.2.1986).
EP 728 743 (Bayer AG; appl. 14.2.1996; D-prior. 27.2.1995).
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Patent
Submitted
Granted
Phenylsulfonamid substituted pyridinealken- and aminooxyalkan-carboxylic-acid derivatives. [EP0471259]
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