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CAS Name: (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-O-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-b-D-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one
Additional Names:N-methyl-11-aza-10-deoxo-10-dihydroerythromycin A; 9-deoxo-9a-methyl-9a-aza-9a-homoerythromycin A
Molecular Formula: C38H72N2O12
Molecular Weight: 748.98
Percent Composition: C 60.94%, H 9.69%, N 3.74%, O 25.63%
Literature References: Semi-synthetic macrolide antibiotic; related to erythromycin A, q.v. Prepn: BE892357; G. Kobrehel, S. Djokic, US4517359 (1982, 1985 both to Sour Pliva); of the crystalline dihydrate: D. J. M. Allen, K. M. Nepveux, EP298650; eidem, US6268489 (1989, 2001 both to Pfizer). Antibacterial spectrum: S. C. Aronoff et al.,J. Antimicrob. Chemother.19, 275 (1987); and mode of action: J. Retsema et al.,Antimicrob. Agents Chemother.31, 1939 (1987). Series of articles on pharmacology, pharmacokinetics, and clinical experience: J. Antimicrob. Chemother.31, Suppl. E, 1-198 (1993). Clinical trial in prevention of Pneumocystis carinii pneumonia in AIDS patients: M. W. Dunne et al.,Lancet354, 891 (1999). Review of pharmacology and clinical efficacy in pediatric infections: H. D. Langtry, J. A. Balfour, Drugs56, 273-297 (1998).
Azithromycin was discovered 1980 by Pliva, and approved for medical use in 1988. It is on the World Health Organization’s List of Essential Medicines, the safest and most effective medicines needed in a health system. The World Health Organization classifies it as critically important for human medicine. It is available as a generic medication and is sold under many trade names worldwide. The wholesale cost in the developing world is about US$0.18 to US$2.98 per dose. In the United States, it is about US$4 for a course of treatment as of 2018. In 2016, it was the 49th most prescribed medication in the United States with more than 15 million prescriptions.
Azithromycin is used to treat many different infections, including:
Prevention and treatment of acute bacterial exacerbations of chronic obstructive pulmonary disease due to H. influenzae, M. catarrhalis, or S. pneumoniae. The benefits of long-term prophylaxis must be weighed on a patient-by-patient basis against the risk of cardiovascular and other adverse effects.
Community-acquired pneumonia due to C. pneumoniae, H. influenzae, M. pneumoniae, or S. pneumoniae
Uncomplicated skin infections due to S. aureus, S. pyogenes, or S. agalactiae
Urethritis and cervicitis due to C. trachomatis or N. gonorrhoeae. In combination with ceftriaxone, azithromycin is part of the United States Centers for Disease Control-recommended regimen for the treatment of gonorrhea. Azithromycin is active as monotherapy in most cases, but the combination with ceftriaxone is recommended based on the relatively low barrier to resistance development in gonococci and due to frequent co-infection with C. trachomatis and N. gonorrhoeae.
Genital ulcer disease (chancroid) in men due to H. ducrey
Acute bacterial sinusitis due to H. influenzae, M. catarrhalis, or S. pneumoniae. Other agents, such as amoxicillin/clavulanate are generally preferred, however.
Acute otitis media caused by H. influenzae, M. catarrhalis or S. pneumoniae. Azithromycin is not, however, a first-line agent for this condition. Amoxicillin or another beta lactam antibiotic is generally preferred.
Pharyngitis or tonsillitis caused by S. pyogenes as an alternative to first-line therapy in individuals who cannot use first-line therapy
Azithromycin has relatively broad but shallow antibacterial activity. It inhibits some Gram-positive bacteria, some Gram-negative bacteria, and many atypical bacteria.
A strain of gonorrhea reported to be highly resistant to azithromycin was found in the population in 2015. Neisseria gonorrhoeae is normally susceptible to azithromycin, but the drug is not widely used as monotherapy due to a low barrier to resistance development. Extensive use of azithromycin has resulted in growing Streptococcus pneumoniae resistance.
Aerobic and facultative Gram-positive microorganisms
No harm has been found with use during pregnancy. However, there are no adequate well-controlled studies in pregnant women.
Safety of the medication during breastfeeding is unclear. It was reported that because only low levels are found in breast milk and the medication has also been used in young children, it is unlikely that breastfed infants would suffer adverse effects. Nevertheless, it is recommended that the drug be used with caution during breastfeeding.
Azithromycin appears to be effective in the treatment of COPD through its suppression of inflammatory processes. And potentially useful in asthma and sinusitis via this mechanism. Azithromycin is believed to produce its effects through suppressing certain immune responses that may contribute to inflammation of the airways.
Most common adverse effects are diarrhea (5%), nausea (3%), abdominal pain (3%), and vomiting. Fewer than 1% of people stop taking the drug due to side effects. Nervousness, skin reactions, and anaphylaxis have been reported.Clostridium difficile infection has been reported with use of azithromycin. Azithromycin does not affect the efficacy of birth control unlike some other antibiotics such as rifampin. Hearing loss has been reported.
Occasionally, people have developed cholestatic hepatitis or delirium. Accidental intravenous overdose in an infant caused severe heart block, resulting in residual encephalopathy.
In 2013 the FDA issued a warning that azithromycin “can cause abnormal changes in the electrical activity of the heart that may lead to a potentially fatal irregular heart rhythm.” The FDA noted in the warning a 2012 study that found the drug may increase the risk of death, especially in those with heart problems, compared with those on other antibiotics such as amoxicillin or no antibiotic. The warning indicated people with preexisting conditions are at particular risk, such as those with QT interval prolongation, low blood levels of potassium or magnesium, a slower than normal heart rate, or those who use certain drugs to treat abnormal heart rhythms.
Azithromycin is an acid-stable antibiotic, so it can be taken orally with no need of protection from gastric acids. It is readily absorbed, but absorption is greater on an empty stomach. Time to peak concentration (Tmax) in adults is 2.1 to 3.2 hours for oral dosage forms. Due to its high concentration in phagocytes, azithromycin is actively transported to the site of infection. During active phagocytosis, large concentrations are released. The concentration of azithromycin in the tissues can be over 50 times higher than in plasma due to ion trapping and its high lipid solubility. Azithromycin’s half-life allows a large single dose to be administered and yet maintain bacteriostatic levels in the infected tissue for several days.
Following a single dose of 500 mg, the apparent terminal elimination half-life of azithromycin is 68 hours.Biliary excretion of azithromycin, predominantly unchanged, is a major route of elimination. Over the course of a week, about 6% of the administered dose appears as unchanged drug in urine.
A team of researchers at the pharmaceutical company Pliva in Zagreb, SR Croatia, Yugoslavia, — Gabrijela Kobrehel, Gorjana Radobolja-Lazarevski, and Zrinka Tamburašev, led by Dr. Slobodan Đokić — discovered azithromycin in 1980. It was patented in 1981. In 1986, Pliva and Pfizer signed a licensing agreement, which gave Pfizer exclusive rights for the sale of azithromycin in Western Europe and the United States. Pliva put its azithromycin on the market in Central and Eastern Europe under the brand name Sumamed in 1988. Pfizer launched azithromycin under Pliva’s license in other markets under the brand name Zithromax in 1991. Patent protection ended in 2005.
It is available as a generic medication. The wholesale cost is about US$0.18 to US$2.98 per dose. In the United States it is about US$4 for a course of treatment as of 2018. In India, it is about US$1.70 for a course of treatment.
In 2010, azithromycin was the most prescribed antibiotic for outpatients in the US, whereas in Sweden, where outpatient antibiotic use is a third as prevalent, macrolides are only on 3% of prescriptions.
^World Health Organization (2019). World Health Organization model list of essential medicines: 21st list 2019. Geneva: World Health Organization. hdl:10665/325771. WHO/MVP/EMP/IAU/2019.06. License: CC BY-NC-SA 3.0 IGO.
^Zarogoulidis, P.; Papanas, N.; Kioumis, I.; Chatzaki, E.; Maltezos, E.; Zarogoulidis, K. (May 2012). “Macrolides: from in vitro anti-inflammatory and immunomodulatory properties to clinical practice in respiratory diseases”. European Journal of Clinical Pharmacology. 68 (5): 479–503. doi:10.1007/s00228-011-1161-x. ISSN1432-1041. PMID22105373.
^Mori F, Pecorari L, Pantano S, Rossi M, Pucci N, De Martino M, Novembre E (2014). “Azithromycin anaphylaxis in children”. Int J Immunopathol Pharmacol. 27 (1): 121–6. doi:10.1177/039463201402700116. PMID24674687.
^Dart, Richard C. (2004). Medical Toxology. Lippincott Williams & Wilkins. p. 23.
^Tilelli, John A.; Smith, Kathleen M.; Pettignano, Robert (2006). “Life-Threatening Bradyarrhythmia After Massive Azithromycin Overdose”. Pharmacotherapy. 26 (1): 147–50. doi:10.1592/phco.2006.26.1.147. PMID16506357.
^Baselt, R. (2008). Disposition of Toxic Drugs and Chemicals in Man (8th ed.). Foster City, CA: Biomedical Publications. pp. 132–133.
^Hicks, LA; Taylor TH, Jr; Hunkler, RJ (April 2013). “U.S. outpatient antibiotic prescribing, 2010”. The New England Journal of Medicine. 368 (15): 1461–1462. doi:10.1056/NEJMc1212055. PMID23574140.
^Hicks, LA; Taylor TH, Jr; Hunkler, RJ (September 2013). “More on U.S. outpatient antibiotic prescribing, 2010”. The New England Journal of Medicine. 369 (12): 1175–1176. doi:10.1056/NEJMc1306863. PMID24047077.
Substances Referenced in Synthesis Path CAS-RN Formula Chemical Name CAS Index Name 76801-85-9 C37H70N2O12 2-deoxo-9a-aza-9a-homoerythromycin A 1-Oxa-6-azacyclopentadecan-15-one, 13-[(2,6-dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-2-eth- yl-3,4,10-trihydroxy-3,5,8,10,12,14-hexamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-hexopyranosyl]oxy]-, [2R-(2 R*,3S*,4R*,5R*,8R*,10R*,11R*,12S*,13S*,1 4R*)]-
Molnupiravir (development codes MK-4482 and EIDD-2801) is an experimental antiviral drug which is orally active (can be taken orally) and was developed for the treatment of influenza. It is a prodrug of the synthetic nucleoside derivative N4-hydroxycytidine, and exerts its antiviral action through introduction of copying errors during viral RNA replication. Activity has also been demonstrated against coronaviruses including SARS, MERS and SARS-CoV-2.
The drug was developed at Emory University by the university’s drug innovation company, Drug Innovation Ventures at Emory (DRIVE). It was then acquired by Miami-based company Ridgeback Biotherapeutics, who later partnered with Merck & Co. to develop the drug further.
In April 2020, a whistleblower complaint by former Head of US Biomedical Advanced Research and Development Authority (BARDA) Rick Bright revealed concerns over providing funding for the further development of molnupiravir due to similar drugs having mutagenic properties (producing birth defects). A previous company, Pharmasset, that had investigated the drug’s active ingredient had abandoned it. These claims were denied by George Painter, CEO of DRIVE, noting that toxicity studies on molnupiravir had been carried out and data provided to regulators in the US and UK, who permitted safety studies in humans to move forward in the spring of 2020. Also at this time, DRIVE and Ridgeback Biotherapeutics stated they planned future safety studies in animals.
After being found to be active against SARS-CoV-2 in March 2020, molnupiravir was tested in a preliminary human study for “Safety, Tolerability, and Pharmacokinetics” in healthy volunteers in the UK and US. In June 2020, Ridgeback Biotherapeutics announced it was moving to Phase II trials to test the efficacy of the drug as a treatment for COVID-19. Two trials of small numbers of hospitalized and non-hospitalized patients in the US and the UK were underway in July. In late July 2020, and without yet releasing any medical data, Merck, which had been partnering with Ridgeback Biotherapeutics on developing the drug, announced its intention to move molnupiravir to late stage trials beginning in September 2020. On October 19 2020, Merck began a one year Stage 2/3 trial focused on hospitalized patients.
A 1L round bottom flask was charged with uridine (25 g, 102.38 mmol) and acetone (700 mL). The reaction mixture was allowed to stir at rt. The slurry was then treated with sulfuric acid (0.27 mL, 5.12 mmol). Stirring was allowed to continue at rt for 18 hours. The reaction was quenched with 100 mL of trimethylamine and was used in the next step without further pruficication.
A 1L round bottom flask was charged with the reaction mixture from the previous reaction. Triethylamine (71.09 mL, 510.08 mmol) and 4-dimethylaminopyridine (0.62 g, 5.1 mmol) were then added. The flask was cooled using an ice bath and then 2-methylpropanoyl 2-methylpropanoate (17.75 g, 112.22 mmol) was slowly added. The reaction mixture was allowed to stir at rt until the reaction was complete. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in 600 mL ethyl acetate and washed with saturated aqueous bicarbonate solution x 2, water x 2 and brine x 2. The organics were dried over sodium sulfate and concentrated under reduced pressure to yield a clear colorless oil. The crude product was used in the next step without further purification.
A 1L round bottom flask was charged with the crude product from above (36 g, 101.59 mmol) and MeCN (406.37 mL). The reaction mixture was allowed to stir until all the starting material was dissolved. Next, 1,2, 4-triazole (50.52 g, 731.46 mmol) was added followed by the addition of N,N-diethylethanamine (113.28 mL, 812.73 mmol). The reaction mixture was allowed to stir at rt until all solids dissolved. The reaction was then cooled to 0°C using an ice bath. Phosphorous oxychloride (24.44 mL, 152.39 mmol) was added slowly. The slurry that formed was allowed to stir under argon while slowly warming to rt. The reaction was then allowed to stir until complete by TLC (EtOAc). The reaction was then quenched by the addition of lOOmL of water. The slurry then became a dark colored solution, which was
then concentrated under reduced pressure. The residue was dissolved in DCM and washed with water and brine. The organics were then dried over sodium sulfate, filtered, and concentrated under reduced pressure. The product was purified by silica gel chromatography (2 x 330 g columns). All fractions containing product were collected and concentrated under reduced pressure.
A 500 mL round bottom flask was charged with the product from the previous step (11.8 g, 29.11 mmol) and isopropyl alcohol (150 mL). The reaction mixture was allowed to stir at rt until all solids dissolved. Next, hydroxylamine (1.34 mL, 43.66 mmol) was added and stirring continued at ambient temperature. When the reaction was complete (HPLC) some solvent was removed under high vacuum at ambient temperature. The remaining solvent was removed under reduced pressure at 45°C. The resulting residue was dissolved in EtOAc and was washed with water and brine. The organics were dried over sodium sulfate, filtered, and concentrated under reduced pressure to yield oil. Crystals formed upon standing at rt. The crystals were collected by filtration, washed with ether x 3, and dried in vacuo to provide the product as a white solid.
A 200 mL round bottom flask was charged with the product from the previous step (6.5 g, 17.6 mmol) and formic acid (100 mL, 2085.6 mmol). The reaction mixture was allowed to stir at rt overnight. The progress of the reaction was monitored by HPLC. The reaction mixture was concentrated under reduced pressure at 42°C to yield a clear, pale pink oil. Next, 30 mL of ethanol was added. Solvent was then removed under reduced pressure. MTBE (50 mL) was added to the solid and heated. Next, isopropyl alcohol was added and heating was continued until all solid material dissolved (5 mL). The solution was then allowed to cool and stand at rt.
A solid started to form after about lhr. The solids were collected by filtration, washed with MTBE, and dried in vacuo to yield the EIDD-2801 as a white solid. The filtrate was concentrated under reduced pressure to yield a sticky solid, which was dissolved in a small amount of isopropyl alcohol with heating. The solution was allowed to stand at rt overnight. A solid formed in the flask, which was collected by filtration, rinsed with isopropyl alcohol and MTBE, and dried in vacuo to an additional crop of desired product.
EIDD-2801 (25 g) was dissolved in 250 mL of isopropyl alcohol by heating to 70°C to give a clear solution. The warm solution was polish filtered and filtrate transferred to 2L three neck flask with overhead stirrer. It was warmed back to 70°C and MTBE (250 mL) was slowly added into the flask. The clear solution was seeded and allowed to cool slowly to rt with stirring for 18 hrs. The EIDD-2801 solid that formed was filtered and washed with MTBE and dried at 50°C under vacuum for l8hours. The filtrate was concentrated, redissolved in 50 mL isopropyl alcohol and 40 mL MTBE by warming to give clear solution and allowed to stand at rt to give a second crop of EIDD-2801.
Example 11: General synthesis for Deuteration
The lactone 389 (0.0325 mol) was added to a dry flask under an argon atmosphere and was then dissolved in dry THF (250 mL). The solution as then cooled to -78°C and a DIBAL-D solution in toluene (0.065 mol) was dropwise. The reaction was allowed to stir at -78°C for 3-4 hours. The reaction was then quenched with the slow addition of water (3 mL). The reaction was then allowed to stir while warming to rt. The mixture was then diluted with two volumes of diethyl ether and was then poured into an equal volume of saturated sodium potassium tartrate solution. The organic layer was separated, dried over MgSCri. filtered, and concentrated under reduced pressure. The residue was purified on silica eluting with hexanes/ethyl acetate. The resulting lactol 390 was then converted to an acetate or benzolyate and subjected to cytosine coupling conditions and then further elaborated to N-hydroxycytidine.
A new route to MK-4482 was developed. The route replaces uridine with the more available and less expensive cytidine. Low-cost, simple reagents are used for the chemical transformations, and the yield is improved from 17% to 44%. A step is removed from the longest linear sequence, and these advancements are expected to expand access to MK-4482 should it become a viable drug substance.
To a 20 mL vial was added N-hydroxycytidine acetonide ester 5 (0.25 g, 96% purity) followed by formic acid (4 mL). The resultant solution was stirred at room temperature for 4 h 20 min. Solvent was removed under reduced pressure and fresh EtOH (5 mL) was added. The resultant solution was again concentrated under vacuum to afford an oil. Methyl tert-butyl ether and IPA (5 mL each) were successively added as described earlier for preparation of compound 4 and concentrated to give 0.205 g of crude material (77% assay yield, 79% purity). This material was purified by silica gel column chromatography in 8 % MeOH/ Chloroform to afford 130 mg of EIDD-2801 as a solid (60% isolated yield corrected for purity, 98% purity) 1H NMR (600 MHz, CD3OD): δ 6.91 (d, J = 8.2 Hz, 1H), 5.82 (d, J = 4.8 Hz, 1H), 5.61 (d, J = 8.2 Hz, 1H), 4.29 (d, J = 3.6 Hz, 2H), 4.14 (t, J = 4.9 Hz, 1H), 4.08 (p, J = 4.9 Hz, 2H), 2.62 (septet, J = 7.0 Hz, 1H), 1.19 (d, J = 7.0 Hz, 6H); 13C NMR (151 MHz, CD3OD): δ 178.6, 151.81, 146.44, 132.04, 99.84, 90.74, 82.88, 74.67, 71.80, 65.23, 35.45, 27.49, 19.65, 19.61.
One-Pot Transamination/Deprotection of 4 to EIDD-2801: To acetonide ester 4 (1.03 g, 77% Purity) in a 100 mL single neck round bottom flask was added hydroxylamine sulfate (1.09 g, 3.2 equiv.) followed by 40% IPA (20 mL prepared by mixing 12 mL of water and 8 mL of 99.5% IPA. The resultant solution was heated to 78˚C (internal temperature 72-73 ˚C) for 23 h upon which time HPLC showed the formation of EIDD-2801. Solvent was removed on a rotary evaporator and isopropanol (20 mL) was then added. The resulting slurry was sonicated for 5 minutes. The insoluble residue was then filtered and the filtrate concentrated under reduced pressure to afford crude material. (1.34 g, 38% purity, 69% assay yield). The resultant material was purified by silica gel chromatography (5-6% MeOH/DCM) to provide pure EIDD-2801 as two fractions (0.26 g, >99% purity, 36% corrected yield) as an yellow solid and 0.27 g (69.5% purity, 26% corrected yield) as a pinkish solid. The lower purity material was subjected to a second column purification again using 7% MeOH/ DCM to afford 0.137 g of material with 90% purity by NMR. The combined yield thus was estimated to be 53%. The 1H NMR spectrum of the product thus obtained matched the one obtained in the sequential approach as outlined above.
C. Oliver Kappe, Doris Dallinger, University of Graz, Austria, and colleagues have developed an improved synthesis of EIDD-2801 from uridine (pictured below) by strategically reordering the synthetic steps. The reaction sequence starts with the activation of uridine with 1,2,4-triazole and continues with a telescoped acetonide protection/esterification and a telescoped hydroxyamination/acetonide deprotection. Telescoped reaction sequences consist of two or more than one one-pot procedures that are performed back-to-back without a work-up step in-between. A continuous flow process was used for the final acetonide deprotection, which improved selectivity and reproducibility.
^Toots M, Yoon JJ, Hart M, Natchus MG, Painter GR, Plemper RK (April 2020). “Quantitative efficacy paradigms of the influenza clinical drug candidate EIDD-2801 in the ferret model”. Translational Research. 218: 16–28. doi:10.1016/j.trsl.2019.12.002. PMID31945316.
Emory, collaborators testing antiviral drug as potential treatment for coronaviruses
An antiviral compound discovered at Emory University could potentially be used to treat the new coronavirus associated with the outbreak in China and spreading around the globe. Drug Innovation Ventures at Emory (DRIVE), a non-profit LLC wholly owned by Emory, is developing the compound, designated EIDD-2801.
In testing with collaborators at the University of North Carolina at Chapel Hill and Vanderbilt University Medical Center, the active form of EIDD-2801, which is called EIDD-1931, has shown efficacy against the related coronaviruses SARS (Severe Acute Respiratory Syndrome)- and MERS-CoV (Middle East Respiratory Syndrome Coronavirus). Some of the data was recently published in Journal of Virology.
EIDD-2801 is an oral ribonucleoside analog that inhibits the replication of multiple RNA viruses, including respiratory syncytial virus, influenza, chikungunya, Ebola, Venezuelan equine encephalitis virus, and Eastern equine encephalitis viruses.
“We have been planning to enter human clinical tests of EIDD-2801 for the treatment of influenza, and recognized that it has potential activity against the current novel coronavirus,” says George Painter, PhD, director of the Emory Institute for Drug Development (EIDD) and CEO of DRIVE. “Based on the drug’s broad-spectrum activity against viruses including influenza, Ebola and SARS-CoV/MERS-CoV, we believe it will be an excellent candidate.”
“Our studies in the Journal of Virology show potent activity of the EIDD-2801 parent compound against multiple coronaviruses including SARS and MERS,” says Mark Denison, MD, the Stahlman Professor of Pediatrics and director of pediatric infectious diseases at Vanderbilt University School of Medicine. “It also has a strong genetic barrier to development of viral resistance, and its oral bioavailability makes it a candidate for use during an outbreak.”
“Generally speaking, seasonal flu is still a much more common threat than this coronavirus, however, novel emerging coronaviruses represent a considerable threat to global health as evidenced by the new 2019-nCoV,” said Ralph Baric, PhD, an epidemiology professor at the University of North Carolina’s Gillings School of Global Public Health. “But the reason the new coronavirus is so concerning is that it’s much more likely to be deadly than the flu – fatal for about one in 25 people versus one in 1,000 for the flu.”
The development of EIDD-2801 has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases (NIAID), under contract numbers HHSN272201500008C and 75N93019C00058, and from the Defense Threat Reduction Agency (DTRA), under contract numbers HDTRA1-13-C-0072 and HDTRA1-15-C-0075, for the treatment of Influenza, coronavirus, chikungunya, and Venezuelan equine encephalitis virus.
About DRIVE: DRIVE is a non-profit LLC wholly owned by Emory started as an innovative approach to drug development. Operating like an early stage biotechnology company, DRIVE applies focus and industry development expertise to efficiently translate discoveries to address viruses of global concern. Learn more at: http://driveinnovations.org/
Emory-discovered antiviral is poised for COVID-19 clinical trials
The nucleoside inhibitor has advantages over Gilead’s remdesivir but has yet to be tested in humans
Asmall-molecule antiviral discovered by Emory University chemists could soon start human testing against COVID-19, the respiratory disease caused by the novel coronavirus. That’s the plan of Ridgeback Biotherapeutics, which licensed the compound, EIDD-2801, from an Emory nonprofit.
EIDD-2801 works similarly to Gilead Sciences’ remdesivir, an unapproved drug that was developed for the Ebola virus and is being studied in five Phase III trials against COVID-19. Both molecules are nucleoside analogs that metabolize into an active form that blocks RNA polymerase, an essential component of viral replication.
But remdesivir can only be given intravenously, meaning it would be difficult to deploy widely. In contrast, EIDD-2801 can be taken in pill form, says Mark Denison, a coronavirus expert and director of the infectious diseases division at Vanderbilt Medical School. Denison partnered with Emory and researchers at the University of North Carolina to test the compound against coronaviruses.
EIDD-2801 has other promising features. Many antivirals work by introducing errors into the viral genome, but, unlike other viruses, coronaviruses can fix some mistakes. In lab experiments, EIDD-2801 “was able to overcome the coronavirus proofreading function,” Denison says.
He also notes that while remdesivir and EIDD-2801 both block RNA polymerase, they appear to do it in different ways, meaning they could be complementary.
Unlike remdesivir, EIDD-2801 lacks human safety data. Ridgeback founder and CEO Wendy Holman says she expects the US Food and Drug Administration to give the green light for a Phase I study in COVID-19 infections within “weeks, not months.”
Percent Composition: C 67.59%, H 8.19%, Cl 11.08%, N 13.14%
Literature References: Prepd by the condensation of 4,7-dichloroquinoline with 1-diethylamino-4-aminopentane: DE683692 (1939); H. Andersag et al.,US2233970 (1941 to Winthrop); Surrey, Hammer, J. Am. Chem. Soc.68, 113 (1946). Review: Hahn in Antibioticsvol. 3, J. W. Corcoran, F. E. Hahn, Eds. (Springer-Verlag, New York, 1975) pp 58-78. Comprehensive description: D. D. Hong, Anal. Profiles Drug Subs.5, 61-85 (1976). Comparative clinical trial with dapsone in rheumatoid arthritis: P. D. Fowler et al.,Ann. Rheum. Dis.43, 200 (1984); with penicillamine: T. Gibson et al.,Br. J. Rheumatol.26, 279 (1987).
Percent Composition: C 41.91%, H 6.25%, Cl 6.87%, N 8.15%, P 12.01%, O 24.81%
Properties: Bitter, colorless crystals. Dimorphic. One modification, mp 193-195°; the other, mp 215-218°. Freely sol in water; pH of 1% soln about 4.5; less sol at neutral and alkaline pH. Stable to heat in solns of pH 4.0 to 6.5. Practically insol in alcohol, benzene, chloroform, ether.
Chloroquine is a medication used primarily to prevent and to treat malaria in areas where that parasitic disease is known to remain sensitive to its effects. A benefit of its use in therapy, when situations allow, is that it can be taken by mouth (versus by injection). Controlled studies of cases involving human pregnancy are lacking, but the drug may be safe for use for such patients.[verification needed] However, the agent is not without the possibility of serious side effects at standard doses, and complicated cases, including infections of certain types or caused by resistant strains, typically require different or additional medication. Chloroquine is also used as a medication for rheumatoid arthritis, lupus erythematosus, and other parasitic infections (e.g., amebiasis occurring outside of the intestines). Beginning in 2020, studies have proceeded on its use as a coronavirusantiviral, in possible treatment of COVID-19.
Distribution of malaria in the world:
♦ Elevated occurrence of chloroquine- or multi-resistant malaria
♦ Occurrence of chloroquine-resistant malaria
♦ No Plasmodium falciparum or chloroquine-resistance
♦ No malaria
In treatment of amoebic liver abscess, chloroquine may be used instead of or in addition to other medications in the event of failure of improvement with metronidazole or another nitroimidazole within 5 days or intolerance to metronidazole or a nitroimidazole.
Chloroquine-induced itching is very common among black Africans (70%), but much less common in other races. It increases with age, and is so severe as to stop compliance with drug therapy. It is increased during malaria fever; its severity is correlated to the malaria parasite load in blood. Some evidence indicates it has a genetic basis and is related to chloroquine action with opiate receptors centrally or peripherally.
Unpleasant metallic taste
This could be avoided by “taste-masked and controlled release” formulations such as multiple emulsions.
This manifests itself as either conduction disturbances (bundle-branch block, atrioventricular block) or Cardiomyopathy – often with hypertrophy, restrictive physiology, and congestive heart failure. The changes may be irreversible. Only two cases have been reported requiring heart transplantation, suggesting this particular risk is very low. Electron microscopy of cardiac biopsies show pathognomonic cytoplasmic inclusion bodies.
Chloroquine has not been shown to have any harmful effects on the fetus when used for malarial prophylaxis. Small amounts of chloroquine are excreted in the breast milk of lactating women. However, this drug can be safely prescribed to infants, the effects are not harmful. Studies with mice show that radioactively tagged chloroquine passed through the placenta rapidly and accumulated in the fetal eyes which remained present five months after the drug was cleared from the rest of the body. Women who are pregnant or planning on getting pregnant are still advised against traveling to malaria-risk regions.
There is not enough evidence to determine whether chloroquine is safe to be given to people aged 65 and older. Since it is cleared by the kidneys, toxicity should be monitored carefully in people with poor kidney functions.
Chloroquine is very dangerous in overdose. It is rapidly absorbed from the gut. In 1961, a published compilation of case reports contained accounts of three children who took overdoses and died within 2.5 hours of taking the drug. While the amount of the overdose was not stated, the therapeutic index for chloroquine is known to be small. One of the children died after taking 0.75 or 1 gram, or twice a single therapeutic amount for children. Symptoms of overdose include headache, drowsiness, visual disturbances, nausea and vomiting, cardiovascular collapse, seizures, and sudden respiratory and cardiac arrest.
An analog of chloroquine – hydroxychloroquine – has a long half-life (32–56 days) in blood and a large volume of distribution (580–815 L/kg). The therapeutic, toxic and lethal ranges are usually considered to be 0.03 to 15 mg/l, 3.0 to 26 mg/l and 20 to 104 mg/l, respectively. However, nontoxic cases have been reported up to 39 mg/l, suggesting individual tolerance to this agent may be more variable than previously recognised.
Chloroquine’s absorption of the drug is rapid. It is widely distributed in body tissues. It’s protein binding is 55%.[ It’s metabolism is partially hepatic, giving rise to its main metabolite, desethylchloroquine. It’s excretion os ≥50% as unchanged drug in urine, where acidification of urine increases its elimination It has a very high volume of distribution, as it diffuses into the body’s adipose tissue.
Accumulation of the drug may result in deposits that can lead to blurred vision and blindness. It and related quinines have been associated with cases of retinal toxicity, particularly when provided at higher doses for longer times. With long-term doses, routine visits to an ophthalmologist are recommended.
Chloroquine is also a lysosomotropic agent, meaning it accumulates preferentially in the lysosomes of cells in the body. The pKa for the quinoline nitrogen of chloroquine is 8.5, meaning—in simplified terms, considering only this basic site—it is about 10% deprotonated at physiological pH (per the Henderson-Hasselbalch equation) This decreases to about 0.2% at a lysosomal pH of 4.6.Because the deprotonated form is more membrane-permeable than the protonated form, a quantitative “trapping” of the compound in lysosomes results.
Mechanism of action
Hemozoin formation in P. falciparum: many antimalarials are strong inhibitors of hemozoin crystal growth.
The lysosomotropic character of chloroquine is believed to account for much of its antimalarial activity; the drug concentrates in the acidic food vacuole of the parasite and interferes with essential processes. Its lysosomotropic properties further allow for its use for in vitro experiments pertaining to intracellular lipid related diseases, autophagy, and apoptosis.
Inside red blood cells, the malarial parasite, which is then in its asexual lifecycle stage, must degrade hemoglobin to acquire essential amino acids, which the parasite requires to construct its own protein and for energy metabolism. Digestion is carried out in a vacuole of the parasitic cell.
Hemoglobin is composed of a protein unit (digested by the parasite) and a heme unit (not used by the parasite). During this process, the parasite releases the toxic and soluble molecule heme. The heme moiety consists of a porphyrin ring called Fe(II)-protoporphyrin IX (FP). To avoid destruction by this molecule, the parasite biocrystallizes heme to form hemozoin, a nontoxic molecule. Hemozoin collects in the digestive vacuole as insoluble crystals.
Chloroquine enters the red blood cell by simple diffusion, inhibiting the parasite cell and digestive vacuole. Chloroquine then becomes protonated (to CQ2+), as the digestive vacuole is known to be acidic (pH 4.7); chloroquine then cannot leave by diffusion. Chloroquine caps hemozoin molecules to prevent further biocrystallization of heme, thus leading to heme buildup. Chloroquine binds to heme (or FP) to form the FP-chloroquine complex; this complex is highly toxic to the cell and disrupts membrane function. Action of the toxic FP-chloroquine and FP results in cell lysis and ultimately parasite cell autodigestion.  Parasites that do not form hemozoin are therefore resistant to chloroquine.
Since the first documentation of P. falciparum chloroquine resistance in the 1950s, resistant strains have appeared throughout East and West Africa, Southeast Asia, and South America. The effectiveness of chloroquine against P. falciparum has declined as resistant strains of the parasite evolved. They effectively neutralize the drug via a mechanism that drains chloroquine away from the digestive vacuole. Chloroquine-resistant cells efflux chloroquine at 40 times the rate of chloroquine-sensitive cells; the related mutations trace back to transmembrane proteins of the digestive vacuole, including sets of critical mutations in the P. falciparum chloroquine resistance transporter (PfCRT) gene. The mutated protein, but not the wild-type transporter, transports chloroquine when expressed in Xenopus oocytes (frog’s eggs) and is thought to mediate chloroquine leak from its site of action in the digestive vacuole. Resistant parasites also frequently have mutated products of the ABC transporterP. falciparum multidrug resistance (PfMDR1) gene, although these mutations are thought to be of secondary importance compared to Pfcrt. Verapamil, a Ca2+ channel blocker, has been found to restore both the chloroquine concentration ability and sensitivity to this drug. Recently, an altered chloroquine-transporter protein CG2 of the parasite has been related to chloroquine resistance, but other mechanisms of resistance also appear to be involved. Research on the mechanism of chloroquine and how the parasite has acquired chloroquine resistance is still ongoing, as other mechanisms of resistance are likely.
Chloroquine has antiviral effects. It increases late endosomal or lysosomal pH, resulting in impaired release of the virus from the endosome or lysosome – release requires a low pH. The virus is therefore unable to release its genetic material into the cell and replicate.
Chloroquine also seems to act as a zinc ionophore, that allows extracellular zinc to enter the cell and inhibit viral RNA-dependent RNA polymerase.
In Peru the indigenous people extracted the bark of the Cinchona plant trees and used the extract (Chinchona officinalis) to fight chills and fever in the seventeenth century. In 1633 this herbal medicine was introduced in Europe, where it was given the same use and also began to be used against malaria. The quinoline antimalarial drug quinine was isolated from the extract in 1820, and chloroquine is an analogue of this.
Chloroquine was discovered in 1934, by Hans Andersag and coworkers at the Bayer laboratories, who named it “Resochin”. It was ignored for a decade, because it was considered too toxic for human use. During World War II, United States government-sponsored clinical trials for antimalarial drug development showed unequivocally that chloroquine has a significant therapeutic value as an antimalarial drug. It was introduced into clinical practice in 1947 for the prophylactic treatment of malaria.
Society and culture
Resochin tablet package
Chloroquine comes in tablet form as the phosphate, sulfate, and hydrochloride salts. Chloroquine is usually dispensed as the phosphate.
Brand names include Chloroquine FNA, Resochin, Dawaquin, and Lariago.
Chloroquine has been recommended by Chinese, South Korean and Italian health authorities for the treatment of COVID-19. These agencies noted contraindications for people with heart disease or diabetes. Both chloroquine and hydroxychloroquine were shown to inhibit SARS-CoV-2 in vitro, but a further study concluded that hydroxychloroquine was more potent than chloroquine, with a more tolerable safety profile. Preliminary results from a trial suggested that chloroquine is effective and safe in COVID-19 pneumonia, “improving lung imaging findings, promoting a virus-negative conversion, and shortening the disease course.” Self-medication with chloroquine has caused one known fatality.
On 24 March 2020, NBC News reported a fatality due to misuse of a chloroquine product used to control fish parasites.
In October 2004, a group of researchers at the Rega Institute for Medical Research published a report on chloroquine, stating that chloroquine acts as an effective inhibitor of the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro.
Chloroquine was being considered in 2003, in pre-clinical models as a potential agent against chikungunya fever.
^Mittra, Robert A.; Mieler, William F. (1 January 2013). Ryan, Stephen J.; Sadda, SriniVas R.; Hinton, David R.; Schachat, Andrew P.; Sadda, SriniVas R.; Wilkinson, C. P.; Wiedemann, Peter; Schachat, Andrew P. (eds.). Retina (Fifth Edition). W.B. Saunders. pp. 1532–1554 – via ScienceDirect.
^Cortegiani A, Ingoglia G, Ippolito M, Giarratano A, Einav S (March 2020). “A systematic review on the efficacy and safety of chloroquine for the treatment of COVID-19”. Journal of Critical Care. doi:10.1016/j.jcrc.2020.03.005. PMID32173110.
^World Health Organization (2019). World Health Organization model list of essential medicines: 21st list 2019. Geneva: World Health Organization. hdl:10665/325771. WHO/MVP/EMP/IAU/2019.06. License: CC BY-NC-SA 3.0 IGO.
^Ajayi AA (September 2000). “Mechanisms of chloroquine-induced pruritus”. Clinical Pharmacology and Therapeutics. 68 (3): 336. PMID11014416.
^Vaziri A, Warburton B (1994). “Slow release of chloroquine phosphate from multiple taste-masked W/O/W multiple emulsions”. Journal of Microencapsulation. 11 (6): 641–8. doi:10.3109/02652049409051114. PMID7884629.
^Hempelmann E (March 2007). “Hemozoin biocrystallization in Plasmodium falciparum and the antimalarial activity of crystallization inhibitors”. Parasitology Research. 100 (4): 671–6. doi:10.1007/s00436-006-0313-x. PMID17111179.
^Essentials of medical pharmacology fifth edition 2003, reprint 2004, published by-Jaypee Brothers Medical Publisher Ltd, 2003, KD Tripathi, pages 739,740.
^Alcantara LM, Kim J, Moraes CB, Franco CH, Franzoi KD, Lee S, et al. (June 2013). “Chemosensitization potential of P-glycoprotein inhibitors in malaria parasites”. Experimental Parasitology. 134 (2): 235–43. doi:10.1016/j.exppara.2013.03.022. PMID23541983.
^Savarino A, Boelaert JR, Cassone A, Majori G, Cauda R (November 2003). “Effects of chloroquine on viral infections: an old drug against today’s diseases?”. The Lancet. Infectious Diseases. 3(11): 722–7. doi:10.1016/s1473-3099(03)00806-5. PMID14592603.
^Krafts K, Hempelmann E, Skórska-Stania A (July 2012). “From methylene blue to chloroquine: a brief review of the development of an antimalarial therapy”. Parasitology Research. 111 (1): 1–6. doi:10.1007/s00436-012-2886-x. PMID22411634.
^Keyaerts E, Vijgen L, Maes P, Neyts J, Van Ranst M (October 2004). “In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine”. Biochemical and Biophysical Research Communications. 323 (1): 264–8. doi:10.1016/j.bbrc.2004.08.085. PMID15351731.
^Yao X, Ye F, Zhang M, Cui C, Huang B, Niu P, et al. (March 2020). “In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)”. Clinical Infectious Diseases. doi:10.1093/cid/ciaa237. PMID32150618.
^Keyaerts E, Vijgen L, Maes P, Neyts J, Van Ranst M (October 2004). “In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine”. Biochemical and Biophysical Research Communications. 323 (1): 264–8. doi:10.1016/j.bbrc.2004.08.085. PMID15351731.
^Savarino A, Boelaert JR, Cassone A, Majori G, Cauda R (November 2003). “Effects of chloroquine on viral infections: an old drug against today’s diseases?”. The Lancet. Infectious Diseases. 3(11): 722–7. doi:10.1016/S1473-3099(03)00806-5. PMID14592603.
Percent Composition: C 47.73%, H 2.47%, Cl 21.68%, N 8.56%, O 19.56%
Literature References: Prepn: GB824345 (1959 to Bayer), C.A.54, 15822b (1960). See also: E. Schraufstätter, R. Gönnert, US3079297; R. Strufe et al.,US3113067 (both 1963 to Bayer); Bekhli et al.,Med. Prom. SSSR1965, 25.
Properties: Pale yellow crystals, mp 225-230°. Practically insol in water. Sparingly sol in ethanol, chloroform, ether.
Melting point: mp 225-230°
Derivative Type: Ethanolamine salt
CAS Registry Number: 1420-04-8
Additional Names: Clonitrilide
Trademarks: Bayluscid (Bayer)
Molecular Formula: C13H8Cl2N2O4.C2H7NO
Molecular Weight: 388.20
Percent Composition: C 46.41%, H 3.89%, Cl 18.27%, N 10.82%, O 20.61%
Side effects include nausea, vomiting, abdominal pain, and itchiness. It may be used during pregnancy and appears to be safe for the baby. Niclosamide is in the anthelmintic family of medications. It works by blocking the uptake of sugar by the worm.
Side effects include nausea, vomiting, abdominal pain, constipation, and itchiness. Rarely, dizziness, skin rash, drowsiness, perianal itching, or an unpleasant taste occur. For some of these reasons, praziquantel is a preferable and equally effective treatment for tapeworm infestation.
Niclosamide’s metabolic effects are relevant to wide ranges of organisms, and accordingly it has been applied as a control measure to organisms other than tapeworms. For example, it is an active ingredient in some formulations such as Bayluscide for killing lamprey larvae, as a molluscide, and as a general purpose piscicide in aquaculture. Niclosamide has a short half-life in water in field conditions; this makes it valuable in ridding commercial fish ponds of unwanted fish; it loses its activity soon enough to permit re-stocking within a few days of eradicating the previous population. Researchers have found that niclosamide is effective in killing invasive zebra mussels in cool waters.
^World Health Organization (2019). World Health Organization model list of essential medicines: 21st list 2019. Geneva: World Health Organization. hdl:10665/325771. WHO/MVP/EMP/IAU/2019.06. License: CC BY-NC-SA 3.0 IGO.
^“Niclosamide”. International Drug Price Indicator Guide. Archived from the original on 10 May 2017. Retrieved 1 December 2016.
^Weinbach EC, Garbus J (1969). “Mechanism of action of reagents that uncouple oxidative phosphorylation”. Nature. 221 (5185): 1016–8. doi:10.1038/2211016a0. PMID4180173.
^Boogaard, Michael A. Delivery Systems of Piscicides “Request Rejected”(PDF). Archived(PDF) from the original on 2017-06-01. Retrieved 2017-05-30.
^Verdel K.Dawson (2003). “Environmental Fate and Effects of the Lampricide Bayluscide: a Review”. Journal of Great Lakes Research. 29 (Supplement 1): 475–492. doi:10.1016/S0380-1330(03)70509-7.
Percent Composition: C 46.90%, H 2.95%, N 13.67%, O 26.03%, S 10.44%
Literature References: Broad spectrum antiparasitic agent; inhibits pyruvate ferredoxin oxidoreductase. Prepn: J. F. Rossignol, R. Cavier, DE2438037; eidem,US3950351 (1975, 1976 both to S.P.R.L. Phavic); and antiparasitic activity: R. Cavier et al.,Eur. J. Med. Chem. – Chim. Ther.13, 539 (1978). Antibacterial spectrum in vitro: L Dubreuil et al.,Antimicrob. Agents Chemother.40, 2266 (1996). Toxicology: J. R. Murphy, J.-C. Friedmann, J. Appl. Toxicol.5, 49 (1985). Clinical pharmacokinetics: A. Stockis et al.,Int. J. Clin. Pharmacol. Ther.34, 349 (1996). Clinical trial in intestinal protozoan and helminthic infections: H. Abaza et al., Curr. Ther. Res.59, 116 (1998). Review of mechanism of action and clinical experience: H. M. Gilles, P. S. Hoffman, Trends Parasitol.18, 95-97 (2002).
Properties: Light yellow crystalline powder. Crystals from methanol, mp 202°. Poorly sol in ethanol. Practically insol in water. LD50 orally in male, female mice: 1350, 1380 mg/kg; in rats: >10 g/kg (Murphy, Friedmann).
Melting point: mp 202°
Toxicity data: LD50 orally in male, female mice: 1350, 1380 mg/kg; in rats: >10 g/kg (Murphy, Friedmann)
Nitazoxanide alone has shown preliminary evidence of efficacy in the treatment of chronic hepatitis B over a one-year course of therapy. Nitazoxanide 500 mg twice daily resulted in a decrease in serum HBV DNA in all of 4 HBeAg-positive patients, with undetectable HBV DNA in 2 of 4 patients, loss of HBeAg in 3 patients, and loss of HBsAg in one patient. Seven of 8 HBeAg-negative patients treated with nitazoxanide 500 mg twice daily had undetectable HBV DNA and 2 had loss of HBsAg. Additionally, nitazoxanide monotherapy in one case and nitazoxanide plus adefovir in another case resulted in undetectable HBV DNA, loss of HBeAg and loss of HBsAg. These preliminary studies showed a higher rate of HBsAg loss than any currently licensed therapy for chronic hepatitis B. The similar mechanism of action of interferon and nitazoxanide suggest that stand-alone nitazoxanide therapy or nitazoxanide in concert with nucleos(t)ide analogs have the potential to increase loss of HBsAg, which is the ultimate end-point of therapy. A formal phase Ⅱ study is being planned for 2009.
Chronic hepatitis C
Romark initially decided to focus on the possibility of treating chronic hepatitis C with nitazoxanide. The drug garnered interest from the hepatology community after three phase II clinical trials involving the treatment of hepatitis C with nitazoxanide produced positive results for treatment efficacy and similar tolerability to placebo without any signs of toxicity. A meta-analysis from 2014 concluded that the previous held trials were of low-quality and with held with a risk of bias. The authors concluded that more randomized trials with low risk of bias are needed to give any determine if Nitazoxanide can be used as an effective treatment for chronic hepatitis C patients.
Nitazoxanide has gone through Phase II clinical trials for the treatment of hepatitis C, in combination with peginterferon alfa-2a and ribavirin.Romark Laboratories has announced encouraging results from international Phase I and II clinical trials evaluating a controlled release version of nitazoxanide in the treatment of chronic hepatitis C virus infection. The company used 675 mg and 1,350 mg twice daily doses of controlled release nitazoxanide showed favorable safety and tolerability throughout the course of the study, with mild to moderate adverse events. Primarily GI-related adverse events were reported.
A randomised double-blind placebo-controlled study published in 2006, with a group of 38 young children (Lancet, vol 368, page 124-129) concluded that a 3-day course of nitazoxanide significantly reduced the duration of rotavirus disease in hospitalized pediatric patients. Dose given was “7.5 mg/kg twice daily” and the time of resolution was “31 hours for those given nitazoxanide compared with 75 hours for those in the placebo group.” Rotavirus is the most common infectious agent associated with diarrhea in the pediatric age group worldwide.
Teran et al.. conducted a study at the Pediatric Center Albina Patinö, a reference hospital in the city of Cochabamba, Bolivia, from August 2007 to February 2008. The study compared nitazoxanide and probiotics in the treatment of acute rotavirus diarrhea. They found Small differences in favor of nitazoxanide in comparison with probiotics and concluded that nitazoxanide is an important treatment option for rotavirus diarrhea.
Lateef et al.. conducted a study in India that evaluated the effectiveness of nitazoxanide in the treatment of beef tapeworm (Taenia saginata) infection. They concluded that nitazoxanide is a safe, effective, inexpensive, and well-tolerated drug for the treatment of niclosamide- and praziquantel-resistant beef tapeworm (Taenia saginata) infection.
A retrospective review of charts of patients treated with nitazoxanide for trichomoniasis by Michael Dan and Jack D. Sobel demonstrated negative result. They reported three case studies; two of which with metronidazole-resistant infections. In Case 3, they reported the patient to be cured with high divided dose tinidazole therapy. They used a high dosage of the drug (total dose, 14–56 g) than the recommended standard dosage (total dose, 3 g) and observed a significant adverse reaction (poorly tolerated nausea) only with the very high dose (total dose, 56 g). While confirming the safety of the drug, they showed nitazoxanide is ineffective for the treatment of trichomoniasis.
The side effects of nitazoxanide do not significantly differ from a placebo treatment for giardiasis; these symptoms include stomach pain, headache, upset stomach, vomiting, discolored urine, excessive urinating, skin rash, itching, fever, flu syndrome, and others. Nitazoxanide does not appear to cause any significant adverse effects when taken by healthy adults.
Information on nitazoxanide overdose is limited. Oral doses of 4 grams in healthy adults do not appear to cause any significant adverse effects. In various animals, the oral LD50 is higher than 10 g/kg.
The anti-protozoal activity of nitazoxanide is believed to be due to interference with the pyruvate:ferredoxin oxidoreductase (PFOR) enzyme-dependent electron transfer reaction which is essential to anaerobic energy metabolism. PFOR inhibition may also contribute to its activity against anaerobic bacteria.
It has also been shown to have activity against influenza A virus in vitro. The mechanism appears to be by selectively blocking the maturation of the viral hemagglutinin at a stage preceding resistance to endoglycosidase H digestion. This impairs hemagglutinin intracellular trafficking and insertion of the protein into the host plasma membrane.
Nitazoxanide modulates a variety of other pathways in vitro, including glutathione-S-transferase and glutamate-gated chloride ion channels in nematodes, respiration and other pathways in bacteria and cancer cells, and viral and host transcriptional factors.
Following oral administration, nitazoxanide is rapidly hydrolyzed to the pharmacologically active metabolite, tizoxanide, which is 99% protein bound. Tizoxanide is then glucuronide conjugated into the active metabolite, tizoxanide glucuronide. Peak plasma concentrations of the metabolites tizoxanide and tizoxanide glucuronide are observed 1–4 hours after oral administration of nitazoxanide, whereas nitazoxanide itself is not detected in blood plasma.
Roughly 2⁄3 of an oral dose of nitazoxanide is excreted as its metabolites in feces, while the remainder of the dose excreted in urine. Tizoxanide is excreted in the urine, bile and feces. Tizoxanide glucuronide is excreted in urine and bile.
Nitazoxanide is the prototype member of the thiazolides, which is a drug class of structurally-related broad-spectrum antiparasitic compounds. Nitazoxanide is a light yellow crystalline powder. It is poorly soluble in ethanol and practically insoluble in water.
Nitazoxanide was originally discovered in the 1980s by Jean-François Rossignol at the Pasteur Institute. Initial studies demonstrated activity versus tapeworms. In vitro studies demonstrated much broader activity. Dr. Rossignol co-founded Romark Laboratories, with the goal of bringing nitazoxanide to market as an anti-parasitic drug. Initial studies in the USA were conducted in collaboration with Unimed Pharmaceuticals, Inc. (Marietta, GA) and focused on development of the drug for treatment of cryptosporidiosis in AIDS. Controlled trials began shortly after the advent of effective anti-retroviral therapies. The trials were abandoned due to poor enrollment and the FDA rejected an application based on uncontrolled studies.
Subsequently, Romark launched a series of controlled trials. A placebo-controlled study of nitazoxanide in cryptosporidiosis demonstrated significant clinical improvement in adults and children with mild illness. Among malnourished children in Zambia with chronic cryptosporidiosis, a three-day course of therapy led to clinical and parasitologic improvement and improved survival. In Zambia and in a study conducted in Mexico, nitazoxanide was not successful in the treatment of cryptosporidiosis in advanced infection with human immunodeficiency virus at the doses used. However, it was effective in patients with higher CD4 counts. In treatment of giardiasis, nitazoxanide was superior to placebo and comparable to metronidazole. Nitazoxanide was successful in the treatment of metronidazole-resistant giardiasis. Studies have suggested efficacy in the treatment of cyclosporiasis, isosporiasis, and amebiasis. Recent studies have also found it to be effective against beef tapeworm(Taenia saginata).
Nitazoxanide is also under investigation for the treatment of COVID-19.
Nitazoxanide is currently available in two oral dosage forms: a tablet (500 mg) and an oral suspension (100 mg per 5 ml when reconstituted).
^ Jump up to:abcdeStockis A, Allemon AM, De Bruyn S, Gengler C (May 2002). “Nitazoxanide pharmacokinetics and tolerability in man using single ascending oral doses”. Int J Clin Pharmacol Ther. 40 (5): 213–220. doi:10.5414/cpp40213. PMID12051573.
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^ Jump up to:abcdeDi Santo N, Ehrisman J (2013). “Research perspective: potential role of nitazoxanide in ovarian cancer treatment. Old drug, new purpose?”. Cancers (Basel). 5 (3): 1163–1176. doi:10.3390/cancers5031163. PMC3795384. PMID24202339. Nitazoxanide [NTZ: 2-acetyloxy-N-(5-nitro-2-thiazolyl)benzamide] is a thiazolide antiparasitic agent with excellent activity against a wide variety of protozoa and helminths. … Nitazoxanide (NTZ) is a main compound of a class of broad-spectrum anti-parasitic compounds named thiazolides. It is composed of a nitrothiazole-ring and a salicylic acid moiety which are linked together by an amide bond … NTZ is generally well tolerated, and no significant adverse events have been noted in human trials . … In vitro, NTZ and tizoxanide function against a wide range of organisms, including the protozoal species Blastocystis hominis, C. parvum, Entamoeba histolytica, G. lamblia and Trichomonas vaginalis 
^ Jump up to:abcdefRossignol JF (October 2014). “Nitazoxanide: a first-in-class broad-spectrum antiviral agent”. Antiviral Res. 110: 94–103. doi:10.1016/j.antiviral.2014.07.014. PMID25108173. Originally developed and commercialized as an antiprotozoal agent, nitazoxanide was later identified as a first-in-class broad-spectrum antiviral drug and has been repurposed for the treatment of influenza. … From a chemical perspective, nitazoxanide is the scaffold for a new class of drugs called thiazolides. These small-molecule drugs target host-regulated processes involved in viral replication. … A new dosage formulation of nitazoxanide is presently undergoing global Phase 3 clinical development for the treatment of influenza. Nitazoxanide inhibits a broad range of influenza A and B viruses including influenza A(pH1N1) and the avian A(H7N9) as well as viruses that are resistant to neuraminidase inhibitors. … Nitazoxanide also inhibits the replication of a broad range of other RNA and DNA viruses including respiratory syncytial virus, parainfluenza, coronavirus, rotavirus, norovirus, hepatitis B, hepatitis C, dengue, yellow fever, Japanese encephalitis virus and human immunodeficiency virus in cell culture assays. Clinical trials have indicated a potential role for thiazolides in treating rotavirus and norovirus gastroenteritis and chronic hepatitis B and chronic hepatitis C. Ongoing and future clinical development is focused on viral respiratory infections, viral gastroenteritis and emerging infections such as dengue fever.
^ Jump up to:abAnderson, V. R.; Curran, M. P. (2007). “Nitazoxanide: A review of its use in the treatment of gastrointestinal infections”. Drugs. 67(13): 1947–1967. doi:10.2165/00003495-200767130-00015. PMID17722965. Nitazoxanide is effective in the treatment of protozoal and helminthic infections … Nitazoxanide is a first-line choice for the treatment of illness caused by C. parvum or G. lamblia infection in immunocompetent adults and children, and is an option to be considered in the treatment of illnesses caused by other protozoa and/or helminths.
^Shoff WH (5 October 2015). Chandrasekar PH, Talavera F, King JW (eds.). “Cyclospora Medication”. Medscape. WebMD. Retrieved 11 January 2016. Nitazoxanide, a 5-nitrothiazole derivative with broad-spectrum activity against helminths and protozoans, has been shown to be effective against C cayetanensis, with an efficacy 87% by the third dose (first, 71%; second 75%). Three percent of patients had minor side effects.
^ Jump up to:abTeran, C. G.; Teran-Escalera, C. N.; Villarroel, P. (2009). “Nitazoxanide vs. Probiotics for the treatment of acute rotavirus diarrhea in children: A randomized, single-blind, controlled trial in Bolivian children”. International Journal of Infectious Diseases. 13(4): 518–523. doi:10.1016/j.ijid.2008.09.014. PMID19070525.
^ Jump up to:abLateef, M.; Zargar, S. A.; Khan, A. R.; Nazir, M.; Shoukat, A. (2008). “Successful treatment of niclosamide- and praziquantel-resistant beef tapeworm infection with nitazoxanide”. International Journal of Infectious Diseases. 12 (1): 80–82. doi:10.1016/j.ijid.2007.04.017. PMID17962058.
^World Journal of Gastroenterology 2009 April 21, Emmet B Keeffe MD, Professor, Jean-François Rossignol The Romark Institute for Medical Research, Tampa
^Lateef, M.; Zargar, S. A.; Khan, A. R.; Nazir, M.; Shoukat, A. (2008). “Successful treatment of niclosamide- and praziquantel-resistant beef tapeworm infection with nitazoxanide”. International Journal of Infectious Diseases. 12 (1): 80–2. doi:10.1016/j.ijid.2007.04.017. PMID17962058.
^Cynthia Liu, Qiongqiong Zhou, Yingzhu Li, Linda V. Garner, Steve P. Watkins, Linda J. Carter, Jeffrey Smoot, Anne C. Gregg, Angela D. Daniels, Susan Jervey, Dana Albaiu. Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases. ACS Central Science 2020; doi:10.1021/acscentsci.0c00272
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.
In 2014, its efficacy to treat Sjögren syndrome was questioned in a double-blind study involving 120 patients over a 48-week period.
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.
The drug label advises that hydroxychloroquine should not be prescribed to individuals with known hypersensitivity to 4-Aminoquinoline compounds. There are a range of other contraindications and caution is required if patients have certain heart conditions, diabetes, psoriasis etc.
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. For short-term treatment of acute malaria, adverse effects can include abdominal cramps, diarrhea, heart problems, reduced appetite, headache, nausea and vomiting.
One of the most serious side effects is retinopathy (generally with chronic use). 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.
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.
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.
Antimalarials are lipophilic weak bases and easily pass plasma membranes. The free base form accumulates in lysosomes (acidic cytoplasmic vesicles) and is then protonated, 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. Alteration in pH causes inhibition of lysosomal acidic proteases causing a diminished proteolysis effect. Higher pH within lysosomes causes decreased intracellular processing, glycosylation and secretion of proteins with many immunologic and nonimmunologic consequences. These effects are believed to be the cause of a decreased immune cell functioning such as chemotaxis, phagocytosis and superoxide production by neutrophils. 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.
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.
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.
It is frequently sold as a sulfate salt known as hydroxychloroquine sulfate. 200 mg of the sulfate salt is equal to 155 mg of the base.
Brand names of hydroxychloroquine include Plaquenil, Hydroquin, Axemal (in India), Dolquine, Quensyl, Quinoric.
Hydroxychloroquine and chloroquine have been recommended by Chinese and South Korean health authorities for the experimental treatment of COVID-19.In vitro studies in cell cultures demonstrated that hydroxychloroquine was more potent than chloroquine against SARS-CoV-2.
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.
^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.
^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.
Umifenovir (trade names ArbidolRussian: Арбидол, Chinese: 阿比朵尔) is an antiviral treatment for influenza infection used in Russia 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. 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, and it is well known in these two countries. Some of the Russian tests showed the drug to be effective and a direct comparison with Tamiflu showed similar efficiency in vitro and in a clinical setting. In 2007, Arbidol (umifenovir) had the highest sales in Russia among all over-the-counter drugs.
Mode of action
Umifenovir inhibits membrane fusion. 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.
Some evidence suggests that the drug’s actions are more effective at preventing infections from RNA viruses than infections from DNA viruses.
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.
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. Chinese experts claim that preliminary tests had shown that arbidol and darunavir could inhibit replication of the virus. So far without additional effect if added on top of recombinant human interferon α2b spray.
In 2007, the Russian Academy of Medical Sciences stated that the effects of Arbidol (umifenovir) are not scientifically proven.
Russian media criticized lobbying attempts by Tatyana Golikova (then-Minister of Healthcare) to promote umifenovir, and the unproven claim that Arbidol can speed up recovery from flu or cold by 1.3-2.3 days. They also debunked claims that the efficacy of umifenovir is supported by peer-reviewed studies.
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
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 . Produced by Pharmstandard, it is still currently used in Russia and China to treat influenza infections . 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 .
The drug’s anti-influenza mechanism of action is related to arbidol’s ability to bind to the haemagglutinin (HA) protein . 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 .
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) .
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 .
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 . 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 .
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
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
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
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.
Data regarding protein-binding of umifenovir are currently unavailable.
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]
Wang Z, Chen X, Lu Y, Chen F, Zhang W: Clinical characteristics and therapeutic procedure for four cases with 2019 novel coronavirus pneumonia receiving combined Chinese and Western medicine treatment. Biosci Trends. 2020 Feb 9. doi: 10.5582/bst.2020.01030. [PubMed:32037389]
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. On March 17 Chinese officials suggested the drug had been effective in treating COVID in Wuhan and Shenzhen.
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. Other research suggests that favipiravir induces lethal RNA transversion mutations, producing a nonviable viral phenotype. Favipiravir is a prodrug that is metabolized to its active form, favipiravir-ribofuranosyl-5′-triphosphate (favipiravir-RTP), available in both oral and intravenous formulations. Human hypoxanthine guanine phosphoribosyltransferase (HGPRT) is believed to play a key role in this activation process. Favipiravir does not inhibit RNA or DNA synthesis in mammalian cells and is not toxic to them. In 2014, favipiravir was approved in Japan for stockpiling against influenza pandemics. However, favipiravir has not been shown to be effective in primary human airway cells, casting doubt on its efficacy in influenza treatment.
In 2014, Japan approved Favipiravir for treating viral strains unresponsive to current antivirals.
Some research has been done suggesting that in mouse models Favipiravir may have efficacy against Ebola. Its efficacy against Ebola in humans is unproven. 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. A clinical trial investigating the use of favipiravir against Ebola virus disease was started in Guéckédou, Guinea, during December 2014. 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. The trial design has been criticised by Scott Hammer and others for using only historical controls. The results of this clinical trial were presented in February 2016 at the annual Conference on Retroviruses and Opportunistic Infections (CROI) by Daouda Sissoko and published on March 1, 2016 in PLOS Medicine.
SARS-CoV-2 virus disease
In March 2020, Chinese officials suggested Favipiravir may be effective in treating COVID-19.
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.
Chemical Papers (2019), 73(5), 1043-1051.
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
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
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)
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.
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)
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.
^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.
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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]
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Nature Biotechnology: Coronavirus puts drug repurposing on the fast track [Link]
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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. It is approved in Japan for the treatment of pancreatitis.
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.
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.
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.”
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