ISSN 10193316, Herald of the Russian Academy of Sciences, 2016, Vol. 86, No. 3, pp. 206–212. © Pleiades Publishing, Ltd., 2016. Original Russian Text © O.N. Chupakhin, V.N. Charushin, V.L. Rusinov, 2016, published in Vestnik Rossiiskoi Akademii Nauk, 2016, Vol. 86, No. 6, pp. 546–552. Scientific Session of the General Meeting of the Russian Academy of Sciences Scientific Foundations for the Creation of Antiviral and Antibacterial Preparations Paper by RAS Academician O. N. Chupakhina, RAS Academician V. N. Charushina, and RAS Corresponding Member V. L. Rusinovb* a Postovskii Institute of Organic Synthesis, Ural Branch, Russian Academy of Sciences, Yekaterinburg, Russia b Ural Federal University, Yekaterinburg, Russia email: [email protected], [email protected], [email protected] Received January 22, 2016

Abstract—The results of the Ural scientific school’s organic chemists on the creation of antiviral and antibac terial, including antitubercular, chemopreparations are considered. Basic research data were generalized on the synthesis and study of antiviral activity and the establishment of the metabolism and mechanism of azoloazine series compounds—azaanalogs of adenine and guanine, as well as their nucleosides, which led to the creation of a new family of antiviral substances. One of them, triazavirin, has become a regular therapeutic fixture as an antiflu preparation. The results of synthetic and biological studies on substituted pyrimidines, nucleosides of the benzimidazole and purine series, and other biologically active azaheterocycles are also discussed.

Keywords: triazavirin, triazide and other azoloazine derivatives, protein disulfide isomerase inhibitors, nucle osides of fluorinecontaining benzimidazoles and purines, antibacterial fluoroquinolones, bacterial DNA gyrase inhibitors, kinetic resolution of intermediates, levofloxacin synthesis. DOI: 10.1134/S1019331616030163

Organic chemistry makes a weighty contribution to Antiviral preparations. According to expert esti the creation of synthetic and semisynthetic drugs that mates, viral diseases during outbreaks affect 10–20% dominate in the global pharmaceutical market, most of the country’s population, annual economic damage of which are represented by heterocyclic compounds. from them reaching –P 10 billion. At the same time, the The authors represent one of the oldest Russian arsenal of antiviral drugs at medicine’s disposal is schools in heterocyclic chemistry; Academician I.Ya. rather limited. Along with the foreign preparation Postovskii, who had been educated at the Munich Tamiflu, the Russian market offers , Arbi Higher Technical School (Germany), where he had dol, and, recently, Ingavirin. worked in the laboratory of Nobel Prizewinner H. Fis With account for the high variability of viruses, the cher, stood at its cradle. Postovskii’s scientific school creation of drugs effective against flu and other viral created a family of medicinal preparations—from sul remains topical and one of the most com fidin, the first domestic antibacterial preparation, plicated tasks of medical chemistry. In this context, it which played an exceptionally important role in curing is advisable to recall the discovery of the structure of the wounded during the Great Patriotic War, to antivi DNA—an outstanding achievement of the 20th cen ral triazavirin, which has recently appeared in the tury, which gave an impetus to the development of pharmacy network. rational approaches, including those to the elabora tion of drugs. It is no wonder that targeted synthesis, Today Ural scientists are developing antitumor which has made it possible to obtain antiviral prepara preparations, radioprotectors, and cardiotropic prep tions, such as acyclovir and , is actively being arations. This paper highlights works on the creation conducted in the series of structural analogs of DNA of drugs against infectious diseases, which is a priority and RNA nucleobases, as well as their nucleosides. task of domestic science. The series of azoloazines—structural analogs of DNA and RNA purine bases—served as the basis for the * RAS Academician Oleg Nikolaevich Chupakhin is director for antiviral preparations that are being developed in the science at the Postovskii Institute of Organic Synthesis, RAS Ural Branch. RAS Academician Valerii Nikolaevich Charushin Urals. is director of the same institute and chair of the RAS Ural As a result of the interdisciplinary studies per Branch. RAS Corresponding Member Vladimir Leonidovich Rusinov is director of the Institute of Physics and Technology of formed by the Institute of Organic Synthesis, RAS Ural Federal University. Ural Branch, and Ural Federal University jointly with

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NH2 H9N2), respiratory syncytial , parainfluenza, R adenoviruses, and so on. The efficiency index relative Z N to types A and B viruses is 65–80%. Impor Y CN N tantly, the new preparation is effective at all stages of X N the development of a viral infection under both pro 55 R 2 O phylactic and therapeutic regimens. The high antiviral CO2Et activity of triazavirin was also demonstrated relative to Z Z − Z R NH N 4 R N the agents of tickborne encephalitis and hemorrhagic Y Y Y + fevers [10, 11]. X X X N NH2 N2 N 3 H Triazavirin is a lowtoxic medicinal substance of 1 drug class IV [4, 5]. Clinical studies (phases I–III) R = F, Cl, Br, I, NO2, CN, CO2Et, Ar, Het; established that the use of triazavirin in the etiotropic X = N, CH, CSAlk, CAlk, CAr; therapy of influenza reduces the duration of the main symptoms of the disease (intoxication, fever, and Y = N, CH, CCN, CCOOEt, CNO2; Z = N, CH catarrhal signs), quickly normalizes body temperature in therapeutic groups, and decreases the reisolation Fig. 1. General diagram of azoloazine synthesis. rate of the viruses [8]. The comparative evaluation of the effectiveness of triazavirin and Tamiflu has shown that triazavirin surpasses Tamiflu in a number of the Research Institute of Influenza of the Ministry of parameters. For example, in the group of patients who Health of the Russian Federation (St. Petersburg; used triazavirin, the recovery time and the duration of supervised by RAS Academician O.I. Kiselev), the the temperature response, headache, and myalgias Virology Center of the Research Institute of Microbi were statistically reliably lower than in the group of ology of the Ministry of Defense of the Russian Feder those who used Tamiflu. According to the results of ation (Sergiev Posad, Moscow oblast; supervised by PCRbased diagnostics, the reisolation rate of the S.V. Borisevich, Dr. Sci. (Med.)), and the State Sci Influenza RNA virus on the fifth day of the disease was ence Research and Test Institute of Military Medicine significantly lower in the group that used triazavirin of the Ministry of Defense of the Russian Federation than in the comparison group (Fig. 2). (St. Petersburg; by S.V. Chepur, Dr. Sci. (Med); The high efficiency of triazavirin has been proved V.N. Bykov, Dr. Sci. (Biol.); and A.V. Maksimov, Dr. by clinical trials in many Russian cities. Especially Sci. (Biol.)), a new family of domestic antiviral prepa noteworthy in the creation of the preparation and the rations has been created: (1) pyrazolo, imidazolo, organization of clinical trials is the colossal role of our 1,2,4triazolo[5,1c]1,2,4triazine7(4H)ones, and colleague, Director of the Institute of Influenza, Rus (2) 7amino azolo[5,1c]1,2,4triazines [1]. sian Ministry of Health, RAS Academician Kiselev, The main methods to construct such biocyclic who died in November 2015. structures include annulation of the azine cycle to the In parallel with clinical studies, the mechanism of azole one, which makes it possible to use (3) a wide triazavirin’s action was investigated. It was established circle of amino azoles and (4) available synthons— that the target of the preparation is the viral protein derivatives of acetic acid or (5) acetonitrile (Fig. 1). hemagglutinin, which was confirmed experimentally Data on their toxicity, metabolism, and mechanism of by the method of surface plasmon resonance. Com action have been obtained. Compounds effective puter simulation of interaction between triazavirin and against diseases caused by influenza, herpes, and tick the hemagglutinin of the pandemic influenza virus borne encephalitis viruses, as well as against those of A/California/04/ 2009(H1N1) was performed. The hemorrhagic fevers, have been identified. The first short sequences CKLRGV, LGK, and FYKLIW were preparation created on the basis of compounds of this identified, which determine the noncovalent interac class—triazavirin (sodium salt of 2methylthio6 tion of influenza virus hemagglutinin with triazavirin nitro[1,2,4]triazolo[5,1c]1,2,4triazine7one, dehy (Fig. 3). drate)—passed the entire cycle of clinical tests as an Experimental data have been obtained that show antiflu drug and, on August 28, 2014, was entered into the important role of interactions of triazavirin with the Drug Register of the Russian Federation. The SH fragments of amino acids and confirm the pres Medsintez Pharmaceutical factory (Novoural’sk, ence of disulfide bonds between the residues of cys Sverdlovsk oblast) and the Ural Center for Biopharma teine in positions 59, 292, 296, and 320 in the tertiary Technologies (Yekaterinburg) organized the commer structure of hemagglutinin. These data imply the par cial production of the drug; since 2014, it has been sold ticipation of both triazavirin and the enzyme responsi through the pharmacy network [2–11]. ble for the formation and isomerization of disulfide Triazavirin is effective against a wide range of influ bonds, socalled protein disulfide isomerase. To prove enza viruses (H1N1, H5N1, H5N2, H7N3, and this version, model peptides HAI (DCNTTCQ),

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(a) (b) 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30

Virus level, % 20 20 10 10 0 0 first day second day Group I: 250 mg × 2 times a day Group II: 250 mg × 3 times a day Group III: placebo

Fig. 2. (a) Normalization of patients’ body temperature Fig. 3. Results of multiple triazavirin docking to H1 and (b) the reisolation rate of influenza RNA viruses after hemagglutinin of the influenza virus. five days of triazavirin therapy by the results of PCRbased The best mutual arrangements of molecules in terms of diagnostics. interaction energy are shown.

O O

NO2 NH2 N N [H] N N H CS . 2H O H CS 3 − N 2 3 N N N N N Na+ H Triazavirin Metabolite

Fig. 4. Metabolic transformation of triazavirin.

HAII (YGNCNTKCQ), and HAIII (LCKLGGIA To study the pharmacokinetics and metabolism of PLHLGKCNamid) were used, each of which con the preparation, 2H and 15Ntagged triazavirin was 2 15 tains two cysteine residues. After the incubation of the synthesized [7]. Sodium salt of [ H3, N3]2meth model peptides with triazavirin in physiological con ylthio6nitro[1,2,4]triazolo[5,1c][1,2,4]triazin ditions, the interaction products were analyzed using 7one, dehydrate, which contains excessive atoms of mass spectrometry. It is shown that triazavirin favors deuterium in the methylthio group and excessive iso the formation of S–S bonds; along with intramolecu topes of nitrogen atoms in positions 1 and 5, as well as lar S–S bonds, intermolecular disulfide bonds form, in the nitro group of the triazavirin molecule, was obtained proceeding from knowingly enriched syn which lead to dimeric structures. Both the monomeric thons (Fig. 5). S–S form and dimeric ones clearly manifest them selves in mass spectra, testifying to the oxidative func Supported by the Ministry of Industry and Trade of tion of triazavirin. These results agree well with phar the Russian Federation, we, jointly with Pharmstan dard JSC, are conducting work on the creation of a macokinetics data, which show that, in playing the new antiviral preparation from the same family of role of the oxidizing agent of thiol groups, triazavirin is azoloazines to which triazavirin belongs. The code transformed from a nitrated derivative into the respec name of the preparation is triazide. By now, preclinical tive amino compound, which exhibits only a weak studies have been completed and the first phase of antiviral effect (Fig. 4). It has also been established clinical trials has begun. experimentally that triazavirin inhibits the enzymatic Speaking about the search for the antiviral action of activity of protein sulfide isomerase, thus disturbing chemical compounds of other series, we would like to the formation of the tertiary structure of hemaggluti note the good prospects of our results, reached jointly nin and the virus’s life cycle. with the Shemyakin and Ovchinnikov Institute of

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NH H2CO3 15 15 N 2 N 15 NH4SCN NH H3CI 2 NH Na NO2 SH H3CS H2NNH 15 N N NH NH2 NH2 O 15 15 15 15 NO Na NO2 N EtOOC 15 N 2 2 NH NO2 N H CS 2H CS . 2H O 3 3 −15N 2 N N+ N N Na+ 15N Fig. 5. Synthesis of triazavirin containing 2H and 15N isotopic tags.

O Cl N HN NH−CH(CH3)2 O N Cl N HO HO O O MARIBAVIR OH X OH OH

UP F F F R N R N HO R N O O F N N N O OH H F F − Purine nucleoside HO HO O O + O OH X phosphorylase

OH X OH X Fig. 6. Synthesis of maribavir analogs.

F F

F O recombinant F O HO uridine O phosphorylase E. coli N CH HO N CH 3 + OPO3H2 3 N N N OH N H N N N H N N N 2 H 2 HO O O HO HN OH O N HO 1bDarabinofuranosyluracil O (AraU) HO

OH Fig. 7. Synthesis of nucleosides of 2aminopurine.

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Fluoroquinolones basics of the synthesis of fluoroquinolones—the most effective antibacterial preparations—are very impor 1962 Quinolones tant. Compounds of this series are characterized by a 1958 Glycopeptides wide spectrum of effects and an exceptionally high level of activity. The mechanism of their action is asso 1950 Aminoglycosides ciated with inhibiting bacterial DNA gyrase. Fluoro quinolones are the only class of preparations that can 1940 Penicillins Semisynthetic βlactams (penicillins, cephalosporins) compete with βlactam antibiotics. In addition, they 1936 Sulfonamides are the least vulnerable in terms of the resistance of 1930 1940 1950 1960 1970 1980 1990 2000 microorganisms [16, 17]. Antibiotics Synthetic drugs The Institute of Organic Synthesis, RAS Ural Branch, and Ural Federal University have performed Fig. 8. Main benchmarks in the creation of preparations a voluminous cycle of works on the synthesis of the against bacterial infections. most important class of synthetic antibacterial prepa rations of the fluoroquinolone series, as well as their heteroanalogs in the series of bi, tri, and polycyclic Bioorganic Chemistry, RAS, on the enzymatic transg fluorinecontaining heterocycles [15–19]. Jointly lycosylation of azaheterocycles (RAS Academician with the Zelinskii Institute of Organic Chemistry, RAS A.I. Miroshnikov), which yielded methods of synthe (RAS Academician O.M. Nefedov) and the Boreskov sizing nucleosides based on fluorinecontaining benz Institute of Catalysis, RAS Siberian Branch (RAS imidazoles (analogs of maribavir) (Fig. 6) [12, 13], as Academician V.N. Parmon), a universal platform was well as in the series of 2aminopurine derivatives created to obtain initial fluoroarenes, key intermedi (Fig. 7) [14]. The obtained substances demonstrate a ates, and fluoroquinolones based on them, which has significant level of activity relative to herpes viruses. passed approval at the pilot plant of the Volgograd Antibacterial preparations. To control bacterial Branch of the Institute of Catalysis, RAS Siberian infections, world science has created various classes of Branch. drugs, obtained using both biotechnologies and meth The new generation of antibacterial — ods of organic synthesis: from sulfonamides and peni fluoroquinolones of the 21st century—is represented cillins to aminoglycosides, cephalosporins, and enan by a number of enantiomerically pure preparations, tiomerically pure fluoroquinolones of the fourth gen such as levofloxacin and moxifloxacin. The Postovskii eration (Fig. 8). Under the challenges that face Russia, Institute of Organic Synthesis has developed scientific including the potential threat of bioterrorism, works foundations for the kinetic resolution of racemic on the creation of the scientific and technological intermediates used in the synthesis of levofloxacin and

F OCH F OCH O 3 O 3 Cl F NH CH3 (S) F N

O O CH3 (S) * CH3 CH3 (R, S) (R) + F OCH O 3

F N

O CH3 (S) CH3 (S) F F + F NH F NH F O O CH3 CH3 (R) (S) F NH O CH (S) 3

Fig. 9. Key stage of the synthesis of enantiomerically pure levofloxacin.

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Activity relative to tuberculosis mycobacteria Minimal inhibiting concentration relative to tuberculosis mycobacteria (mg/mL) Compound H37Rv M. Avium M. Terrae MDR 0.7 0.7 0.7 1.5 S EtOOC N N H C N N 3 H Enantiomer 10.7 0.7 0.7 0.7 Enantiomer 20.7 0.3 0.3 0.7 1.5 1.5 1.5 3.1

EtOOC N N H C N N 3 H Isoniazid 0.1 0.1 0.1 – other enantiomerically pure preparations (Fig. 9). The effective against multidrugresistant mycobacterial technology of obtaining levofloxacin is realized on a strains. The solution of this problem is exceptionally pilotproduction basis. Jointly with the Engelhardt important for Russian health care. Institute of Molecular Biology, RAS, we have shown its compliance with high quality standards, including in experiments on the inhibition of bacterial DNA REFERENCES gyrase (RAS Corresponding Member S.N. Kochet 1. V. L. Rusinov, E. N. Ulomskii, O. N. Chupakhin, and kov). As a result of studies made jointly with the V. N. Charushin, “Azolo[5,1c]1,2,4triazines as a Zakusov Institute of Pharmacology (supervised by new class of antiviral compounds,” Russ. Chem. Bull. 7 RAS Academician S.B. Seredenin), a new dosage (5), 985 (2008). form of levofloxacin has been created. 2. O. N. Chupakhin, V. L. Rusinov, E. N. Ulomskii, et al., RF Patent No. 2294936 (2007). The epidemiological situation in Russia shows the 3. S. Ya. Loginova, S. V. Borisevich, V. A. Maksimov, et exceptional importance of the survey work targeted at al., “Studies on triazavirin’s antiviral activity in relation finding efficient inhibitors of protein kinases of tuber to flu agent A(H5N1) in a cell culture,” Antibiot. Khi culosis mycobacteria and creating effective antituber mioter. 52, 18 (2007). cular preparations on this basis, which would affect 4. I. Karpenko, S. Deev, O. Kiselev, et al., “Antiviral prop multidrugresistant causative agents. These studies are erties, metabolism, and pharmacokinetics of a novel being conducted by the Institute of Organic Synthesis azolo 1,2,4,triazine derived inhibitor of influenza A jointly with the Vavilov Institute of General Genetics and B virus replication,” Antimicrobial Agents Che (the department of V.N. Danilenko, Dr. Sci. (Biol.)) motherapy 54, 2017 (2010). and the Ural Research Institute for Phthisiopul 5. S. Ya. Loginova, S. V. Borisevich, V. L. Rusinov, et al., “Assessing the toxicity of a new domestic chemoprepa monology (Yekaterinburg; supervised by S.N. Skorny ration triazavirin,” Antibiot. Khimioter. 57, 8 (2012). akov, Dr. Sci. (Med.)) [20, 21]. In particular, the Insti tute of Organic Synthesis has obtained derivatives of 6. S. Ya. Loginova, S. V. Borisevich, V. A. Maksimov, et al., “Medicinal efficacy of the new domestic prepara the pyrimidine series with a high antitubercular activ tion triazavirin in relation to flu agent A (H5N1),” ity, which are promising for indepth study. Note that, Antibiot. Khimioter. 56, 10 (2011). to synthesize biologically active pyrimidines, the 7. T. S. Shestakova, I. A. Khalymbadzha, S. L. Deev, et al., methodology of direct C–H functionalization of the “Synthesis of the [2H,15N]labeled ‘triaz pyrimidine ring under the action of nucleophilic avirin,’” Russ. Chem. Bull. 60 (4), 729 (2011). reagents was used [22]. The antitubercular activity of 8. O. I. Kiselev, E. G. Deeva, T. I. Mel’nikova, et al., enantiomerically pure derivatives of condensed pyri “New antiviral preparation ‘triazavirin’: Results of the midines was also studied (table). These data create the secondphase clinical studies,” Vopr. Virusol. 57 (6), 9 basis for the further targeted search for substances (2012).

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