Isosteric ribavirin analogues: Synthesis and antiviral activities

Article abstract of DOI:10.1016/j.bmcl.2017.11.029

The novel isosteric ribavirin analogues were synthesized by two different ways. Some of them showed significant antiviral action against hepatitis C virus (HCV), herpes simplex (HCV-1) and influenza A virus comparable to that of ribavirin itself. The data obtained confirm the proposed theory of the ribavirin possible antiviral activity mechanism related with bioisosterism.

Full text of DOI:10.1016/j.bmcl.2017.11.029

Accepted Manuscript
Isosteric ribavirin analogues: synthesis and antiviral activities.
Nikolay I. Zhurilo, Mikhail V. Chudinov, Andrey V. Matveev, Olga S.
Smirnova, Irina D. Konstantinova, Anatoly I. Miroshnikov, Alexander N.
Prutkov, Lyubov E. Grebenkina, Natalya V. Pulkova, Vitaly I. Shvets
PII:
S0960-894X(17)31116-2
https://doi.org/10.1016/j.bmcl.2017.11.029
BMCL 25435
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 July 2017
10 November 2017
14 November 2017
Please cite this article as: Zhurilo, N.I., Chudinov, M.V., Matveev, A.V., Smirnova, O.S., Konstantinova, I.D.,
Miroshnikov, A.I., Prutkov, A.N., Grebenkina, L.E., Pulkova, N.V., Shvets, V.I., Isosteric ribavirin analogues:
synthesis and antiviral activities., Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/
j.bmcl.2017.11.029
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Isosteric ribavirin analogues: synthesis and antiviral activities.
Nikolay I. Zhuriloa, Mikhail V. Chudinov a†, Andrey V. Matveev a, Olga S. Smirnovab,
Irina D. Konstantinovab, Anatoly I. Miroshnikovb, Alexander N. Prutkovc, Lyubov E. Grebenkinac, Natalya V.
Pulkovaс, Vitaly I. Shvetsa
a Lomonosov Institute of Fine Chemical Tehnologies, Moscow Technological University, Vernadskogo Pr. 78,
119454, Moscow, Russia.
b Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya
St. 16/10, 117997 GSP, Moscow B-437, Russia.
c Moscow Politechnical University, Bolshaya Semenovskaya Str., 38, 107023, Moscow, Russia.
† Corresponding author: mikle@irims.ru
Abstract
The novel isosteric ribavirin analogues were synthesized by two different ways. Some of them showed significant
antiviral action against hepatitis C virus (HCV), herpes simplex (HCV-1) and influenza A virus comparable to that of
ribavirin itself. The data obtained confirm the proposed theory of the ribavirin possible antiviral activity
mechanism related with bioisosterism.
Ribavirin (Virazole, 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a nucleoside analogue with a broad
antiviral activity spectrum. It is active against influenzia virus, HCV, RSV, HSV etc. 1-6
.
Ribavirin action mechanism is still not clarified, however, an existing hypothesis assume the heterocyclic base
of ribavirin, 1,2,4-triazole-3-carboxamide (TKA), as a mimic of guanosine purine cycle. This steric similarity allows
to ribavirin molecule to incorporate into the viral genome and cause irreversible mutations7.
.

Figure 1. Structure of guanosine and some isosteric antivirals8.
A wide variety of antiviral nucleoside analogues with corresponding structure mojeties are known for the
moment (see Fig.1).
1,2,4-Triazole-3-carboxamide and other 1,2,4-triazole derivatives are substrates for purine nucleoside
phosphorylases (NPs) in a manner similar to natural purine nucleosides. We showed earlier that various
substituted amides of 1,2,4-triazole-3-carboxylic acid are also NP substrates9 and demonstrate antiviral activity.
1,2,4-Oxadiazole ring is a common isosteric replacement of the amide group in drug design10. However,
ribavirin analogues with carboxamide fragment substituted by isosteric 1,2,4-oxadiazole ring were not described
before. Here we present a synthetic pathway to this type of ribavirin analogues as well as their antiviral activity.
There are three possible approaches to the synthesis of these compounds: i) ribavirin direct modification;
ii) chemical or iii) enzymatic ribozylation of the corresponding heterocyclic bases. The first method allows
obtaining only beta-ribosides, while the second one enables to get a set of analogues bearing various sugar
mojeties. Enzymatic ribosylation is limited by the NP substrate specificity, while the chemical one is limited by
the heterocyclic base stability in the ribosylation reaction conditions.
The first approach is presented on the Scheme 1. Starting from ribavirin a protected amidoxime 3, a starting
material for the ribavirin analogues 5a-d synthesis, was obtained by several step pathway. Thus, compound 4a
was obtained by heating of 3 in triethylformate, while derivatives 4b-d were synthesized by the amidoxime 3
reaction with the corresponding acyl chlorides.

Schema 1. Synthesis of isosteric ribavirin analogues 5a-d from ribavirin. Reagents and condition: (a) 2,2-
dimethoxypropane, p-toluenesulfonic acid (2% mol), DMF, 2 hr, 100 oC; (b) trityl chloride, pyridine, 1 hr, 5 oC,
57% yield; (c) trifluoroacetic anhydride, pyridine, CH2Cl2, 12 hr, 0-5 oC, 70% yield; (d) NH2OH (aq), MeOH, 12 hr, rt,
96% yield; (e) triethyl ortoformate, p-toluenesulfonic acid (10% mol), 3 hr, reflux, 96% yield; (f) acyl chloride,
triethyl amine, CH2Cl2, 1.5 hr, rt, 73-89% yields; (g) trifluoroacetic acid-water (9:1), 40 min, rt, 48-73% yields.
Also heterocyclic bases 8a-d were synthesized for the experiments with glycosylation and enzymatic
transglycosilation reactions aiming to obtain isosteric ribavirin analogues (Scheme 2).
Schema 2. Synthesis of isosteric TKA bases. Reagents and condition: (a) trifluoroacetic anhydride, pyridine,
dioxane, 10 min, -5 oC, 72 % yield; (b) hydroxylamine (50% in water), ethanol, 12hr, rt, 98% yield ; (c) triethyl
ortoformate or triethyl ortoacetate, p-toluenesulfonic acid (2.5% mol), 3 hr, reflux, 68-71 % yields ; (d) acyl
chloride, triethyl amine, CH2Cl2, 1.5 hr, 0oC ->rt, then tetrabutyl ammonium fluoride (TBAF), toluene, 4 hr, reflux,
68-71 %.

It was shown that the reaction of the base 8a with ribose tetraacetate in standard conditions11 yields mainly
the derivative 9 (Scheme 3), consequently, this approach is inapplicable for the preparative synthesis of isosteric
ribavirin analogues.
Schema 3. Reaction of heterocyclic base 8a with ribose tetraacetate. Reagents and condition: (a) bis-p-
nitrophenyl phosphate (3% mol), 160oC, 15 min.
The opposite was observed for the enzymatic transglycosylation of the nucleosides: all bases 8 regardless of
the substituent size in the oxadiazole ring appeared to be good substrates for NP. This quite unexpectable
behaviour was in accordance to a theory based on previous experiments in our laboratory9,12: the substituent size
in the 3d position of 1,2,4-triazole ring has mininimal effect on the substrate properties.
Schema 4. Synthesis of isosteric ribavirin analogues via transglycosylation reaction.
Moreover, glycosylation of compound 8d bearing bulky phenyl substituent proceeds notably faster than for
compounds 8a-c (Fig. 2).

Figure 2. Dependence of bases 8a-d conversion into riboside 5a-d. Reagents and condition: base (1 mM) and Urd
(2 mM) in 1.0 ml of the buffer (pH 7.0; 4 mM KH2PO4), the recombinant E. coli enzymes (70 e. u. of PNP and 90 e.
u. of UP), 50 oC, 48 hr.
Synthesis of 2-deoxyribosides 10a-d proceeded faster than that of ribosides, with a 95-98% conversion in 1 h,
while riboside synthesis requires from 2 to 24 hours (Fig. 3).
Figure 3. Dependence of bases 8a-d conversion into 2’-deoxyriboside 10a-d. Reagents and condition: base (1
mM) and dUrd (2 mM) in 1.0 ml of the buffer (pH 7.0; 4 mM KH2PO4), the recombinant E. coli enzymes (70 e. u. of
PNP and 90 e. u. of UP), 50 oC, 3 hr.
The nucleosides 5a-d and 10a-d formation was confirmed by HPLC and LC-MS data. Nucleosides 5b
and 10b were obtained in a sufficient amount for antiviral activity studies.

The antiviral activity and cytotoxicity was estimated in vitro on influenza A virus H5N1
(A/duck/Novosibirsk/56/05) and HCV in SPEV cells as well as in Vero E6 cells infected with herpes
simplex virus (type 1, L2 strain), and the prevention of the development of the virus-induced cytopathic
effect (CPE)13 was evaluated. Cytotoxicity was estimated by monolayer condition using optical methods.
The ribavirin analogues 5a-c and 10b showed no cytotoxicity against Vero E6 cells up to a concentration
of 1000 µg/ml, although compound 5d showed moderate cytotoxicity (CC50=380 µg/ml). All compounds
showed no cytotoxicity against SPEV cells up to a concentration of 150 µg/ml (CC50= 75 µg/ml for
ribavirin).
Riboside 5d exhibits a significant selective activity against HCV (IC50=8.8 µg/ml, 12.5 µg/ml for
ribavirin). Compound 5a showed activity against HSV-1 comparable to the ribavirin activity at a
multiplicity of infection (MOI) of 0.01 PFU/cell (IC50=250 µg/ml, 125 µg/ml for ribavirin). Riboside 5b
exhibits a moderate activity against influenza A virus (IC50=50 µg/ml, 12.5 µg/ml for ribavirin).
Compounds 5c and 10b exhibit no antiviral activity.
Thus, we proposed two ways to efficient synthesis of the new ribavirine analogues: chemical and chemo-
enzymatic. The preliminary antiviral activity studies for some of them have shown significant antiviral action
comparable to that of ribavirin itself. The data obtained confirm the proposed theory of the ribavirin possible
antiviral activity mechanism related with bioisosterism.
Acknowledgements
The work was supported by the grant no.20.9895.2017 of the Ministry of Education and Science of the
Russian Federation. Also this work was supported by the Moscow Technological University grant for the support
of young scientists and a grant of the president of Russian Federation for the leading scientific school state
support (7946.2016.11). We thank V.L. Andronova, G.A. Galegov and P.G. Deryabin from Ivanovsky Research
Institute of Virology (Moscow, Russia) for their assistance in the antiviral evaluation. We thank R.S. Esipov and T.I.
Muravyova (IBCH RAS) for kindly providing for experiments enzymes nucleoside phosphorylases.
Supplementary data
Supplementary data (experimental procedures and spectroscopic characterizations data of the compounds (1-
9)) associated with this article can be found in the Electronic Supplementary Information (ESI).
Notes and references
1. Lee N.; Qureshi S. T. Crit. Care Clin. 2013, 29,1045.
2. Burrows F. S.; Carlos L. M.; Benzimra M.; Marriott D. J.E.; Havryk A. P.; Plit M. L. et al. J. Heart Lung Transplant.
2015, 34, 958.
3. Hartline J.; Mierek C.; Knutson T.; Kang C. Am. J. Emerg. Med. 2013, 31, 978.

4. McCormick J.B.; King I.J.; Webb P.A.; Scribner C.L.; Craven B.B. et al. Engl. J. Med. 1986, 314, 20.
5. Terzano C.; Petroianni A.; Ricci A. Curr. Therap. Res. 2004, 65, 90.
6. Castells L.; Rimola A.; Manzardo C.; Valdivieso A. et al. J. Hepatol. 2015, 62, 92.
7. Crotty S.; Cameron C.; Andino R. J. Mol. Med. 2002, 80, 86.
8. Zeidler J.; Baraniak D.; Ostrowski T. Eur. J. Med. Chem. 2015, 97, 409.
9. Konstantinova I.D.; Chudinov M.V.; Fateev I.V.; Matveev A.V.; Zhurilo N.I. et al. Bioorg. Khim. 2013, 39, 53.
10. Patani G. A.; LaVoie E. J. Chem. Rev. 1996, 96, 3147.
11. Chudinov M.V.; Matveev A.V.; Prutkov A.N.; Konstantinova I.D.; Fateev I.V. et al. Mend Comm, 2016, 26, 214.
12. Fateev I.V.; Kharitonova M. I.; Antonov K. V.; Konstantinova I. D.; Stepanenko V.N. et al. Chem. Eur. J. 2015,
21, 13401.
13. Fedorov I. I.; Kazmina E.M.; Gurskaya G.V.; Jasko M.V.; Zavodnic V.E.; Balzarini J. et al. J. Med. Chem. 1997,
40, 486.