Design, synthesis, and biological evaluation of novel coxsackievirus B3 inhibitors

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Abstract

The synthesis and SAR study of a novel class of coxsackievirus B3 (CVB3) inhibitors are reported. These compounds could be considered as the 6-chloropurines substituted at position 9 with variously substituted bicyclic scaffolds (bicyclo[2.2.1]heptane/ene—norbornane or norbornene). The synthesis and biological evaluation of 31 target compounds are described. Several of the analogues inhibited CVB3 in the low micromolar range (0.66–2 μM). Minimal or no cytotoxicity was observed.

Graphical abstract

We report synthesis and results of the anti-coxsackievirus B3 screening of the novel 6-chloropurine derivatives.

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Introduction

Coxsackieviruses are non-enveloped, single-stranded (+) RNA viruses belonging to the Picornaviridae family.1 Coxsackieviruses are subdivided into two subgroups—CVA and CVB, based on their pathology to newborn mice. Infections with coxsackieviruses, and in particular with CVB3, are the most common cause of viral myocarditis. Moreover, these viruses are reported to be associated with the development of pancreatitis, meningitis, and encephalitis.2 Other diseases associated with coxsackievirus infections are hand-foot-and-mouth disease and hemorrhagic conjunctivitis.

There is currently no approved antiviral therapy for the treatment of picornaviral infections in man or animals. Numerous compounds were reported in the past as selective inhibitors of enteroviruses.3 For example, pleconaril entered clinical trials but despite the promising results, it was not approved by the FDA for the treatment of common cold symptoms in HRV-infected patients.4 Rupintrivir, another compound tested in the clinical trials,5 failed in natural infection study in patients.6 A large series of promising novel compounds based on substituted 5-nitro-5-phenoxybenzonitriles was recently discovered. Most of the compounds examined in the study exerted significant activity against CVB3 comparable with the parental analogue MDL-860 (6.5 ± 4.9 μM),7 and some of them were also tested against other picornaviruses. The compound 2-(2-chlorophenoxy)-5-nitrobenzonitrile possessed broad-spectrum anti-enterovirus activity including activity against Echo 9 at micromolar level unlike the parental compound MDL-860. Anti-enterovirus activity in micromolar range was also reported for compounds derived from 2,6-dihalophenyl-substituted 1H,3H-thiazolo[3,4a]benzimidazoles.8

Recently, we reported a series of carbocyclic nucleoside analogues with antiviral activity against coxsackieviruses. These compounds carry bicycloalkanes,9 bicycloheteroalkanes10 or tricycloheteroalkanes11 instead of the glycone part of the nucleoside molecule (compounds 16, Fig. 1). The compounds were primarily designed as the conformationally locked analogues of carbocyclic nucleosides.9 Some of the analogues also exhibited antiviral activity against HIV-1 and HIV-2 on T-lymphocyte (CEM) cells.9(b), 9(c), 11(a) Activity against CVB3 was in the micromolar range with minimal or no cytotoxicity in Vero cells (Table 1).

This study involves the synthesis of a second generation of this compound class with bicyclic scaffold (variously substituted bicyclo[2.2.1]heptane) connected with a nucleobase (6-chloropurine) at position 2. Our concept was based on the simplification of the previously reported compounds with activity against CVB3.9, 10, 11 We examined which substitutions on the bicyclic part of the molecule are necessary for antiviral activity.

Section snippets

Synthetic chemistry

The first group of target compounds were synthesized by the Mitsunobu reaction (Method A). 6-Chloropurine was coupled with the appropriate alcohol (Table 2). The source of these alcohols is reported in the Supplementary data. Only few derivatives were prepared by construction of the nucleobase at the aminogroup on the corresponding scaffold by a coupling with 4,6-dichloropyrimidin-5-amine and the subsequent ring closure reaction12 (Method B, Table 3, source of the amines—see Supplementary data

Results and discussion

The structures of the prepared compounds were determined by 1H and 13C NMR spectroscopies. Complete assignment of all 1H and 13C signals is based on combination of 1H, 13C APT, H,H-COSY, H,C-HSQC, and H,C-HMBC experiments. The position of the hydroxy or keto group in compounds 25 and 26 was confirmed using HMBC spectra. The hydrogen atom in position 2 (which is easily recognized by its typical chemical shift 4.5–5.5 ppm) exhibits a three-bond correlation cross-peak with carbon 6 and there is

Conclusion

In summary, we have synthesized a novel class of coxsackievirus inhibitors derived from a bicyclo[2.2.1]heptane scaffold. Two methodologies were employed for introduction of the chloropurine moiety to the scaffold: (i) the Mitsunobu reaction of the chloropurine nucleobase with appropriate alcohols and (ii) a built-up strategy from appropriate amines. All compounds were evaluated for antiviral activity against coxsackievirus B3. Most analogues showed activity in the low micromolar range with

Chemistry

Melting points were determined on a Büchi B-540 apparatus. NMR spectra (δ, ppm; J, Hz) were measured on a Bruker Avance II-600 and/or Bruker Avance II-500 instruments (600.1 or 500.0 MHz for 1H and 150.9 or 125.7 MHz for 13C) in hexadeuterated dimethyl sulfoxide and referenced to the solvent signal (δ 2.50 and 39.70, respectively). Mass spectra were measured on a LTQ Orbitrap XL (Thermo Fisher Scientific) using electrospray ionization (ESI) and a GCT Premier (Waters) using EI. Column

Acknowledgments

This work is a part of the research project Z4 055 0506. It was supported by the ‘Centre for New Antivirals and Antineoplastics’ (Ministry of Education, Youth and Sports of the Czech Republic, 1M0508) and by Gilead Sciences, Inc. (Foster City, CA, U.S.A.). Armando M. De Palma is a postdoctoral fellow of the FWO-Vlaanderen. The author (M.S.) would like to thank to Ms. Eliška Procházková for her crucial role in preparing of the manuscript.

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