Elsevier

Bioorganic & Medicinal Chemistry

Volume 23, Issue 22, 15 November 2015, Pages 7131-7137
Bioorganic & Medicinal Chemistry

Synthesis and evaluation of C-5 modified 2′-deoxyuridine monophosphates as inhibitors of M. tuberculosis thymidylate synthase

https://doi.org/10.1016/j.bmc.2015.09.053Get rights and content

Abstract

A series of 5′-monophosphates of 5-substituted 2′-deoxyuridine analogs, which recently demonstrated in vitro substantial suppression of two strains of Mycobacterium tuberculosis growth (virulent laboratory H37Rv and multiple resistant MS-115), has been synthesized and evaluated as potential inhibitors of M. tuberculosis thymidylate synthases: classical (ThyA) and flavin dependent thymidylate synthase (ThyX). A systematic SAR study and docking revealed 5-undecyloxymethyl-2′-deoxyuridine 5′-monophosphate 3b, displaying an IC50 value against ThyX of 8.32 μM. All derivatives lack activity against the ThyA. It can be assumed that the mechanism of action of 3b may be partially associated with the inhibition of the ThyX.

Introduction

Tuberculosis (TB) is one of the most serious problems of modern health care. At present, Mycobacterium tuberculosis infected about one-third of the world’s population and mortality from TB runs at 2 million people annually.1 TB is a crucial opportunistic infection for HIV-infected persons, which significantly increases the risk of progression of HIV infection. Furthermore, HIV infection increases the likelihood of activation of latent forms of M. tuberculosis2, 3 Nowadays, antitubercular therapy is a long course of at least six months including multicomponent drug cocktails and the increasing incidences of tuberculosis caused by drug resistant strains (MDR, XDR) complicate it substantially.4 The drug development for TB therapy acting on fundamentally new targets is the priority task of medicine.5 The genome sequencing of M. tuberculosis6 has created the basis for the search of such targets.

Natural and synthetic compounds with antitubercular activity belong to different classes of organic substances: phenols, quinones, peptides, steroids, alkaloids, terpenes etc. Therapy for viral infections is often based on using natural nucleoside derivatives.7 However, nucleosides have not been found to be potential antitubercular agents until the last decade. Recent reports have appeared on several groups of modified nucleosides displaying a remarkable antimycobacterial activity in vitro.8, 9, 10, 11, 12 One type of the promising pyrimidine nucleoside derivatives has lengthy 1-alkinyl substituents at the C-5 position of nucleobase moiety.9 Recently we reported about a new set of antitubercular active C-5 modified nucleosides with various lengthy substituents introduced via ether or (1,2,3-triazol-1-yl)methyl linkers.12, 13 The most active compounds 5-dodecyloxymethyl-2′-deoxyuridine and 5-[4-decyl-(1,2,3-triazol-1-yl)methyl]-2′-deoxyuridine showed significant activity (MIC99: 20 and 10 μg/mL, respectively) against M. tuberculosis virulent laboratory strain H37Rv as well as drug resistant MS-115 strain. Unfortunately, the mechanism of action and the main target of the compounds are still undisclosed.

In the most eubacteria and eukaryotic cells de novo synthesis of dTMP is carried out by thymidylate synthase (TS or ThyA) catalyzing the reductive methylation of dUMP to dTMP.14 However, it has been shown that several microorganisms including Bacillus, Helicobacter and Mycobacteria families contain another TS—ThyX which shows no structural similarity with ThyA and catalyzes dTMP formation via quite different mechanism.15 Since ThyX is rare in eukaryotes and is absent in humans, it can be considered as an attractive target for the design of specific ThyX inhibitors to combat tuberculosis. Recently, 5-alkyl and 5-aryl-derivatives of 2′-deoxyuridine 5′-monophosphates, as well as 6-aza-2′-deoxyuridine 5′-monophosphates and acyclic nucleoside 5′-phosphonates that contain 5-alkynyl uracil have been revealed as selective inhibitors of ThyX with IC50 values from 0.9 to 30 μM.16, 17, 18 However, 5′-modified nucleotides did not exhibit any antimycobacterial activity even at high concentrations (up to 250 μM).13, 19

The aim of this work was the synthesis and evaluation of 2 types of the 5′-monophosphates of 2′-deoxyuridine derivatives with lengthy alkyl substituents at the C-5 position, introduced via ether (including 3′-azido-2′,3′-dideoxy-5-dodecyloxymethyluridine with additional 3′-azido group) or (1,2,3-triazol-1-yl)methyl linkers. These compounds demonstrated substantial suppression of M. tuberculosis growth,12, 13 (there is no inhibition of ThyA) Additionally 5′-monophosphate of 5′-(2,5,8,11-tetraoxadodecyl)-2′-deoxyuridine was synthesized. Choice of hydrophilic substituent of the latter (one with a chain of 12 atoms at the C-5 position of the pyrimidine base) corresponds to nucleosides, with the highest antitubercular activity.9, 12, 13

Section snippets

Chemistry

Preparation of nucleoside 5′-monophosphates starting from 3′-unprotected nucleosides is usually carried out using Yoshikawa procedure (phosphorus(V) oxychloride/triethyl phosphate).20 In our case, the selective phosphorylation at 5′-hydroxy group led to disappointing results with low yields of the expected products.

To perform the phosphorylation of 5-alkyloxymethyl-2′-deoxyuridines 1ad, which were prepared according to the methods described previously,21 a tert-butyldimethylsilyl (TBDMS) group

Conclusion

Previously we described the antimycobacterial activity of C-5 modified nucleosides with various lengthy substituents introduced via ether or triazolyl linkers.12, 13 Herein we reported a facile synthesis of modified nucleotides as potential inhibitors of the mycobacterial enzyme ThyX. The method is based on using combination of protecting groups: acetyl and tert-butyldimethylsilyl followed by phosphorylation. 1,3-Dipolar cycloaddition reaction between proper 1-acetylenes and

Experimental section

The reactions were performed with the use of commercial reagents (Acros, Aldrich and Fluka); anhydrous solvents were purified according to the standard procedures. Column chromatography was performed on Silica Gel 60 0.040–0.063 mm (Merck, Germany) columns (elution with CHCl3/MeOH) and LiChroprep RP-2 (0.025–0.040 mm). Thin layer chromatography (TLC) was performed on Silica Gel 60 F254 aluminum-backed plates (Merck, Germany). Preparative chromatography was performed on Silica Gel 60 F254

Acknowledgements

Molecular modeling and physicochemical analysis were supported by the Russian Science Foundation, project No 14-50-00060. Chemical synthesis described in this paper was supported by ‘Molecular and cellular biology’ Program of the Presidium of the RAS and the Russian Foundation for Basic Research [Grant number 14-04-00755].

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