Synthesis and anti-trypanosomal evaluation of novel N-branched acyclic nucleoside phosphonates bearing 7-aryl-7-deazapurine nucleobase

Highlights

• Synthesis of novel aza-ANPs with 7-aryl-7-deazapurine nucleobase.

• Anti-trypanosomal activity with EC₅₀ values 0.58–6.8 μM.

• Improved selectivity index of the most potent compound (SI = 16.4) compared to tubercidin (SI = 0.40).

• TbrAPRT1 inhibitors with low micromolar K_i values.

Abstract

A series of novel 7-aryl-7-deazaadenine-based N-branched acyclic nucleoside phosphonates (aza-ANPs) has been prepared using the optimized Suzuki cross-coupling reaction as the key synthetic step. The final free phosphonates 15a-h were inactive, due to their inefficient transport across cell membranes, but they inhibited Trypanosoma brucei adenine phosphoribosyltransferase (TbrAPRT1) with K_i values of 1.7–14.1 μM. The corresponding phosphonodiamidate prodrugs 14a-h exhibited anti-trypanosomal activity in the Trypanosoma brucei brucei cell-based assay with EC₅₀ values in the range of 0.58–6.8 μM. 7-(4-Methoxy)phenyl-7-deazapurine derivative 14h, containing two phosphonate moieties, was the most potent anti-trypanosomal agent from the series, with EC₅₀ = 0.58 μM and SI = 16. Finally, phosphonodiamidate prodrugs 14a-h exerted low micromolar cytotoxicity against leukemia and/or cancer cell lines tested.

Introduction

Nucleosides bearing 7-deazapurine (pyrrolo[2,3-d]pyrimidine) as a nucleobase are structural analogues of biogenic purine nucleosides [1]. The only structural difference is the replacement of N7 nitrogen with carbon. Due to this substitution, the five-membered ring of 7-deazapurine (the pyrrole ring) becomes electronically richer compared to imidazole and, thus, can more easily participate in potential cation-π and π-π interactions [1].

Several 7-deazapurine nucleosides with diverse biological properties have been found in nature [2]. Among the most thoroughly studied representatives are tubercidin (1a, Fig. 1) and its derivatives toyocamycin (1b) and sangivamycin (1c), natural products isolated from Streptomyces spp. These analogues have been scrutinized for their antibiotic, antifungal, antiviral and/or anticancer activities [1]. Tubercidin (1a), for example, displays both antiviral [3,4] and anti-trypanosomal [5] properties. However, because of its notoriously high toxicity to mammalian cells, its use in clinical practice is negligible.

In view of this, several recent studies have focused on the development of tubercidin analogues, namely 7-(het)aryl-7-deazapurine nucleosides (e.g. compounds 2 and 3, Fig. 1) [[6], [7], [8], [9], [10]]. Several newly prepared derivatives exhibited potent cytotoxic [6], anti-mycobacterial [7] or antiparasitic [[8], [9], [10]] effects. In particular, derivatives substituted at C7 position with a five-membered heterocycle were found to be highly cytotoxic [6]. In contrast, derivatives with bulkier substituents, such as phenyl or pyridine, were less cytotoxic [5].

In addition to the relatively extensive study of 7-substituted 7-deazapurine derivatives, other articles have focused on additional modifications of either nucleobase [11,12] or carbohydrate moiety [5,13,14]. However, to our best knowledge, the fundamental cyclic sugar moiety was always preserved in the aforementioned reports.

Design of our current series of compounds was based on the structure of acyclic nucleoside phosphonates (ANPs) [15,16]. ANPs are a group of nucleotide analogues with a broad spectrum of biological activities, including antiviral, antibacterial, antiparasitic, and cytostatic [17]. ANPs consist of a heterocyclic base connected to a phosphonate group through various aliphatic linkers that mimic the sugar moiety [18,19]. The main advantages of ANPs (represented by compound 4, adefovir, Fig. 1) are the enzymatic and chemical stability of the phosphonate group (compared to phosphate), the absence of the hydrolysable glycosidic bond (present in natural nucleos(t)ides), and the increased flexibility of the acyclic linker, which enables the compounds to adopt a suitable conformation [18,19]. The linkers in ANPs can be unbranched, as in adefovir (Fig. 1), or branched, in which case the nitrogen atom is the convenient branching point, as previously demonstrated for the so-called aza-ANPs (e.g., analogues 5, Fig. 1) [[20], [21], [22], [23], [24]]. The introduction of nitrogen as the branching atom within the aliphatic linker enables the attachment of a new chain with additional functional groups, thereby opening possible new interactions of potential inhibitors with the enzyme active site.

Here, we designed a novel series of aza-ANPs, compounds 6 (Fig. 1), based on the combination of aza-ANPs 5 (employing the aliphatic linkers) with the above-mentioned 7-aryl-7-deazapurine nucleosides 2 and 3 (using the modified adenine nucleobase). As side chains for the attachment to the branching nitrogen atom, aliphatic linkers bearing various functional groups (namely hydroxyl, cyano group, and phosphonate moiety) were selected, which were previously successfully employed for the design and synthesis of potent antimalarial agents based on the inhibition of 6-oxopurine phosphoribosyltransferases [[20], [21], [22]].

Novel aza-ANPs 6, prepared in the form of phosphonodiamidate prodrugs, were evaluated for their potential anti-trypanosomal and cytotoxic activities.