Anti-HIV-Active Nucleoside Triphosphate Prodrugs

Abstract

We disclose a study on nucleoside triphosphate (NTP) analogues in which the γ-phosphate is covalently modified by two different biodegradable masking units and d4T as nucleoside analogue that enable the delivery of d4TTP with high selectivity in phosphate buffer (pH 7.3) and by enzyme-triggered reactions in human CD4 + Tlymphocyte CEM cell extracts. This allows the bypass of all steps normally needed in the intracellular phosphorylation. These TriPPPronucleotides comprising an acyloxybenzyl (AB; ester) or an alkoxycarbonyloxybenzyl (ACB; carbonate) in combination with an ACB moiety are described as NTP delivery systems. The introduction of these two different groups led to the selective formation of γ-(ACB)-d4TTPs by chemical hydrolysis and in particular by cell extract enzymes. γ-(AB)-d4TTPs are faster cleaved than γ-(ACB)-d4TTPs. In antiviral assays, the compounds are highly active against HIV-1 and HIV-2 in wild-type CEM/O cells and more importantly in thymidine kinase-deficient CD4 + T-cells (CEM/TK − ).

γ‐Non‐Symmetrically Dimasked TriPPPro Prodrugs as Potential Antiviral Agents against HIV

“…D4TDP 1 d increased constantly to an amount of 15 % after 3 h incubation and 32 % after 9 h incubation. Thus, by introducing the hydrophilic PEG‐bearing acyloxybenzyl‐mask, the “normal” lipophilic acyloxybenzyl‐mask was cleaved preferably in these hydrolysis studies, which is in agreement with the results obtained from the studies as reported before [29] …”

“…As an example, when Tri PPP ro‐compounds 3 bf was totally consumed after incubation of 480 h, the ratio of 1 t : 1 d : 1 m was 14 : 11 : 1. Both intermediates γ‐(ab‐C15)‐d4TTP 5 f and γ‐(AB−C4)‐d4TTP 5 b were formed in almost identical concentrations and thus, the hydrolysis proceeded without marked selectivity, which was in agreement with the results obtained from the studies of γ‐(ACB−C4,ACB−C12)‐d4TTPs in PB, but in contrast with results as γ‐(AB−C2,ACB−C16)‐d4TTPs [29] . Compared to the half‐lives in PB of symmetric γ‐(AB−C4,AB−C4)‐d4TTP (t 1/2 =22 h) and γ‐(AB−C9,AB−C9)‐d4TTP (t 1/2 =44 h), the half‐life of all non‐symmetric Tri PPP ro‐compounds studied here showed half‐lives close to that of γ‐(AB−C9,AB−C9)‐d4TTP, e. g. γ‐(AB−C4,AB−C14)‐d4TTP 3 be ; t 1/2 =45 h, although most of them comprise also one short acyloxybenzyl moieties [26] .…”

“…In addition, both intermediates 5 f and 5 b were observed in low concentration. Thus, as above and in contrast to our previous work on the Di PP ro‐compounds 6 [23] and γ‐(AB,ACB)‐d4TTPs [29] no selective cleavage of the different masks was observed in the hydrolysis in CEM cell extracts. In our previous work, the half‐lives of symmetric γ‐(AB−C4,AB−C4)‐d4TTP and γ‐(AB−C9,AB−C9)‐d4TTP in CEM/0 cell extracts were t 1/2 =0.43 h and t 1/2 =2.8 h, respectively.…”

“…D4TMP 1 m was prepared applying the method described by Sowa and Ouchi [30] . For the synthesis of γ‐(AB,ab)‐d4TTPs 3 , the H ‐phosphonate route was used as disclosed previously by us [27,29] . Briefly, diphenyl hydrogen phosphonate (DPP) was selectively reacted with two different acyloxybenzyl alcohols 7 which led to H ‐phosphonates 8 in moderate yields ranging from 44 % to 52 %.…”

“…The prodrugs comprised two different ACB‐moieties or a combination of an ACB and an AB mask at γ‐phosphate. For the latter compounds it has proven that the acyloxybenzyl‐mask was faster cleaved to give almost selectively the γ‐ACB‐NTP‐intermediates in chemical hydrolysis studies and also in cell extracts [29] …”