Potent Anti‐HIV Activity of Alkyl‐Modified DiPPro‐Nucleotides

Two convergent approaches for synthesizing a new class of nucleoside diphosphate prodrugs bearing different nucleoside analogs are reported herein. The DiPPro‐nucleotides comprise an acyloxybenzyl group in combination with a lipophilic alkyl residue at the β‐phosphate or β‐phosphonate group, respectively. They are selectively cleaved to form their corresponding β‐alkylated nucleoside diphosphate derivatives in chemical and biological hydrolysis studies. In contrast, there is a selective but slow cleavage observed in the hydrolysis of the DiPPro‐compounds bearing two different, nonbioreversible alkyl moieties in human CD4+ T‐lymphocyte CEM/0 cell extracts. In these studies, the delivery of nucleoside monophosphates rather than nucleoside diphosphates is being observed, most likely due to a pure chemical phosphoranhydride cleavage of the β‐phosph(on)ate moiety. The antiviral evaluation of these two types of prodrugs reveals that these compounds exhibit marked anti‐HIV efficacy in HIV‐2‐infected thymidine kinase‐deficient CD4+ CEM T‐cells (CEM/TK−), with significantly better activities (up to 6700‐fold) against HIV‐2 replication than the parent nucleosides. Primer extension assays demonstrate that the β‐dialkylphosphate‐modified nucleoside derivatives, β‐monoalkylated‐diphosphates, and nucleoside diphosphates serve as substrates for HIV reverse transcriptase for the viral DNA elongation.


Introduction
For many decades, nucleoside analogs such as 1 (Figure 1) have been an important part of the treatment of several viral infections caused by the human immunodeficiency virus (HIV), herpes virus (herpes simplex virus, varicella-zoster viruses, and cytomegalovirus), hepatitis B virus and hepatitis C virus (HCV), influenza, and recently the severe acute respiratory syndrome coronavirus 2. [1][2][3][4][5][6][7][8][9] Nucleoside analogs still serve as cornerstones of current antiviral and antitumoral chemotherapies. [2]][16] These NTPs are substrates for the viral RNA-dependent DNA polymerase of HIV (HIV-RT) [2,3] involved in the early phase of the infection either by blocking RT's enzymatic function or incorporation of the nucleotide analogs into the viral DNA followed by chain termination which results in the inhibition of HIV replication.Previous studies have shown that one or more steps in the phosphorylation pathways can be rate-limiting due to the specificity of the metabolizing cellular kinases, which results in low or no biological activity [15,17,18] or adverse effects. [19,20]As examples, for the nucleoside analogs d4T 1a [14,21] or AZT 1b [15,22] the limiting phosphorylation step is the conversion of d4T 1a to d4TMP 2a but in the case of AZT from AZTMP 2b to AZTDP 3b mediated by thymidine kinase (TK) or thymidylate kinase (TMP-K), respectively.

Chemical and Enzymatic Stabilities of DiPPro-Compounds 8 and 9
All DiPPro-compounds 8 and 9 were studied in different media with respect to their stabilities and potential hydrolysis products.The studies of DiPPro-compounds 8 and 9 were conducted by means of reversed-phase RP18-HPLC.The half-lives of DiPPro-compounds 8 and 9 in PBS, PLE, and CEM cell extracts are summarized in Table 1.The assumed chemical and enzymatic hydrolysis pathways of DiPPro-compounds 8 are summarized in Scheme 2 (lower section).

Hydrolysis in Human Plasma
Based on the phosphorylation profiles, nucleoside analogs 1b,c,d, h are phosphorylated the corresponding mono-, di-, and finally triphosphates.However, the action of antiviral active abacavir (ABC, 1e) is transformed into carbovir-triphosphate (CBVTP). [10]ccording to a report, [65] heparin-stabilized plasma mimics closest the serum (gold standard), whereas citrate-stabilized plasma differed markedly from serum, largely due to the addition of the complexing chelator citrate that "fishes out" divalent ions as Mg 2þ or Ca 2þ .In order to compare effects caused by the different plasma preparations, in the work presented here seven (AZT or ABC)-DiPPro-compounds 8b,e and 9b,e were incubated in both heparin-stabilized human or citrate-stabilized human plasma.The half-lives for DiPPro-compounds 8bw,8bx,8ex comprising a biodegradable moiety were found to be lower in heparinstabilized human plasma than in citrate-stabilized human plasma.This my point to a contribution to the compound's stability of the divalent ions still available in the heparin-stabilized plasma samples either directly by interaction with the charged diphosphate moieties or that plasma enzymes present depending on these ions loose activity in the citrate-stabilized samples.In contrast, the stabilities of DiPPro-compounds 8by,8bz,9b,9e bearing only noncleavable alkyl moieties were found to be very high and not influenced by the human plasma preparation (Table 2).

Dialkyl-Modified NDP Compounds 8by,8ez
The half-lives determined for dialkylated diphosphate derivatives 8by,8ez (t 1/2 > 8 h, Table 2) bearing two alkyl groups were very high.As hydrolysis products starting from the DiPPro-nucleotides 8by,8ez, a small amount of nucleoside analogs was detected (Figure 5B and S37-S39, Supporting Information).Additionally, almost no formation of NMPs was detected using these dialkylated diphosphate derivatives in contrast to the studies in PBS.

Antiviral Evaluation
DiPPro-nucleotides 8 and 9 and the corresponding parent nucleosides were investigated for their in vitro anti-HIV activity in HIV-1 and HIV-2-infected wild-type CEM/0 cells and in HIV-2-infected mutant TK-deficient (CEM/TK À ) cells.The antiviral and cytostatic data are displayed in Table 3.As anticipated, nucleoside analogs 1b,1d-f were devoid of any antiretroviral activity (EC 50 > 100 μM, Table 3) in CEM/TK À cells because of the lack of their intracellular kinase-catalyzed activation. [15,22]Their corresponding NDP derivatives 3 (e.g., AZTDP (EC 50 = 40.9μM)) also exhibited no antiviral activity in CEM/TK À cell cultures because of the high polarity and dephosphorylation.Assay conditions are summarized in the Experimental Section (Supporting Information).
It was concluded from these studies that DiPPro-nucleotides 8 studied here delivered successfully nucleotide metabolites (NMPs and alkylated diphosphate analogs 9,10) inside cells with demonstrated potent antiviral activity.The promising antiviral data of the DiPPro-compounds 8 bearing different nucleoside analogs demonstrate the general applicability and high potential of the DiPPro-approach described here.Therefore, these advanced DiPPro-compounds 8 offer high potential and expectations in the development of future antiviral chemotherapies.
As anticipated, most of the C18-NDPs 9 exhibited lower activities against HIV-2 replication in cultures of infected wild-type CEM/0 cells as compared to the antiviral evaluation of the parent nucleoside analogs 1, which might be due to their high polarity which hampered cell membrane penetration.Nevertheless, the antiviral activity determined for C18-ABCDP 9e (EC 50 : 0.044 μM/HIV-1; EC 50 : 0.026 μM/HIV-2) in wild-type CEM/0 cells was 210-fold and 130-fold, respectively, better as compared to the parent nucleoside ABC 1e for unknown reasons.Interestingly and somewhat surprisingly, all mono-alkylated DiPPro-compounds 9 bearing a C18 aliphatic chain also showed moderate to marked antiviral activity against HIV-2 in CEM/TK À cells.With C18-ABCDP 9e (EC 50 < 0.001 μM/HIV-2) the antiviral potency in CEM/TK À cells was also considerably improved by >2000-fold as compared to ABC 1e, indicating a successful uptake into cells of the mono-alkylated nucleoside diphosphates 9.In addition to their anti-HIV activity in virus-infected cell cultures, DiPPro-compounds 8 exhibited only slightly higher cellular cytotoxicity than the parent nucleosides 1 whereas most monoalkylated DiPPro-compounds 9 (CC 50 > 100 μM) were endowed with minimal cellular cytotoxicity.

Primer Extension Assays
As disclosed above, DiPPro-compounds 8 showed significant anti-HIV activity in HIV-1/2-infected wild-type CEM/0 cells and in HIV-2-infected CEM/TK À cells.Taken the results from the hydrolysis studies, the formation of monoalkylated nucleoside diphosphates 9,10 (for the DiPPro-compounds 8bw-fw and 8bx-fx) and NMPs 2 (for the DiPPro-compounds 8by-fy and 8bz-fz) was detected in PBS as well as in CEM/0 cell extracts, which is a striking difference to the doubly, bioreversibly modified DiPPro-compounds 5 (Scheme 2).Previously we have shown that (alkyl)-d4TDPs 9a were substrates for HIV-RT. [60]To shed further light into these results, primer extension assays with HIV-RT and two different human DNA-polymerases α and γ were performed here as well.As controls in these primer extension assays, the four canonical dNTPs were added to the polymerases  (positive control (þ lane)) and a further experiment without the polymerases (negative control (-lane)) was performed.Additionally, TTP was used as the reference compound because TTP was accepted not only by HIV-RT but also for DNA polymerases α, γ as a substrate (Figure 6).
As expected, with HIV-RT full extension of the primer to the 30mer proceeded (þ lane).No extension was observed (-lane) without HIV-RT.Interestingly, HIV-RT recognize (C4;C18)-AZTDP 8by and C18-AZTDP 9b as substrate which was concluded from the appearance (though weak) of the corresponding n þ 1 bands.Furthermore, it appears that the ABC derivatives 8ey and 9e 2 may also serve as substrates for HIV-RT, albeit with low affinity.As also can be seen in Figure 6A,B, not only the triphosphates of the antiviral active nucleoside analogs such as  AZT, ABC or FddU and FLT were well incorporated into the primer but also their diphosphate derivatives.
The analogous experiments using DNA polymerase α showed no incorporation of the alkylated NDP compounds studied here (Figure 7A,B).In contrast to AZTTP, interestingly two triphosphate analogs proved to be substrates: FddUTP and FLTTP (Figure 7A).
All new alkylated compounds proved inactive in being substrates for DNA polymerase γ (Figure 8).

Conclusion
Here, a class of lipophilic nucleoside diphosphate compounds 8 comprising different nucleoside analogs is disclosed.The synthesis of DiPPro-nucleotides 8,9 was performed by using the H-phosphonate and/or H-phosphinate chemistry.The stability of DiPPro-compounds 8 was dependent on the present nucleoside analogs and the different types of masking groups.It was shown that the AB-moiety in DiPPro-compounds 8bw-fw and 8bx-fx was selectively cleaved to form monoalkylated NDP derivatives 9,10 by chemical hydrolysis in PBS and particularly in CEM/0 cell extracts, with PLE as well as in human plasma.The formation of nucleoside monophosphates 2 from DiPProcompounds 8by-fy and 8bz-fz in PBS containing PLE was purely chemically driven.As compared to the PLE hydrolysis, small amounts of nucleoside monophosphates were observed in the case of DiPPro-compounds 8by-fy and 8bz-fz in CEM/0 cell extracts, most probably due to the presence of phosphatases.From these studies, it was concluded that the cleavage of DiPPro-compounds 8 proceeded similar to the hydrolysis pathways for DiPPro-AZTDPs 8bw-bz in different chemical and biological media.
Hence, it was convincingly shown that the DiPPro-technology can provide a high potential to be used in antiviral chemotherapies compared to the β-(AB;AB)-nucleoside diphosphates [45] and γ-(AB;ACB or AB)-nucleoside triphosphates. [49,52]Highly active prodrugs 8 may be further studied in terms of their PK/PD properties as well as for their in vivo activity in the future.
in CD4 þ T-lymphocytes: 50% effective concentration; values are the mean AE SD of n = 2-3 independent experiments; b) Cytotoxicity: 50% cytostatic concentration or compound concentration required to inhibit CD4 þ T-cell (CEM) proliferation by 50%; values are the mean AE SD of n = 2-3 independent experiments.

Table 1 .
Hydrolysis half-lives of DiPPro-NDPs 8 and alkyl-NDPs 9 in PBS, PLE, and CEM/0 cell extracts as well as retention times.

Table 3 .
Antiviral activity and cytotoxicity profile of DiPPro-compounds 8,9 and NDPs in comparison with the parent nucleoside analogs 1.