Prodrugs of γ‐Alkyl‐Modified Nucleoside Triphosphates: Improved Inhibition of HIV Reverse Transcriptase

Abstract The development of nucleoside triphosphate prodrugs is one option to apply nucleoside reverse transcriptase inhibitors. Herein, we report the synthesis and evaluation of d4TTP analogues, in which the γ‐phosphate was modified covalently by lipophilic alkyl residues, and acyloxybenzyl prodrugs of these γ‐alkyl‐modified d4TTPs, with the aim of delivering of γ‐alkyl‐d4TTP into cells. Selective formation of γ‐alkyl‐d4TTP was proven with esterase and in CD4+‐cell extracts. In contrast to d4TTP, γ‐alkyl‐d4TTPs proved highly stable against dephosphorylation. Primer extension assays with HIV reverse transcriptase (RT) and DNA‐polymerases α, β or γ showed that γ‐alkyl‐d4TTPs were substrates for HIV‐RT only. In antiviral assays, compounds were highly potent inhibitors of HIV‐1 and HIV‐2 also in thymidine‐kinase‐deficient T‐cell cultures (CEM/TK−). Thus, the intracellular delivery of such γ‐alkyl‐nucleoside triphosphates may potentially lead to nucleoside triphosphates with a higher selectivity towards the viral polymerase that can act in virus‐infected cells.


Introduction
Over the last decades av ariety of nucleoside analogues have been applied in antitumor and antiviral therapy and still play an important role in combatting HIV,herpes viruses,and hepatitis Ba nd hepatitis Cv iral infections. [1,2] Thet argets of these nucleoside analogue drugs are the virus-encoded DNAor RNAp olymerases,s uch as HIV reverse transcriptase (RT) [3,4] or the hepatitis C-encoded RNA-dependent RNApolymerase NS5B. [5] To date,s everal nucleoside analogues have been approved as HIV reverse transcriptase inhibitors (NRTIs) [6] and they are used as the backbone of the combined antiretroviral therapy (cART). However,the antiviral activity of nucleoside analogues,such as 3'-deoxy-2',3'-didehydrothymidine 1 (d4T), athymidine (3)analogue,isdependent on the intracellular phosphorylation by host cell kinases into their nucleoside triphosphates (NTPs), for example,d 4TTP 2t via the nucleoside mono-(2m,h ere:d 4TMP) and the diphosphate (2d,h ere:d 4TDP). [7,8] This stepwise transformation into the corresponding triphosphates,such as 2t,often occurs insufficiently by the involved kinases due to structural differences to the natural nucleosides (for example,TMP 4m, TDP 4d,and TTP 4t). Furthermore,limitations,such as poor biological half-lives due to catabolic elimination, variable bioavailability after oral administration or selection of drug resistance,c ounteract the use of nucleoside analogues. [9] To overcome some of these hurdles,p rodrugs of the first phosphorylated metabolite (nucleoside monophosphate, NMP;S cheme 1) have been explored in the past. [10][11][12][13][14][15][16] This resulted in orally applicable forms of af ew antiviral NMPs that are used as powerful drugs.R ecently,w eh ave reported asuccessful NDP-delivery approach using lipophilic,but still partially charged, NDP prodrugs (DiPPro approach; Scheme 1). [17][18][19] Fort he delivery of the ultimately active nucleoside analogue triphosphate several approaches using nanoparticles or nanogels (nanocarriers) were tried. [20] This was mainly done because it was thought that it is impossible to design chemical NTP prodrugs. [21] However, we recently reported the first chemical delivery system for NTPs through aprodrug technology (TriPPPro approach 5;S cheme 1). [22][23][24][25] We proved for d4T 1 and other nucleoside analogues that the corresponding Tr i PPPro compounds even retained pronounced anti-HIV activity in thymidine-kinase-deficient T-Scheme 1. Metabolism of nucleoside analoguessuch as d4T 1 and the corresponding nucleotide prodrugs thereof. cell cultures (CEM/TK À ), whereas the parent d4T 1 was virtually inactive in these cells due to the lack of phosphorylation. Them embrane permeability was achieved by two covalently but bioreversibly attached 4-acceptor-substituted benzyl esters at the g-phosphate group.T he enzyme-driven cleavage of both masks from TriPPPro compounds 5 by an initial cleavage of the acyloxy ester moiety and asubsequent spontaneous cleavage of the remaining part of the mask led to the formation of d4TTP 2t (Supporting Information, Scheme SI1). Thecellular uptake of these compounds was proven by ausing fluorescent nucleoside analogue. [24] Thed evelopment of NTP prodrugs is highly desirable because this would lead to the bypass of all steps of phosphorylation and would in principle maximize the intracellular concentration of the ultimately bioactive NTP. [26] However,t here are two important unsolved issues.T he first drawback can be related to the intracellular metabolic instability of NTPs due to fast enzymatic dephosphorylation. Assuming an efficient delivery of NTPs from the prodrug form, arapid enzymatic dephosphorylation would counteract the original idea of producing high intracellular concentrations of the NTPs because rephosphorylation is often inefficient in the case of nucleoside analogues. [7,8] Thus, am etabolic stabilization of the triphosphate unit is needed in order to keep the concentration as high as possible. Secondly,a na sh igh as possible selectivity of the nucleoside analogue triphosphate for viral polymerases and not for cellular polymerases,s uch as DNAp olymerases a, b,a nd g, should be achieved. It was observed previously that pyrimidine nucleoside triphosphates,i ncluding d4TTP inhibited DNApolymerase b and even more DNApolymerase g,while not being as ubstrate for DNAp olymerase a. [27] Particularly, the inhibition of the mitochondrial DNAp olymerase g is crucial and often associated with marked toxicity effects. [28,29] Although the described compounds were not fully characterized, Krayevsky et al. reported that the replacement of the g-phosphate group by am ethyl-or aphenylphosphonate moiety had almost no effect on the substrate properties towards retroviral RT,b ut abolished their ability to function towards DNAp olymerases a and b. [30,31] Moreover,t he authors reported that such compounds led to an increase in stability of the triphosphate unit in human serum (about 10fold). [32] However,their compounds proved to be completely inactive in antiviral tests and no prodrug forms were studied. Thus,w ed ecided to explore lipophilic prodrugs of g-alkyl-NTPs.Afurther motivation that such an approach is worth exploring was obtained from our own previous work. In contrast to Tr i PPPro compounds 5 (unpublished results and refs. [33] and [34]), the intermediate that comprised only one masking group (Supporting Information, Scheme SI1) proved to be asubstrate for HIV-RTand d4TMP was incorporated by HIV-RTi naprimer extension assay into the DNAs trand. Although to al esser extent than the Tr i PPPro derivatives 5, the monomasked d4TTP derivative comprising al ipophilic octadecyloxybenzyl moiety surprisingly proved to be anti-HIV active in the cell assay using CEM/TK À cells (EC 50 = 1.5 mm). [34] These observations guided us to design NTP prodrugs 6 that comprise different length g-alkyl chains in combination with ab iodegradable acyloxybenzyl group.T he aim was the intracellular delivery of g-alkyl-NTPs 7 (Figure 1).

Results and Discussion
Synthesis of g-(AB,Alkyl)-d4TTP Prodrugs 6 Title compounds 6 were synthesized using the H-phosphonate route (Scheme 2). [24] First, d4TMP 2m was prepared from d4T 1 as described before. [35] Thed 4TMP 2 NH 4 + salt was isolated and converted into its acid form by aD owex 50WX8 (H + )i on exchange and then titrated with tetra-nbutylammonium hydroxide solution to pH 7f ollowed by freeze drying.
Thehighly hygroscopic di(nBu 4 N) + salt of d4TMP 2mwas rigorously dried before use.N ext, diphenyl H-phosphonate (DPP) was reacted with alcohols 8 and 4-acyloxybenzyl alcohols 9 to form mixed H-phosphonates 10. [36] This H-phosphonate route offered two main advantages as compared to the phosphoramidite method:i )H-phosphonates 10 were found to be stable at À20 8 8Cfor more than two years and ii)d4TMP was easier to synthesize and additionally more stable than the corresponding d4TDP.N ext, compounds 10 were reacted with N-chlorosuccinimide (NCS). Thef ormed corresponding phosphorochloridates were further reacted with tetra-n-butylammonium phosphate to give pyrophosphates 11 in almost quantitative yields.Due to their chemical lability,pyrophosphates 11 were quickly purified by extraction and immediately used in the next step.T he final reaction was started with an activation of pyrophosphates 11 (1.3 equiv) with trifluoroacetic acid anhydride (TFAA) followed by N-methylimidazole and then coupled with d4TMP 2m (1 equiv) to form g-(AB,alkyl)-NTPs 6 (n-Bu 4 N + form). After ar eversed-phase (rp) column chromatography step and aDowex 50WX8 (NH 4 + )ion exchange step,followed by as econd rp-column chromatography step, g-alkyl-NTP prodrugs (NH 4 + form) 6 were isolated as diastereomeric mixtures reliably in 33-63 %y ield (Scheme 2). These diaste-

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Research Articles 22064 www.angewandte.org reomeric mixtures were inseparable by chromatography and indistinguishable in the 31 P-NMR spectra.
Synthesis of g-Alkyl-d4TTPs 7a nd g-Alkyl-TTP 15 To synthesize g-alkyl-d4TTPs 7,t he b-cyanoethyl group was used during the synthesis as protection group for the gphosphate group.A fter the coupling reaction of compounds 12 w-z and d4TMP 2m to give the g-protected nucleoside triphosphates 13 w-z,t he crude reaction product was stirred in amixture of 40 %nBu 4 N + OH À (water solution) and acetonitrile at room temperature to yield target compounds 7w-z.T he reaction was monitored by HPLC and stopped after 8-20 hours. g-Alkyl-d4TTPs 7w-z were isolated in 15-46 %y ield (Scheme 3). By using the same method which led to g-alkyl-d4TTP 7, g-alkyl-TTPs 15 were synthesized as well for the primer extension experiment. [37] Stability Studies g-(AB,alkyl)-NTP prodrugs 6 and g-alkyl-NTPs 7 were incubated in aqueous phosphate buffer (PBS,25mm,pH7.3), or were exposed to pig liver esterase (PLE) in phosphate buffer (PBS) and to human CD4 + T-lymphocyte cell extracts to study their stability and the product distribution. The hydrolysis mixtures were analyzed by means of analytical rp-18-HPLC.T he calculated half-lives of prodrugs 6 ( Table 1, t 1/ 2 )reflect the removal of the bioreversible AB group to yield galkyl-NTPs 7.P ossible hydrolysis pathways and products are summarized in Scheme 4.

Chemical Stability in Aqueous Phosphate Buffer (PBS, pH 7.3)
In PBS,t he half-lives of g-(AB,alkyl)-d4TTP prodrugs 6 were found to be between 94 ha nd 269 hw ithout showing ac lear trend. Thel owest half-life was observed for compound 6cw comprising short alkyl residues in the AB group and as the alkyl moiety (94 h, Table 1). As hydrolysis products,apredominant formation of g-alkyl-d4TTPs 7 and av ery small amount of d4TDP 2m was detected (approximately 3%). After consumption of the starting material, no further increase of d4TDP concentration was observed. This supports the hypothesis that only the double-g-esterified d4TTP prodrugs 6 were prone to ab ond breakage between the g-a nd b-phosphate.A sa ne xample,t he hydrolysis of compound 6cz is shown in Figure 2A.T he formed g-alkyl-d4TTPs 7 proved to be entirely stable at pH 7.3 in PBS (Table 1). In contrast to previous studies with Tr i PPPro-NTPs 5 containing as econd cleavable mask (first generation NTP prodrugs), [23][24][25] no d4TTP was detected in these studies using 6 or 7.Aside reaction occurred during the studies using [a] Yields are given for the conversion of 2m to 6. compounds 7 that led to the formation of thymine by the cleavage of the glycosidic bond as reported before. [19,22] As shown in Figure SI1 in the Supporting information, this is the main reason for the decrease of 7z in PBS hydrolysis and the calculated half-life of 7z is about 1500 h.

Hydrolysis Study Using Esterase
Next, the enzymatic stability of prodrugs g-(AB-C4,alkyl-C18)-d4TTP 6cz and g-(AB-C15,alkyl-C4)-d4TTP 6ew was examined by incubation with PLE, which is used as am odel enzyme for studying the esterase activation of the prodrugs,in PBS,p H7.3 at 37 8 8C. Thec leavage of the acyloxybenzyl masking unit in 6cz occurred readily under the experimental

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Research Articles 22066 www.angewandte.org conditions (t 1/2 = 8.1 min) leading selectively to the g-alkyl-d4TTP derivative.F or prodrug 6ew containing al ong alkyl chain mask, the half-life was found to be 25.5 min. This was in full agreement with the results from the studies of Tr i PPPro compounds 5 described before [23,24] and proved as ignificant contribution of the enzyme.I nt hese studies,n oc leavage of the alkyl residue in compounds 7w,z was observed. This proves our initial concept of introducing an enzyme (esterase)-stable group to the g-phosphate unit.
Incubation of Compounds 6, 7, and 15 in Cell Extracts:A Comparison with the TriPPPro NTPs 5 Thes tability of prodrugs 6 and the g-alkyl-NTPs 7 was further investigated in human CD 4 + T-lymphocyte CEM cell extracts.T he half-lives of prodrugs 6 were found to be significantly lower than the half-lives in PBS buffer (up to 500-fold;T able 1). Thus,anenzymatic cleavage reaction took place.H alf-lives as low as 0.4 h( 6az)t o5 .2 h( 6dz)w ere determined. Here,c ompounds 6az and 6bz comprising an acetyl-or ap ropanoylester moiety,r espectively,w ere found to be the least stable compounds (t 1/2 of 0.4 and 1.6 h, respectively). This is in accordance to our previous results for the DiPPro- [17,18] or the Tr i PPPro-compounds 5. [22,23] In all cases we observed the predominant formation of g-alkyl-d4TTPs 7 and d4TDP 2d in very low concentration (Figure 2B)b ut no NTP formation was detected, in contrast to the studies using the previously described Tr i PPPro compounds 5. [23,24] However,t he difference to the compounds bearing long lipophilic acyl groups was not very pronounced (2-to 3-fold). In the case of g-(AB-C4,alkyl-C18)-d4TTP 6cz, g-C18-d4TTP 7z was detected as the main product. After 8h incubation, the ratio of g-C18-d4TTP 7z and d4TDP 2d was 5:1. This was in sharp contrast to the studies performed with Tr i PPPro compounds 5. [23,24] There,i twas almost impossible to detect significant concentrations of d4TTP due to its fast dephosphorylation to form first d4TDP and finally d4TMP 2m.E ven after very long incubation periods (up to 30 h), compounds 7 proved to be entirely stable,for example, The hydrolysis experimentso fg-alkyl-NTP 6,7 were performed in aqueous2 5mm phosphate buffer (PBS, pH 7. 96 %ofthe thymidine bearing compound 15 was still detected after 30 h. Thef ormation of thymine or d4TTP 2t from g-C18-d4TTP 7z was not detected (Supporting Information, Figure SI2).

Antiviral Evaluation
g-(AB,alkyl)-d4TTP prodrugs 6 and the g-alkyl-d4TTPs 7 were evaluated for their ability to inhibit the replication of HIV in HIV-1-or HIV-2-infected wild-type CEM/0 as well as mutant thymidine-kinase-deficient CEM cell cultures (CEM/ TK À ). Forcomparison, two Tr i PPPro derivatives 5 were also included in this assay.T he results are summarized in Table 1. As can be seen, all compounds showed virtually similar or even slightly better activities against HIV-1 and HIV-2 than the parent nucleoside d4T 1.B ecause the lipophilicity of compounds 6 is similar due to the combination of the introduced aliphatic alkyl groups in the masking unit as well as in the stable g-alkyl moiety,t he antiviral activity did not differ much. More importantly almost all prodrugs 6 were also highly potent in CEM/TK À cell cultures whereas d4T 1 lacked any relevant anti-HIV activity in this thymidinekinase-deficient cell line (EC 50 :1 50 mm). Only prodrug 6ey (C15/C15) lost some of the activity displayed in the wild-type CEM cell cultures compared to the other prodrugs,while 6ew (C15/C4) was surprisingly found to be inactive in the HIV-2infected CEM/TK À cell line.A lso,p rodrug 6cw (C4-AB and C4 chain) showed amarked loss of activity in the CEM/TK À cell cultures.This might be due to an insufficient lipophilicity of the compound combined with ar elatively fast cleavage of the bioreversible moiety that led to the formation of shortchain g-C4-d4TTP 7w.C ompound 7w was almost inactive in this assay.I ts hould also be noticed that none of the prodrugs 6 were significantly more cytotoxic than the parent d4T 1 (Table 1). Interestingly,also the g-C18-alkyl-d4TTP 7z was highly potent against HIV-2-infected CEM/TK À cell cultures (EC 50 :0 .055 mm). Surprisingly,c ompound 7z is the most active compound of all the listed derivatives (2700-fold more active than d4T), although this compound is more negatively charged than the prodrugs.H owever,c om-pound 7z is highly lipophilic due to the C18-chain. Also,i n the case of the Tr i PPPro compounds 5,w ef ound for the intermediates comprising aC 17 aliphatic chain, antiviral activity in HIV-2-infected CEM/TK À cells (1.5 mm). [34] However,t he antiviral activity of compound 7z was even improved by 30-fold. Consequently,itseems that even one long aliphatic chain provides enough lipophilicity to enable the cellular uptake of the compounds.T his is supported by the results obtained for compounds 7w (C4 chain) and 7x (C11 chain). Both compounds (as well as 6ew)s howed am arked loss in antiviral activity in CEM/TK À cell cultures (35 mm and 16 mm,respectively,T able 1). Most probably their lipophilicity is not sufficient to enable an effective uptake into the cells.As expected, both thymidine-containing compounds 15 w and 15 z as well as d4TTP were found to be inactive in the CEM/ TK À cells.T he two TriPPPro compounds bearing either two C8 chains (5a)oramixture of ashort C4 and along C17 alkyl group (5b)were also active in the CEM/TK À cells.Again, the compound bearing the C17 chain proved to be more active than the second one (C8 chain). However,both do not reach the excellent activity of compound 7z.

Primer Extension Assays
All compounds described here showed significant antiviral activity against HIV-1-and HIV-2-infected wild-type CEM/O cell cultures.T his activity was also virtually retained in CEM/TK À cell cultures.T aking the results from the hydrolysis studies,i tc an be concluded that the delivered galkyl-d4TTPs 7 were responsible for the inhibitory effect of these compounds because we have proven that no d4TTP was formed. This is astriking difference to the formerly reported Tr i PPPro compounds 5. [23,24] Further studies showed that the g-dimasked Tr i PPPro compounds 5 were not substrates for DNAp olymerases,s uch as HIV-RTo rD NA polymerase b (unpublished results).
To shed more light on these results,p rimer extension assays were performed to investigate the substrate properties of the g-alkyl-triphosphates for three different DNAp olymerases:r everse transcriptase (RT; an HIV-encoded RNA-

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Research Articles 22068 www.angewandte.org dependent DNAp olymerase), cellular DNAp olymerases a, b,a nd the mitochondrial DNAp olymerase g,w hich is often responsible for toxic side-effects caused by nucleoside triphosphate analogues. [28,29] Tw od ifferent set-ups have been used. First, am ixture of dATP,d CTP,d GTP,a nd g-alkyl-TTP (N*) or -d4TTP (T*) was used as am ixture.T he results obtained from these experiments were compared to those in which just g-alkyl-TTP (N) or d4TTP (T) was used. In each experiment aprimer extension without added polymerase (negative control, -lane) and an experiment in which all four canonical NTPs were added (positive control, + lane) were used as controls.
Studies Using g-Alkyl-TTPs 15 and g-Alkyl-d4TTP 7with HIV-1 Reverse Transcriptase (RT) Figure 3s ummarizes the results of the primer extension using HIV-RTand 60 minutes incubation time.Noelongation occurred without HIV-RT( lane 1). Full extension was observed when all four canonical triphosphates were present (lane 2). Replacement of TTP 4t by g-C18-TTP 15 z also led to full extension of the primer proving that HIV-RTaccepted the alkylated TTP as asubstrate (lane 3). This was confirmed by the single incorporation assay using just g-C18-TTP without the other three canonical triphosphates (n + 1b and; lane 4). Next, g-C18-d4TTP 7z was used in combination with the other three canonical triphosphates (lane 5). Only the n + 1b and was detected because d4TMP was incorporated and acted as an immediate chain terminator.L ane 6s hows the result obtained when d4TTP 2t and the three other nucleoside triphosphates were used. Again, only the n + 1b and is visible.T hus,b oth g-C18-alkylated nucleoside triphosphates were accepted by HIV-RTasasubstrate,a nd were properly incorporated in the growing DNAc hain.
Next, an identical series of experiments was conducted using 15 minutes of incubation time (Supporting Information, Figure SI3). Thecontrol experiments showed identical results as above (lane 1,2,7). However,i nc ontrast to the first set of experiments only av ery weak spot for the full extension product was detected when g-C18-TTP 15 z was used (lane 3). When g-C4-TTP 15 w was used, again clearly the full extension product (lane 4) was detected. Thus,t he shorter alkyl chain led to ac ompound with much better substrate properties for HIV-RT. Single incorporation experiments proved this as well (lane 5). g-C4-d4TTP 7w in the mixture with the other three nucleoside triphosphates led to as ingle incorporation and an immediate chain termination. An incubation time of 30 minutes showed also for g-C18-TTP 15 z incorporation and elongation with strong stalls for incompletely elongated primers (Supporting Information, Figure SI4, lane 3). Again, single incorporation of g-C18-TTP 15 z proceeded but some of the starting primer was still present (lane 4). Incorporation of d4TMP from g-C18-d4TTP 7z again led to an immediate chain termination (lane 5).
Finally,acomparison of the three g-alkyl-modified d4TTP was performed (Supporting Information, Figure SI5). Different incubation periods were studied as well. Thef irst two lanes are the control reactions.Inall cases,the conversion of the primer to the n + 1product continued over time.Again, g-C4-d4TTP 7w and g-C11-d4TTP 7x were found to be better substrates for HIV-RTcompared to g-C18-d4TTP 7z.
Studies Using g-Alkyl-TTPs 15 and g-Alkyl-d4TTP 7with Human DNA Polymerase b (Pol b) Ther esults obtained for HIV-RTw ere compared to primer extension assays using human DNAp olymerase b (Figure 4).
Studies Using g-Alkyl-TTP 15 and g-Alkyl-d4TTP 7with Human DNA Polymerase g (Pol g) In contrast to the results obtained with DNApolymerase b,a ll compounds 15 (thymidine analogue) and 7 (d4TTP analogue) as well as d4TTP itself were not substrates for the mitochondrial DNAp olymerase g (Supporting Information, Figure SI6). Only the primer was detected after the assay. Thus,t his polymerase does not tolerate any modification at the g-phosphate moiety.
Studies Using g-Alkyl-TTP 15 and g-Alkyl-d4TTP 7with Human DNA Polymerase a (Pol a) More importantly,a ll g-modified compounds 7 and 15 were also not substrates for DNApolymerase a,which is one of the most important replication polymerases.I nc ontrast, nucleoside analogues without a g-phosphate alkyl residue were incorporated by the polymerase in our assays.(Supporting Information, Figure SI7).
Consequently,nucleoside triphosphates modified at the gposition are substrates for HIV-RT, while they fail to act as substrates when the modification has as ufficiently high size/ length. Thus,b yt hese structural changes,d 4TTP,w hich was originally as ubstrate for DNAp olymerase a and b,c an be converted into an on-substrate for this enzyme,w hile still being asubstrate for HIV-RT.

Conclusion
In summary,w eh ave disclosed as econd generation of lipophilic nucleoside triphosphate prodrugs which differ from the previously described TriPPPro derivatives [23][24][25] by comprising anon-cleavable moiety in addition to abiodegradable prodrug moiety at the g-phosphate group.A sn on-cleavable group lipophilic alkyl residues of different lengths were attached in addition to the previously used acyloxybenzylprodrug moiety.T he synthesis was achieved in satisfying chemical yields using H-phosphonate chemistry.W eh ave proven that the prodrug group was selectively cleaved to give g-alkyl-modified nucleoside triphosphates by chemical hydrolysis (slow process) or by enzymes present in cell extracts (fast process). The g-alkyl-nucleoside triphosphates proved to be stable in cell extracts for at least 30 hw hile the corresponding nucleoside triphosphates d4TTP or TTP were rapidly enzymatically dephosphorylated. In contrast to d4T itself,a ll these compounds showed marked antiviral activity against HIV-2 in thymidine-kinase-deficient cell cultures (CEM/TK À cells) indicating the successful cell membrane passage of these compounds.T hus,a lthough such secondgeneration nucleoside triphosphate prodrugs are still negatively charged at the phosphate groups,o bviously the modification at the g-phosphate group by one lipophilic, bioreversible moiety and the stable g-alkyl group gives the molecule sufficient lipophilicity to penetrate the cell membrane. g-Alkylated-TTPs were substrates for HIV-RTa s shown in primer extension assays,while they proved not to be substrates for the cellular DNApolymerases a, b,and g.T he corresponding d4TTP derivatives were also found to be substrates for HIV-RTbut were still not substrates for DNA polymerases depending on the alkyl chain length. Thus,these results point to an increased selectivity of the compounds to different polymerases with amarked preference for the viral enzyme HIV-RT.
Thus,h ere we have disclosed the development of an advanced prodrug concept for NTP derivatives that delivers g-alkyl-NTPs with high selectivity by an enzyme-triggered mechanism that then allows i) the bypass of all phosphorylation steps usually needed for the activation of an ucleoside analogue,i i) the delivery of compounds which act as substrates for av iral polymerase (RT) but not substrates for cellular DNAp olymerases,a nd iii)which show very high stability against dephosphorylation of the triphosphate unit compared to natural NTPs.W ea re currently working on whether this increased selectivity towards the viral polymerase is ag eneral property of these g-modified compounds by using different nucleoside analogues and different viral polymerases.
Please note:M inor changes have been made to this manuscript since its publication as an AcceptedArticle.T he Editor.