The in vitro potency of GMP-grade stampidine (CAS 217178-62-6) was examined against 3 clinical HIV-1 isolates and 6 recombinant HIV-1 clones with multi-NRTI 'resistance (NRTI: nucleoside reverse transcriptase inhibitors). GMP-grade stampidine active drug substance (Lot #'s MPR-M0008.00-01 and MPR-M0008.01-01) as well as GMP-grade stampidine extracted from the clinical stampidine capsules (GMP-Grade Clinical Batch, Pharmaceutical Service Lot Number 159I0601) were highly potent and exhibited nanomolar IC50 values against clinical HIV-1 isolates as well as recombinant HIV-1 clones with multi-NRTI resistance containing common patterns of reverse transcriptase mutations responsible for NRTI resistance.

There is still a need for direct determination (i.e. without dephosphorylation) of nucleoside reverse transcriptase inhibitor (NRTI) triphosphorylated nucleotides in the peripheral-blood mononuclear cells (PBMCs) of HIV-positive patients. The objective of this paper was first to improve our previously described direct liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for stavudine triphosphate (d4T-TP). Preparation of PBMCs was modified to reduce degradation of d4T-TP during cell preparation and to simplify this step for routine use in clinical units. The performance of several HPLC columns was compared in order to improve the stability of peak shape over time. The SMT C(18) column was replaced by a Supelcogel ODP-50, thereby reducing two-fold the concentration of the first standard. Various internal standards were compared to optimize peak shape and remove an interfering peak in LC. 2-Chloroadenosine 5prime prime or minute-triphosphate was chosen as the most appropriate internal standard. Substitution of the narrowbore column by a microbore column (150 x 0.32 mm) is also presented and discussed. Secondly, this improved method was successfully applied to the simultaneous determination of d4T-TP, dideoxyadenosine triphosphate (ddA-TP) and lamivudine triphosphate (3TC-TP) in PBMCs, which is useful in view of the common use of NRTI combinations. The method was subsequently applied to clinical samples from HIV-positive patients receiving antiretroviral therapy containing d4T, ddl and/or 3TC. This method can be used simply and routinely on approximately 200 samples per week, using commercially available instruments and with a simple cell lysis as sample treatment.

New substituted-aryl phosphoramidate derivatives of the anti-HIV drug d4T were synthesized as membrane-soluble intracellular prodrugs for the free bioactive phosphate to establish relationship(s) between compound structure and in vitro antiviral activity. The majority of compounds demonstrated an elevation of in vitro potency relative to that of the parent nucleoside, and unlike d4T, all retained full activity in thymidine kinase-deficient cells. The compound bearing a p-chloro aryl group (8e) expressed nanomolar activity in vitro, a 14-fold increase in activity relative to that of the unsubstituted phosphoramidate (100-fold compared to d4T). An assay using pig liver esterase was used to establish the stability of the compounds to enzymatic degradation. While there was no apparent correlation between in vitro activity and half-life of enzymatic degradation, there was a close correlation between compound lipophilicity, determined by octanol/water partition coefficient, and in vitro potency. We suggest that substitutions made to the aryl moiety of the aryl phosphoramidate of d4T that result in enhancing lipophilicity may serve to increase the cellular uptake of the prodrug by passive diffusion, leading to the expression of antiviral potency at reduced prodrug concentrations.

d4T-5'-[p-Bromophenyl methoxyalaninyl phosphate](d4T-pBPMAP), a novel phenyl phosphate derivative of 2',3'-didehydro-2',3'-dideoxythymidine (d4T) that has an enhanced ability to undergo hydrolysis due to the electron withdrawing properties of its single bromo substituent at the para-position of the phenyl moiety, was found to yield substantially more of the key metabolite alaninyl d4T monophosphate (A-d4T-MP) than the unsubstituted d4T-5'-phenyl methoxyalaninyl phosphate or para-methoxy substituted d4T-5'-phenyl methoxyalaninyl phosphate. d4T-pBPMAP was tested for its anti-HIV-1 activity in peripheral blood mononuclear cells (PBMNC) and thymidine kinase (TK)-deficient CEM T-cells. d4T-pBPMAP was 12.6-fold more potent than the parent compound d4T in inhibiting p24 production (IC50 values: 44 nM vs 556 nM) and 41.3-fold more potent than d4T in inhibiting the reverse transcriptase (RT) activity (IC50 values: 57 nM vs 2355 nM) in HIV-1-infected TK-deficient CEM cells. Similarly, d4T-pBPMAP was more potent than the unsubstituted or para-methoxy substituted phenyl methoxyalaninyl phosphate derivatives of d4T. d4T-pBPMAP did not exhibit any detectable cytotoxicity to PBMNC or CEM cells at concentrations as high as 10,000 nM. Notably, d4T-pBPMAP was capable of inhibiting the replication of a zidovudine (ZDV/AZT)-resistant HIV-1 strain as well as HIV-2 in PBMNC at nanomolar concentrations. To our knowledge, this is the first demonstration that the potency of the d4T-aryl-phosphate derivatives can be substantially enhanced by introducing a single para-bromo substituent in the aryl moiety.

We report the design, synthesis and antiviral evaluation of a series of lipophilic, masked phosphoramidate derivatives of the anti-human immunodeficiency virus (HIV) nucleoside analogue d4T, designed to act as membrane-soluble prodrug forms for the free nucleotide. In particular, we report a series of 12 novel compounds with systematic variation in the structure of the carboxylate ester function. In order to rationalize the changes in antiviral action with variation of this moiety we applied our recently developed 31P NMR-based assay for carboxyesterase lability to this series. However, no clear positive correlation emerged, indicating that, at least within this series, factors other than simple esterase lability may be the major determinants of antiviral potency.

Three aryl phosphate derivatives of 2',3'-didehydro-2',3'-dideoxythymidine (d4T) were tested for their anti-human immunodeficiency virus (HIV) activity in peripheral blood mononuclear cells (PBMC) and thymidine kinase (TK)-deficient CEM T cells. Compared to the parent compound d4T, the lead compound d4T-5'-[p-bromophenyl methoxyalaninylphosphate] with a para-bromo substituent in the aryl moiety was 12.6-fold more potent in inhibiting p24 production (IC50 values: 44 nM versus 556 nM) and 41.3-fold more potent in inhibiting the reverse transcriptase (RT) activity (IC50 values: 57 nM versus 2355 nM) in HIV-infected TK-deficient CEM cells. None of the compounds exhibited any detectable cytotoxicity to PBMC or CEM cells at concentrations as high as 10,000 nM. To our knowledge, this is the first demonstration that the potency as well as selectivity index of the d4T aryl phosphate derivatives in TK-deficient cells can be substantially enhanced by introducing a single para-bromo substituent in the phenyl moiety.

Novel chain-extended nucleoside phosphoramidates of the anti-human immunodeficiency virus (HIV) drug d4T (stavudine) have been prepared as possible membrane-permeable prodrugs of the bio-active free 5'-monophosphates. Phosphorochloridate chemistry gave the target compounds in moderate to high yields, and all materials were fully characterized by spectroscopic and analytical methods. The compounds are related to the previously reported phenyl methoxyalaninyl derivative of d4T, which was shown to be a potent and selective inhibitor of HIV. In this study the amino acid nitrogen and ester moieties were separated by methylene spacers of between two and six carbon atoms. In vitro evaluation of these compounds indicated an almost complete lack of anti-HIV activity, the compounds being several orders of magnitude less potent than the corresponding alpha-amino acid derivatives. The reasons for the virtual lack of anti-HIV activity appear to involve poor enzyme-mediated hydrolysis.

Nucleoside analogue prodrugs are dependent on efficient intracellular stepwise phosphorylation to their triphosphate form to become therapeutically active. In many cases it is this activation pathway that largely determines the efficacy of the drug. To gain further understanding of the determinants for efficient conversion by the enzyme thymidylate kinase (TMPK) of clinically important thymidine monophosphate analogues to the corresponding diphosphates, we solved the crystal structures of the enzyme, with either ADP or the ATP analogue AppNHp at the phosphoryl donor site, in complex with TMP, AZTMP (previous work), NH2TMP, d4TMP, ddTMP, and FLTMP (this work) at the phosphoryl acceptor site. In conjunction with steady-state kinetic data, our structures shed light on the effect of 3'-substitutions in the nucleoside monophosphate (NMP) sugar moiety on the catalytic rate. We observe a direct correlation between the rate of phosphorylation of an NMP and its ability to induce a closing of the enzyme's phosphate-binding loop (P-loop). Our results show the drastic effects that slight modifications of the substrates exert on the enzyme's conformation and, hence, activity and suggest the type of substitutions that are compatible with efficient phosphorylation by TMPK.

Removal of 2',3'-didehydro-3'-deoxythymidine-5'-monophosphate (d4TMP) from a blocked DNA chain can occur through transfer of the chain-terminating residue to a nucleotide acceptor by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). ATP-dependent removal of either d4TMP or 3'-azido-3'-deoxythymidine-5'-monophosphate (AZTMP) is increased in AZT resistant HIV-1 RT (containing D67N/K70R/T215F/K219Q mutations). Removal of d4TMP is strongly inhibited by the next complementary deoxynucleoside triphosphate (50% inhibitory concentration [IC(50)] of approximately 0.5 microM), whereas removal of AZTMP is much less sensitive to this inhibition (IC(50) of >100 microM). This could explain the lack of cross-resistance by AZT-resistant HIV-1 to d4T in phenotypic drug susceptibility assays.

The administration of CycloSaligenyl 3'-azido-2',3'-dideoxythymidine monophosphate (CycloSal-AZTMP) to CEM cells resulted in a concentration- and time-dependent conversion to the 5'-monophosphate (AZTMP), 5'-diphosphate (AZTDP), and 5'-triphosphate (AZTTP) derivatives. High ratios of AZTMP/AZTTP were found in the CEM cell cultures treated with CycloSal-AZTMP. The intracellular T(1/2) of AZTTP in CEM cell cultures treated with either AZT and CycloSal-AZTMP was approximately 3 h. A variety of human T- and B-lymphocyte cell lines efficiently converted the prodrug to the AZT metabolites, whereas peripheral blood lymphocytes and primary monocyte/macrophages showed at least 10-fold lower metabolic conversion of the prodrug. CycloSal-AZTMP failed to generate marked levels of AZT metabolites in thymidine kinase-deficient CEM/TK(-) cells, an observation that is in agreement with the substantial loss of antiviral activity of CycloSal-AZTMP in CEM/TK(-) cells. The inability of CycloSal-AZTMP to generate AZTMP in CEM/TK(-) cells is presumably due to a relatively high hydrolysis rate of AZTMP to the parent nucleoside AZT, combined with the inability of CEM/TK(-) cells to phosphorylate AZT to AZTMP through the cytosolic salvage enzyme thymidine kinase.

A QSAR study, involving the use of calculated physical properties (TSAR), and the use of a neural network approach (TSAR), has been performed concerning the anti-HIV activity and cytotoxic effects of a series of d4T phosphoramidate derivatives with varying L-alanine esters. Models were obtained which allow reliable predictions for the anti-HIV activity, and cytotoxicity, of these derivatives.