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Antiviral Chemistry & Chemotherapy 13:197–203 Effects of aryl substituents on the anti-HIV activity of the arylphosphoramidate derivatives of stavudine Fatih M Uckun4*, P Samuel2, S Qazi3, C Chen2, S Pendergrass4 and TK Venkatachalam1 1Departments of Chemistry, Drug Discovery Program, Parker Hughes Cancer Center, Parker Hughes Institute St. Paul, Minn., USA 2Department of Pharmaceutical Sciences, Parker Hughes Cancer Center, Parker Hughes Institute St. Paul, Minn., USA, 3Department of Bioinformatics, Parker Hughes Cancer Center, Parker Hughes Institute St. Paul, Minn., USA 4Department of Virology, Parker Hughes Cancer Center, Parker Hughes Institute St. Paul, Minn., USA *Corresponding author: Tel: +651 796 5450; Fax: +1 651 796 5493; E-mail: [email protected] We compared the anti-HIV activity of 13 phenyl phosphoramidate derivatives. The rate of chemi- phosphate derivatives of stavudine (2′,3′-didehy- cal hydrolysis under alkaline conditions (but not dro-2′,3′-dideoxythymidine/d4T) by examining the lipophilicity) predicted the potency of the their ability to inhibit HIV-1 replication in human compounds. peripheral blood mononuclear cells. Our results show that the introduction of electron-withdraw- Keywords: d4T, HIV, aryl phosphate, hydrolysis, ing substituents enhances the activity of these AIDS Introduction According to the most recent estimates, 36.1 million peo- (Uckun et al., 2000; Vig et al., 1998). The presence of a sin- ple worldwide are infected with HIV and more than 16 000 gle para-bromine group in the phenyl moiety of stampidine new infections occur daily (Sepkowitz, 2001; Greene, contributes to its ability to undergo rapid hydrolysis yield- 1991). More than 18 anti-retroviral drugs are now available ing the key active metabolite alaninyl-stavudine- for clinical use, and have led to significant reductions in monophosphate (ala-STV-MP) (Venkatachalam et al., morbidity and mortality for HIV-infected individuals 1998). STAMP has been shown to inhibit the replication (Sepkowitz, 2001; Greene, 1991). Reverse transcriptase of HIV-1 strain HTLV-IIIB, HIV-2, as well as the ZDV- (RT), a vital enzyme of HIV, responsible for the reverse resistant HIV-1 strain RT-MDR in human peripheral transcription of retroviral RNA to proviral DNA, is one of blood mononuclear cells at nanomolar concentrations the most important molecular targets in contemporary (Uckun et al., 2002). In preliminary studies, we found that treatment programmes against AIDS (Greene, 1991). stampidine is substantially more potent than stavudine in Stavudine/d4T is a pyrimidine nucleoside analogue used inhibiting HIV-1 replication in thymidine kinase-deficient in the treatment of human HIV infection. It inhibits viral T-cells (Venkatachalam et al., 1998; Uckun et al., 2002). reverse transcriptase as do zidovudine (ZDV/AZT), Whereas stavudine inhibited HIV-1 replication with an didanosine (ddI), zalcitabine (ddC) and lamivudine (3TC), IC50 value of 20 nM and a selectivity index (SI) of 100, which comprise the family of nucleoside analogue reverse stampidine inhibited HIV-1 replication with an IC50 value transcriptase inhibitors (NRTIs) (Balzarini et al., 1989). of ≤1 nM and an SI of ≥30 000 (Uckun et al., 2002). We The 5′-triphosphates of these NRTIs, which are generated recently investigated the in vivo pharmacokinetics and intracellularly by the action of nucleoside and nucleotide metabolism of this promising new anti-HIV agent in mice kinases, are potent inhibitors of HIV-1 RT (Balzarini et al., (Uckun et al., 2002). Stampidine was found to form two 1989). The rate-limiting step for the generation of the active metabolites, namely ala-STV-MP and stavudine, bioactive stavudine metabolite stavudine-triphosphate is with favourable pharmacokinetics after systemic adminis- the conversion of stavudine to its monophosphate deriva- tration (Uckun et al., 2002). Our recent studies provided evi- tive (Balzarini et al., 1989). In an attempt to overcome the dence that stampidine is a highly potent inhibitor of primary dependence of stavudine on intracellular nucleoside kinase clinical HIV-1 isolates with a genotypic and/or phenotypic activation, we prepared stampidine (STAMP)/HI-113, NRTI-resistant or non-nucleoside reverse transcriptase stavudine-5′-[p-bromophenyl methoxyalaninyl phos- inhibitor-resistant profile. In the present study, we prepared phate], a novel aryl phosphate derivative of stavudine 12 additional alaninyl phenyl phosphate derivatiives of ©2002 International Medical Press 0956-3202/02/$17.00 1 FM Uckun et al. stavudine and compared their anti-HIV activity. Our find- which uses a murine monoclonal antibody (mAb) to HIV ings establish the rate of chemical hydrolysis as the primary core protein coated onto microwell strips to which the anti- predictor of anti-HIV potency for these novel stavudine gen present in the test culture supernatant samples binds. derivatives. Percent viral inhibition was calculated by comparing the p24 values from untreated infected cells (that is, virus con- Materials and methods trols). Chemicals Partition coefficients All chemicals were purchased from Aldrich (Milwaukee, The octanol/water partition coefficient was determined by Wis, USA), with the exception of d4T, which was synthe- the shake flask method. The phosphoramidate analogues sized in-house. All syntheses were performed under a were added to 2 ml of water and 2 ml of octanol in a glass nitrogen atmosphere. 1H, 13C, and 31P NMR were obtained vial. The mixture was shaken for 4 h at room temperature. on a Varian Mercury 300 instrument at ambient tempera- The two phases were carefully separated and filtered ture in CDCl3.FT-IR spectra were recorded on a Nicolet through a Millipore filter and analysed by HPLC. The par- Protege 460 spectrometer. MALDI-TOF mass spectra tition coefficient was calculated using the ratio of the area were obtained by using a Finnigan MAT 95 system. UV under the curve for octanol and water, respectively. spectra were recorded by a Beckmann UV-VIS spectropho- tometer (Model 3DU 74000) with a cell path length of 1 Statistical analysis cm. High performance liquid chromatography (HPLC) The IC50 values were calculated from each set of triplicate purification was achieved by using a reverse-phase wells using non-linear regression modelling of the expo- × Lichrospher column (250 4 mm, Hewlett-Packard, RP- nential form of the linearized equation. The average IC50 18, Cat #79925) and an isocratic flow (1 ml/min) consist- values were log10 transformed to homogenize the variances ing of water (70%) and acetonitrile (30%). The alkaline within each group. Unpaired t-tests were performed in chemical hydrolysis was conducted at room temperature order to test for differences between the mean IC50 values with sodium hydroxide (1 ml of 0.05N) and 3 ml of for different compound groups. Hydrolysis rates were methanol solution containing 10 mg of the substrates in a determined by fitting single exponential decay equations to Teflon lined reaction vial. The solution was stirred using a the disappearance of the compound in alkali conditions. magnetic stirrer and an aliquot of the reaction mixture was The IC50 values of the compounds were correlated to the injected into HPLC. The disappearance of the starting log transformed hydrolysis rate constants by fitting a linear material was monitored as a function of time. The rate of model ( JMP Software, SAS Institute Inc.). P values less unimolecular reaction was obtained using first order rate than 0.05 were deemed significant. equation. HPLC runs were done with varying interval of time and measuring the disappearance of the substrate peak Physical constants for new compounds with time. 5′-[3-Dimethylaminophenylmethoxyalaninylphos- phate]-2′,3′-didehydro-3′-deoxythymidine (DDE 599). ° 1 δ In vitro assays of anti-HIV-1 activity Yield: 0.83 g (18%); mp: 61–62 C; H NMR (CDCl3) Normal human peripheral blood mononuclear cells from 9.93 (s, 1 H), 7.27 (br m, 1 H), 7.04 (m, 1 H), 6.97 (m, 1 HIV-negative donors were cultured 72 h in RPMI 1640 H), 6.44 (m, 3 H), 6.24 (m, 1 H), 5.81 (m, 1 H), 4.94 (m, supplemented with 20% (v/v) heat inactivated fetal bovine 1 H), 4.24 (s, 2 H), 4.08 (m, 1 H), 3.92 (m, 1 H), 3.64* (m, serum, 3% interleukin-2, 2mM L-glutamine, 25mM 3 H), 2.86 (s, 6 H), 1.77* (m, 3 H), 1.28* (m, 3 H); 13C δ HEPES, 2g/l NaHCO3, 50 mg/ml gentamicin, and NMR (CDCl3) 173.7*, 163.9*, 151.3*, 150.8*, 135.5*, 4 mg/ml phytohaemagglutinin prior to exposure to HIV-1 132.9*, 129.5*,126.9*, 111.0*, 108.8*, 107.2*, 103.7*, 89.3*, at a multiplicity of infection (m.o.i.) of 0.1 during a 1 h 84.4*, 66.7*, 66.1*, 52.3*, 49.9*, 40.2, 20.7, 12.2; 31P NMR ° δ adsorption period at 37 C in a humidified 5% CO2 atmos- (CDCl3) 3.32, 2.70; IR (KBr) n 3448, 3050, 2952, 1691, -1 λ phere. Subsequently, cells were cultured in 96-well 1506, 1450, 1247, 1143, 999 cm ; UV(MeOH) max 203, × 6 microtitre plates (100 ml/well; 2 10 cells/ml) in the pres- 206, 21, 258 nm; FAB MS m/z 531.1619 (C22H29N4O8P + ence of various concentrations of d4T phosphoramidates Na+); HPLC tR 3.36 min. and aliquots of culture supernatants were removed from the wells on the 7th day after infection for p24 antigen assays, 5′-[2,6-Dimethoxyphenylmethoxyalaninylphosphate]- as previously described (Uckun et al., 1998). The p24 2′,3′-didehydro-3′-deoxythimidine (DDE 600). Yield: ° 1 δ enzyme immunoassay (EIA) used was the unmodified 0.60 g (13%); mp: 51–53 C; H NMR (CDCl3) 9.78 (s, kinetic assay commercially available from Coulter 1 H), 7.38 (br d, 1 H), 6.95 (m, 3 H), 6.48 (m, 3 H), 6.29 Corporation/Immunotech, Inc.

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