(3) in Progress

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

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. (Westbrooke, Me., USA), (m, 1 H), 5.81 (m, 1 H), 4.36 (m, 3 H), 4.02 (m, 2 H), 3.74

Figure 1.

λ (m, 6 H), 3.63* (m, 3 H), 1.74* (d, 3 H), 1.29* (m, 3 H); cm–1; UV(MeOH) max 215, 267 nm; FAB MS m/z 13 δ + C NMR (CDCl3) 173.7*, 163.9*, 151.7*, 150.8*, 578.0105 (C20H22BrClN3O8P + H ); HPLC tR 18.63, 135.7*, 133.1*, 128.4, 126.8*, 125.0*, 110.9*, 104.8*, 89.2, 20.63 min. 84.6*, 66.8, 55.8*, 52.2*, 49.7*, 49.4*, 21.0*, 11.8*, 31P δ η ′ ′ NMR (CDCl3) 4.97, 4.28; IR (KBr) 3432, 3072, 2950, 5-[2-Bromophenylmethoxyalaninylphosphate]-2 ,3 - -1 λ ′ 1691, 1483, 1261, 1112, 931 cm ; UV(MeOH) max 210, didehydro-3 deoxythymidine (DDE 605). Yield: 0.36 g + ° 1 δ 267 nm; FAB MS m/z 526.1570 (C22H28N3O10P + H ); (19%); mp: 45–46 C; H NMR (CDCl3) 9.55 (s, 1 H), HPLC tR 6.55 min. 7.47 (m, 2 H), 7.24 (m, 2 H), 6.99 (m, 2 H), 6.29 (m, 1 H), 5.88 (m, 1 H), 5.00 (m, 1 H), 4.35 (m, 2 H), 4.02 (m, 2 H), 5′[5′-[3-Bromophenylmethoxyalaninylphosphate]- 3.66 (s, 3 H), 1.80* (m, 3 H), 1.30* (m, 3 H); 13C NMR 2′,3′-didehydro-3′deoxythymidine (DDE 602). Yield: (CDCl3) d 173.6*, 163.8*, 150.8, 147.3*, 135.4 *, 133.0*, ° 1 δ 0.67 g (14%); mp: 47–48 C; H NMR (CDCl3) 9.65 (s, 128.5*, 127.2*, 126.1*, 121.3 *, 114.4*, 111.3*, 89.6*, 84.3*, 31 δ 1 H), 7.34–7.11 (m, 5 H), 6.97 (m, 1 H), 6.26 (m, 1 H), 67.2*, 52.5, 50.1*, 29.6, 20.8*, 12.4; P NMR (CDCl3) 5.87 (m, 1 H), 4.98 (m, 1 H), 4.26 (m, 3 H), 3.93 (m, 1 H), 2.98, 2.37; IR (KBr) η 3432, 3072, 2954, 1685, 1475, 1245, 13 -1 λ 3.67* (m, 3 H), 1.76* (m, 3 H), 1.32* (m, 3 H); C NMR 1089, 933 cm ; UV(MeOH) max 207, 267 nm; FAB MS δ + (CDCl3) 173.5*, 163.8*, 150.6*, 135.4, 132.8*, 130.6, m/z 544.0469 (C20H23BrN3O8P + H ); HPLC tR 8.37, 128.0, 127.3*, 123.3*, 122.3*, 118.8*, 111.1, 89.5*, 84.4*, 9.23 min. 31 δ 67.2, 66.6, 52.6, 50.0*, 20.7*, 12.3*, P NMR (CDCl3) 3.36, 2.74; IR (KBr) η 3432, 3070, 2954, 1685, 1473, 1247, 5′-[2-Chlorophenylmethoxyalaninylphosphate]-2′,3′- -1 λ ′ 941 cm ; UV(MeOH) max 208, 213, 267 nm; FAB MS didehydro-3 deoxythymidine (DDE 606). 2.10 g (47%); + ° 1 δ m/z 544.0486 (C20H23BrN3O8P + H ); HPLC tR 10.30, mp: 4345 C; H NMR (CDCl3) 9.80 (s, 1 H), 7.39 (m, 10.65 min. 1 H), 7.29 (m, 1 H), 7.20 (m, 1 H), 7.13 (m, 1 H), 7.01 (m, 4-Bromo-2-chlorophenylmethoxyalaninylphosphate]- 1 H), 6.92 (m, 1 H), 6.24 (m, 1 H), 5.81 (m, 1 H), 4.94 (m, 2′,3′-didehydro-3′-deoxythymidine (DDE 603). Yield: 1 H), 4.28 (m, 3 H), 3.96 (m, 1 H), 3.59* (m, 3 H), 1.72* ° 1 δ 13 δ 0.89 g (17%); mp: 51–52 C; H NMR (CDCl3) 9.52 (s, (m, 3 H), 1.25* (m, 3 H); C NMR (CDCl3) 173.5*, 1 H), 7.52 (s, 1 H), 7.32 (m, 2 H), 7.22 (m, 1 H), 6.99 (m, 163.8*, 150.8, 145.9*, 135.3*, 132.7*, 130.0, 127.5*, 127.0*, 1 H), 6.29 (m, 1 H), 5.90 (m, 1 H), 5.00 (m, 1 H), 4.33 (m, 124.8*, 121.2*, 111.0*, 89.3*, 84.3*, 66.9, 52.3, 49.8*, 20.5, 31 δ η 2 H), 4.19 (m, 1 H), 4.01 (m, 1 H), 3.67 (s, 1 H), 1.79* (m, 12.1*, P NMR (CDCl3) 3.23, 2.67; IR (KBr) 3209, 3 H), 1.31* (m, 3 H); 13C NMR (CDCl3) δ 173.5*, 163.8*, 3070, 2952, 1691, 1481, 1245, 1035, 931 cm-1; λ 150.8, 145.5*, 135.3, 132.8*, 130.9, 127.3*, 126.2*, 122.7*, UV(MeOH) max 214, 215, 219, 267 nm; FAB MS m/z + 117.8*, 113.3*, 89.6*,), 84.3*, 67.5*, 67.1, 52.6, 50.1, 20.8*, 500.1028 (C20H23ClN3O8P + H ); HPLC tR 7.62, 8.32 31 δ η 12.3*; P NMR (CDCl3) 3.11, 2.54; IR (KBr) 3415, min. 3222, 3072, 2952, 1691, 1475, 1245, 1085, 1035, 929

Antiviral Chemistry & Chemotherapy 13:3 3 FM Uckun et al.

Table 1. Physiochemical properties and biological activity of phosphoramidate derivatives of d4T

–1 DEE number Substituents Partition (log P) Hydrolysis (min )IC50 HTLV IIIB (nm ±SE) 113 4-Br 1.21 0.0210 <1 598 4-OMe 0.39 0.0102 4 ±2 599 3-N(CH3)2 0.76 0.0058 25 ±16 600 2-diOMe 0.40 0.0029 6 ±3 601 4-CN 0.05* 0.1199 1 ±0 602 3-Br 1.12 0.0338 2 ±2 603 4-Br,2-Cl 1.81 0.1500 1 ±1 604 4-F 0.54 0.0117 1 ±0 605 2-Br 0.95 0.0336 2 ±1 606 2-Cl 0.84 0.0370 3 ±1 607 H 0.38 0.0082 2 ±1 608 2.5-diCl 1.41 0.1840 1 ±0 609 4-Cl 0.64 0.0216 1 ±0 Three physiochemical properties; partition coefficient (octanol/water), alkali hydrolysis rate, and biological activity are shown for each of the d4T derivatives. The ability of the compounds to inhibit virus replication are expressed as inhibition constants determined from three inde- pendent experiments (±SE). *The value from 4-CN substituent is inaccurate because clear separation was not obtained between octanol and water.

5′-[2,5-Dichlorophenylmethoxyalaninylphosphate]- by comparing the p24 antigen levels from the test 2′,3′-didehydro-3′ deoxythymidine (DDE 608). Yield: substance-treated infected cells with those from vehicle- ° 1 δ 0.68 g (30%); mp: 42–44 C; H NMR (CDCl3) 9.43 (s, treated infected cells. 1 H), 7.45 (m, 1 H), 7.25 (m, 2 H), 7.04 (m, 1 H), 6.99 (m, All compounds (see Table 1) with a mono-halo substi- 1 H), 6.32 (m, 1 H), 5.88 (m, 1 H), 4.99 (m, 1 H), 4.32 (m, tution at the para position, including the previously 3 H), 4.00 (m, 1 H), 3.67 (s, 3 H), 1.77* (m, 3 H), 1.33* described compound 113/stampidine with a 4-Br substitu- 13 δ (m, 3 H); C NMR (CDCl3) 173.5*, 163.8, 150.8, tion, compound 604 with a 4-F substitution, and com- 146.4*, 135.3, 132.7*, 130.7, 127.4, 125.8, 123.7*, 121.7*, pound 609 with a 4-Cl substitution as well as compound 111.2*, 89.6*, 84.3*, 67.1*, 52.6, 50.1, 29.6, 20.7*, 12.3*; 31P 601 substituted with the electron drawing CN group at the δ η NMR (CDCl3) 3.24, 2.60; IR (KBr) 3423, 3205, 3072, para position, and compounds 603 and 608 with double -1 λ 2954, 1691, 1475, 1245, 1093, 946 cm ; UV(MeOH) max halo substitutions had an IC50 value of only 1 nM or less. 211, 216, 220, 268 nm; FAB MS m/z 534.0581 Compounds with mono-halo substitutions at the 2- or 3- + (C20H22Cl2N3O8P + H ); HPLC tR 13.18 min. positions were less active (mean IC50=2.3 ±0.3) than compounds with mono-halo substitutions at the 4-position

Results and discussion (mean IC50=1.0 ±0.0 nM, P<0.001). Compounds substituted with electron donating groups, including compounds The target phosphoramidate derivatives of stavudine were 598, 599, and 600, also appeared to be less active than com- synthesized according Figure 1. The anti-HIV activity of pounds with mono-halo substitutions at the 4-position the compounds was examined by evaluating their ability to (mean IC50=11.7 ±6.7 nM, P=0.017). Thus, the presence of inhibit HIV replication in peripheral blood mononuclear electron withdrawing groups seems to enhance the anti- cells using previously described procedures (Uckun et al., HIV activity of this group of nucleoside analogues. A larg- 1998). Percent inhibition of viral replication was calculated er series of experiments will be needed to accurately confirm

Figure 2. Literature proposed metabolic pathway of arylphosphate derivatives of stavudine

In the first step, compound B is generated by carboxyesterase by hydrolysis of the methylester. The subsequent step involves an intramolecu- lar cyclization at the phosphorous center with simultaneous elimination of the phenoxy group to form the cyclic intermediate (D). In the presence of water, this intermediate is converted into the active metabolite E (McIntee et al., 1997). the statistical significance of the observed differences in Figure 2 depicts the literature proposed metabolic pathway

IC50 values since the vast majority of the compounds were of arylphosphate derivatives of d4T. The presence of elec- very active. tron withdrawing substituents at the para position of the We next set out to determine if the anti-HIV potency of phenyl moiety is likely to increase the hydrolysis rate of the the aryl phosphate derivatives of stavudine can be predict- phenoxy group in the metabolite precursor B generated by ed from their lipophilicity or hydrolysis rates. Contrary to the carboxyesterase-dependent first step of the metabolic the hypothesis of Siddiqui et al. (1999), the lipophilicity of pathway of these aryl phosphate derivatives. In our earlier the aryl phosphate derivatives of stavudine did not correlate publication (Venkatachalam et al., 1998) we had postulated with their biological activity against HIV-1 (R2=0.06, that the electronic effect induced by the electron withdraw- t=0.86, P=0.4). Compounds with similar or identical parti- ing substituents would result in enhanced hydrolysis of the tion coefficients had a wide range of IC50 values (Table 1). phenoxy group C yielding D and subsequently E, the pre- For example, compounds 600 and 607 had the same parti- cursors of the key metabolite Ala-d4T-MP. Chemical tion coefficient as compound 508. Yet, their IC50 values hydrolysis using alkaline conditions showed an increase in were 50% higher (6 nM vs 4 nM) and 50% lower (2 nM vs the amount of Ala-d4T-MP formation when electron

4 nM), respectively, than the IC50 value of compound 598. withdrawing groups were present in the structure of these

Whereas 2-Br, 2-Cl, and N(CH3)2 substitutions in the phosphoramidate derivatives (Venkatachalam et al., 1998). phenyl ring resulted in increased lipophilicity, as reflected Because of its enhanced susceptibility to hydrolysis yielding by 2- to 2.5-fold higher partition coefficients, they did not substantially greater amounts of A-d4T-MP (the key pre- increase the anti-HIV potency and in the case of 3- cursor of the active d4T-TP metabolite), compounds con-

N(CH3)2 substitution caused a >10-fold loss in activity. taining electron withdrawing groups in their structure were

Antiviral Chemistry & Chemotherapy 13:3 5 FM Uckun et al.

Figure 3. Relationship between hydrolysis, chemical structure and anti-HIV potency

(a) (b)

(a) The disappearance of the starting material was fitted to a single exponential (k values shown in Table 1). Electron withdrawing substituents (E–H) showed faster rates of hydrolysis than electron donating substituents (A,B). (b) Data were log transformed to normalize residuals. The solid line represents the best fit line and the dotted line represents the 95% confidence. There was a significant negative 2 relationship between the rate of hydrolysis and IC50 values (R =0.42, t = –2.8, P=0.017). postulated to be a more potent anti-HIV agents than com- References pounds without such substitutions. This hypothesis is strongly supported by the experimental data presented in Balzarini, J, Heredewijn, P, De Clercq E (1989). Differential patterns ′ ′ ′ ′ Table 1 and Figure 3. Addition of electron withdrawing of intracellular metabolism of 2 ,3 -didhydro-2 ,3 -dideoxythymidine and 3′-azido-2′,3′-dideoxythymidine, two potent anti-human groups increased the rate of hydrolysis (Figure 3a) and immunodeficiency virus compounds. Journal of Biological Chemistry potency of the compound (Figure 3b). The three com- 264:6127–6133. pounds with electron donating substitutions (viz, 3- Greene WC (1991). The molecular biology of human immunodeficiency virus Type-1 infections. New England Journal of Medicine N(CH3)2, di-OMe and 4-OMe), had the slowest rates of hydrolysis and were the least potent. There was an inverse 324:308–317. linear relationship between log transformed values for the McIntee EJ, Remmel RP, Schinazi RF, Abraham TW & Wagner 10 CR (1997). Probing the mechanism of action and decomposition rate of hydrolysis and the IC50 values (R2=0.42, t=–2.8, of aminoacid phosphomonoester amidates of antiviral necleoside P=0.017). prodrugs. Journal of Medicinal Chemistry 40:3323–3331. Sepkowitz KA (2001). AIDS the first 20 years. New England Journal Conclusions of Medicine 344:1764–1772. Siddiqui AQ, McGuigan C, Ballatore C, Zuccotto F, Gilbert IH, De The antiviral activities of new phosphoramidate derivatives Clercq E & Balzarini J (1999). Journal of Medicinal Chemistry 42:4122–4128. of stavudine were examined. The presence of electron with- Uckun FM, Chelstrom LM, Tuel-Ahlgren L, Dibirdik I, Irvin JD, drawing groups was found to enhance the anti-HIV activ- Chandan-Langlie M & Myers DE. (1998). TXU(Anti-CD7)- ity of this group of nucleoside analogues. All compounds Pokeweed Antiviral Protein as a Potent Inhibitor of Human with a mono-halo substitution at the para position and Immunodeficiency Virus. Antimicrobial Agents and Chemotherapy 42:383–388. compounds 603 and 608 with double halo substitutions Uckun FM & Vig R (2000) Aryl phosphate derivatives of D4T hav- had an IC50 value of only 1 nM. Compounds with mono- ing anti-HIV activity. US Patent Number 6,030,957. Issue date: 2- halo substitutions at the 2- or 3-positions were less active. 29-2000. US Patent Number 6,350,736. Issue date: 2-26-2002. Compounds substituted with electron donating groups Uckun FM, Qazi S, Pendergrass S, Venkatachalam TK, Mao C, were the least active. The rate of chemical hydrolysis under Richman D (2002) Stampidine is a potent inhibitor of NRTI- alkaline conditions (but not the lipophilicity) predicted the resistant primary clinical HIV-1 isolates with B- and non-B sub- types. Antimicrobial Agents and Chemotherapy (in press). potency of the compounds. Uckun FM, Chen CL, Lisowski E, Mitcheltree GC, Venkatachalam TK, Erbeck D, Chen H & Waurzyniak (2002) Toxicity and phar-

macokinetics of stampidine in mice and rats. Arzneimittelforschung Vig R, Venkatachalam T K & Uckun FM (1998). D4T-5′-[p-bro- Drug Research (in press). mophenyl methoxyalaninyl phosphate] as a potent and non-toxic Venkatachalam TK, Tai HL, Vig R, Chen CL, Jan S & Uckun F anti-human immunodeficiency virus agent. Antiviral chemistry and (1998). Enhanced effects of a mono-bromo substitution at the para Chemotherapy 9:445. position of the phenyl moiety on the metabolism and anti-HIV activity of D4T-phenyl methoxyalaninyl phosphate derivatives. Biorganic Medicinal Chemistry Letters 8:3121.

Received 25 April 2002; accepted 17 June 2002

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