Synthesis and pharmacological evaluation of pyrazolo[4,3-c]quinolinones as high affinity GABAA-R ligands and potential anxiolytics.

The synthesis, in vitro ligand binding study and in vivo Elevated Plus Maze test (EPM) of a series of pyrazolo[4,3-c]quinolin-3-ones (PQs) are reported. Multistep synthesis of PQs started from anilines and diethyl 2-(ethoxymethylene)malonate to give the quinolin-4-one nucleus, via the Gould-Jacobs reaction. These quinolinones were transformed to 4-chloroquinolines, which react with aryl-hydrazines affording the final compounds. PQs exhibited different potency in displacing specific [3H]Flunitrazepam binding from the benzodiazepine binding site at the γ-aminobutyric acid receptor (GABAA-R) depending on the substitution of the pyrazoloquinolone nucleus. PCA helped determine how different substituents contributed to the differential behavior of the PQs studied. Compounds with high affinity for the GABAA-R were tested regarding their anxiolytic properties in Wistar adult male rats using the Elevated Plus Maze (EPM). Thus, PQs with a p-methoxy phenyl group at N-1 (7b-ii and 7c-ii) displayed a remarkable anxiolytic activity at low doses (0.5-1.0 mg/kg). Meanwhile, PQs featuring an unsubstituted phenyl (7b-i) or p-fluoro phenyl group (7b-iii) at the N-1 showed anxiogenic effects in the EPM test.

Development of a receptor model for efficient in silico screening of HIV-1 integrase inhibitors.

Integrase (IN) is a key viral enzyme for the replication of the type-1 human immunodeficiency virus (HIV-1), and as such constitutes a relevant therapeutic target for the development of anti-HIV agents. However, the lack of crystallographic data of HIV IN complexed with the corresponding viral DNA has historically hindered the application of modern structure-based drug design techniques to the discovery of new potent IN inhibitors (INIs). Consequently, the development and validation of reliable HIV IN structural models that may be useful for the screening of large databases of chemical compounds is of particular interest. In this study, four HIV-1 IN homology models were evaluated respect to their capability to predict the inhibition potency of a training set comprising 36 previously reported INIs with IC50 values in the low nanomolar to the high micromolar range. Also, 9 inactive structurally related compounds were included in this training set. In addition, a crystallographic structure of the IN-DNA complex corresponding to the prototype foamy virus (PFV) was also evaluated as structural model for the screening of inhibitors. The applicability of high throughput screening techniques, such as blind and ligand-guided exhaustive rigid docking was assessed. The receptor models were also refined by molecular dynamics and clustering techniques to assess protein sidechain flexibility and solvent effect on inhibitor binding. Among the studied models, we conclude that the one derived from the X-ray structure of the PFV integrase exhibited the best performance to rank the potencies of the compounds in the training set, with the predictive power being further improved by explicitly modeling five water molecules within the catalytic side of IN. Also, accounting for protein sidechain flexibility enhanced the prediction of inhibition potencies among the studied compounds. Finally, an interaction fingerprint pattern was established for the fast identification of potent IN inhibitors. In conclusion, we report an exhaustively validated receptor model if IN that is useful for the efficient screening of large chemical compounds databases in the search of potent HIV-1 IN inhibitors.

Preformulation studies of novel 5'-O-carbonates of lamivudine with biological activity: solubility and stability assays.

As a part of preformulation studies, the aim of this work was to examine the solubility and stability of a series of 5'-O-carbonates of lamivudine with proven antihuman immunodeficiency virus activity. Solubility studies were carried out using pure solvents (water, ethanol and polyethylene glycol 400 [PEG 400]), as well as cosolvents in binary mixture systems (water-ethanol and water-PEG 400). These ionizable compounds showed that their aqueous solubility is decreasing as the carbon length of the substituent moiety increases, but being enhanced as the pH was reduced from 7.4 to 1.2. Thus, 3TC-Metha an active compound of the series, with an intrinsic solubility at 25 °C of 17 mg/mL, was about 70 times more soluble than 3TC-Octa (0.24 mg/mL), and at pHs of 1.2, 5.8 and 7.4 had intrinsic solubilities of 36.48, 19.20 and 15.40 mg/mL, respectively. In addition, the solubility was enhanced significantly by using ethanol and PEG 400 as cosolvents. A stability study was conducted in buffer solutions at pH 1.2, 5.8, 7.4 and 13.0 and in human plasma at 37 °C. Stability-indicating high-performance liquid chromatography procedure was found to be selective, sensitive and accurate for these compounds and good recovery, linearity and precision were also observed.

Development and validation of a stability indicating method for seven novel derivatives of lamivudine with anti-HIV and anti-HBV activity in simulated gastric and intestinal fluids.

A simple micellar liquid chromatography (MLC) method has been developed and validated for use in stability indicating studies of lamivudine and its carbonate derivatives with proved activity against human immunodeficiency and hepatitis B viruses (HIV and HBV, respectively), in simulated gastric (SGF) and intestinal (SIF) fluids samples. The optimized method involves a C18 column thermostated at 30°C, UV detection at 272 nm, a flow rate of 1.0 mL min(-1) and a micellar mobile phase composed by 0.15M sodium dodecyl sulphate (SDS) - 4% (v/v) 1-butanol - 0.01 M KH2PO4-Na2HPO4 (pH 7), using zidovudine (AZT) as internal standard. Validation under Food and Drug Administration (FDA) guideline of the analytical parameters include: linearity (r(2)>0.9996), LODs (1.6 × 10(-7)-6.9 × 10(-6)M) and LOQ (1 × 10(-5)M), intra (0.02-1.48%) and inter-day precision (0.04-1.66%) expressed as relative standard deviation (R.S.D.), and robustness parameters (less than 1.98%). Using this method, recoveries ranging from 92.9 to 119% were obtained for the eight substances. Thus, this method provides a simple, sensitive, accurate and precise assay for the determination of all compounds that can be readily adaptable to routine use by clinical laboratories with standard equipment. In addition, we evaluated the stability of carbonates of lamivudine in buffer pH 1.2 and 6.8; SGF (pH 1.2) and SIF one (pH 6.8), all as indicated in United States Pharmacopeia (USP) 32. Finally, this chromatographic method was applied to stability studies which resulted in all the compounds following a pseudo-first-order kinetics, and in the determination of its kinetic constant and half-life time.

Synthesis, biological evaluation and molecular modeling of 4,6-diarylpyrimidines and diarylbenzenes as novel non-nucleosides HIV-1 reverse transcriptase inhibitors.

A series of novel 4,6-diarylpyrimidines (4,6-DAPY) and diarylbenzenes (DABE) compounds were synthesized and evaluated as inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were 8b, 8d, 14b and 18 (EC(50) = 0.049, 0.381, 0.599 and 0.398 µM, respectively), with HIV-1 inhibitory activity improved or similar to nevirapine (NVP, EC(50) = 0.097 µM) and delavirdine (DEV, EC(50) = 0.55 µM). The other compounds displayed moderate activity (8c, EC(50) = 5.25 µM) or were inactive (8a and 14a) against HIV-1 replication. Molecular modeling studies were performed with the synthesized compounds in complex with the wild-type reverse transcriptase (RT). A correlation was found between the anti-HIV activity and the electrostatic energy of interaction with Lys101 residue. These findings enrich the SAR of these Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) families.

Intestinal permeability of lamivudine (3TC) and two novel 3TC prodrugs. Experimental and theoretical analyses.

Lamivudine (3TC) is an antiviral drug with a widely demonstrated clinical efficacy used to treat Acquired Immunodeficiency Syndrome (AIDS) in humans. However, since the rapid emergence of resistant viral strains has limited its effect, several new strategies such as the design of prodrugs have been applied to try to optimize its pharmacotherapeutic properties. The present study deals with the intestinal permeation of 3TC and two novel prodrugs of 3TC, namely 3TC-Etha and 3TC-Buta, by using rat intestinal segments and applying the gut sac in vitro technique. An adequate bioanalytical method (sample preparation and quantitative analysis) was fully developed and validated for the quantitation of these three compounds. A low permeability coefficient (P(app) 0.408 ± 0.049 × 10(-4) cm/min) was found for 3TC if compared to that previously reported for zidovudine (2.38 ± 0.12 × 10(-4) cm/min), while no statistically significant differences were observed in its apical-to-basal or basal-to-apical permeabilities. Our data suggests that 3TC permeates through the intestinal tissue by passive diffusion, with no intervention of efflux mechanism during this process as determined applying the gut sac technique. Regarding the prodrugs, both 3TC-Etha and 3TC-Buta were able to increase 3TC permeability (2 and 10 times, respectively), but none of these compounds were capable of crossing the intestinal tissue in their intact forms. In the case of 3TC-Etha, the permeability of the intact compound (P(app) 0.093 ± 0.010 × 10(-4) cm/min) was impaired by a P-glycoprotein (P-gp) mediated efflux, evidenced by the higher permeability coefficient (6.933 ± 0.586 × 10(-4) cm/min) determined in the presence of verapamil on the apical side of the enterocyte. In contrast, 3TC-Buta was not subjected to efflux mechanisms on the apical side of the enterocyte, but was efficiently converted to 3TC by enzymatic hydrolysis during the permeation process. In the light of these results, molecular modeling (docking and molecular dynamics) techniques were applied to study further the structural features that may confer the different behaviors of these two compounds with respect to P-gp mediated efflux. The results also highlight the potential of combining experimental and theoretical studies in the design of 3TC prodrugs with enhanced intestinal permeation properties.

Preformulation studies of Zidovudine derivatives: Acid dissociation constants, differential scanning calorimetry, thermogravimetry, x-ray powder diffractometry and aqueous stability studies.

As part as of the preformulation studies of new 5'-OH derivatives of zidovudine, compounds 2-6, their acid dissociation constants, Differential Scanning Calorimetry (DSC) and Thermogravimetry (TG) curves, X-Ray Powder diffractograms and aqueous stability are reported. A sensitive technique such as differential scanning potentiometry was used to determine the pKa constants of the above mentioned compounds. In addition, pKa values were calculated from theoretical methods, and no significant differences with those of experimental ones were observed. X-Ray Powder Diffractometry data demonstrated that compounds 2-4 were crystalline while 5 and 6 were amorphous. DSC analysis indicated that all of them presented an exothermic decomposition peak above 150 °C which is accompanied by a weight loss in the respective TG curves. The stability of these compounds in aqueous medium at different pH values was investigated, using a validated High Performance Liquid Chromatography (HPLC) method, which demonstrated to be rapid, selective, sensitive, accurate and stability-indicating. Good recovery, linearity and precision were also achieved. For all compounds the aqueous hydrolysis followed a pseudo-first-order kinetics, depending on pH and the union existing between AZT and the associate moiety. The hydrolysis was catalyzed by hydroxide ion in the 7.4-13.2 pH range, while all compounds exhibited pH-independent stability from acidic to neutral media (pHs 1.0-7.4).

P-glycoprotein limits the absorption of the anti-HIV drug zidovudine through rat intestinal segments.

Zidovudine (AZT) was the first drug approved for the treatment of Acquired Immunodeficiency Syndrome (AIDS) in humans, and although its clinical efficacy has been demonstrated, suboptimal pharmacokinetic aspects still remain a concern. To assess the basis of its highly variable oral bioavailability, this work deals with the study of AZT intestinal absorption by applying the gut sac technique. Permeation through the rat jejunum and ileum segments was analyzed at different drug concentrations and gut regions, with higher apparent permeability coefficients (P(app)) being found for the proximal regions of the small intestine compared to distal ones. Bi-directional permeation assays demonstrated that AZT is subjected to efflux mechanisms in distal regions of small intestine, which are blocked by verapamil (VER), thus demonstrating a P-glycoprotein (P-gp) mediated mechanism. The efficiency of AZT efflux increased in the distal ileum as consequence of exposure to AZT, with the amount of drug permeating from the mucosal to the serosal side diminishing after 35 min. Molecular modeling techniques were applied to analyze the binding mode of AZT to P-gp, which was compared to that of VER and AZT-Ac, a novel prodrug of AZT. The energy required for their solvation was found to constitute a critical feature in their binding to this efflux protein. The present work updates the impact of P-gp in AZT oral bioavailability, highlighting the need for further study of the dynamic nature of its expression at intestinal level.

Stability-indicating micellar liquid chromatography method for three novel derivatives of zidovudine in aqueous and simulated gastric and intestinal fluids matrices.

This work studies the stability of three new anti-HIV agents which were obtained by the association of zidovudine (AZT) with different amino acids, such as leucine (AZT-Leu) and valine (AZT-Val), and one with an acid group (AZT-Ac). Before commercialisation, their stability in different matrices - simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8), both as the USP 32 Guideline indicates, and buffers (pH 1.2 and 6.8) - must be studied. To this end, a new stability-indicating micellar liquid chromatography (MLC) method has been optimised and validated. Measurements were based on the disappearance of reagents and the appearance of the only degradation product (AZT). This optimised and validated method used a C18 column and a mobile phase containing 0.05 M sodium dodecyl sulphate-1% (v/v)1-butanol-0.01 M NaH(2)PO(4) (pH 3.0) at 30°C, and a flow rate of 1 mL min(-1). Under these conditions, retention times were 1.4, 3.6, 6.3 and 9.5 min for AZT-Ac, AZT, AZT-Val and AZT-Leu, respectively. Calibrations better than 0.9995, intra- and inter-day precisions below 1.08% and good recoveries (94.47-116.52%) and robustness (RSD less that 1.08%) were obtained and were adequate to analyse the four compounds. Finally, this MLC method was applied to achieve stability studies which resulted in the evidence that all the compounds followed a pseudo-first-order kinetics, and in the determination of their kinetic constants and half-life time. A reference method, applied in the same studies, validated the MLC method reported herein.

Rational approaches for the design of effective human immunodeficiency virus type 1 nonnucleoside reverse transcriptase inhibitors.

The binding of several classes of nonnucleoside reverse transcriptase inhibitors (NNRTIs) to wild-type (wtRT) and K103N mutant (mRT) human immunodeficiency virus type 1 (HIV-1) reverse transcriptase is studied by molecular dynamics and energy decomposition techniques. The imidoylthiourea (ITU), diaryltriazine (DATA), and diarylpyrimidine (DAPY) NNRTIs studied maintain the hydrogen bond with Lys101 during the 3 ns molecular dynamics trajectories. When bound to mRT, all the DAPYs studied establish hydrogen bonds with Glu138; among these, those of the potent inhibitors TMC120 and TMC125 are water-mediated. The molecular interactions of the NNRTIs in the binding pocket are correlated to the drugs' potency. Quantitative free energy analyses show a linear relationship between the van der Waals energetic component and the potency against wtRT. The molecular basis of the interaction between NNRTIs and RT presented here provide quantitative approaches for the design of novel effective anti-HIV drugs.

Antiviral activity of 5'-O-carbonate-2',3'-dideoxy-3'-thiacytidine prodrugs against hepatitis B virus in HepG2 2.2.15 cells.

The antiviral activities of lamivudine (3TC; 2',3'-dideoxy-3'-thiacytidine) and six 5'-O-carbonates of 3TC were determined by inhibition of hepatitis B virus (HBV) replication in HepG2 2.2.15 cells. HBV DNA in cell supernatants was quantified by real-time polymerase chain reaction (PCR). The results showed that 3TC-Etha was six times more active than 3TC and that 3TC-Buta, 3TC-Hexa and 3TC-Octa were approximately three times more active than 3TC. In contrast, 3TC-Penta and 3TC-Metha showed anti-HBV activity similar to that of the parent compound 3TC. In conclusion, 5'-O-carbonates of 3TC appear to be promising candidates as anti-HBV compounds. This modification could optimise the use of 3TC, a well-tolerated, effective and inexpensive drug, in monotherapy or combined therapy for chronic HBV infections as well as human immunodeficiency virus (HIV)/HBV co-infections.

Synthesis and anti-HIV activity of novel 2',3'-dideoxy-3'-thiacytidine prodrugs.

We report here the synthesis of a novel series of 5'-O-carbonates of 3TC, using different aliphatic alcohols and N,N-carbonyldiimidazol. Its antiviral activity was determined in peripheral blood mononuclear cells (PBMCs) showing some carbonate derivatives with an activity similar to or better than 3TC, except 3TC-Metha and 3TC-2Pro with less activity. In vitro assays in PBMCs have demonstrated that cytotoxicity increases as the carbon chain length of the alcohol moiety increases, showing compounds with a normal chain length of n=2-5 good selective index, compared to the parent drug. Thus, this work is an important contribution leading to the suppression of HIV replication.

Binding to human serum albumin of zidovudine (AZT) and novel AZT derivatives. Experimental and theoretical analyses.

This work presents the binding of AZT and nine novel AZT derivatives to human serum albumin (HSA), both defatted (HSA(D)) and complexed with fatty acids (HSA(FA)). The bound fractions and binding site were determined by applying an ultrafiltration procedure, with an increased affinity for the majority of these derivatives to HSA(D) being found with respect to that of AZT, while only one derivative exhibited an increased affinity for HSA(FA). By means of computational methods, we observed that specific electrostatic interactions are responsible for the increased affinity for HSA(D), while the presence of fatty acids complexed to HSA caused an intense electrostatic repulsion with negatively charged ligands located in Sudlow site I, thus diminishing their bound fractions. A strong relationship between the calculated energetic components and the observed experimental affinity was identified.

Determination of liposome permeability of ionizable carbamates of zidovudine by steady state fluorescence spectroscopy.

In the present paper the relative permeabilities of AZT-Pyp and AZT-Ethy across a phospholipid bilayer were estimated by the means of fluorescence spectroscopy. The center of spectral mass of both non-encapsulated AZT-derivatives (AZT-der) emission spectra increased as a function of the illumination time inside the spectrofluorimeter cell. This phenomenon was even more evident when drugs were incubated under an UV mercury lamp, suggesting its photolytic origin. AZT-der were protected from photolysis inside liposomes and decomposed upon irradiation when they were free in the aqueous phase. The time-dependent decrease in the fluorescence intensity at a constant wavelength was fitted to a two-exponential equation and the values of rate constants for permeability and photolysis were calculated. It was concluded that AZT-Pyp but not AZT-Ethy diffused across the bilayer. This behavior correlated with the molecular volumes of AZT-Pyp (379.6A(3)) and AZT-Ethy (450.5A(3)), determined from the minimum energy conformations but not with previously reported logP values. These results reinforce the concept that not only lipophilicity but also membrane structure and AZT-der molecular size had a critical influence in passive diffusion across bilayers and may help in future refinements of other AZT-der molecular design.

Quantitative plasma determination of a novel antiretroviral derivative of zidovudine by solid-phase extraction and high-performance liquid chromatography.

A solid-phase extraction methodology, followed by high-performance liquid chromatography (HPLC) quantification with UV absorbance detection (lambda = 267 nm), was developed in order to study the stability of 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (AZT-Iso), a novel derivative of the antiretroviral AZT, in different matrixes. The half-lives (t1/2) for AZT-Iso were 1.19, 1.13 and 0.30 h for human, rat and rabbit plasma, respectively, and 14.91 and 25.49 h for potassium phosphate buffer (pH 7.4) and human serum albumin solution, respectively. The HPLC method proved to be selective, sensitive and accurate. Good recovery, linearity and precision were achieved using p-fluorophenol as an internal standard. The validity of this method was tested using synthetic mixtures of the intact drug with its decomposition products. In conclusion, the method presented is applicable to in vivo pharmacokinetics studies of AZT-Iso in rats.

3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine: a novel antiretroviral analog of zidovudine. III. Solubility studies.

The pH-solubility behavior and solubility of 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (AZT-Iso), an antiretroviral derivative of zidovudine with important biological activity, was studied in water, ethanol, ethanol: water, and n-octanol. The N-pyridine pKa value was determined from its pH-solubility profile, which was in accordance with that of the experimental value of methyl isonicotinate. Also, the ethanol cosolvency in ethanol:water mixtures at 25 degrees C was studied, and log-linear and nonlinear solubilization models were applied to the experimental solubility AZT-Iso data, which allowed us to predict its solubility in those solvent mixtures at a determined content of cosolvent.

3'-Azido-3'-deoxy-5'-O-isonicotinoylthymidine: a novel antiretroviral analog of zidovudine. II. Stability in aqueous media and experimental and theoretical ionization constants.

Degradation of 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (AZT-Iso), an antiretroviral derivative of zidovudine, was investigated in buffer pH 7.4, mu = 300 mOsm at 37, 50 and 60 degrees C, and in water (pH 6.6, 37 degrees C), giving zidovudine (AZT) and isonicotinic acid (INA) as products. The rate constants were determined by reversed-phase HPLC showing pseudo-first-order kinetics related to the residual amount of AZT-Iso. In this way, the studied compound was demonstrated to be 153 times more stable in water than in buffer solution at 37 degrees C. The analytical method was conveniently validated demonstrating to be a rapid and accurate stability-indicating technique. In addition, experimental and theoretical values of pKa were determined.

Conformational studies of novel antiretroviral analogs of zidovudine.

Conformational properties of three novel zidovudine analogs, namely 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (AZT-Iso, 2), (-)-trans-(5S,6S)-5-bromo-6, 5'-epoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (3) and (+)-trans-(5R,6R)-5-bromo-6,5'-epoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (4), have been investigated by AM1 calculations and NMR studies, and compared with those of the parent nucleoside (AZT, 1). Based on the results obtained the following correlation may be established, a) AZT and AZT-Iso exhibit a conformational behavior analog to other pyrimidinic nucleosides, displaying a dynamic equilibrium in solution where the two conformers (North and South) undergo a constant transformation. b) Compounds 3 and 4 show a different conformational profile. The estimate of the pseudorotation phase angle reveals the rigid structures of the latter compounds, which do not evidence conformational equilibrium in solution; the azide group being the only group free to rotate. c) Diastereoisomers 3 and 4 exhibit an extra conformational parameter compared with other pyrimidinic nucleosides: the chair or boat conformation in the third ring formed between the sugar and the base. In all cases, a reasonable correlation was obtained between theoretical and NMR spectroscopic data.

Antiretroviral activity and cytotoxicity of novel zidovudine (AZT) derivatives and the relation to their chemical structure.

Zidovudine (AZT) was the first nucleoside analogue licensed for the treatment of HIV infection. Efforts have continuously been made to improve the therapeutic characteristics of this drug, most of them focussed on prodrugs design. Here we describe the anti-HIV-1 activity and cytotoxicity of six novel AZT derivatives namely 3'-azido-3'-deoxy-5'-O-oxalyl-N-valinethymidine, 3'-azido-3'-deoxy-5'-O-oxalyl-N-leucinethymidine, 3'-azido-3'-deoxy-5'-O-oxalyl-N-isoleucinethymidine, 3'-azido-3'-deoxy-5'-O-oxalyl-N-phenylalaninethymidine, 3'-azido-3'-deoxy-5'-O-oxalylthymidine acid, 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine and 5-chloro-6-hydroxy-5,6-dihydro-3'-azido-3'-deoxythymidine which were perfectly characterized. AZT-Val, AZT-Leu, AZT-iLeu, AZT-Phen, AZT-Ac and AZT-Iso have shown a similar or higher selectivity index than that of AZT itself, in one or both of the different cell cultures used (PBMC and MT2). However, AZT-ClOH showed no anti-HIV activity. These results suggest that using amino acids in the design of AZT derivatives improves AZT activity.

3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine, a new prodrug of zidovudine. Synthesis, solid state characterization, and anti HIV-1 activity.

Synthesis, solid state characterization and anti HIV-1 activity of 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (2), a new prodrug of zidovudine (AZT, 1), are described. Two solid forms of 2 prepared by crystallization from ethyl acetate-petroleum ether (form alpha) and from a melt sample of form alpha (amorphous form) were characterized by X-ray diffractometry, infrared spectroscopy, differential scanning calorimetry (DSC) and thermogravimetry (TGA) techniques. The novel nucleoside exhibited antiviral activity against standard and resistant strain panels of HIV-1 as well as cytotoxicity similar to that of AZT.