TSAO derivatives, inhibitors of HIV-1 reverse transcriptase dimerization: recent progress.

There is an urgent need for the development of new and safer drugs for the treatment of HIV (human immunodeficiency virus) infection, active against the currently resistant viral strains or directed to novel targets in the viral replicative cycle that may be useful for multiple drug combination. TSAO derivatives are a peculiar group of highly functionalized nucleosides that belong to the so-called nonnucleoside RT inhibitors (NNRTIs). HIV-1 reverse transcriptase (RT) is a key enzyme that plays an essential and multifunctional role in the life cycle of the virus and thus represents a key target for antiviral chemotherapeutic intervention. The dimeric form of the enzyme is absolutely required for all enzymatic activities. Thus, the process of dimerization and subsequent maturation into the p66/p51 heterodimer is essential for a fully functional RT and constitutes a target for therapeutic intervention, however to date such agents have not been developed. TSAO molecules are a peculiar group of non-nucleoside RT inhibitors that exert a unique selectivity for HIV-1 through a specific interaction with the p51 subunit of HIV-1 RT. They interact at the p66/p51 heterodimer interface of the enzyme. They were the first small non peptidic molecules shown to interfere with the dimerization process of the enzyme. This review covers the recent work within this family of compounds aimed at enhancing their interaction with the dimer interface of HIV-1 RT.

Chemical, biochemical and genetic endeavours characterizing the interaction of sparsomycin with the ribosome.

Sparsomycin interaction with the ribosome and characteristics of the drug binding site in the particle were studied using chemical modification of the drug, affinity labeling methods and isolation of drug resistant mutants. The structure-function relationship studies, performed with a large number of drug derivatives, indicate that the drug interacts with the ribosome by its western and eastern moieties. The uracil ring, in the western end of the drug molecule, probably forms hydrogen bonds with the rRNA, while the apolar CH3-S-CH3 group in the eastern end interacts with a hydrophobic ribosomal domain that affinity labeling results seem to indicate is formed by protein. An increase in lipophilicity in this part of the antibiotic results in a dramatic increase in the inhibitory activity of the drug. The sparsomycin binding site is not accessible in free ribosomes, but the presence of an N-blocked amino acyl-tRNA at the P-site turns the particles capable of reversible interaction with the drug. After failure using Escherichia coli, a sparsomycin-resistant mutant was obtained by direct mutagenesis on Halobacterium halobium, a species with a unique copy of rRNA genes, stressing the role of rRNA on the drug interaction site.

3'-Spiro nucleosides, a new class of specific human immunodeficiency virus type 1 inhibitors: synthesis and antiviral activity of [2'-5'-bis-O-(tert-butyldimethylsilyl)-beta-D-xylo- and -ribofuranose]-3'-spiro-5"-[4"-amino-1",2"-oxathiole 2",2"-dioxide] (TSAO) pyrimidine nucleosides.

A series of 3'-spiro nucleosides have been synthesized and evaluated as anti-HIV-1 agents. Reaction of O-mesylcyanohydrins of furanos-3'-ulosyl nucleosides with base afforded [1-[2',5'-bis-O- (tert-butyldimethylsilyl)-beta-D-xylo- and -ribofuranosyl]]-3'-spiro-5"- [4"-amino-1",2"-oxathiole 2",2"-dioxide] derivatives of thymine, uracil and 4-N-acetylcytosine 11 and 12. Desilylation of 11 and 12 gave the full deprotected 3'-spiro xylo- and ribofuranosyl nucleosides 13 and 14 or the partially 5'-O-deprotected-3'-spiro beta-D-xylo- and -ribo-nucleosides 15 and 16, or 2'-O-deprotected-3'-spiro beta-D-ribo-nucleoside 17. 2'-Deoxygenation of 17 afforded 2'-deoxy-3'-spiro beta-D-erythro-pentofuranosyl derivative 18. These 3'-spiro derivatives were evaluated for their anti-HIV-1 activity. All 3'-spiro nucleosides having a xylo configuration did not show any anti-HIV-1 activity. 3'-Spiro ribo-nucleosides with none or only one silyl group at C-2' or C-5' or the 2'-deoxy derivative were also inactive at subtoxic concentrations. However, 3'-spiro ribo-nucleosides having two silyl groups at C-2' and C-5' were potent and selective inhibitors of HIV-1. None of the nucleoside analogues that showed anti-HIV-1 activity proved inhibitory to the replication of HIV-2 or SIV.

TSAO analogues. Stereospecific synthesis and anti-HIV-1 activity of 1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro -5''- (4''-amino-1'',2''-oxathiole 2'',2''-dioxide) pyrimidine and pyrimidine-modified nucleosides.

Several analogues of a new lead for anti-HIV-1 agents [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-thymine] -3'-spiro-5''-(4''-amino-1'',2''-oxathiole 2'',2''-dioxide) (TSAO) modified at positions N-3, O-4 and C-5 of the thymine moiety, have been prepared and evaluated as inhibitors of HIV-1 replication. A new stereoselective synthetic procedure is described. Reaction of 1,2-di-O-acetyl-5-O-benzoyl-3-C-cyano-3-O-mesyl-D-ribofuranose with pyrimidine bases, followed by treatment with Cs2CO3 afforded stereoselectively, beta-D-ribofuranosyl-3'-spiro nucleosides. 2',5'-O-Deacylation and subsequent treatment with tert-butyldimethylsilyl chloride gave the TSAO derivatives. Only those analogues having a tBDMSi group at both the C-5' and C-2' positions of the ribose moiety showed potent anti-HIV-1 activity. The activity ranged from 0.060 microM to 1.0 microM. Introduction of an alkyl or alkenyl function at N-3 of the thymine ring markedly decreased cytotoxicity without affecting the antiviral activity. While markedly active against HIV-1, the TSAO derivatives had no activity against HIV-2 or SIV. They represent the first example of nucleoside analogues with an intact ribose moiety that discriminate between HIV-1 and other retroviruses.

TSAO analogues. 3. Synthesis and anti-HIV-1 activity of 2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl 3'-spiro-5''-(4''-amino-1'',2''-oxathiole 2'',2''-dioxide) purine and purine-modified nucleosides.

Several purine and purine-modified analogues of the new lead anti-HIV-1 agent [[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl] thymine]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole 2'',2''-dioxide) (TSAO-T) have been prepared and evaluated as inhibitors of HIV-1-induced cytopathicity. Reaction of O-mesylcyanohydrins of furanos-3'-ulosyladenine with Cs2CO3 afforded beta-D-xylo- and ribofuranosyladenine 3'-spiro nucleosides. Reaction of 1,2-di-O-acetyl-5-O-benzoyl-3-C-cyano-3-O-mesyl-D-ribofuranose with purine bases, followed by treatment with Cs2CO3, stereoselectively afforded beta-D-ribofuranosyl 3'-spiro nucleosides. 2',5'-O-Deacylation and subsequent treatment with tert-butyldimethylsilyl chloride gave the required TSAO derivatives. The 3'-spiro nucleosides with a xylo configuration did not show any anti-HIV activity. However, the purine ribo 3'-spiro nucleosides were potent and selective inhibitors of HIV-1 with a 50% effective concentration in the range of 0.1-1 microM and a selectivity index ranging from 2 to 3 orders of magnitude. Introduction of an alkyl function at N-1 of the purine moiety markedly decreased cytotoxicity without affecting antiviral activity.

Novel series of TSAO-T derivatives. Synthesis and anti-HIV-1 activity of 4-, 5-, and 6-substituted pyrimidine analogues.

Several 4-, 5-, and 6-substituted pyrimidine analogues of the new anti-HIV-1 lead compound [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]thymine] -3'-spiro-5''-(4''-amino-1'',2''-oxathiole 2'',2''-dioxide) (TSAO-T) have been prepared and evaluated as inhibitors of HIV-1 and HIV-2 replication in cell cultures. Reaction of 1,2-di-O-acetyl-5-O-benzoyl-3-C-cyano-3-O-mesyl-D-ribofuranose with 5-substituted pyrimidine bases, followed by treatment with Cs2CO3, afforded, stereoselectively, beta-D-ribofuranosyl-3'-spironucleosides. 2',5'-O-Deacylation and subsequent treatment with tert-butyldimethylsilyl chloride gave the TSAO-5-substituted pyrimidine derivatives. Reaction of 5-halogen-TSAO derivatives with nucleophiles gave 6-substituted-TSAO analogues. Treatment of TSAO-pyrimidine analogues with POCl3/1,2,4-triazole and methylamine or di-methylamine afforded the 4-substituted pyrimidine compounds. Several substituted TSAO-thymine, TSAO-uracil, and TSAO-cytosine derivatives were found to be superior to their unsubstituted TSAO congeners with regard to their antiviral and/or cytotoxic properties.

1,2,3-Triazole-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D- ribofuranosyl]-3'-spiro-5"-(4"-amino-1",2"-oxathiole 2",2"-dioxide) (TSAO) analogues: synthesis and anti-HIV-1 activity.

Several 4- or 5-monosubsituted and 4,5-disubstituted 1,2,3-triazole analogues of the anti-HIV-1 lead compound [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D- ribofuranosyl]thymine]-3'-spiro-5"-(4"-amino-1",2"-oxathiole 2",2"-dioxide) (TSAO-T) have been prepared and evaluated as inhibitors of HIV-1-induced cytopathicity. These analogues have been prepared by 1,3-diplar cycloaddition of [2,5-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]- 3-spiro-5'-(4'-amino- and 4'-(N-acetylamino)-1',2'-oxathiole 2',2'-dioxide) (TSAO) azides to various substituted acetylenes. Several 4- and 5-substituted 1,2,3-triazole-TSAO analogues proved superior to the unsubstituted derivative by 1-2 orders of magnitude. In particular the 5-substituted amido-, (methylamido)-, and (dimethylamido)-1,2,3-triazole derivatives of TSAO were endowed with potent anti-HIV-1 activity (50% effective concentration: 0.056-0.52 microM). They show a similar resistance spectrum as previously noted for TSAO-T and related derivatives.

Synthesis and anti-HIV activity of [AZT]-[TSAO-T] and [AZT]-[HEPT] dimers as potential multifunctional inhibitors of HIV-1 reverse transcriptase.

In an attempt to combine the HIV-inhibitory capacity of 2',3'-dideoxynucleoside (ddN) analogues and non-nucleoside reverse transcriptase (RT) inhibitors (NNRTI), we have designed, synthesized, and evaluated for their anti-HIV activity several dimers of the general formula [ddN]-(CH2)n-[NNRTI]. These dimers combine in their structure a ddN such as AZT and a NNRTI such as TSAO-T and HEPT linked through an appropriate spacer between the N-3 of the thymine base of both compounds. The [TSAO-T]-(CH2)n-[AZT] dimers proved markedly inhibitory to HIV-1. Also, if AZT was replaced by thymidine in the dimer molecules, potent anti-HIV-1 activity was observed. However, although the compounds proved inhibitory to HIV-1, they were less potent inhibitors than the parent compounds from which they were derived. None of the dimers were endowed with anti-HIV-2 activity. In contrast with the TSAO-T monomers, none of the TSAO-T-containing dimers proved markedly cytotoxic to the cells. There was a clear trend toward decreased antiviral potency with lengthening the methylene spacer in the [TSAO-T]-(CH2)n-[AZT] dimers.

Abasic analogues of TSAO-T as the first sugar derivatives that specifically inhibit HIV-1 reverse transcriptase.

With the aim of assessing the role that the thymine base of TSAO-T may play in the interaction of TSAO compounds with HIV-1 reverse transcriptase (RT), we have designed, synthesized, and evaluated for their anti-HIV-1 activity a series of 3-spiro sugar derivatives substituted at the anomeric position with nonaromatic rings or with amine, amide, urea, or thiourea moieties that mimic parts or the whole thymine base of TSAO-T. Also, a dihydrouracil TSAO analogue and O-glycosyl 3-spiro sugar derivatives substituted at the anomeric position with methyloxy or benzyloxy groups have been prepared. Compounds substituted at the anomeric position with an azido, amino, or methoxy group, respectively, were devoid of marked antiviral activity (EC50: 10-200 microM). However, the substituted urea sugar derivatives led to an increase in antiviral potency (EC50: 0.35-4 microM), among them those urea derivatives that mimic most closely the intact TSAO-T molecule retained the highest antiviral activity. Also, the dihydrouracil TSAO derivative retained pronounced anti-HIV-1 activity. None of the compounds showed any anti-HIV-2 activity. The results described herein represent the first examples of sugar derivatives that interact in a specific manner with HIV-1 RT. Molecular modeling studies carried out with a prototype urea derivative indicate that a heteroaromatic ring is not an absolute requirement for a favorable interaction between TSAO-T and HIV-1 RT. Urea derivatives, which can mimic to a large extent both the shape and the electrostatic potential of a thymine ring, can effectively replace this nucleic acid base when incorporated into a TSAO molecular framework with only moderate loss of activity.

Identification of a putative binding site for [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)thymine (TSAO) derivatives at the p51-p66 interface of HIV-1 reverse transcriptase.

A binding site for TSAO-m(3)T at the interface between the p66 and p51 subunits of HIV-1 reverse transcriptase (RT) and distinct from that of "classical" HIV-1 non-nucleoside inhibitors is proposed. The feasibility of the binding mode was assessed by carrying out nanosecond molecular dynamics simulations for the complexes of TSAO-m(3)T with reduced models of both the wild-type enzyme and a more sensitive R172A mutant. The molecular model is in agreement with a previous proposal, with known structure-activity and mutagenesis data for this unique class of inhibitors, and also with recent biochemical evidence indicating that TSAO analogues can affect enzyme dimerization. The relative importance of residues involved in dimer formation and TSAO-RT complex stabilization was assessed by a combination of surface area accessibility, molecular mechanics, and continuum electrostatics calculations. A structure-based modification introduced into the lead compound yielded a new derivative with improved antiviral activity.

Metabolism and pharmacokinetics of the anti-HIV-1-specific inhibitor [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3-N- methyl-thymine]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dio xide).

[1-[2',5'-Bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3-N- methyl-thymine]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''- dioxide) (TSAO-m3T) is a potent, selective and specific inhibitor of human immunodeficiency virus type 1 replication in vitro. Uptake of TSAO-m3T by human CEM cells is drug concentration-dependent and increased proportionally with increasing initial extracellular TSAO-m3T concentrations up to 20 micrograms/mL. Within 6 hr of incubation, the cells were almost completely saturated with the test compound; further incubation up to 72 hr did not markedly increase the intracellular concentration of the compound. No intracellular metabolic conversion of TSAO-m3T was observed in CEM, MT-4 or MOLT-4 cells. Upon intravenous bolus administration of TSAO-m3T to mice at 0.75 mg/kg, TSAO-m3T was rapidly cleared from the plasma in a mono-exponential manner (half-life: 22 min; distribution volume: 9.5 L/kg; total body clearance: 17.8 L/hr/kg). TSAO-m3T mainly accumulated in the lungs, followed by the heart, kidney and liver. Significant amounts of different metabolites of TSAO-m3T were detected in most tissues, the liver, kidney and spleen being the organs that showed the most extensive metabolism. The principal metabolites identified were TSAO-m3T derivatives in which the t-butyldimethylsilyl moiety at C-2' and/or C-5' had been split off. The free base N3-methylthymine was not detected.

Exploring the role of the 5'-position of TSAO-T. Synthesis and anti-HIV evaluation of novel TSAO-T derivatives.

Various analogues of the anti-HIV-1 agent TSAO-T, [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]thymine]-3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide) have been synthesized in which the 5'-TBDMS group has been replaced by alkyl-, alkenyl- or aromatic ether groups, substituted amines, carbamoyl or (thio)acyl groups. The compounds synthesized were evaluated for their inhibitory effect on HIV-1 and HIV-2 replication in cell culture. Replacement of the 5'-TBDMS group by an acyl, aromatic or a cyclic moiety markedly diminish or even eliminate the anti-HIV activity. However, the presence at that position of an alkyl or alkenyl chain, partially retain antiviral activity. These observations suggest that the 5'-TBDMS group of the TSAO molecule plays a crucial role.

TSAO-T analogues bearing amino acids at position N-3 of thymine: synthesis and anti-human immunodeficiency virus activity.

Novel analogues of the anti-HIV-1 lead compound [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]thymine]- 3'-spiro-5'-(4"-amino-1",2"-oxathiole-2',2'-dioxide) (TSAO-T) bearing different amino acids at position N-3 of thymine were prepared and evaluated as inhibitors of HIV replication. The synthesis of the target compounds was accomplished by coupling of the appropriate TSAO intermediate with a conveniently protected (L) amino acid in the presence of BOP and triethylamine, followed by deprotection of the amino acid moiety. Several TSAO derivatives, bearing at N-3 position of the thymine base an L-amino acid retaining the free carboxylic acid, acquired activity against HIV-2, in addition to their inhibitory effect on HIV-1.

An approach towards the synthesis of potential metal-chelating TSAO-T derivatives as bidentate inhibitors of human immunodeficiency virus type 1 reverse transcriptase.

Novel derivatives of the potent human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitor TSAO-T have been designed, synthesized and tested for their in vitro antiretroviral activity against HIV. These TSAO-T derivatives have been designed as potential bidentate inhibitors of HIV-1 RT, which combine in their structure the functionality of a non-nucleoside RT inhibitor (TSAO-T) and a bivalent ion-chelating moiety (a beta-diketone moiety) linked through an appropriate spacer to the N-3 of thymine of TSAO-T. Some of the new compounds have an anti-HIV-1 activity comparable to that of the parent compound TSAO-T, but display a markedly increased antiviral selectivity. There was a clear relationship between antiviral activity and the length of the spacer group that links the TSAO molecule with the chelating moiety. A shorter spacer invariably resulted in increased antiviral potency. None of the TSAO-T derivatives were endowed with anti-HIV-2 activity.

4"-H-TSAO-T, a novel prototype in the HIV-1 specific TSAO family.

The first TSAO derivative that lacks the amino group at the 3'-spiro moiety has been prepared. This molecule retained its HIV-1 specificity (NNRTI characteristic) but did not select for any of the classical NNRTI-specific mutations in the NNRTI binding pocket, including 138-Lys (TSAO resistant strain).

"Second generation" of TSAO compounds directed against HIV-1 TSAO-resistant strains.

A "second generation" of TSAO molecules directed against TSAO-resistant strains have been prepared. The presence of two neighboring carbonyl groups at the 4" position of the 3'-spiro moiety seems to be important for the anti-HIV-1 activity against both wild type and TSAO-resistant strains. NMR conformational studies in solution and theoretical calculations of the novel compounds have also been carried out.

Interaction of the antibiotic sparsomycin with the ribosome.

Phenol-alanine-sparsomycin, a derivative of sparsomycin carrying a p-hydroxy-benzyl function easily labeled by iodination, has been used to study the interaction of this drug with the ribosome. Our study indicated that the binding of the drug to the ribosome is sensitive to trichloracetic acid and is equally affected by disintegration of the particle after RNase and protease treatments. The ribosome is not irreversibly inactivated, and the chemical structure of the drug is not affected by interaction with the particle. These data are not compatible with the proposed covalent association of sparsomycin with the ribosome by G. A. Flynn and R. J. Ash (Biochem. Biophys. Res. Commun. 114:1-7, 1983); therefore, the antibiotic must inhibit protein synthesis through a reversible interaction with the ribosome.

Biochemical and kinetic characteristics of the interaction of the antitumor antibiotic sparsomycin with prokaryotic and eukaryotic ribosomes.

Using 125I-labeled phenol-alanine sparsomycin, an analogue of sparsomycin having higher biological activity than the unmodified antibiotic, we studied the requirements and the characteristics of its interaction with the ribosome. The drug does not bind to either isolated ribosomal subunits or reconstituted whole ribosomes. For sparsomycin binding to 70S and 80S ribosomes, the occupation of the peptidyltransferase P-site by an N-blocked aminoacyl-tRNA is a definitive requirement. The sparsomycin analogue binds to bacterial and yeast ribosomes with Ka values of around 10(6) M-1 and 0.6 x 10(6) M-1, respectively, but its affinity is probably affected by the character of the peptidyl-tRNA bound to the P-site. Chloramphenicol, lincomycin, and 16-atom ring macrolides compete with sparsomycin for binding to bacterial ribosomes, but streptogramins and 14-atom ring macrolides do not. Considering the reported low affinity of puromycin for bacterial ribosomes, this antibiotic is also a surprisingly good competitor of sparsomycin binding to these particles. In the case of yeast ribosomes, blasticidin is a relatively good competitor of sparsomycin interaction, but anisomycin, trichodermin, and narciclasin are not. As expected, puromycin is a poor competitor of the binding in this case. The results from competition studies carried out with different sparsomycin analogues reveal, in some cases, a discrepancy between the drug ribosomal affinity and its biological effects. This suggests that some intermediate step, perhaps a ribosomal conformational change, is required for the inhibition to take place.

[2',5'-Bis-O-(tert-butyldimethylsilyl)]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide) (TSAO) derivatives of purine and pyrimidinenucleosides as potent and selective inhibitors of human immunodeficiency virus type 1.

The [2',5'-bis-O-(tert-butyldimethylsilyl)]-3'-spiro-5''-(4''-amino- 1'',2''-oxathiole-2'',2''-dioxide) (TSAO) derivatives of ribofuranosylthymine, uridine, 5-bromouridine, 5-methylcytidine, inosine, and adenosine are potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) but not of other retroviruses (HIV-2, simian immunodeficiency virus, or Moloney murine sarcoma virus). The 50% effective concentration (EC50) of the most active TSAO congeners for inhibition of HIV-1 replication ranged from 0.034 to 0.44 microgram/ml. The 50% cytotoxic concentration (CC50) affecting the viability of MT-4 cells ranged from 2.35 to 18 micrograms/ml. The TSAO thymine derivative proved to be a highly selective inhibitor of HIV-1 reverse transcriptase but not of HIV-2 reverse transcriptase and DNA polymerase alpha. Introduction of an alkyl or alkenyl function at N3 of the thymine ring markedly decreased cytotoxicity but did not affect the antiviral activity of the compounds. The most potent (EC50, 0.034 microgram/ml) and most selective (CC50/EC50, 4088) inhibitor of HIV-1 replication proved to be the N3-methyl derivative of (1-[2',5'-bis-O-(tert-butyldimethylsilyl)beta-D-ribofuranosyl]thymine)- 3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide). This compound should be considered as a promising drug candidate for the treatment of HIV-1 infections.

Suppression of the breakthrough of human immunodeficiency virus type 1 (HIV-1) in cell culture by thiocarboxanilide derivatives when used individually or in combination with other HIV-1-specific inhibitors (i.e., TSAO derivatives).

Five structurally related thiophene and furane analogues of the oxathiin carboxanilide derivative NSC 615985 (UC84) (designated UC10, UC68, UC81, UC42, and UC16) were identified as potent inhibitors of HIV-1 replication in cell culture and HIV-1 reverse transcriptase activity. These compounds were markedly active against a series of mutant HIV-1 strains, containing the Leu-100-->Ile, Val-106-->Ala, Glu-138-->Lys, or Tyr-181-->Cys mutations in their reverse transcriptase. However, the thiocarboxanilide derivatives selected for mutations at amino acid positions 100 (Leu-->Ile), 101 (Lys-->Ile/Glu), 103 (Lys-->Thr/Asp) and 141 (Gly-->Glu) in the HIV-1 reverse transcriptase. The compounds completely suppressed HIV-1 replication and prevented the emergence of resistant virus strains when used at 1.3-6.6 microM--that is, 10- to 25-fold lower than the concentration required for nevirapine and bis(heteroaryl)piperazine (BHAP) U90152 to do so. If UC42 was combined with the [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1",2"- oxathiole-2",2"-dioxide)]-beta-D-pentofuranosyl (TSAO) derivative of N3-methylthymine (TSAO-m3T), virus breakthrough could be prevented for a much longer time, and at much lower concentrations, than if the compounds were used individually. Virus breakthrough could be suppressed for even longer, and at lower drug concentrations, if BHAP was added to the combination of UC42 with TSAO-m3T, which points to the feasibility of two- or three-drug combinations in preventing virus breakthrough and resistance development.