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.

Identification of a novel family of nucleosides that specifically inhibit HIV-1 reverse transcriptase.

N-3-Benzyloxycarbonylmethyl- and N-3-carboxymethyl-TBDMS-substituted nucleosides were synthesized and evaluated for activity against HIV replication. It was found that the N-3-carboxymethyl-TBDMS-substituted nucleosides were specific inhibitors of HIV-1 replication. They should be considered as members of a novel and original class of NNRTIs.

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.

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.

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.

Exploitation of the low fidelity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the nucleotide composition bias in the HIV-1 genome to alter the drug resistance development of HIV.

The RNA genome of the lentivirus human immunodeficiency virus type 1 (HIV-1) is significantly richer in adenine nucleotides than the statistically equal distribution of the four different nucleotides that is expected. This compositional bias may be due to the guanine-to-adenine (G-->A) nucleotide hypermutation of the HIV genome, which has been explained by dCTP pool imbalances during reverse transcription. The adenine nucleotide bias together with the poor fidelity of HIV-1 reverse transcriptase markedly enhances the genetic variation of HIV and may be responsible for the rapid emergence of drug-resistant HIV-1 strains. We have now attempted to counteract the normal mutational pattern of HIV-1 in response to anti-HIV-1 drugs by altering the endogenous deoxynucleoside triphosphate pool ratios with antimetabolites in virus-infected cell cultures. We showed that administration of these antimetabolic compounds resulted in an altered drug resistance pattern due to the reversal of the predominant mutational flow of HIV (G-->A) to an adenine-to-guanine (A-->G) nucleotide pattern in the intact HIV-1-infected lymphocyte cultures. Forcing the virus to change its inherent nucleotide bias may lead to better control of viral drug resistance development.

Site-directed mutagenesis of human immunodeficiency virus type 1 reverse transcriptase at amino acid position 138.

TSAO derivatives represent a class of nonnucleoside reverse transcriptase inhibitors (NNRTIs) that consistently select for the Glu138Lys resistance mutation in HIV-1 reverse transcriptase (RT). Seven RT mutants (i.e., Ala, Asp, Gln, Gly, Lys, Phe, and Tyr) were constructed by site-directed mutagenesis. The mutant Glu138Asp, Glu138Lys, Glu138Gln, Glu138Ala, and Glu138Gly RTs retained marked catalytic activity. In contrast, the Glu138Phe and Glu138Tyr RT mutants showed poor RNA-dependent DNA polymerase activity (30 and 4% of wild-type, respectively). TSAO derivatives lost their inhibitory activity against all mutant enzymes, except against the closely related Glu138Asp RT mutant that remained as sensitive to TSAOs as did wild-type RT. Other NNRTIs, including delavirdine, emivirine, and UC-781, and the NRTI ddGTP retained pronounced inhibitory activity against all mutant enzymes. When the amino acid mutations at position 138 of RT were introduced in recombinant virus clones, the sensitivity/resistance spectrum obtained toward the TSAOs and other NNRTIs was similar to those observed for the isolated recombinant mutant enzymes. The Glu138Lys RT mutant virus had the most marked resistance to TSAOs, followed by the Glu138Gln, Glu138Phe, Glu138Gly, Glu138Tyr, and Glu138Ala virus mutants. The Glu138Asp RT mutant virus kept full sensitivity to the TSAO derivatives. Mixtures of Glu138Lys RT mutant virus with the other virus clones mutated at the 138 position resulted in all cases, except for the Glu138Asp and Glu138Gly RT mutant viruses, in an outgrowth of the Glu138Lys RT mutant virus. Since the Glu138Lys RT proved most resistant to TSAO derivatives, was among the most catalytically efficient enzymes, and resulted in highly replication-competent virus, our data explain why the Glu138Lys RT mutant virus strains but not virus strains containing other amino acids at position 138 invariably emerge in cell cultures under TSAO drug pressure.

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.

Novel TSAO derivatives modified at positions 3" and 4" of the spiro moiety.

We have explored the introduction of different functional groups at positions 3" and 4" of the spiro moiety of TSAO-T. Alkylation of this spiro moiety afforded mixtures of N and/or C-alkylated derivatives, while acylation occurs, exclusively, on the amino group. Position 3" has been selectively functionalized by halogenation followed by Stille-cross coupling reaction with organostannanes under a variety of experimental conditions.

Novel series of [ddN]-[TSAO-T] heterodimers as potential bi-functional inhibitors of HIV-1 RT. Studies in the linker and ddN region.

Novel series of [ddN]-(CH2)n-[TSAO-T] heterodimers have been prepared and tested for their anti-HIV-1 and HIV-2 activity. The most active compound of this series was the [d4T]-(CH2)3-[TSAO-T] heterodimer (EC50 = 0.018 +/- 0.03 microM).

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.

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.

Marked inhibitory activity of non-nucleoside reverse transcriptase inhibitors against human immunodeficiency virus type 1 when combined with (-)2',3'-dideoxy-3'-thiacytidine.

Human immunodeficiency virus type 1 (HIV-1)-infected CEM cells were treated (as single agents or in combination) with (minus)-2', 3'-dideoxy-3'-thiacytidine (3TC) and the following HIV-1-specific non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs): 2', 5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5'-(4'-amino-1',2'-oxathi ole)-2',2'-dioxide derivative of 3-methylthymidine (TSAO-m3T), the thiocarboxanilides UC10 and UC42, bis(heteroaryl)piperazine (BHAP) derivative U90152, and the 1-(2-hydroxyethoxymethyl)-6-(phenylthio)thymine (HEPT) derivative 5-isopropyl-1-ethoxymethyl-6-benzyluracil (MKC-442). When used individually, the compounds led to the emergence of HIV-1 strains containing the following mutations in the RT: Glu138 to lysine for TSAO-m3T, Met184 to valine for 3TC, Lys103 to threonine/asparagine for the thiocarboxanilides, and Tyr181 to cysteine for BHAP and MKC-442. When 3TC was combined with TSAO-m3T, UC10, UC42, BHAP, or MKC-442, breakthrough of virus was markedly delayed or even suppressed. For these drug combinations, the concentrations of the individual drugs could be lowered by > or = 25-50-fold to suppress virus breakthrough compared with the individual use of the compounds. The concomitant presence of the Lys138 and Ile/Val184 mutations was found in the RT of the mutant viruses that emerged with combination therapy of the lowest concentrations of 3TC with either the lowest concentrations of TSAO-m3T or UC10 (approximately 0.5-3-fold the EC50 value). These virus strains retained high sensitivity to other NNRTIs such as BHAP or HEPT. The virus mutants that arose in the presence of combinations of the lowest concentrations of 3TC with either BHAP or HEPT predominantly contained the Cys181 mutation in the RT. In one case, the Ile181 mutation was found. The latter mutations, particularly the Ile181 mutation, resulted in markedly decreased sensitivity to the NNRTIs but not to 3'-azido-2', 3'-dideoxythymidine or 3TC.

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.

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.

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.

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.

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.

Human immunodeficiency virus type 1 (HIV-1) strains selected for resistance against the HIV-1-specific [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro- 5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)]-beta-D-pentofurano syl (TSAO) nucleoside analogues retain sensitivity to HIV-1-specific nonnucleoside inhibitors.

We recently reported that a newly discovered class of nucleoside analogues--[2',5'-bis-O-(tert-butyldimethylsilyl)- 3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)]-beta-D - pentofuranosyl derivatives of pyrimidines and purines (designated TSAO)--are highly specific inhibitors of human immunodeficiency virus type 1 (HIV-1) and targeted at the nonsubstrate binding site of HIV-1 reverse transcriptase (RT). We now find that HIV-1 strains selected for resistance against three different TSAO nucleoside derivatives retain sensitivity to the other HIV-1-specific nonnucleoside derivatives (tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO), 1-[(2-hydroxyethoxy)methyl]-6-phenylthiothymine, nevirapine, and pyridinone L697,661, as well as to the nucleoside analogues 3'-azido-3'-deoxythymidine, ddI, ddC, and 9-(2-phosphonylmethoxyethyl)adenine. Pol gene nucleotide sequence analysis of the TSAO-resistant and -sensitive HIV-1 strains revealed a single amino acid substitution at position 138 (Glu-->Lys) in the RT of all TSAO-resistant HIV-1 strains. HIV-1 RT in which the Glu-138-->Lys substitution was introduced by site-directed mutagenesis and expressed in Escherichia coli could not be purified because of rapid degradation. However, HIV-1 RT containing the Glu-138-->Arg substitution was stable. It lost its sensitivity to the TSAO nucleosides but not to the other HIV-1-specific RT inhibitors (i.e., TIBO and pyridinone). Our findings point to a specific interaction of the 4''-amino group on the 3'-spiro-substituted ribose moiety of the TSAO nucleosides with the carboxylic acid group of glutamic acid at position 138 of HIV-1 RT.