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TSAO derivatives are potent and selective HIV-1 reverse transcriptase (HIV-1 RT) inhibitors. Although, structurally, they can be considered as highly functionalized nucleosides, they inhibit their target enzyme (HIV-1 RT) similarly to the so-called Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI).1-6 The different families of NNRTI, including TSAO, interact with the HIV-1 RT in a highly hydrophobic pocket, close but different from the active site, and several complexes of NNRTIs-HIV-1 RT have been resolved by X-ray crystallography.7-15

The structures of two novel 3′-spiro nucleosides analogues of the potent human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) inhibitor TSAO-m3T, in solution, as derived from NMR spectroscopy are described. In these TSAO analogues the spiro amino oxathioledioxide moiety has been replaced by spiro amino oxazolone or spiro amino oxathiazoledioxide moieties. A comparative study based on theoretical calculations of the hydrophobicity, the solvation free energies and molecular electrostatic potentials (MEP) of the three compounds is also described. No significant conformational differences were detected in solution between TSAO-m3T and its analogues that might account for the differencesobserved in their inhibitory activity against HIV-1 RT. The calculated hydrophobicity (log P) values, dipole moments and the electrostatic contributions to the solvation free energies of the three spiro ring systems were also similar. However, the differences found in the calculated MEPs of the spiro systems between TSAO-m3T and its analogues suggest that the different electrostatic surroundings of the 4″-amino group of the spiro moiety in the analogues may be responsible for a detrimental electrostatic interaction of the spiro rings with the Glu-B138 of RT.

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.

Novel 3'-spiro nucleoside analogues 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 anti-retroviral activity against HIV-1. In these TSAO analogues the spiro amino-oxathioledioxide moiety was replaced by other spiro moieties that maintained an NH group at the same position as the 4″-NH2group in the prototype compound TSAO-T. Anti-HIV-1 activity, although around 100-fold less pronounced than that of the parent TSAO-m3T derivative, was observed for the spiro oxazolone derivative. The spiro oxathiazoledioxide compound also showed antiviral activity. The corresponding β-D-xylofuranosyl analogues were devoid of antiviral activity; this is in accordance with the behaviour of TSAO-m3T. None of the test compounds were inhibitory to HIV-2 replication. The markedly decreased potency of the spiro oxathiazoledioxide and oxazolone compounds against HIV-1 replication is in agreement with their decreased anti-HIV-1 RT activity.

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 vitroantiretro-viral 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 β-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.

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.

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.

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.

Human immunodeficiency virus type 1 (HIV-1)-infected CEM cells were treated by the HIV-1-specific inhibitors bis-heteroarylpiperazine (BHAP), 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1 H)-on e (TIBO) R82913, nevirapine, and the N3-methylthymine derivative of [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro- 5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide) (TSAO-m3T), as single agents or in combination, at escalating concentrations. When used individually, the compounds led to the emergence of drug-resistant virus strains within two to five subcultivations. The resulting strains were designated HIV-1/BHAP, HIV-1/TIBO, HIV-1/Nev, and HIV-1/TSAO-m3T, respectively. The mutant viruses showed the following amino acid substitutions in their reverse transcriptase (RT): Leu-100-->Ile for HIV-1/BHAP; Lys-103-->Asn for HIV-1/TIBO; Val-106-->Ala for HIV-1/Nev; and Glu-138-->Lys for HIV-1/TSAO-m3T. Both the Tyr-181-->Cys and Val-106-->Ala mutations were found in another mutant emerging following treatment with nevirapine at escalating concentrations. The BHAP-resistant virus remained fully sensitive to the inhibitory effects of nevirapine and TSAO-m3T, whereas the TSAO-m3T-resistant virus remained fully sensitive to the inhibitory effects of nevirapine and BHAP. When different pairs of nonnucleoside RT inhibitors (i.e., BHAP plus TSAO-m3T, nevirapine plus TSAO-m3T, TIBO plus TSAO-m3T, nevirapine plus TIBO, and BHAP plus nevirapine) were used, resistant virus emerged as fast as with single-drug therapy. In all cases the Tyr-181-->Cys mutation appeared; the virus showed markedly reduced sensitivity to all HIV-1-specific inhibitors but retained sensitivity to 2',3'-dideoxynucleoside analogs such as zidovudine, ddC, and ddI. Our findings argue against simultaneous combination of two different nonnucleoside RT inhibitors that are unable to inhibit HIV-1 mutant strains containing the Tyr-181-->Cys mutation when administered as single drugs.

A series of pyrimidine nucleoside analogues containing [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5''-(4''-amino- 1'',2''-oxathiole-2'',2''-dioxide)]-beta-D-ribofuranose as the pentose were found to inhibit human immunodeficiency virus type 1 [HIV-1(IIIB)] replication at a concentration of 0.06-0.8 microM but were not cytotoxic at a 1000- to 10,000-fold higher concentration. These nucleoside derivatives were also effective against various other HIV-1 strains, including those resistant to 3'-azido-3'-deoxythymidine, but not against HIV-2, simian immunodeficiency virus, Moloney murine sarcoma virus, or other RNA or DNA viruses. They proved to be highly specific inhibitors of the RNA-dependent DNA polymerase function of the HIV-1 reverse transcriptase, showing no marked inhibition of the HIV-1 reverse transcriptase-associated DNA-dependent DNA polymerase activity, HIV-2 reverse transcriptase, DNA polymerase alpha, herpes simplex virus 1 DNA polymerase, or Thermus aquaticus DNA polymerase.