Vol. 36, No. 5ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1992, p. 1073-10800066-4804/92/051073-08$02.00/0Copyright C) 1992, American Society for Microbiology

[2' ,5 '-Bis-O-(tert-Butyldimethylsilyl)]-3' -Spiro-5"-(4"-Amino-1",2"-Oxathiole-2",2"-Dioxide) (TSAO) Derivatives of Purine andPyrimidine Nucleosides as Potent and Selective Inhibitors

of Human Immunodeficiency Virus Type 1JAN BALZARINI,l* MARIA-JESUS PEREZ-PEREZ,2 ANA SAN-FELIX,2 SONSOLES VELAZQUEZ,

MARIA-JOSE CAMARASA,2 AND ERIK DE CLERCQ'Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven,

Belgium, 1 and Instituto de Quimica Medica, 28006 Madrid, Spain2Received 10 January 1992/Accepted 3 March 1992

The [2',5'-bis-O-(tert-butyldimethylsilyl)1-3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide) (TSAO) deriv-atives of ribofuranosylthymine, uridine, 5-bromouridine, 5-methylcytidine, inosine, and adenosine are potentand 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%o effective concentration (EC50) ofthe most active TSAO congeners for inhibition of HIV-1 replication ranged from 0.034 to 0.44 ig/ml. The 50%cytotoxic concentration (CC50) affecting the viability of MT-4 cells ranged from 2.35 to 18 p,g/ml. The TSAOthymine derivative proved to be a highly selective inhibitor of HIV-1 reverse transcriptase but not of HIV-2reverse transcriptase and DNA polymerase a. Introduction of an alkyl or alkenyl function at N3 of the thyminering markedly decreased cytotoxicity but did not affect the antiviral activity of the compounds. The most potent(EC50, 0.034 ,ug/ml) and most selective (CC50/EC50, 4088) inhibitor of HIV-1 replication proved to be theN3-methyl derivative of {l-[2',5'-bis-O-(tert-butyldimethylsilyl)13-D-ribofuranosylJthymine}-3'-spiro-5"-(4"-ami-no-l',2"-oxathiole-2',2"-dioxide). This compound should be considered as a promising drug candidate for thetreatment of HIV-1 infections.

Recently, different classes of compounds have been foundto inhibit human immunodeficiency virus type 1 (HIV-1) butnot HIV-2 or simian immunodeficiency virus (SIV) replica-tion. These highly specific HIV-1 inhibitors include the6-substituted acyclouridine derivatives {i.e., 1-[(2-hydroxy-ethoxy)methyl]-6-phenylthiothymine (HEPT)} (1-4, 27),benzodiazepinone and benzodiazepinthione (TIBO) deriva-tives [i.e., tetrahydroimidazo(4,5,1-jk)(1,4)-benzodiazepin-2(1H)-one and tetrahydroimidazo(4,5,1-jk)(1,4)-benzodiaz-epin-2(1H)-thione] (17, 21), dipyridodiazepinones (i.e., BI-RG-587) (16, 19), pyridinones (i.e., L-697,639 and L-697,661)(14), and bis(hetero)arylpiperazine (BHAP) (24). All theseclasses of compounds seem to be targeted at the HIV-1reverse transcriptase (RT) (2, 9, 11, 13, 14, 19, 21, 27, 28).We have now identified a novel class of compounds, i.e.,[2',5'-bis-O-(tert-butyldimethylsilyl)]-3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide) (TSAO) derivatives of pyrimi-dine and purine nucleosides, that selectively inhibit thereplication of HIV-1 but not that of HIV-2, SIV, or otherretroviruses.

MATERIALS AND METHODS

Compounds. The compounds were synthesized accordingto a procedure that is described elsewhere (8b, 21a, 21b).Stock solutions were prepared in 20 mg of dimethyl sulfoxide(100%) per ml, and further dilutions of the test compoundswere made in 10% serum-containing culture medium. 3'-Azido-2',3'-dideoxythymidine (AZT) was purchased fromSigma Chemical Company (St. Louis, Mo.) and TIBO

* Corresponding author.

(R82150) was obtained from Zhang Hao (National CancerInstitute, Bethesda, Md.).

Cells. Human T-4 lymphocyte MT-4 cells were kindlyprovided by N. Yamamoto (Yamaguchi University, Ya-maguchi, Japan). MT-4 cells were cultivated in RPMI 1640medium supplemented with 10% fetal calf serum, 2 mML-glutamine, and 0.075% NaHCO3.

Viruses. The origin and preparation of Moloney murinesarcoma virus (MSV), HIV-1 (strain human T-cell lympho-tropic virus type IIIB [HTLV-IIIB]; provided by R. C. Galloand M. Popovic [National Cancer Institute]), HIV-2 (strainlymphadenopathy-associated virus type 2 [ROD], providedby L. Montagnier [Pasteur Institute, Paris, France]), andSIV (strain MAC251, molecular clone BK28, provided by C.Bruck [RIT-Smith-Kline, Rixensart, Belgium]) have beendescribed previously (10, 12, 15, 22).

Antiretrovirus assays. The methodology of the anti-HIVassays has been described previously (6). Briefly, MT-4 cells(5 x 105 cells per ml) were suspended in fresh culturemedium and infected with HIV-1, HIV-2, or SIV at 100 timesthe infective dose for 50% of the cell cultures per milliliter ofcell suspension. Then, 100 ,ul of the infected cell suspensionwas transferred to microplate wells and mixed with 100 ,ul ofthe appropriate dilutions of the test compounds, and themixture was further incubated at 37°C. After 5 days, thenumber of viable cells was determined in a blood cell-counting chamber by trypan blue staining for both virus- andmock-infected cells. The 50% effective concentration (EC50)and 50% cytotoxic concentration (CC50) were defined as thecompound concentrations required to reduce by 50% thenumber of viable cells in the virus- and mock-infected cellcultures, respectively.C3H/3T3 cells were seeded at 20,000 cells per ml into wells

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ANTIMICROB. AGENTS CHEMOTHER.

of tissue culture cluster plates (48 wells per plate). Followinga 24-h incubation, cell cultures were infected with 80 focus-forming units (FFU) of MSV for 120 min, after which theculture medium was replaced by 1 ml of fresh mediumcontaining appropriate concentrations of the test compound.After 6 days, transformation of the cells was examinedmicroscopically.

Radiochemicals. The radiolabeled nucleosides [methyl-3H]deoxythymidine (dThd) (specific radioactivity, 40 Ci/mmol), [5-3H]deoxyuridine (dUrd) (specific radioactivity, 27Ci/mmol), and [5-3H]deoxycytidine (dCyd) (specific radioac-tivity, 18.2 Ci/mmol) and the radiolabeled amino acid L-[4,5-3H]leucine (specific radioactivity, 52 Ci/mmol) were ob-tained from the Radiochemical Centre (Amersham,England); [5-3H]uridine (Urd) (specific radioactivity, 21 Ci/mmol) and [5-3H]cytidine (Cyd) (specific radioactivity, 26Ci/mmol) were obtained from Moravek Biochemicals Inc.(Brea, Calif.).

Inhibition of the incorporation of radiolabeled DNA, RNA,or protein precursors into TCA-insoluble MT-4 cell material.The incorporation of [methyl-3H]dThd, [5-3H]dUrd, [5-3H]dCyd, [5- H]Urd, [5- H]Cyd and L-[4,5-3H]Leu into tri-chloroacetic acid (TCA)-insoluble MT-4 cells was measuredin the wells of a 96-well microtiter plate (Falcon-3072;Becton Dickinson, Paramus, N.J.). To each well were added105 MT-4 cells, 0.25 p,Ci of the radiolabeled precursor, and agiven amount of the test compound. The cells were allowedto proliferate for 20 to 24 h at 37°C in a humidified,C02-controlled atmosphere. At the end of this incubationperiod, the contents of the wells (200 p,l) were transferredonto 25-mm glass fiber filters (type A/E; Gelman InstrumentCompany, Ann Arbor, Mich.) mounted on a Millipore 3025sampling manifold apparatus. The filters were washed onceeach with 2 and 5 ml of cold NaCl-Pi (phosphate-bufferedsaline), 2 and 5 ml of cold 10% TCA, and 5 and 7.5 ml of cold5% TCA and twice with 1 ml of cold ethanol.

Anti-HIV-1 activity of test compounds upon delayed addi-tion to HIV-1-infected MT-4 cells. MT-4 cells were suspendedin culture medium, infected with HIV-1, and transferred tomicroplate wells as described above. Then, 100 RI of theappropriate dilutions of the test compounds were added tothe HIV-1-infected cell cultures at the time of infection (0 h)or at 8, 16, 24, 40, or 48 h after infection. After 5 days, thenumber of viable cells was measured and the EC50s andCC50s of the test compounds were determined as describedabove.

Toxicity of test compounds after 2 h of incubation. MT-4cells (3.5 x 105 cells per ml) were exposed to 100, 20, 4, 0.8,or 0.16 ,ug of the test compounds per ml for 2 h at 37°C andsubsequently washed with culture medium. The pretreatedcells were seeded in microplate wells at 70 x 103 cells per200 p,l of culture medium per well and allowed to grow for 3days at 37°C. Control cultures were exposed to similarconcentrations of the test compounds during the entireincubation period. Then, the cells were counted in a CoulterCounter (Harpenden Hertz, England), and the CC50s weredetermined as described above.

Determination of P.. Partition of the test compoundsbetween 1-octanol (Merck, Darmstadt, Germany) and 10mM potassium phosphate buffer, pH 7.5 (Merck), wasmeasured as previously described (5). Briefly, a 50 FMconcentration of the test compound in potassium phosphatebuffer was thoroughly mixed with an equal volume of1-octanol for 30 min at room temperature. Then, the mixturewas further equilibrated at room temperature, and UVabsorption was measured for the aqueous and alcoholic

liquid phases. The partition coefficient (Pa) was calculated asthe ratio of the compound concentration present in the1-octanol phase to the compound concentration present inthe aqueous phase.RT assay. Enzyme assays were carried out with 0.2 mM

poly(C) oligo(dG) and various concentrations of [3H]dGTP(4, 6, 10, 20, and 40 p,M) as the substrate in a reactionmixture (40 p.l) containing 50 mM Tris-HCl (pH 7.8); 5 mMdithiothreitol; 300 mM glutathione; 500 p.M EDTA; 150 mMKCl; 5 mM MgCl2; 1.25 p.g of bovine serum albumin; 0.03%Triton X-100; 10 p.l of compound 9 (see Table 1) at 100, 20,4, or 0 p,g/ml; and 1 p.l of the reverse transcriptase (RT)preparation (p66; kindly provided by Philip J. Barr, Chiron).

RESULTS

Anti-HIV-1 activity of purine and pyrimidine nucleosideanalogs. A series of purine and pyrimidine nucleoside ana-logs containing substitutions at the 2', 3', and 5' positions ofthe ribose moiety were evaluated for their inhibitory effectson HIV-1-induced cytopathicity in MT-4 cells. Substitutionsincluded a spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide)group (both in R [nbo] or S xyloJ configuration) at C-3'and/or an O-tert-butyldimethylsilyl (tBDMS), O-benzoyl(OBz), or O-acetyl (OAc) group at C-2' and/or C-5'. Thepyrimidine moiety was thymine, uracil, 5-bromouracil, 5-ethyluracil, cytosine, 5-methylcytosine, N3-alkyl- or N3-alkenyl-substituted thymine or uracil, or N4-acetyl (Ac)-substituted cytosine (Table 1). The purine moiety wasadenine, N6-monomethoxytrityl-adenine or hypoxanthine(linked to the ribose moiety via its N9 or N7 atom) (Table 2).From the data in Table 1, it is clear that the presence of the

3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide) group inthe nucleosides with the nbo configuration is a prerequisitefor anti-HIV-1 activity. The 3'-spironucleosides with thexylo configuration resulted in a loss of antiviral activity(compare compounds 9, 18, and 28 with compounds 10, 19,and 29, respectively). Also, replacement of the spiro moietyby a 3'-OH group resulted in inactivity (compound 6).A second requirement for the anti-HIV-1 activity is the

presence of an O-tBDMS group at both the C-2' and C-5'positions of the ribose. Replacement of one of the O-tBDMSgroups by an OBz, OAc, OH, or H substituent resulted in acomplete loss of antiviral activity (compare compounds 2, 3,4, 5, 7, and 8 with compound 9).

Thus, from the antiviral data obtained for compounds 1 to10, it is clear that the simultaneous presence of the 3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide) group in the nboconfiguration and both the 2',5'-bis-O-tBDMS groups areessential for antiviral activity. It is also obvious that thepresence of the tBDMS groups at C-2' and C-5', but not thatof the 3'-spiro substituent (in nbo or xylo configuration), isresponsible for the cytotoxic activity of the compounds (notethe lack of toxicity of compounds 1 and 2 and the pro-nounced toxicity of compounds 6, 9, 10, 18, 19, 28, and 29).However, the cytotoxicity of the thymine analogs is

markedly decreased by introducing a lipophilic entity at N3of the thymine ring. Indeed, introduction of a methyl (com-pound 12), ethyl (compound 13), allyl (compound 14), ordimethylallyl (compound 16) group at N results in a 10- to40-fold decrease in cytotoxicity. The N3-alkyl derivativesbut not the N3-alkenyl derivatives completely retain theiractivity against HIV-1 (compare compounds 12, 13, 14, and16 with compound 9). As a result, compounds 12 and 13 areamong the most potent and selective inhibitors of HIV-1.The TSAO ribose (not containing a pyrimidine moiety) is

1074 BALZARINI ET Al-

SPECIFIC ANTI-HIV-1 ACTIVITY OF TSAO DERIVATIVES 1075

TABLE 1. Anti-HIV-1 activity of 3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide) pyrimidine furanosyl nucleosides in MT-4 cellsa

R3. R20Compound BaeR 2 R. Sugar con- EC50b CC5 bBaseX4 X5 X; R5' RT R' 00 I

no. figuration (Lg/ml) (Gg/ml)

1 T OH CH3 OH OH Spiro Xylo >500 >500 .12 T OH CH3 OH OH Spiro Ribo > 100 > 1003 T OH CH3 OH O(Si) Spiro Ribo >40 106 + 17 <2.54 T OH CH3 O(Si) OH Spiro Ribo >20 45 ± 2.6 <2S T OH CH3 O(Si) H Spiro Erythro >0.8 1.5 + 0.04 <26 T OH CH3 O(Si) O(Si) OH Ribo >1.6 5.7 ± 1.7 <3.67 T OH CH3 OBz O(Si) Spiro Ribo >8 20 + 2.3 <2.58 T OH CH3 OBz OAc Spiro Ribo > 100 > 1009 T OH CH3 O(Si) O(Si) Spiro Ribo 0.034 ± 0.015 7.7 ± 1.5 22710 T OH CH3 O(Si) O(Si) Spiro Xylo >4 18 ± 16 <4.511 T OH CH3 OH H O(Si) Erythro >20 34 ± 9.4 <212 3-Me-T OH CH3 Me O(Si) O(Si) Spiro Ribo 0.034 ± 0.060 139 ± 4.4 4,08813 3-Et-T OH CH3 Et O(Si) O(Si) Spiro Ribo 0.073 ± 0.034 73 ± 53 1,00014 3-Al-T OH CH3 Al O(Si) O(Si) Spiro Ribo 0.141 ± 0.10 .200 .1,41815 3-Al-T OH CH3 Al O(Si) OH Spiro Ribo >8 32 ± 27 <416 3-DMAI-T OH CH3 DMAI O(Si) O(Si) Spiro Ribo 0.239 ± 0.036 73 ± 1.0 25217 5-Et-U OH C2H5 O(Si) O(Si) Spiro Ribo 0.038 3.2 8218 U OH H O(Si) O(Si) Spiro Ribo 0.114 ± 0.023 8.3 ± 0.55 7319 U OH H O(Si) O(Si) Spiro Xylo >1.6 3.1 ± 0.5 <220 U OH H OH O(Si) Spiro Xylo >100 221 ± 5.0 1121 5-Br-U OH Br O(Si) O(Si) Spiro Ribo 0.206 ± 0.152 2.35 ± 1.67 1122 U OH H -N-4"-6-cyclo O(Si) O(Si) Spiro Ribo 3.8 ± 2.6 10 ± 4.0 2.823 3-Al-U OH H Al O(Si) O(Si) Spiro Ribo 0.364 ± 0.206 5.31 ± 1.38 1524 C NH2 H O(Si) O(Si) Spiro Ribo 0.439 ± 0.001 .200 .45625 C NHAc H O(Si) O(Si) Spiro Ribo 0.097 ± 0.043 7.5 ± 2.2 7726 C NHAc H O(Si) O(Si) Spiro Xylo >4 7.1 ± 2.7 <1.827 5-Me-C NH2 CH3 O(Si) O(Si) Spiro Ribo 0.072 ± 0.022 17.7 ± 0.50 246

a Spiro, 3'-spiro-5"-(4'-amino-1",2"-oxathiole-2",2"-dioxide); O(Si), O-(tert-butyldimethylsilyl); Me, methyl; Et, ethyl; Al, allyl; DMAI, dimethylallyl; Br, bromo;NHAc, N4-acetyl; Bz, benzoyl.

b Data represent the means ± standard deviations of 2 to 5 independent experiments.' SI, selectivity index, i.e., the ratio of CC50 to EC50.

1,000-fold less effective as an antiviral agent (EC5O, 21 ,ug/ml)and totally devoid of cytotoxicity at 200 ,ug/ml (compound 35[Table 3]).The nature of the pyrimidine moiety is less critical for

antiviral activity than the nature of the sugar moiety. Sub-stitution of thymine by uracil (compound 18), cytosine(compound 24), 5-methylcytosine (compound 27), 5-bromo-uracil (compound 21), or 5-ethyluracil (compound 17) did notlead to a marked decrease in anti-HIV-1 activity (Table 1).Only a few purine (adenine and hypoxanthine) derivatives

have been synthesized and evaluated for anti-HIV activity(Table 2). Compounds 28, 33, and 34 proved to be potentinhibitors of HIV-1 replication in MT-4 cells. Their EC50s(0.092 to 0.162 ,ug/ml) were only fourfold higher thanthose of the most active pyrimidine nucleoside analogs, buttheir CC50s (7.3 to 8.7 ,ug/ml) were comparable to those ofthe unsubstituted thymine (compound 9), uracil (compound18), and N4-acetylcytosine (compound 25) derivatives. Thus,

the selectivity indexes of compounds 28, 33, and 34 wereinferior to those of the most active pyrimidine derivatives(>100).The compounds (listed in Table 1) that were inhibitory to

HIV-1 (HTLV-IIIB) were also found to be active against theHE strain of HIV-1 at a similar EC50 (data not shown). Themost selective compound (compound 12) has also beenevaluated for its inhibitory effect against viral antigenexpression in HIV-1 (HTLV-IIIB)-infected MT-4 cells (26).At concentrations ranging between 0.25 and 30 ,uM, com-pound 12 proved completely inhibitory to virus antigenexpression, as assessed by indirect immunofluorescence (8)(data not shown).The active compounds 9, 12, 14, 18, 24, and 27 were also

examined for their antiviral activity in primary peripheralblood lymphocytes and were found to be inhibitory to HIV-1(HTLV-IIIB) replication at EC50s ranging between 0.027 and1.36 ,uM. The selectivity indexes of the compounds in

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ANTIMICROB. AGENTS CHEMOTHER.

TABLE 2. Anti-HIV-1 activity of 3'-spiro-5"-(4"-amino-1",2"-oxathiole-2,2"-dioxide) purine furanosylnucleoside derivatives in MT-4 cells"

J'

U r

as e

£2

o oCompound Base X6 Rs, RT RX Sugar con- EC50b CC5ob S

no. ase X figuration (4/ml) (6fml) S'no.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

28 A NH2 O(Si) O(Si) Spiro Ribo 0.162 ± 0.030 7.3 ± 3.4 4529 A NH2 O(Si) O(Si) Spiro Xylo >1.6 3.9 + 0.49 <230 A NH2 OH O(Si) Spiro Xylo >200 2200 <131 A NH-MMTr O(Si) O(Si) Spiro Xylo >100 163 ± 131 <1.632 A NH-MMTr O(Si) O(Si) Spiro Ribo >100 164 ± 47 <1.633 Hx (via N9) OH O(Si) O(Si) Spiro Ribo 0.092 ± 0.061 8.3 ± 1.0 9034 Hx (via N7) OH O(Si) O(Si) Spiro Ribo 0.101 ± 0.003 8.7 ± 0.1 86

I Abbreviations: see Table 1, fcotnote a; MMTr, monomethoxytrityl; Hx, hypoxanthine (linked to ribose via N9 in compound 33 and via N7 in compound 34).b Data represent the means ± standard deviations of 2 to 5 independent experiments.c SI, selectivity index (ratio of CC50 to EC50).

peripheral blood lymphocyte cells were comparable to thoseobtained in the human T4 cell lines (e.g., MT-4). In addition,compounds 9 and 12 were highly effective against the clinicalHIV-1 isolates A012G762-3 (8) and 336 (25). The end point ofantiviral activity in these assays was determined by thequantitation of the HIV-1 p24 antigen (26).

Activity of the purine and pyrimidine nucleoside analogsagainst retroviruses other than HIV-1. None of the testcompounds listed in Tables 1 to 3 proved effective againstHIV-2 [strain LAV (ROD)], SIV (strain MAC251), or MSVin MT-4 cells (HIV-2 and SIV) or C3H/3T3 cells (MSV) at

TABLE 3. Anti-HIV-1 activity of TSAI

%s. .

subtoxic concentrations (data not shown). Compounds 9, 12,14, 16, 17, 24, and 27 were also tested and found to beinactive against another HIV-2 strain (EHO).

Inhibitory effects ofthe test compounds on the incorporationof radiolabeled precursor molecules into MT-4 celi DNA,RNA, or protein. The most active TSAO nucleoside deriva-tives, including the thymine (compound 9), N3-methylthy-mine (compound 12), N3-allylthymine (compound 14), N3-dimethylallylthymine (compound 16), uracil (compound 18),cytosine (compound 24), and 5-methylcytosine (compound27) derivatives, were evaluated for their inhibitory effects on

O furanose derivatives in MT-4 cells"

0 @

Compound R s

RSugar con- EC,0b CC5yb SICno. R2 3 5 figuration (pg/ml) (IJg/mI)35 O(Si) Spiro O(Si) Ribo 21 ± 8.5 >200 >9.736 O(Si) Spiro O(Si) Xylo >200 .200

I For abbreviations, see Table 1, footnote a.b Data represent the means + standard deviations of 2 to 5 independent experiments.I SI, selectivity index (i.e., ratio of CC50 to EC50)-

1076 BALZARINI ET AL.

IAtt.l

SPECIFIC ANTI-HIV-1 ACTIVITY OF TSAO DERIVATIVES 1077

TABLE 4. Inhibitory effects of TSAO nucleoside derivatives on the incorporation of radiolabeled precursorsinto MT-4 cell DNA, RNA, or protein

Compound ICsoa (ig/ml)no. [methyl-3H]dThd [5-3H]dUrd [5-3H]dCyd [5-3HlUrd [5-3H]Cyd L-[4,5-3H]Leu

9 13 ± 0.8 9.1 ± 0.7 16 ± 1.7 11 ± 0.5 8.0 ± 2.8 11.6 ± 2.512 >100 >100 >100 >100 >100 >10014 >100 >100 >100 >100 >100 >10016 >100 >100 >100 >100 >100 >10018 38 ± 25 12 ± 0.7 18 ± 0.1 11 ± 1.7 11 ± 2.2 14 ± 2.724 >100 >100 >100 >100 >100 >10027 14" 9.9b 14b 8.9b llb 13b

a IC50, 50% inhibitory concentration, or concentration required to inhibit the incorporation of radiolabeled precursor into either DNA, RNA, or protein. Data

renresent the means ± standard deviations of two independent experiments.Data represent the IC50s for a single experiment.

DNA, RNA, and protein syntheses. Most compounds thatwere not cytotoxic at 100 ,ug/ml to MT-4 cells, on the basisof cell proliferation (Table 1), were also not inhibitory to theincorporation of dThd, dUrd, or dCyd into DNA, the incor-poration of Urd or Cyd into RNA, or the incorporation ofleucine into protein at a concentration of 100 ,ug/ml (Table 4).On the contrary, the compounds that proved inhibitory toMT-4 cell proliferation (i.e., compounds 9, 18, and 27) werealso inhibitory to the incorporation of the precursors intoDNA, RNA, or protein (Table 4). Thus, neither the thyminederivative (compound 9) nor the uracil (compound 18) or

el 3iox

E0

2

x

I

V-

0 4 23 51

Inhbor concentration (pg/mil)

5-methylcytosine (compound 27) derivative showed prefer-ential inhibition of the incorporation of any of the precursors(dThd, dUrd, dCyd, Urd, Cyd, and Leu).

Antiviral and cytotoxic activity of the test compounds upondelayed addition to HIV-1-infected MT-4 cells. Compounds 9,12, and 17, as well as the reference compounds AZT andTIBO (R82150), were added to HIV-1-infected MT-4 cells atdifferent times (6, 17, 24, 41, or 48 h) after infection.HIV-1-induced cytopathicity was determined at 5 days afterinfection. Delay of addition of the test compounds by 17 to24 h resulted in a 10-fold decrease of the antiviral activities of

I 1.125 1.1t11/S (jM-,)

FIG. 1. Double reciprocal (Lineweaver-Burk) plot for HIV-1 RT (p66) inhibition by compound 9 with [3H]dGTP as the variable substrateand poly(C) oligo(dG) as the template primer. Km for dGTP was 3.1 ,uM. The concentrations of compound 9 were 100 (*), 20 (0), 4 (O), or0 (O) ,ug/ml.

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ANTIMICROB. AGENTS CHEMOTHER.

compounds 9, 12, and 17 and a two- to fivefold decrease inthe antiviral activities ofAZT and TIBO (data not shown). Ifthe addition of the compounds was delayed for 41 to 48 h, theantiviral activities of compounds 9, 12, and 17, as well asthose of AZT and TIBO, were almost totally lost. However,the toxicity of compound 9 for MT-4 cells was not affectedby the time of addition of the compound to the cells. In fact,exposure of the MT-4 cells to the test compounds (i.e.,compounds 9, 18, and 27) for a period as short as 24 or even6 h proved as inhibitory to MT-4 cell proliferation as 3 or 5days of exposure. This contrasts with the behavior of AZT,which becomes less cytostatic the shorter its exposure timeto MT-4 cells (data not shown).

Lipophilicity of the test compounds. The Pa values weredetermined for a number of test compounds listed in Tables1 to 3. AZT was included for comparison. The Pa values forAZT and compound 2 were 1.12 and 0.117, respectively.Compounds 3, 4, 6, 7, 9, 12, 13, 14, 18, 24, 27, and 33 werefound to be present for more than 95% in the n-octanolphase. Consequently, the Pa values could not be accuratelydetermined. They were invariably higher than 20.

Inhibitory effect of compound 9 on HIV-1 RT (p66) activity.The prototype compound (compound 9) showed a markedinhibition of the RNA-dependent DNA polymerase activityof HIV-1 RT. It inhibited the poly(C)- oligo(dG)-directedDNA synthesis with [3H]dGTP as the substrate at a concen-tration of 17 ,M. The inhibition was noncompetitive withrespect to [3H]dGTP (Fig. 1). Its Ki value was 17 ,uM, andthe KJ/Km ratio was 5.4. Also, compound 9 proved non-competitively inhibitory to HIV-1 RT with respect topoly(C). oligo(dG) as the template primer (data not shown).

DISCUSSION

The TSAO derivatives of purine and pyrimidine nucleo-side analogs represent a novel class of potent and selectiveinhibitors of HIV-1 replication. These compounds are notactive against HIV-2 (strains ROD and EHO), SIV (strainMAC251) and MSV, and in this respect, they behave muchlike the HEPT (3), TIBO (21), dipyridodiazepinone (nevi-rapine) (19), pyridinone (14), and BHAP (24) derivatives,which are also highly specific for HIV-1. Structure-activitystudies revealed that the simultaneous presence of the 3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide) substituentand the tert-butyldimethylsilyl groups at C-2' and C-5' ofthe ribose is essential for antiviral activity. In addition,the 3 '-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide) sub-stituent has to be in the nbo configuration; the xylo config-uration makes the molecules completely inactive as antiviralagents.

In contrast with the HEPT derivatives, whose activitystrictly depends on the presence of the thymine or 5-alkyl-uracil moiety (3, 4), the TSAO nucleoside derivatives maycontain a variety of pyrimidine or purine bases, includingthymine, uracil, 5-ethyluracil, 5-bromouracil, cytosine,5-methylcytosine, adenine, or hypoxanthine. Introduction ofan alkyl moiety at N3 of the thymine ring reduces the toxicityof the thymine derivatives while keeping the antiviral activ-ity unaffected. As a result, the N3-alkyl derivatives of theTSAO-thymine analogs acquire the highest selectivity index(CC50/EC50 > 1,000). Neither the antiviral activities nor thecytotoxic activities of the compounds could be reversed byaddition of the natural pyrimidine nucleosides (i.e., dThdand 2'-dCyd) (8). These observations suggest that the test

compounds do not interfere with pyrimidine nucleotidemetabolism per se.At the highest concentrations tested, the compounds

proved cytotoxic. Exposure of MT-4 cells to relatively highconcentrations of compound 9 for a period as short as6 h resulted in a destruction of the cells, an effect thathas not been observed for other nucleoside analogs, suchas AZT. Also, the fact that the compounds are equally in-hibitory to DNA, RNA, and protein syntheses points to adirect toxic effect on the cells. Because of their lipophilicnature (Pa > 20), the test compounds may well interact withthe cellular membrane, thereby affecting the integrity of thecells.The high Pa values of the nucleoside analogs suggest that

they might easily cross the blood-brain barrier, a propertythat should be beneficial in light of the observations that HIVhas marked tropism for the central nervous system. Becauseof their lipophilic nature, the test compounds most likelypenetrate into the cells by passive diffusion rather than byfacilitated transport.The HEPT, TIBO, BI-RG-587 (nevirapine), and pyridi-

none (L-697,639 and L-697,661) derivatives have beenshown to specifically interact with the HIV-1 RT (2, 3, 11,16, 19, 24, 28). For several TSAO nucleosides, we have alsonoted a specific interaction with HIV-1 RT (Fig. 1) but notwith HIV-2 RT, cellular DNA polymerase a, or herpessimplex virus type 1 DNA polymerase (data not shown). Anextensive study on the kinetics of the TSAO derivativesagainst HIV-1 RT will be published elsewhere (7). In agree-ment with an antiviral action targeted at the RT were theobservations that the antiviral activity of the compounds wasmarkedly reduced when the compounds were added 17 to 24h after infection and was virtually annihilated when theywere added 40 to 48 h after infection. Recently, Nunberg etal. (20) and Richman et al. (23) demonstrated the rapid invitro emergence of HIV-1 mutants resistant to the pyridi-nones (L-697,639). These mutant virus strains are alsocross-resistant to the other specific HIV-1 RT inhibitorsTIBO and nevirapine (BI-RG-587). These findings indicatethat these different classes of compounds may share acommon binding site on the HIV-1 RT. We recently isolatedHIV-1 mutants that are resistant to TSAO (compounds 9, 12,and 13). Surprisingly, these mutant HIV-1 strains retainedtheir sensitivity to TIBO (R82150) and nevirapine (8a). Thesedata suggest subtle differences in the interactions of thedifferent HIV-1-specific compounds with HIV-1 RT.

In conclusion, the TSAO nucleoside derivatives representa novel class of nucleoside analogs which, akin to HEPT,TIBO, dipyridodiazepinone (BI-RG-587), pyridinone (L-697,639 and L-697,661), and BHAP derivatives (1-4, 14, 19, 21,24), are specific inhibitors of HIV-1 replication and aretargeted at the HIV-1 RT (2, 7-8a, 11, 13, 14, 24, 28).

ACKNOWLEDGMENTS

We thank Miette Stuyck, Ann Absillis, and Lizette van Berck-elaer for excellent technical assistance and Christiane Callebaut fordedicated editorial help. We are indebted to P. J. Barr (Chiron) forproviding the recombinant (p66) HIV-1 RT and Stephen Hughes(NCI, NIH) for providing the recombinant HIV-2 RT.

This research was supported in part by the AIDS Basic ResearchProgramme of the European Community and by grants from theBelgian Fonds voor Geneeskundig Wetenschappelijk Onderzoek(projects 3.0097.87 and 3.0026.91), the Belgian GeconcerteerdeOnderzoeksacties (project 90/94-2), and the Spanish Programa Na-cional de Investigacion y Desarrollo Farmaceutico (project FAR88-01606/1).

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