Journal of Medicinal Chemistry, 1992, Vol. 35, No. 16 2989
Anti-HIV-1 TSAO Analogues Scheme I
B
0-0JC 1
5 a,b,c
2 R=H 3 R=Ms
-
4
B
5 a,b,c
B
U
a : B = Thymin-1-yl b : B = Uracil-1-yl c : B = 5-Ethyluracil-I -yl
6 a,b,c
a well-established p r o c e d ~ r e . ~Treatment ~-~ of the dose 1 with sodium cyanide in a two-phase ethyl ether/water (12) Balzarini, J.; Van Aerschot, A.; Pauwels, R.; Baba, M.; Schols, D.; Herdewijn, P.; De Clercq, E. 5-Halogeno-3’-fluoro-2’,3’-dideotyuridinea as inhibitors of human immunodeficiency virus (HIV): potent and selective anti-HIV activity of 3’-fluoro2’,3’-dideoxy-5-chlorouridine. Mol. Pharmacol. 1989, 35, 571-677. (13) Chu, C. K.; Schinazi, R. F.; Ahn, M. K.; U b ,G. U.;Gu, Z. P. Structureactivity relationship of pyrimidine nucleosides as antiviral agents for human immunodeficiency virus type 1in peripheral blood mononuclear cells. J.Med. Chem. 1989,32, 612-617. (14) Lin,T. S.; Shen,Z. Y.;August, E. M.; Braukovan, V.;Yang,H.; Ghezzouli, I.; Prusoff, W. H. Synthesis and antiviral activity of several 2,5‘-anhydro analogues of 3‘-azido-3‘-deoxythymidine, 3’-azido-2’,3’-dideoxyuridine, 3’-azido-2’,3’-dideoxy-bhalouridines, and 3’-deoxythymidine against human immunodeficiency virus and Rauscher-murine Leukemia virus. J. Med. Chem. 1989,32,1891-1895. (15) Herdewijn, P.; Balzarini, J.; De Clerq, E.; Pauwels, R.; Baba, M.; Broder, S.; Vanderhaeghe, H. 3’-Subatituted 2‘,3‘-dideoxynucleoside analogues as potential anti-HIV (HTLVIII/LAV) agents. J. Med. Chem. 1987,30,1270-1278. (16) Pauwels, R.; Balzarini,J.; Schols, D.; Baba, M.; Desmyter, J.; Rosenberg, I.; Holy, A.; De Clercq, E. Phosphonylmethoxyethyl purine derivatives, a new class of anti-human immune deficiency virus agents. Antimicrob. Agents Chemother. 1988, 32, 1035-1030. (17) Balzarii, J.; Naeaens, L.; Herdewijn, P.; Rosenberg, I.; Holy, A.; Pauwels, R; Baba, M.; Johns, D. G.; De Clercq, E. Marked in vivo anti-retrovirus activity of 9-(2-phosphonylmethoxyethyl)adenine, a selective anti-HIV agent. Proc. Natl. Acad. Sci. U.S.A. 1989,86, 332-336. (18) Balzarini,J.; Naeaens, L.; Slachmuylders, J.; Niphuie, H.; Rosenberg, I.; Holy, A.; Schellekens, H.; De Clercq, E. 942Phosphonylmethoxyethy1)adenine(PMEA) effectively inhibits retrovirus replication in vitro and simian immunodeficiency virus infection in rhesus monkeys. AIDS 1991,5,21-28. (19) Balzarini, J.; Holy, A.; Jindrich, J.; Dvorakova, H.; Hao, Z.; Snoeck, R.; Herdewijn, P.; Johns, D. G.; De Clercq, E. 9[(2RS)-3-fluoro-2-phoephonylmethoxypropyl]derivativesof purinea: A class of highly selective antiretroviral agents in vitro and in vivo. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4961-4965. (20) Balzarini,J.; Naeaens, L.;De Clercq, E. Anti-retrovirus activity of 9-(2-phoephonylmeth01yethyl)adenine (PMEA) in vivo increases when it is l a frequently a d m i t e r e d Znt. J. Cancer 1990,46,337-340.
7 a,b,c
8 a,b,c
Scheme I1
9a
1Oa
system, in the presence of NaHC03, afforded the kinetically c o n t r ~ l l e d ~ n’bo-cyanohydrin l?~~ 2 (Scheme I). For(21) Naesens, L.; Balzarini, J.; Rosenberg, I.; Holy, A,; De Clercq, E. 9-(2-Phosphonylmethoxyethyl)-2,6-diaminopurine(PMEDAP): A Novel agent with anti-human immunodeficiency virus activity in vitro and potent anti-Moloney Murine Sarcoma virus activity in vivo. Eur J. Clin. Microbiol. Infect. Dis. 1989,8,1043-1048. (22) Naesens, L.; Balzarini, J.; De Clercq, E. Single-dose administration of 9-(2-phoephonylmethoxyethyl)adenine@‘MEA) and 9-(2-ph~phonylmethoxyethyl)-2,6-diaminopurine (PMEDAP) in the prophylaxis of retrovirus infection in vivo. Antiuiral Res. 1991, 16, 53-61. (23) Balzarini, J.; Hao, Z.; Herdewijn, P.; Johns, D. G.; De Clercq, E. Intracellular metabolism and mechanism of anti-retrovim action of 9-(2-phoephonylmethoxyethyl)adenine,a potent anti-human immunodeficiency virus compound. Proc. Natl. Acad. Sci. U.S.A. 1991,88, 1499-1503. (24) Hao, Z.; Conney, D. A.; Farquhar, D.; Pemo, C. F.; Zhang, K.; Masood, R.; Wilson, Y.; Hartman, N. R.; Balzarini, J.; Johns, D. G. Potent DNA chain termination activity and selective inhibition of human immunodeficiency virus reverse transcrintase bv 2’.3’-dideoxvuridine-5‘-tri~hos~hate. Mol. Phar- malol. 1990, 37, 167-163. (25) Furman, P. A.; Fyle, J. A.; St Clair, M. H.; Weinhold, K.; Rideout. J. L.: Freeman. G. A.: Nusinoff-Lehrman, S.: Bolognesi, D. P.; Brbder, S.; Mitsuya, H.; Barry, D. W.; PhAphosylation of 3’-azido-3’-deoxythyidine and selective interaction of the 5’-triphosphate with human immunodeficiency vim reverse transcriptase. A.oc. Natl. Acad. Sci. U.S.A.1986,83, 8333-8337. (26) St. Clair, M. H.; Richards, C. A.; Spector, T.; Weinhold, K. J.; Miller, W. H.; Langlois, A. J.; Furman, P. A. 3’42ido-3’deoxythymidine triphosphate as an Inhibitor and Subatrate of purified human Inmunodeficiency Virus Reverse Transcrip tase. Antimicrob. Agents Chemother. 1987, 31, 1972-1977. (27) PCez-PBrez, M. J.; San-FBlix, A.; Balzarini, J.; De Clercq, E.; Camarasa, M. J. Tetrahedron Lett. 1992,33, 3029-3032. (28) Camarasa, M. J.; PBrez-PBrez, M. J.; San-FBlix, A.; Balzarini, J.; De Clercq, E. J. Med. Chem., in press.
2990 Journal of Medicinal Chemistry, 1992, Vol. 35, No. 16
P&ez-P&ez e t al.
Scheme 111 NH,
8a R = C H 3 8b R = H
mation of 2 is in agreement with the approach of the cyanide ion from the sterically less hindered @-faceof the dose 1, oppbsite to the 1,2-O-isopropylidenegroup. The cyanohydrin 2 was not isolated. It was transformed without further purification to the cyano mesylate 3. The absolute configuration of 3 was assumed to be identical to that of the corresponding cyanohydrin 2 since, in previous mesylations of cyanohydrins, no epimerization was 0bserved.3~*~~~*~~ Hydrolysis of the 1,2-0-isopropylidene group of 3, with aqueous trifluoroacetic acid, followed by reaction with acetic anhydride/pyridine afforded a (1.5:l) mixture of the two anomeric forms (aand 8) of the diacetate derivative 4 in 95% yield. Condensation of 1,2bis-0-acetyl cyano mesylate 4 with silylated bases using trimethylsilyl triflate as condensing reagent, as described by Vorbriiggen,@resulted in 3’-cyano mesylate nucleosides (29) Although the oxathiole ring has priority over the nucleoside syatem, double primes have been used in the numbering of the oxathiole ring in order to k e e the ~ same numberinn swtem for all the nucleosides described in thii paper. Balzarini, J.; PCrez-PCrez, M. J.; San-F€lix, A.; Schols, D.; Pemo, C. F.;Vandamme, A. M.;Camarasa, M.J.; De Clercq, E. Roc. Natl. Acad. Sci. U.S.A. 19?2,89,4392-4396. Calvo-Ma&, A.; Camarasa, M.J.; Diaz-Ortiz, A; De las Heras, F. G. Novel aldol-type cyclocondensation of 0-mesyl (methylsulphony1)cyanohydrins. Application to the stereospecific syntheses of branched-chain sugars. J. Chem. Soc., Chem. Commun. 1988, 1114-1115. Phx-PCrez, M.J.; Camarasa, M.J.; Dhz-Ortiz, A.; San FClix, A.; De las Heras, F. G.Stereospecific synthesis of branchedchain sugars by a novel aldol-type cyclocondensation. Carbohydr. Res. 1991, 216, 399-411. Hollemberg, D. H.; Klein, R. S.; Fox,J. J. Pyridinium chlorochromate for the oxidation of carbohydrates. Carbohydr. Res. 1978,67,491-494. Levene, P. A.; Raymond, A. L. Derivatives of monoacetone xylose. J. Biol. Chem. 1933,102,317-330. Bourgeoie, J. M.; Synthese de eucres amin& ramifi6s 11. S y n t l k et reactions de spiro-aziridines. Helv. Chim. Acta 1974,57,2563-2557. Thang, T. T.;Winternitz, F.;Lagrange, A.; Oleste,A.; Lukas, G. Stereoepecific acceaa to branched-chain carbohydrate synthons. Tetrahedron Lett. 1980,21,4495-4498. Yoshimura, J.; Aqeel, A.; Hong, N.; Sato, K.; Hashimoto, H. Syntheses of Prubranitrose and methyl a-D-tetrorithose by cyanomesylation of hexopyranosid-3-uloses. Carbohydr. Res. 1986,155, 236-246. 1
-
5 in good yields. Thus, the glymylation of 4 with thymine, uracil, and 5-ethyluracilmafforded the 3’-cyano mesylates of thymine 5a (77%), uracil 5b (78%) and 5-ethyluracil 5c (93%), respectively. Due to the presence of a 2-0-acyl participating group, the products obtained were exclusively @-anomem Coupling constant values were, in the range of Jl#g = 6.0-7.0 Hz,which is in reasonably good agreement with literature data for other 8-~-ribo-3’-C-branchednuc l e o s i d e ~ ,and ~ > ~further ~ corroborates the @-anomeric configuration of cyano mesylates 5a-c. Treatment of cyano mesylate Sa with CszC03afforded the spiro derivative 6a in 65% yield. Deprotection of 6a with saturated methanolic ammonia, gave the fully deprotected nucleoside 7aa in 66% yield, which, by reaction with tert-butyldimethylsilyl chloride yielded the 2’,5’bis-0-silylated nucleoside 8a.28 Selective cleavage of the 5’-0-silyl ether of 8a (Scheme 11)followed by bemylation with benzoyl chloride/pyridine gave the 5’-0-benzoyl-2’0-silyl spiro nucleoside 10a (80%). The spiro nucleosides 8b and & were prepared from the corresponding cyano mesylates 5b and 5c, in an overall yield of 24% and 32%, reapectively. The reaction sequence was similar to that described for the synthesis of 8a, and no attempts were made at isolating the spiro nucleoside intermediates 6b, 6c, 7b, and 7c. Structures of the new compounds were assigned on the basis of the corresponding analytical and spedtroscopic data, and by comparison of such data with those of the previously reported 8a and 8b whose structure had been (38) Vorbniggen, H.; Krolikiewicz, K.; Bennua, B. Nucleoside Synthesis with Trimethyleilyl Triflate and Perchlorate as Catalysts. Chem. Ber. 1981,114,1234-1256. (39) Kaul, R.; Kiefer, G.; Erhardt, S.; Hempel, B. 2J4C-l(2’Deoxy-~-~-ribofuranosyl)-5-ethyluracil: Synthesis and Biotransformation in Rats. J. Pharm. Sci. 1980, 69, 631-534. (40) Beigelmau, L.N.; Gurekaya, G. U.; Taakina, E.N.; Mikhailov, 5. N. Epimerhtion during the acetolysis of 3-O-acetyl-5-0benzoyl-l,2-O-isopropylidene-3-C-methyl-~-~-ribof~anose. Synthesis of 3’-C-methylnucleoeides with the O-D-ribo and 8D-arabino-configurations.Carbohydr. Res., 1988,181,7748. (41) Walton, E.; Jenkins, S. R.; Nutt, R.F. et al. Branched-Chain Sugar Nucleosides. V. Synthesie and Antiviral Properties of Several Branched-Chain Sugar Nucleosides. J. Med. Chem. 1969,12,306-309.
Anti- HIV-1 TSAO Analogues
Journal of Medicinal Chemistry, 1992, Vol. 35, No.16 2991
unequivocally determined* in an earlier paper of this seTable I. Inhibitory Effects of 3’-spiro Oxathiole Dioxide Nucleoside Analogues on HIV-1-Induced Cytopathicity in MT-4 ries. As described in previous reports in this ~ e r i e s , * *the ~ ~ + ~ ~Cells disappearance, in the ‘H NMR spectra, of the signal CCwc (pM) SId compdo ECwb (pM) corresponding to the mesyl group and the presence of two >200 >200 6a singlets at b 6.93-7.17 assigned to NH2-4” and at 6 0.060 f 0.027 14 f 2.0 227 8a 0.206 f 0.042 15 f 1.0 73 8b 5.60-5.74 assigned to H-3’’ indicate the formation of the 0.066 f 0.010 8c 5.5 f 1.0 82 spiro aminooxathiole dioxide ring. The values of J1f,2t= >80 229 f 37 9a 15 36 f 4.2 80 >lo53 followed by treatment with an excess of concentrated amBI-RG-587 0.128 f 0.041 >80 >625 monia afforded the cytidine analogues 11 and 12 in 67% “Data represent the mean values of at least three to five indeand 84% yield, respectively. pendent experiments. * 50% Effective concentration or compound Finally, selective N-3-all&tionG of the spiro nucleosides concentration required to inhibit HIV-1-induced cytopathicity in MT-4 cells by 50%. “0% Cytotoxic concentration or compound 8a and 8b afforded the N-3-substituted derivatives 13-17. concentration required to reduce MT-4 cell viability by 50%. Reaction of 8a with methyl iodide in the presence of podSelectivity index or ratio of CCw to ECw. tassium carbonate gave 3-methy1-3’-spiroderivative 13 in 55% yield. the proposed structures and with those reported for other Attachment of the methyl group to the N-3 and not to N-3-substituted p y r i m i d i n e ~ . ~ ~ * ~ ~ l ~ either the oxygen atoms of the C 4 groups of thymine Biological Results or to the NH2-4/’or (2-3’’ of the spiro oxathiole moiety was established as follows: (a) the ‘H NMR spectrum showed A number of pyrimidine 3’-spiro-~’’-(4”-amino-~’‘,~‘’the disappearance of the signal at 10.32 ppm assigned to oxathiole 2’’,2”-dioxide) pyrimidine nucleoside analogues the NH-3 and the presence of a new singlet at 3.26 ppm were evaluated for their inhibitory activity against HIV-1 corresponding to the 3-N-CH3. (b) No modification was (IIIB)-inducedcytopathicity in human MT-4 lymphocyte observed at the signals of the protons of the spiro oxathiole cells (Table I). Only those nucleoside analogues that moiety, thus indicating that no methylation had occurred contain a tert-butyldimethylsilyl (tBDMS) group at both at this moiety. (c) The I3C NMR showed no changes in the C-5’ and C-2’ position of the ribose moiety showed the chemical shifts of C-2 and C-4 with respect to those potent anti-HIV-1 activity, irrespective of the nature of observed for 8a, used as starting material. the pyrimidine base (i.e. thymine, uracil, or cytosine). The These carbons appear in derivative 13 at 152 ppm (C-2) anti-HIV-1activity ranged from 0.060 pM to 1.0 pM. The and at 163ppm (C-4) and are in agreement with the values thymine derivative 8a (ECSO:0.060pM) proved superior observed for C-2 and C-4 in thymidine,* confirming that to the uracil derivative 8b (ECSO:0.206 pM) and cytosine the methyl group was not attached to them. 1.0 pM). However, due to ita marked derivative 12 Similarly, reaction of 8a with ethyl iodide, d y l bromide, lower toxicity, the cytosine derivative 12 proved more seand of 8b with allyl and 4-bromo-2-methyl-2-butene lective as an anti-HIV-1 agent than the corresponding bromide afforded the 3-N-ethyl-, 3-N-allyl-, and 3-N-(3thymine and uracil derivatives (selectivityindexes: 1360, methyl-2-butenyl)spiro nucleosides of thymine, 14,15, and 227, and 73, respectively). 16 in 77%, 65%, and 70% yield, respectively, and the Introduction of a methyl group at C-5 of the cytosine 3-N-allyl spiro derivative of uracil 17 in 89% yield. Their ring (11) enhanced the antiviral activity by 6-fold but also analytical and spectroscopic data were in agreement with markedly increased the toxicity; thus the selectivity index of compound 11 was 246. Also, the 5ethyluracil derivative (42) Rosenthal, A.; Cliff, B. L.Synthesis of an Analog of the Nu8c showed an antiviral activity comparable to that of the cleoside Moiety of Polyoxine. Cwbohydr. Res. 1980,79,63-77. parent thymine derivative 8a, but was slightly more cy(43) T o m e n d , L. B. Nuclear Magnetic Resonance Spectroscopy totoxic than 8a. Interestingly, introduction of an alkyl in the Study of Nucleic Acid Componenb and Certain Related moiety at 3-N of the thymine ring (as in compounds 13 and Derivatives. In Synthetic Procedures in Nucleic Acid Chem14) did not alter the antiviral potency of the parent comistry; Lorbach, W. W., Tipaon, R. S., W.; Wiley-Interecience: New York-London, 1973; Vol. 2, pp 267-398. pound, but markedly decreased the cytotoxicity. Conse(44) (a) Van Aerschot, A.; hreraert, D.; Balzarini,J.; Augustyns, K.; quently, the selectivity index went up to 4088 and 1O00, Jie, L.; Janssen, G.; Peetera, 0.; B l a h , N.; De Ranter, C.; De respectively. Substitution of an alkenyl function at 3-N Clercq, E.; Herdewijn, P. Synthesis and Anti-HIV Evaluation of the thymine ring [as in 3-N-allylthymine 15 and 3-Nof 2’,3’-Dideoxyribo-5-chloropyrimidineAnalogues: Reduced 161 decreased the toxicity of the (dimethylally1)thymine Toxicity of 5-Chlorinated 2’,3-Dideoxynucleoeides. J. Med. parent compound but also weakened the antiviral activity Chem. 1990,33,1833-1839. (b) Divakar, K. J.; h s e , C. B. 4(1,2,4-Triazol-l-y1)- and 4(3-nitro-l,2,4triazol-l-yl)-1-(8-D- by 4 to 7-fold. Surprieiily, the 3-N-allyluracilderivative 2,3,5-tri-O-acetylarabinofuranosyl)pyrimidin-2(1H)-ones. Valuable Intermediatee in the Synthesis of Derivatives of 1~-~Arabinofuranosyl)cytosine (Ara-C). J. Chem. Soc., Perkin Trans. I 1982, 1171-1176. (45) sasaki, T.; Minamoto, K.; Suzuki, H. Elimination Reactions on the Di-and Trimesylated Derivativen of N-3-Benyluridine. J. Org. Chem. 1973,38,598-607. (46) Jones, A. J.; Grant, D. M.; Wmkley, M. W.; Robins, R. K. Carbon-13 Magnetic Resonance. XVII. Pyrimidine and Pu1970, 92, 4079-4087. rine Nucleosides. J. Am. Chem. SOC.
(47) Yamamoto,I.; Kimura, T.; Tateoka, Y.; Watanabe, K.; Ho, I. K. N-Substituted oxopyrimidines and Nucleosides: Structure-Activity Relationship for Hypnotic Activity as Central Nervous System Depresant. J. Med. Chem. 1987, 30, 2227-2231. (48) Cook, A. F.; Moffat, J. G. Carbodiimide-Sulfoxide Reactions. VI. Syntheses of 2’- and 3’-Ketouridines. J. Am. Chem. SOC. 1967,89,2697-2705.
2992 Journal of Medicinal Chemistry, 1992, Vol. 35, No.16
17 was slightly less active and more cytotoxic than its unsubstituted counterpart 8b. While markedly active against HIV-1, the silylated 3'spiro-5"-(4"-amino-l",2"-0xathiole 2",2"-dioxide) derivatives had no activity against HIV-2 (ROD) or HIV-2 @NO) or simian immunodeficiency virus (SIV)(data not shown). They represent the first example of nucleoside analogues with an intact ribose moiety that discriminate between HIV-1 and other retroviruses, In this respect, the novel nucleoside analogues resemble the non-nucleoside anaTIB0,62953 BI-RG-587 (nevirapin),"~~~ logues HEPT,49-S* L-697,639,Mand BHLW.~' As noted for these compounds,Sg62our data also indicate that the 2',5'-silylated (49) Baba, M.; Tanaka, H.; De Clercq, E.; Pauwels, R.; Balzarini, J.; Schols, D.; N h h i m a , H.; Pemo, C. F.; Walker, R. T.; Miyasaka, T. Highly Specific Inhibition of Human Immunodeficiency V i Type 1 by a Novel &Substituted Acyclouridine Derivative. Bbchem. Biophys. Res. Commun. 1989, 166,1375-1381. (50) Baba,M.; De Clercq, E.; Iida, S.; Tanaka, H.; Nitta, I.; Ubamwa, M.: Takashima H.; Sekiya, K.; Umezu, K.; Nakashima, H.; Shigeta, S.; Walker, R.T.;-Miy& T. Anti-human Immunodeficiency Virus Type 1Activities and Pharmacokinetics of Novel &Substituted Acyclouridine Derivatives. Antimicrob. Agents Chemother. 1990,34,2358-2363. (51) Tanaka, H.; Baba,M.; Hayakawa, H.; sakamalu',T.; Miyasaka, T.; ubarrawa, M.; Takeehima, H.; Sekiya, K.; Nitta, I.; Shigeta, S.; Walker, R. T.; Balzarini, J.; De Clercq, E. A New Class of HIV-1-Specific &Substituted Acyclouridine Derivatives: Synthesis and Anti-HIV-1 Activity of 5- or &Substituted Analoguea of l-[(2-Hydmxyethoxy)methyl]-&(phenylthio)thymi11e (HEPT). J. Med. Chem. 1991,34,344-357. (52) Pauwels, R.; Andries, K.; Desmyter, J.; Schols, D.; Kukla, M. J.; Breslin, H. J.; Raeymaeckers, A.; Van Gelder, J.; Woestenborghe,R.; Heykanta, J.; Schellekens, K.; Janaeen, M. A. C.; De Clercq, E.; Janaeen, P. A. J. Potent and Selective Inhibition of HIV-1 Replication in Vitro by a Novel Series of TIBO Derivatives. Nature 1990,343,470-474. (53) Kukla, M. J.; Breslin, H. J.; Pauwels, R.; Fedde, C. L.; Miranda, M.; Scott, M. K.; Sherrill, R. G.; Raeymaekers, A,; Van Gelder, J.; Andries, K.; Janssen, M. A. C.; De Clercq, E.; Janssen, P. A. J. Synthesis and Anti-HIV-1 Activity of 4,5,6,7-Tetrahydro-5-methylimidazo[ 4,5,1-jk] [ 1,4]benzodiezepin-2(lfl)-one (TIBO) Derivatives. J. Med. Chem. 1991, 34,746-751. (54) Merluzzi, V. J.; Hargrave, K. D.; Labadia, M.; Grozinger, K.; Skoog, M.; Wu, J. C.; Shih, C. K.; Eckner, K.; Hattox, S.; Adams, J.; h e n t h a l , A. S.; Faanes, R.; Eckner, R. J.; Koup, R. A.; Sullivan, J. L. Inhibition of HIV-1 Replication by a Nonnucleoeide Revem Transcriptase Inhibitor. Science 1990, 250,1411-1413. (55) Koup, R. A.; Merluzzi, V. J.; Hargrave, K. D.; Adams, J.; Grozinger, K.; Eckner, R. J.; Sullivan, J. L. Inhibition of Human Immunodeficiency Virue Type 1(HIV-1) Replication by the Dipyridodhpinone BI-RG587. J. Infect. Dis 1991,163, 966-970. (56) Goldman, M. E.; Nunberg, J. H.; O'Brien, J. A.; Quintero, J. C.; Schleif, W. A.; Freund, K. F.; Gaul, S. L.; Saari, W. S.; Wai, J. S.; Hoffman, J. M.; Anderson, P. S.; Hupe, D. J.; Emini, E. A; Stem. A. M. Pyridinone Derivatives: Specfic Human Immunodeficiency Virus Type 1Reverse Transcriptase Inhibitors with Antiviral Activity. Roc. Natl. Acad. Sci. U.S.A. 1991,88, 6863-6867. (57) Romero, D. L.; Busso, M.; Tan, C. K.; Reuaeer, F.; Palmer, J. R.; Poppe, 5. M.; Aristoff, P. A.; Downey, K. M.; So, A. G.; Resnick, L.; Tarpley, W. G. Nonnucleoside Reverse Transcriptase Inhibitors that Potently and Specifically Block Human Immunodefkiency V i Type-1 Replication. Proc. Natl. Acad. Sci. U.S.A. 1991,88, 8806-8810. (58) Baba,M.; De Clercq, E; Tauaka, H.; Ubaeawa, M.; Takaehima, H.; Sekiya, K.;Nitta, I.; Umezu, K.; Nakashima, H.; Mori, S.; Shigeta, S.; Walker, R. T.; Miyaaaka, T. Potent and Selective Inhibition of Human Immunodeficiency Virus Type 1(HIV-1) by 5-Ethyl-6-phenylthiouracil Derivatives through Their Inaction with the HIV-1 Reverse Transcriptase. Proc. Not1 Acad. Sci. U.S.A. 1991,88,2356-2360.
Pkrez-Perez et al.
3'-spiro-5''-(~''-amino-l",~''-oxathiole 2" ,2"-dioxide) derivatives are inhibitory to HIV-1 reverse transcriptase but not HIV-2 reverse t r a n ~ c r i p t a s e . ~ ~ ~ ~ In conclusion, nucleoeide analogues containing a silyl group at both (2-2' and C-5', and a spiro-5"-(4"-amino1",2"-0xathiole 2",2"-dioxide) group at C-3',proved to be potent and selective anti-HIV-1 agents that should be further pursued for their therapeutic potential in the treatment of HIV-1 infections. Experimental Section Chemical Procedures. Melting pointa were measured with a Reichert-Junt Kofler micro hot stage apparatus and are uncorrected. Microanalyses were obtained with a Heareus CHN0-RAPIDinstrument. *H NMR spectra were recorded with a Varian EM-390, a Varian XL-300, and a Bruker AM-200 spectrometer operating at 90,300,and 200 MHz,and '9c NMR spedra with a Bruker WP-WSY, a Bruker AM-200, and a Varian XI.-300 spectrometer operating at 20, 50, and 75 MHz, with Me,Si as internal standard. IR spectra were recorded with a Shimadzu IR-435 Spectrometer. Analytical TLC was performed on silica gel & FmI(Merck). Flash column chromatcgraphy waa performed with silica gel 60 (230-400 mesh) (Merck). Proximities were established conventionally on the basis of using NOE effecte. 5 - 0 -Benzoyl-Q-C-cyano-1,2-O 4sopropylidene-3-0-mesyl-a-Bribofuranose (3). A mixture of 5-O-benzoyl-l,2-O-isopropylidene-a-~-erythro-pentofuranos-3-ul~e (1Im (1.3 g, 4.5 mmol), water (13 mL), ethyl ether (26 mL), sodium hydrogen carbonate (0.85 g, 10.2 mmol), and sodium cyanide (0.85 g, 10.2 "01) was stirred vigorously at room temperature for 4 h. Ethyl ether (50 mL) was added, the organic phase was separated, and the aqueous phase was washed with ethyl ether (2 X 30 mL). The combined ethereal phases were dried over NadO,, filtered, and evaporated to dryness. The residue (the cyanohydrin 2), was dissolved in dry pyridine (10 mL). T o this solution was added The mixture was stirred at mesyl chloride (1.9 mL, 25.5 "01). 8-10 "C for 16 h, poured into ice and water, and extracted with chloroform (2 X 50 mL). The combined extracta were washed with 1N HCl(50 mL), aqueous sodium hydrogen carbonate (50 (59) Cohen, K. A.; Hopkins, J.; Ingraham, R. H.; Pargellia, C.; Wu, J. C.; Palladino, D. E. H.; Kinkade, P.; Warren, T. C.; Rogers, S.; Adams, J.; Farina, P. R.; Grob, P. M. Characterization of the Binding Site for Nevirapine (BI-RG587), a Nonnucleoside Inhibitor of Human Immunodeficiency V i m Type-1 Reverse Transcriptase. J. Biol. Chem. 1991,266,14670-14674. (60)Frank, K. B.; Noll, G. J.; Connell, E. V.; Sim, I. S. Kinetic Interaction of Human Immunodeficiency V i Type 1 Reverse Transcriptase with the Antiviral Tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepine-2(lH)-thioneCompound, R. 82150. J. Biol. Chem. 1991,266, 14232-14236. (61) Debyser, Z.;Pauwels, R.;Andries, K.; Desmyter, J.; Kukla, M.; Janaeen, P. A. J.; De Clercq, E. An Antiviral Target on Reverse T"xiptase of Human Immunodeficiency Virus Type 1Revealed by Tetrahydroimidazo-[4,5,l-jk] [1,4]-benzodiazepin-2(lH)-one and -thione Derivatives. Proc. Natl. Acad. Sci. U. S.A. 1991,88,1451-1455. (62) White, E. L.; Burkheit, R. W., Jr.; Roes, L. J.; Germany, J. M.; Andries, K.; Pauwels, R.; Janssen, P. A. J.; Shannon, W. M.; Chirigoe, M. A. A TIBO derivtive, R 82913, is a Potent Inhibitor for HIV-1 Reverse Transcriptase with Heteropolymer Templates. Antioiral Rea. 1991,16,257-266. (63) Balzarini, J.; PBrez-PBrez, M. J.; San-FBlix, A.; Camarm, M. J.; B a t h u t , I. C.; Barr, P. J.; De Clercq, E. Kinetics of Inhibition of Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptaseby the Novel HIV-1 Specific Nucleoeide Analogue 2',5'-Bie-0-( tert-Butyldimethylsilyl)-3'-spir0-5''-(4"amino-1".2"-oxathiole-2".2"-dioxide) - thvmine (TSAO-T). J. Biol. Chem., in press. (64) Balzarini, J.; PBrez-PCrez, M. J.; San-FBlix, A; Vekquez, S.; Camarasa, M. J.; De Clercq, E. 2',5'-bis-O-(tert-Butyldimethylsilyl)-3'-spiro-5~~-(4"-~ino-1",2'~-oxathiole-2".2"-di-
oxide) (TSAO) Derivatives of Purine and Pyrimidine Nucleosides aa Potent and Selective Inhibitors of Human Immunodeficiency V i Type. Antimicrob. Agents Chemother. 1992, 36,1073-1080.
J. Med. Chem. 1992,35,2995-3002 (d, 1 H, H-5), 6.15 (d, 1 H, H-l’),6.45 (bs, 2 H, NHs), 7.79
(8,
1
H, H-6). Anal. (CzsH6N308SSiz)C, H, N. Antiretrovirus Assays. HIV-1 was originaUy obtained from the culture Supernatant of the persistently HIV-infected H9 cell line (H9/H”LV-IIIB),s6.which waa kindly provided by R. C. Gallo and M. Popovic (National Institutes of Health, Bethesda, MD). Virus stocks were prepared from the supematanta of HIV-l-infected MT-4 cells. MT-4 cells are human T-lymphocyte cells transformed by HTLV-1 and highly susceptible to the cytopathic effect of HIV. The methodology of the anti-HIV assays has been described previously.”~ Briefly, MT-4 cells (5 X 106 cells/mL) were suspended in fresh culture medium and infected with HIV-1 at 100 tima the 50% cell culture infective dose (CCIDd per “ i t e r of cell suspension. Then 100 p L of infected cell suspension was transferred to microtiter plate wells and mixed with 100 p L of the appropriate dilutions of test compounds. After 5 days, the number of viable cells for both virus-infectedand mock-infected cell cultures was determined in a blood-cell-counting chamber by trypan blue staining. The 50% effective concentration (ECw) (65) Popovic, M.; Sangadharan, M. G.; Read, E.; Gallo, R. G. Detection, Isolation, and Continuous Production of Cytopatic Retroviruses (HTLV-III) from Patients with AIDS and PreAIDS. Science 1984,224,497-500.
2996
and 50% cytotoxic concentration (CC,) were d e f i e d as the compound concentrations required to reduce by 50% the number of viable cells in the virus-infected and mock-infected cell culturea, respectively.
Acknowledgment. We thank Maria J e s h Moreno, Ann Abeillis, and Lizette van Berckelaer for their excellent technical assistance. We thank Mr. Francisco Caballero for processing the manuscript. We also thank the Resi-
dencia de Estudiantes and Ayuntamiento de Madrid for a grant to M.J.P. This research was supported in part b y the Programa Nacional de Investigaci6n y Desarrollo Farmadutim of Spain (Project FAR8&01606/1), the AIDS Basic Research Programme of the European Community, and by grants from the Belgian Fonds vmr Geneeskundig Wetenschappelijk Onderzoek (Projects 3.0097.87 and 3.0026.91), and the Belgian Geconcerteerde Onderzoeksacties (Project 90/94-2). Registry No. 1,6698-46-0; 3, 142131-02-0;a-4,142102-69-0; &4,142102-70-3; Sa, 142102-714,Sb, 142102-72-5; Sc, 142102-73-6, 6a, 142102-747;7a, 141781-162;8a, 141781-17-1;8b,141845-83-2; &, 142102-75-8;k,141684-47-1;lOa, 142102-76-9; 11,14210277-0; 12,142102-78-1; 13,142102-79-2;14,142102-8&5;15,142102-81-6, 16,142102-82-7; 17,142102-83-8; thymine, 65-71-4; uracil, 66-22-8.
Template-Directed Design of a DNA-DNA Cross-Linker Based upon a Bis-Tomaymycin-Duplex Adduct Jeh-Jeng Wang, G. Craig Hill, and Laurence H. Hurley* College of Pharmacy, The Drug Dynamics Institute, University of T e r m a t Austin, Austin, Teras 78712. Received March 13, 1992
A template-directed approach to the design of a DNA-DNA interstrand cross-linker based upon the structure of a bis-tomaymycin-duplex adduct has been carried out. Tomaymycin is a member of the pyrrolo[l,4]bendiazepinea
antitumor antibiotics. In a previous study (F.L. Boyd et al., Biochemistry 1990,29,2387-2403), we have shown that two tomaymycin molecules can be covalently bound to a 12-mer duplex molecule, where the drug molecules are on opposite strands six base-pairs apart, and the stereochemistry at the drug bonding site, and orientation in the minor groove, was defiied by high-field NMR. This bis-tomaymycin 12-mer duplex adduct maintains the self-complementarity of the duplex and a B-type structure. In the present study we have shown using high-field NMR that this same 12-mer sequence can be truncated by two base pairs so that the two tomaymycin-modified guanines are now only four base-pairs apart, the two species of tomaymycin molecules are still bound with the same stereochemistry and orientation, and the 10-mer duplex adduct maintains ita self-complementarity. In a second 10-mer duplex we have shown that changing the bonding sequence from 5’CGA to 5’AGC does not significantly affect the structure of the bis-tomaymycin-duplex adduct. However, when the sequence is rearranged so that the drugs point in a tail-to-tailorientation rather than in the previous head-bhead configuration, there are more than one species of tomaymycin bound to DNA, and, as a consequence, the bis-tomaymycin 10-mer duplex adduct loses ita self-complementarity. Last, we have used the 10-mer duplex containing the 5’CGA sequence, in which the tomaymycin molecules are oriented head to head, to design an interstrand cross-linking species in which the two drug molecules are linked together with a flexible linker molecule.
Introduction Anthramycin, tomaymycin (I),and sibiromycin are the best known examples of the naturally occurring pyrrolo[1,4]benzodiazepines (P[1,4]B).’” These antibiotics react covalently with DNA to form an N2-guanine adduct that lies within the minor groove of DNA (Figure l).495 The P[1,4]Bs are not only specific for N 2 of guanine, but are only reactive with guanines in certain sequences, and therefore show sequence selectivity.6J The most favored sequences for bonding are 5’PuGPu with 5’PyGPu and 5’PuGPy of intermediate reactivity ( P u = purine; P y = pyrimidine), while 5 T y G P y sequences show the least reactivity. In principle, there are four species of covalently *Address correspondence to this author or call (512)471-4841.
bound adducts that can occur as the two 11senantiomers, in which the aromatic ring of the drug lies either to the (1) Remere, W. A. In The Chemistry of Antitumor Antibiotics; Wiley: New York, 1988, Vol. 2, pp 28-92. (2) Thurston, D. E.; Hurley, L. H. A Rational Bash for Development of Antitumor Agents in the Pyrrolo(l,4)benzodiapine
Group. Drugs Future 1984,8,967-971.
( 3 ) Remere, W. A.; Barkley, M. D.; Hurley, L. H. Pyrrolo(l,4)benzodiazepines Unraveling the Complexity of the Structum of the Tomaymycin-DNAAdducta in Various Sequences Using
Fluorescence, ‘H-NMR, and Molecular Modeling. A chapter in Nucleic Acid Targeted Drug Design; Perun, T., Propst, C., W.; Marcel Dekker, Inc.: New York, 1992, in press. (4) Hurley, L. H.;Petrusek, R. Proposed Structure of the Anthramycin-DNA Adduct. Nature (London) 1979, 282, 629.
0022-262319211835-2995$03.00/0 Q 1992 American Chemical Society
