J.Med. Chem. 1991,34, 1383-1394 (8, 26 H, (CH2)13), 1.57 (m, 2 H, CH2CH20),2.8 (m, 2 H, CHzP), 3.38-3.78 (m, 8 H, CH30CHCH20CH2),4.38 (bs, 2 H, POCH2), 5.09 (m, 2 H, CH2N), 8.03 (m, 2 H, pyridine), 8.40 (m, 1 H, pyridine), 9.49 (d, 2 H, pyridine). Anal. (CnH&NOSP~1H20) C, H, N.
Acknowled~ent*The authors would like to thank Dr. Michael M a x , Zhao-Qinq W a g , and Shag-Yong Chen for the preparation of several candidate ComPoUds. This research was supported in part by the National Institutes of Health research grants CA 42216 and CA 12197 and BRS p a n t RR 05404 and by the Tissue Culture Core Laboratory Facility of the Cancer Center of Wake Forest 7 University. Registry No. 2, 131973-30-3;3, 131933-48-7; 4, 127642-24-4; 5, 112989-00-1;6, 112989-01-2;7, 88876-07-7;8, 112989-02-3;9,
1383
103304-64-9;10, 103304-65-0;11, 112989-09-0; 12, 131933-49-8; 13,22598-16-9;14,131973-31-4;15,131933-50-1; 16,131933-51-2; 16 trityl derivative, 131933-60-3; 16 2-bromoethyl phosphate derivative, 131933-61-4; 17, 131933-52-3; 18, 124581-78-8; 19, 124581-94-8; 20, 124581-81-3;21, 124581-79-9;22, 131933-53-4; 23,23248-47-7;24,131933-54-5; 25,111-57-9; 26,82755-92-8;27, 131933-56-7; 28,119980-186; 29,119980-19-7;30,92758-87-7; 31, 131933-57-8;32, 126614-08-2;33, 126614-06-0;34, 131933-58-9; 35,126614-21-9;36,131933-59-0;36 dimethyl ester, 131933-63-6; 37,131933-64-7;Et-l&OMe, 70641-51-9; Et-18-OEt, 78858-43-2; 92758-87-7; AZT, 30516-87-1; l-0-hexadecyl-2-0-ethylglycerol, rac-l-O-tosyl-2-O-ethylglycerol, 131973-32-5;rac-3-(hexadecylthio)-2-ethoxy-l-bromopropane,124581-76-6; N,N-dimethylN-(2,3-dihydroxypropyl)amine, 98923-15-0; 2-(octadecanamido)ethyl 2'-bromoethyl phosphate, 131933-62-5; 1-(octadecyloxy)-2-iodoethanethane, 90339-56-3; N-@-hydroxyethyl)pyridinium bromide, 31678-16-7;reverse transcriptase, 9068-38-6.
Receptor-Based Design of Novel Dihydrofolate Reductase Inhibitors: Benzimidazole and Indole Derivatives Kwasi A. Ohemengt and Barbara Roth*J Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709. Received September 19, 1990
Although many thousands of inhibitors of the enzyme dihydrofolate reductase (DHFR) have been synthesized, all of the very active compounds have been 2,4-diaminopyrimidines or very close analogues. This paper describes 2,4-diamino-6-benzylbenzimidazole(3b) and the corresponding indole (4), as well as more complex tri- and tetracyclic derivatives (5 and 6). These were designed on the basis of molecular modeling to the known X-ray structure of Escherichia coli DHFR, in an effort to determine whether one could drastically alter the diamino configuration by placing one amino substituent in a 5-membered nitrogen-containing ring and the second in the ortho position of a fused ring and still inhibit DHFR significantly. Although the electronics and bond angles are quite different from that of a 2,4-diaminopyrimidine, the pK, values are in an appropriate range, and hydrogen-bond distances appear to be quite reasonable. The most active compound, 4, was very unstable and active only in the lo-' M range. Dihydroindenoimidazole derivatives such as 6 showed quite a good fit to the enzyme by modeling studies, but had low activity. Since the most active compound made was 2 orders of magnitude weaker as an inhibitor of bacterial we concluded that such a ring system was unlikely DHFR than the unsubstituted 5-benzyl-2,4-diaminopyrimidine, to produce the high inhibitory potency of trimethoprim (l),even with greatly improved hydrophobic contacts. Thus the 2,4-diaminopyrimidine system remains unparalleled to date for the competitive inhibition of this enzyme. Successful inhibitors of dihydrofolate reductase (DHFR,
EC 1.5.1.3), such as trimethoprim (1) and methotrexate (2), have in almost every case been based on the 2,4-diaminopyrimidine skeleton or on closely allied 1,2,4-triazine or 1,3,5-dihydrotriazine analogues.' Prior to elucidation
bCH,
1 (Trimelhoprim, TMP) COOH
2 WT)O
of the 3-dimensional structure of this enzyme many other Present address: Department of Medicinal Chemistry, The R.W. Johnson Pharmaceutical Research Institute, Route 202, Raritan, N J 08809. Present address: Chemistry Department, University of North Carolina, Chapel Hill, NC 27599.
substituent patterns, as well as other ring systems, were examined for their inhibitory properties, but none possessed the apparent unique properties of this original prototype. The 3-dimensional structures of DHFR from Escherichia coli, Lactobacillus casei, chicken liver, mouse liver lymphoma, and human DHFR have been solved and refined in the presence of several ligand^,^-^ and it is now known that a very complex hydrogen-bonding pattern exists between a protonated diaminopyrimidine and the protein, involving all of the available hydrogen atomsa5 In Roth, B.; Cheng, C. C. In Progress in Medicinal Chemistry; Ellis, G . P., West, G. B., Eds.; Elsevier/North Holland Biomedical Press: Amsterdam, 1982; Vol. 19, pp 269-331. Champness, J. N.; Stammers,D. K.; Beddell, C. R. FEBS Lett. 1986, 199, 61. Matthews, D. A.; Bolin, J. T.; Burridge, J. M.; Filman, D. J.; Voiz, K. W.; Kaufman, B. T.; Beddell,C. R.; Champneea, J. N.; Stammers, D. K.; Kraut, J. J.Biol. Chem. 1985, 260, 381. Matthews, D. A.; Bolin, J. T.; Burridge, J. M.; Filman, D. J.; Volz, K. W.; Kraut, J. J. Biol. Chem. 1985,260, 392. Bolin, J. T.; Filman, D. J.; Matthews, D. R.; Hamlin, R. C.; Kraut, J. J. Biol. Chem. 1982, 257, 13650. Stammers, D. K.; Champness, J. N.; Beddell, C. R.; Dann, J. G.; Eliopoulos, E.; Geddes, A. J.; Ogg, D.; North, A. C. T. FEBS Lett. 1987, 218, 178. Oefner, C.; DArcy, A.; Winkler, F. K. Eur. J.Biochem. 1988, 174, 377.
0022-2623/91/1834-1383$02.50/00 1991 American Chemical Society
1384 Journal of Medicinal Chemistry, 1991, Vol. 34, No. 4
Ohemeng and Roth
Scheme I
1
I
NH&H EtOH
-
TFA,
- l p
CNBr
H2N&
HZN
CHpPh 4
CNBr
H2N
7
II
H
Scheme I1
CHzPh
L5
t
t
a2
33 attempted synthesis) of more complex semirigid derivatives, which are also discussed.
B
a R=C(=O)Ph
k
I
/2.
R = CHzPh
2) NaOEl8nElOH PdlC A under NI
addition, tight binding requires a hydrophobic moiety which fits into an adjacent hydrophobic pocket. The object of the research reported here was to test other types of ring systems which might conceivably fit into the active site of the enzyme by using the same hydrogen-bonding atoms, but in a system which would of necessity involve a different geometry and electron density pattern, and which would also have a hydrophobic moiety. The compounds chosen for initial study were the benzimidazoles 3a and 3b, and the related indole 4. Molecular modeling, chemical synthesis, and enzyme inhibitory data are described below. The results led to the synthesis (or
5 R, R ' = H2
6 R, R' = -S(CH&S
2 R , R ' = -O(CH2)2O
Chemistry The syntheses of compounds 3a,b, and 4 are summarized in Schemes I and 11. It was initially anticipated that the synthesis of compounds 3a and 3b could be accomplished from 2-amino-5-benzoylbenzimidazole (9).* We prepared
Benzimidazole and Indole Derivatives
compound 9 from the commercially available 3,4-diaminobenzophenone (8) and cyanogen bromide. However, several attempts to nitrate 9 were unsuccessful, due to insolubility of the acetylated derivative in appropriate solvents. An alternate route for synthesis of 3a and 3b involved the Friedel-Crafts reaction of 4-chloro-3,5-dinitrobenzoyl chloride (from 11) and benzene to give 12. The highly activated chloro group of 12 was then displaced with ammonia in dimethyl sulfoxide to give 13. Selective reduction of one of the nitro groups was accomplished by the Zinin p r o ~ e d u r e to , ~ give the diamino derivative 14. This was then converted to the benzimidazole 10, followed by reduction of the ketone with trifluoroacetic acid and triethylsilane to give 15. Alternatively the ketone group of 13 could be reduced first, followed by the Zinin reduction of the nitro group. The resultant diamino derivative 17 was then treated with cyanogen bromide to give 15. This procedure gave slightly better yields in the final step. Catalytic reduction of the nitro groups of 10 and 15 gave the products 3a and 3b, respectively. In the case of the aminoindoles, it was considered desirable to establish which tautomer (19a or 19b, Scheme 11) was the predominant structure in solution, due to past controversy on this point.lOJ1 Following the procedure of Pschorr and Hope," o-nitrophenylacetonitrile (18) was reduced with P d / C and hydrogen to the amino derivative, which was then cyclized in deoxygenated sodium ethoxide solution to give a highly unstable compound. This product was isolated under nitrogen and immediately dissolved in deoxygenated deuterochloroform for 'H NMR studies. The 3-H of 2-methylindole has been reported to occur a t 6.05 ppm ( 6 ) in CDC1,,12 and thus we expected a signal in this region if 19a was present in solution. However, in CDCl, a strong signal appeared a t 3.60 ppm which integrated for approximately 2 H , suggesting structure 19b, which has two nonaromatic protons at position 3. A small sharp peak was also present a t 5.60 ppm (about 6% of that a t 3.60) which suggested that a small amount of tautomer 19a was present in solution. Compound 21 was synthesized from 4-methyl-3-nitrobenzoic acid (20) by nitration followed by Friedel-Crafts a ~ y l a t i o n . ' ~Several attempts to brominate the methyl group with the intent to convert the brominated derivative to a nitrile (22) were unsuccessful, probably due to inactivation of the side chain by the neighboring electronwithdrawing groups. The problem was circumvented by first reducing the ketone function of 21 with TFA and triethylsilane to give 23, followed by reaction with dimethylformamide dimethyl acetal to give the enamine, which was hydrolyzed with silica gel and water to give the aldehyde 24. This product was then converted to the oxime, followed by dehydration with acetic anhydride to produce the nitrile 25. The dinitro groups of 25 were reduced catalytically with P d / C and hydrogen, followed by cyclization with sodium ethoxide to give a highly unstable product 4. Attempts to purify the product either by recrystallization or by column chromatography led to polymeric products. The substance was finally isolated as the hydrochloride salt by precipitation with HC1 gas (8) Ram, S.; Skinner, M.; Kalvin, D.; Wise, D. S.; Townsend, L. B.; McCall, J. W.; Worth, D.; Ortwine, D.; Werbel, L. M. J . Med. Chem. 1984,27, 914. (9) Porter, H. K. Org. React. 1973, 20, 455. (10) Kebrle, J.; Hoffmann, K. Helu. Chim.Acta 1956, 39, 116. (11) Pschorr, R.; Hope, G. Chem. Ber. 1910, 43, 2543. (12) Jardine, R. V.; Brown, R. K. Can. J . Chem. 1963, 41, 2067. (13) Chardonnens, L. Helu. Chim.Acta 1929, 22, 649.
Journal of Medicinal Chemistry, 1991, Vol. 34, No. 4
1385
Scheme I11
HN2"
300 "C; MS (M+ + 1)261; N M R (MeaO-d,) 6 1.9 (m, 2, 6-CH2), 2.1 (s,3, CH3CO),3.0 (m, 4-H, 5 and 7-CHz),7.5 (s, 1, 8-H). Anal. (CizHizN403) C, H, N. 2,4-Diamino-1,5,6,7-tetrahydroindeno[5,6-d]imidazole Dihydrochloride (5). A suspension of 29 (1.5 g, 5.7 mmol) was hydrolyzed with 2 N HCl (20 mL) by refluxing for 1.5 h. The solution was chilled and the precipitate isolated and hydrogenated over Pd/C (200 mg) in 50 mL of 95% EtOH until 3 equiv of Hz was consumed. The catalyst was removed and the EtOH evaporated. The residue was crystallized from 50% EtOH plus a few drops of 10 N HCl: wt 0.85 g (57%) of 5; mp 300 "C dec; MS (M+) 188; NMR (MezSO-d6)6 2.49 (m, 2,6-CH2),2.75 (m, 4, 5and 7-CH2),4.88 (br s, 4, NHz, (NH),), 6.63 (e, 1,8-H), 8.33 ( 8 , 2, NHz). Anal. (C10H12N4-2HC1) C, H, N, C1. 3-(4-Chloro-3-nitrophenyl)propionicAcid (31). A solution of 3016 (10 g, 0.044 mol) in deoxygenated MeOH (150 mL) was added to a well-stirred suspension of potassium azodicarboxylate1"18 (17.1 g, 61.6 mmol) in deoxygenated MeOH (150 mL) under N1. A mixture of 8 g of AcOH and 20 mL of MeOH was then added dropwise to the mixture over 30 min, followed by stirring for 6 h. A second mixture of 8 g AcOH and 20 mL of MeOH was added over 20 min and stirring continued for another 18 h. The solution was clarified and the solvents removed, giving a yellow solid. This was dissolved in 50 mL of water and filtered and the pH of the filtrate adjusted to 6 with 2 N H2S04, which resulted in the precipitation of a pink solid: 8.05 g (80%) of 31; mp 65-67 OC (50% EtOH); MS (M+)229; NMR (CDC13) 6 2.72 (t, 2, 2'-CHz), 3.00 (t, 2, 3'-CHz), 7.38 (dd, 1, 6-ArH), 7.47 (d, 1, 5-ArH), 7.74 (d, 1, 2-ArH). Anal. (C9HsC1N04)C, H, N,
c1.
Ethyl 4-Amin6-3-nitrocinnamate (33). Compound 3016was esterified with anhydrous EtOH and HCl, which was slowly bubbled through for 48 h. The solvent was removed, and the reeidue (5.0 g, 0.02 mol) was then added to a solution of anhydrous NH3 in 50 mL of MezSO, prepared by saturating the solvent at 25 "C by bubbling NH3 through for 15 min. The mixture was then heated at 100 "C while continuously adding NH3 for a 48-h period. The solution was then poured into 50 mL of ice/water and the yellow precipitate isolated and recrystallizedfrom i-PrOH 4.65 g (98.5%) of 33 was obtained, mp 135-137 O C ; MS (M+) 236; NMR (CDClS) d 1.33 (t, 3, CH3), 4.26 (q, 2, CHI) 6.30 (d, 1, C=CH), 6.40 (br s, 2, NH2), 6.84 (d, 1,5-ArH), 7.55 (m, 2,6-ArH and ArCH=), 8.27 (d, 1,2-ArH). Anal. (C11H12N204)C, H, N. Ethyl 3-(2-Aminobenzimidazol-5-yl)acrylate(34). A suspension of 33 (10 g, 0.042 mol) in a mixture of 150 mL of 95% DOH, 5 mL of 12 N HCl, and 50 mL of H20 was hydrogenated at 40 psi over 10% Pd/C (3 g) until 3 equiv of Hz was consumed. The product was in solution at the end of this time. The catalyst was removed and the filtrate made basic with NH40H, followed by evaporation under reduced pressure, which produced a syrupy residue. This was added to 100 mL of water, and solid CNBr (6 g) was added in 2-g portions at 30-min intervals while stirring at 25 "C. The mixture was then stirred at 25 "C for 1h, followed by filtration. The filtrate was made basic with NH40H and the waxy precipitate isolated, dissolved in EtOH, and clarified. Removal of the EtOH and purification by chromatography on silica gel, eluting with EtOAc/EtOH (3:1), gave 7 g (72%) of 34: NMR (Me2SO-d6)6 1.25 (t, 3, CH3), 4.15 (4, 2, CHz),6.3 (d, 1,
and 1-NH). Anal. (C12H13N3OZ)C, H, N.
3-(2-Acetamidobenzimidazol-S-yl)propionic Acid (35). A suspension of 7.5 g (0.032 mol) of 34 in 250 mL of 2 N HCL was refluxed for 2 h. Analysis by TLC indicated the complete disappearance of the starting material. The mixture was cooled in ice and the solid (6.4 g) separated. This was reduced in MezSO with 18 g of potassium azodicarboxylate as described for 31.'"18 The product was then refluxed in Ac20 (100 mL) for 3 h and poured into 900 mL of ice water, and the precipitate was isolated. This was dissolved in 100 mL of EtOAc, washed twice with water, and dried over MgS04, followed by removal of the drying agent. Eventually crystals formed on chilling: 2.4 g (28%) of 35; MS (M+ + 1)248; NMR (Meao-d,) 6 2.6 (s, 3, CH3),3.0 (t,2,3-CH,J, 3.2 (t, 2, 2-CHz), 7.62 (m, 3, ArH), 8.2 (br s, 2, (NH),). Anal. (C12H13N303'1.2HZO) C, H, N. &(or 6-)Acetamide-C(or 5-)nitro-l-indanone (38). A solution of Cr03 (26.5 g) in a mixture of 15 mL of HzO and 235 mL of AcOH was prepared by sonicating the suspension for 45 min. This was added dropwise to a cooled solution of 26 (22 g, 0.1 mol) in Ac20 (2.5 L) at such a rate that the temperature remained between 15-20 OC, while stirring mechanically. After the addition was completed, the mixture was stirred at 25 OC for 4 h, poured into 10 L of water, and stirred for 1 h. The solution was then extracted with two 2-L portions of CH2Cl2,and the combined CHZCl2fractions were then concentrated to 500 mL, washed with two 50-mL portions of 10% NaOH followed by water, and then dried (MgSO,). The solvent was removed, leaving a yellow powder, which was purified on a silica gel column, eluted with CHZCl2. Unreacted starting material (2.5 g) was recovered first, followed by 12 g (50%) of 3 8 mp 98-100 OC; MS (M+) 235; NMR (MeaO-de) 6 2.30 (s,3,CH3),2.77(m,2,2-CHz)3.25(m,2,3-CHJ, 8.60 (8, 1, 4-H), 8.94 (8, 1, 7-H), 10.62 (br s, 1, NH). Anal. (CllHloNz04.0.2HzO) C, H, N. 5-Amino-6-nitro-l-indanone (39). A suspension of 38 (10 g, 0.042 mol) in 2 N HCl(200 mL) and EtOH (100 mL) was refluxed for 30 min with stirring. The reaction was cooled to 15 OC and the resultant precipitate isolated and recrystallized from dilute EtOH: 7.9 g (97.5%) of 39; mp 248 "C; MS (M+ + 1) 193; NMR (Me2SO-d6)6 2.52 (m, 2, 2-CH2), 3.00 (m, 2, 3-CHz),7.00 (s, 1, 4-H), 7.93 (9, 2, NH2),8.20 (8, 1,7-H). Anal. (C9HsNZO3)C, H, N. N-[1,5,6,7-Tetrahydro-7-oxoindeno[ 5,6-d]imidazol-2-yl]acetamide (36). A suspension of 39 (13.25g, 0.069 mol) and 10% Pd/C (3.3 g) in 95% EtOH (100 mL) was hydrogenated at 30 psi until 3 equiv of H2was consumed. The catalyst was removed and NH40H (5 mL) added to the filtrate, which was then evaporated to dryness; a yellow solid remained. This was suspended in 150 mL of HzO, and solid CNBr (8 g, 0.075 mol) was added. The mixture was stirred for 30 min, and a second quantity of CNBr (1g) was then added. Stirring was continued for another 20 min, followed by filtration. The filtrate was made alkaline to pH 8 with NH40H, and the resultant precipitate filtered and dried. This was suspended in 200 mL of AczO and stirred at 25 "C for 18 h. The insoluble material was collected, washed well with ether, and dried: 7.3 g (AcOH) of 36 was obtained; mp >300 OC; MS (M+ + 1) 230; NMR (MezSO-d6)6 2.17 (s, 3, CH3), 2.61 (t, 2, 6-CHz),3.12 (t, 2, 5-C&), 7.49 (s, 1,4-H), 7.65 (s, 1,8-H), 11.85 (9, 1, CONH), 12.20 (8, 1, NH). Anal. (C~ZH~~N~O~*~.OACOH) C, H, N. N - (1,5,6,7-Tetrahydro-4-nitro-5-oxoindeno[ 5,6-d 1imidazol-2-y1)acetamide (40). A solution of 36 (0.9 g, 3.1 "01) in CF3COOH (30 mL) was cooled to -7 "C. To this was added dropwise a mixture of 0.5 mL of 70% HN03 and 2 mL of AczO at such a rate that the temperature did not exceed 0 OC. The mixture was then stirred for about 1h, until the temperature rose to room temperature. The solvent was removed, and the residue was washed with water and dried: yield 0.53 g (58.9%) of 40, a yellow powder which melted at 180 "C dec; MS (M+)274; NMR ( M Q O - 4 6 2.07 (s, 3, CHd, 2.70 (m, 2, W H J , 3.18 (m, 2,7-CH,J, C, H, N. 7.7 (s, 1, 8-H). Anal. (C1zHloN404~0.2HN03~0.2H20) N-(1,5,6,7-Tetrahydro-4-nitro-7-oxoindeno[ 5,6-d 1imidazol-2-y1)acetamide (41). A solution of Cr03 (2.6 g) in 500 mL of Ac20 was prepared by sonicating the suspension for 1.5 h. A solution of 29 (2.61 g, 0.01 mol) in Ac20 (500 mL) was prepared by heating the suspension to boiling and filtering. The
J. Med. Chem. 1991,34, 1394-1399
1394
filtrate was then cooled to 10 OC. The Cr03 solution was then added to a m l e d solution of 29 at such a rate that the temperature did not rise above 15 OC. The mixture was then stirred at room temperature for 4 h, diluted to 3 times its volume with icewater, and then cooled to 25 OC. This was followed by extraction three times with EtOAc. The combined fractions were concentrated to 150 mL and washed twice with 10% NaOH and twice with water, followed by drying over MgSO, and concentration to dryness. Analysis by TLC (silica gel, EtOAc) showed the presence of three compounds, with RI values of 0.88,0.43, and 0.35. The mixture was then fractionated on a silica gel column, which was eluted with EtOAc. The first compound eluted was the starting material. This was followed by 0.6 g (22%) of 40 and then 1.2 g (44%) of 41. The latter melted at 249-250 "C: MS (M+ + 1) 275; NMR (MezSO-da)6 2.21 (8, 3,CH3),2.68 (m, 2,6-CHJ, 3.41 (m, 2, 5-CH2),7.91 ( 8 , 1, 8-H). Anal. (C12HloN404),.C, H, N. 2-Amino-6,7-dihydro-8-nitroindeno[5,6-d 11midazol-5(lEI)-one Hydrochloride (42). A suspension of 41 (0.5 g, 1.8 mmol) in 2 N HCl (15 mL) was heated to boiling. The material went into solution after 20 min. After another 15 min the mixture was chilled and the precipitate was isolated and recrystallized from dilute EtOH plus a few drops of HCl: wt 460 mg (97%)of 42; mp 250 "C dec; MS (M+ + 1)233; NMR (MezSO-d6)b 3.52 (m, 2, 6-CHz), 3.76 (m, 2, 7-CHz),7.80 (8, 1, 4-H), 8.82 (br s, 2, NHz). Anal. (C10H8N403-HC1) C, H, N, C1. 2'-Amino-1',5',6',7'-tetrahydro-8'-nitrospiro[ 1,3-dithiolane-2,5'-indeno[5,6-d]imidazole](43). To a solution of 42 (0.4 g, 1.5 mmol) in CF3COOH was added 6 mL of ethanedithiol. The mixture was stirred at room temperature for 2 h, and the solvents were evaporated under vacuum. The resultant syrup was added to 50 mL of EtOAc and a few drops of concentrated NH40H was added to adjust to pH to 8. The solution was shaken three times with 50-mL portions of water and dried over MgS04,and the solvent was removed. The yellowish solid was recrystallized from EtOAc: wt 0.35 g (73%) of 43; mp 240 "C; MS (M+ + 1)309, NMR (Me2SO-d6)b 2.45 (t, 2,6'-CH2), 2.60
(t, 2, 7'-CHz), 3.42 (m, 4, S(CH2)2S),6.70 (8, 2, NH2) 7.32 (8, 1, 4'-H), 11.50 (br s, 1, NH). Anal. (C12H12N402S2) C: H, N, S. ~-Amino-lf~',6',7'-tetrahydro-8'-nit~~pi~[ 1,3-&oxolane2,5'-indeno[5,6-d]imidazole] (44). A solution of 42 (1.0 g, 3.7 mmol) in 10 mL of ethylene glycol was prepared by heating the mixture, and 30 mL of benzene was then added. The biphasic mixture was refluxed for 20 h, with continuous removal of water, with we of a Dean Stark trap. The mixture was then poured into 10 mL of icewater and extracted three times with WmL portions of EtOAc. The combined extracts were washed with water and evaporated to dryness under reduced pressure. The residue waa extracted with EtOAc: wt residue 1.0 g (95%) of 44; mp 300 "C dec; MS (M+ + 1)277; NMR (Me#O-d6) 6 2.25 (t, 2,6-CH2),3.1 (t, 2, 7-CH2),4.05 (m, 4, O(CH2),0), 6.47 (br s, 2, NH2),7.35 (8, 1,4'-H), 11.5 (br s, 1, l'-NH). Anal. (C12H12N404-0.4H20) C, H, N. 2',4'-Diamino- 1',5',6',7'-tetrahydrospiro[ 1,3-dithiolane2,7'-indeno[5,6-d]imidazole] (6). A solution of 43 (0.1 g, 0.3 mmol) in EtOH (20 mL) was hydrogenated over 10% Pd/C (60 mg) at 30 psi until 3 equiv of Hz was consumed. The catalyst was removed and the filtrate evaporated to drynw. The resulting solid was recrystallized from 50% aqueous EtOH to give 45 mg (53%)of 6: mp 210-212 "C dec; MS (FAB)(M+ + 1)279; NMR (Me2SO-d6)6 2.58 (m, 2, 5', and 6'-CHz), 3.40 (m, 4, S(CHz)2S), 4.50 (br s, 2, NHJ, 7.9 (s, 2, NHJ, 8.60 (s, 1, 8'-H), 11.30 (br s, 1, NH). Anal. (C12H14N4S2-0.2 H20) C, H, N, S.
Acknowledgment. We thank Prof. Ernest Eliel for many helpful discussions, and Dr. David Henry for his encouragement of this pursuit. We also acknowledge help from Dr. Lee Kuyper in use of the Evans and Sutherland graphics system and energy calculations. Expert technical assistance was provided by Robert Hunter. The kinetic data were obtained under the supervision of Robert Ferone.
Synthesis and Anti-HIV Activity of 2-,3-, and 4-Substituted Analogues of 1-[ (2-Hydroxyethoxy)methyl]-6-(phenylthio)thymine(HEPT) Hiromichi Tanaka,' Masanori Baba,' Masaru Ubasawa,* Hideaki Takashima,o Kouichi Sekiya,l Issei Nitta,f Shiro Shigeta,* Richard T. Walker," Erik De Clercq,l and Tadashi Miyasaka*J School of Pharmaceutical Sciences, Showa University, Hatanodai 1-5-8, Shinagawa-ku, Tokyo 142, Japan, Fukushima Medical College, Fukushima, 960-12, Japan, Mitsubishi Kasei Corporation Research Center, Yokohama 227, Japan, Department of Chemistry, University of Birmingham, Birmingham, B15 2TT, United Kingdom, and Rega Institute for Medical Research, Katholieke Uniuersiteit Leuven, B-3000 Leuven, Belgium. Received June 22, 1990 Several analogues of a new lead for anti-HIV-1 agents, 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine(HEPT), in which the (2-2, N-3, or C-4 position was modified were synthesized. These involve 2-thiothymine (ll),2-thiouracil (12), 4-thiothymine (17), 4-thiouracil (18), 5-methylcytosine (27), and cytosine (28) derivatives. Preparation of N-3-substituted derivatives (29 and 30) of HEPT was also carried out. Among these analogues, compound 11 exhibited excellent activity against HIV-1 HTLV-IIIB strain with an ECSOvalue of 0.98 pM, which is 7-fold more potent than that of HEPT. Removal of the 5-methyl group in compound 11 results in total loss of activity. Other compounds did not show any anti-HIV-1 activity. The 4-thio derivatives 17 and 18 were found to be rather cytotoxic. When compound 11 was evaluated for its inhibitory effects on another HIV-1 strain, HTLV-IIIW, and two HIV-2 strains, LAV-2RoD and LAV-BmO, it proved equally inhibitory to HTLV-IIIRF,whereas both HIV-2 strains were insensitive to the compound. In the search for more selective and effective agents against human immunodeficiency virus (HIV),'l2 which is the causative agent of the acquired immunodeficiency syndrome (AIDS), a large number of nucleoside analogues have been synthesized and investigated for their antiviral a c t i ~ i t i e s . ~Among ~~ these, 3'-azido-3'-deoxythyidine5 Showa University. Fukushima Medical College. 8 Mitsubishi Kasei Corporation. H University of Birmingham. Rega Institute. f
0022-2623/91/1834-1394$02.50/0
(UT)has already been approved for use for patients with AIDS. 2',3'-Dideoxyinosine (DDI), which is less toxic than ~~
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Barr6-SGoussi,.; Chermann, J. C.; Rey, F.yNugeyre, M. T.; Chamaret, S.; Gruest, J.; Dauguet, C.; Axler-Blin; VBzientBrun, F.; Rouzioux, C.; Rozenbaum, W.;Montagnier, L. Science (Washington, D.C.) 1983,220,868. (2) Gallo, R. C.; Sarin, P. S.; Gelmann, E. P.; Robert-Guroff, M.; Richardson, E.; Kalyanaraman, V. S.; Mann, D.; Sidhu, G.D.; Stahl, R. E.; Zolla-Pazner, S.; Leibowitch, J.; Popovic, M. Science (Washington,D.C.)1983,220,865. (3) De Clercq, E. In Approaches to Antiviral Agents; Harnden, M. R., Ed.; MacMillan: London, 1985; p 57. (1)
0 1991 American Chemical Society
