670
J. Med. Chem. 1984, 27,670-674
saline (0.85% NaCl in distilled water), compounds 3b,8, and 10 were administered in saline plus Tween 80, and compound 6 was administered in saline plus Tween 80 in most experiments (experiments 4-8, Table I) and, also, in suspension in saline.
Acknowledgment. This investigation was supported by Grant R01-CA23127 from the National Cancer Institute. Tests against mouse leukemia P388 in vivo were supported by Contract N01-CM-97309 between the Southern Research Institute and The Division of Cancer
Treatment, National Cancer Institute. Spectrometric and microanalytical determinations were performed by Dr. william c. Coburn, jr., Martha c. Thorpe, ~~i~~ c, Kirk, and Christine G. Richards of this Institute. Registry No. 3b,4239-10-5; 4c, 88343-71-9; 6, 88343-72-0; 7, 88343-73-1; 8, 88343-74-2; 9, 88343-75-3; 10, 61801-29-4; 17, 35329-86-3; 18, 22317-89-1; 2-chloroethano1, 107-07-3; 2-fluoroethanol, 371-62-0; 2-bromoethanol, 540-51-2; methanesulfonyl chloride, 124-63-0.
Synthesis and Antiviral Evaluation of Carbocyclic Analogues of Ribofuranosides of 2-Amino-6-substituted-purinesand of 2-Amino-6-substituted-8-azapurines Y.Fulmer Shealy,* Joe D. Clayton, Gussie Arnett, and William M. Shannon Kettering-Meyer Laboratories, Southern Research Institute, Birmingham, Alabama 35255. Received August 10, 1983
Carbocyclic analogues of ribofuranosides of 2-amino-6-substituted-purines and of 2-amino-6-substituted-8-azapurines were prepared from the 2-amino-6-chloropurine ribofuranoside analogue (2) and the 2-amino-6-chloro-8-azapurine ribofuranoside analogue (9), respectively. Analogues of purine ribofuranosides with the chloro, amino, methylamino, or methylthio group a t position 6, the thioguanosine analogue, and the previously reported guanosine analogue were evaluated in vitrc against herpes simplex virus, type 1 (HSV-1). 8-Azapurine ribofuranoside analogues with the chloro, amino, or methylthio group a t position 6 and the previously reported 8-azaguanosine analogue were also evaluated against HSV-1. The carbocyclic analogue (6) of 2,6-diarninopurine ribofuranoside is highly active against and the HSV-1 and, also, against vaccinia virus. The 2-amino-6-chloropurine, 2-amino-6-(methylamino)purine, 2,6-diamino-S-azapurine derivatives also demonstrated significant activity against HSV-1.
Carbocyclic analogues of 6-substituted-purine ribothat it is active against P388 leukemia in mice.12 Bennett furanosides, including the racemic analogue (C-Ado) of et al.13 reported in 1975 that several members of the initial adenosine, constituted the first group of carbocyclic anagroup of carbocyclic analogues of 6-substituted-purine logues of nucle~sides.l-~The adenosine analogue was ribofuranosides have antiviral activity. Subsequently, it subsequently isolated as the antibiotic aristeromycin was shown that carbocyclic analogues of cytidine and (1’R,2’S93’R,4’R enantiomer, nucleoside n ~ m b e r i n g ) . ~ , ~ arabinofuranosylcytosine ( ~ r a - C ) , ~arabinofuranosyl~-l~ The identity of C-Ado and aristeromycin was reported5and adenine (ara-A),l’ 3-deazaadenosine,18and 5-substitutedwas confirmed in more detail6 after a later synthesis and 2’-deoxyuridineslgalso have antiviral activity. In addition resolution were claimed.’ Other ribofuranoside analogues to the guanosine (3) and 8-azaguanosine (4) analogues,8we had also prepared carbocyclic analogues of ribofuranosides synthesized in the early stages of this work were the carbocyclic analogues of guanosine,s 8-azaguanosine,s and of other 2-aminopurines and 2-amino-8-azapurines (1,2,38-azapurine ribofurano~ides.~Initial biochemical studies triazolo[4,5-d]pyrimidines);we now describe the prepshowed that C-Ado may function either as a substrate for aration, antiviral evaluation, and antineoplastic testing of or as an inhibitor of enzymes involved in the biosynthesis these carbocyclic analogues. de novo or in interconversions of purine nucleotides,lOJ1 Chemistry. The synthesis of the pyrimidine precursor and tests of the 8-azaadenosine analogue in vivo showed (1) of both the 2-aminopurine and 2-amino-8-azapurine analogues was described earlier.8 Previously, the acidcatalyzed reaction of triethyl orthoformate with pyrimidine 1 in dimethylformamide and the use of the total crude (1) Shealy, Y. F.; Clayton, J. D. J . Am. Chem. SOC.1966, 88, 3885-3887. product of this reaction to produce the guanosine analogue (2) Shealy, Y. F.; Clayton, J. D. J . Am. Chem. SOC.1969, 91, (3) were reported.8 In addition to forming the purine ring, 3075-3083. interaction of 1 and triethyl orthoformate should produce (3) Shealy, Y. F.; Clayton, J. D. J . Pharm. Sci. 1973, 62, 1252-1257. (4) Kusaka, T.; Yamamoto, H.; Shibata, M.; Muroi, M.; Kishi, T.; Mizuno, K. J . Antibiot. 1968, 21, 255-263. (5) Kishi, T.; Muroi, M.; Kusaka, T.; Nishikawa, M.; Kamiya, K.; Mizuno, K. Chem. Pharm. Bull. 1972,20,940-946. (6) Shealy, Y. F.; Thorpe, M. C.; Coburn, Jr., W. C.; Clayton, J. D. Chem. Pharm. Bull. 1980,28, 3114-3117. (7) Holg, A. Collect. Czech. Chem. Commun. 1976,41, 2096-2109. (8) Shealy, Y. F.; Clayton, J. D. J . Pharm. Sci. 1973, 62, 1432-1434. (9) Shealy, Y. F.; Clayton, J. D.; O’Dell, C. A. J. Heterocycl. Chem. 1973, IO, 601-605. (10) Bennett, Jr., L. L.; Allan, P. W.; Hill, D. L. Mol. Pharrnacol. 1968, 4, 208-217. (11) Hill, D. L.; Straight, S.; Allan, P. W.; Bennett, Jr., L. L. Mol. Pharmacol. 1971, 7, 375-380. 0022-2623/84/1827-0670$01.50/0
(12) Shealy, Y. F.; Clayton, J. D. J.Pharm. Sci. 1973,62, 858-859. (13) . , Bennett. Jr.. L. L.: Shannon. W. M.: Allan. P. W.: Arnett, G. Ann. N.Y.Acad. Sci. 1975, 255, 342-358. ’ (14) Shealy, Y. F.; O’Dell, C. A. J . Heterocycl. Chem. 1976, 13, 1353-1354. (15) Shealy, Y. F.; O’Dell, C. A. U.S. Patent 4 177 348, Dec 4,1979. (16) Shannon, W. M.; Arnett, G.; Westbrook, L.; Shealy, Y. F.; O’Dell, C. A.; Brockman, R. W. Antimicrob. Agents Chemother. 1981, 20, 769-776. (17) Vince, R.; Daluge, S. J . Med. Chem. 1977, 20, 612-613. (18) Montgomery, J. A.; Clayton, S. J.; Thomas, H. J.; Shannon, W. M.; Arnett, G.; Bodner, A. J.; Kim, I.-K.; Cantoni, G. L.; Chiang, P. K. J. Med. Chem. 1982,25, 626-629. (19) Shealy, Y. F.; O’Dell, C. A.; Shannon, W. M.; Amett, G. J. Med. Chem. 1983,26, 156-161.
0 1984 American Chemical Society
2-Amino-6-substituted-purines and -8-azapurines
HO
HO OH
OH
.2.,
.I.,
H
2
N
bRP
R
:,X=
CH
.5.,
%,X= N
R
R 6_vX a NHZ .7.,, X = NHCH3 2, X = SCH,
2,x = CI
-
IO, X X
a.-.I I,
NH2 SCHs
HOCHe R= HO
OH
derivatives of the hydroxyl and the 2-amino groups. Isolation of pure specimens of the 2-amino-6-chloropurine(2) was not attempted at that time. Subsequently, pure specimens of 2 were isolated by treating the crude product, obtained from the reaction of 1 and triethyl orthoformate in dimethylacetamide, with 50% acetic acid and then with methanolic ammonia to liberate the hydroxyl and amino groups. The thioguanosine analogue ( 5 ) was prepared by treating the 2-amino-6-chloropurine (2) with thiourea in refluxing propanol and isolating the product as the sulfate. Similarly, the 2,6-diaminopurine ribofuranoside analogue (6) and the 6-(methylamino) derivative (7) were isolated as sulfate salts after reactions of 2 with ammonia and methylamine, respectively, at 80 "C. The 2-amino-6(methy1thio)purine (8) was prepared by treating 2 with sodium methyl mercaptide in a mixture of methanol and methanethiol at temperatures in excess of 100 "C. The preparation of the 2-amino-6-chloro-8-aapurine (9) from pyrimidine 1 was described earlier? The carbocyclic analogues (10 and 11) of 2,6-diamino-8-azapurine ribofuranoside and 2-amino-6-(methylthio)purineribofuranoside were prepared from 9 by methods similar to, but less strenuous than, those used to obtain the corresponding purines. Biological Evaluation. A standard method, which has been described previously,l6Vz0for determining the inhibition of virus-induced cytopathogenic effects (CPE) by antiviral compounds was used to evaluate the ribofuranoside analogues against herpes simplex virus, type 1 (HSV-I), and against influenza virus in vitro. A few of these compounds were also tested against vaccinia virus. (20) Shannon, W. M.; Shortnacy, A.; Amett, G.; Montgomery, J. A. J . Med. Chem. 1974, 17, 361-363.
Journal of Medicinal Chemistry, 1984, Vol. 27, No. 5 671
The antiviral activity of each compound was expressed in terms of a virus rating (VR), and the potency was measured as a minimum inhibitory concentration (MICSO). The VR, determined by a modification of the method of Ehrlich et a1.,21is a weighted measurement of antiviral activity which takes into account the degree of inhibition of virus-specific CPE and the degree of cytotoxicity produced by the test compound. A VR greater than or equal to 1.0 indicates definite antiviral activity, a VR of 0.5 to 0.9 indicates marginal to moderate antiviral activity, and a VR less than 0.5 usually indicates no significant antiviral activity. The M E s o is the concentration of the tested compound required to inhibit virus-induced CPE by 50%. The results of antiviral evaluations of the carbocyclic analogues of 2-aminopurine ribofuranosides are summarized in Table I. In tests against strain HF of HSV-1 replicating in human epidermoid carcinoma cells (H.Ep.-2), the carbocyclic analogue (6) of 2,6-diaminopurine ribofuranoside displayed high activity (VR = 4.6) and potency (MIC, = 0.32 mcg/mL). 1-P-D-Arabinofuranosyladenine (ura-A),a known antiviral agent, was the positive control and was less active (VR = 2.0-2.2; MIC5,, = 2.5-10 mcg/ mL). The 2-amino-6-(methylamino)purine(7), 2,6-diamino-8-azapurine (lo), and 2-amino-6-chloropurine (2) derivatives also displayed significant activity (VR = 1.3-1.7, Table I) against strain HF of HSV-1, and the 2-amino-6-chloro-8-azapurine (9) was modestly active (VR = 0.9). Four of the analogues were tested against vaccinia virus replicating in H.Ep.-2 cells (Table I). In this test, the 2,6-diaminopurine ribofuranoside analogue (6) was highly active, the 2-amino-6-chloropurine (2) was significantly active, and the 2-amino-6-chloro-8-azapurine (9) was marginally active. Compounds 2-4 and 6-11 were also tested against influenza virus Ao/PR-8/34 replicating in Madin-Darby canine kidney cells; none showed activity. After these ribofuranoside analogues (2-1 1) had been synthesized, the synthesis of several arabinofuranoside analogues by the same routes was reported.22 Only the carbocyclic analogue of 2,6-diaminopurine arabinofuranoside showed significant activity (VR = 1.5) against strain HF of HSV-1.22Thus, compounds 6 and 10 (ribofuranoside analogues) appear to be considerably more active in this test than are the corresponding arabinofuranoside analogues, and compound 6 also appears to be more active in vitro than is the carbocyclic analogue of arabinof~ranosy1adenine.l~ All of these carbocyclic analogues were evaluated in the same way in these laboratories.17223 The results of tests of compounds 2-1 1 for cytotoxicity to cancer cells in culture and against leukemia L1210 in mice are summarized in Table 11. Compounds 5-7 and 9 were cytotoxic at relatively high concentrations to KB or H.Ep.-2 cells. The guanosine analogue (3) was tested on three dosage schedules against leukemia L1210 in vivo and was found to be without activity in prolonging life span. The toxicity of 3 in vivo appears to be greater than its cytotoxicity indicated. Compounds 2 and 6 were not active when tested q.d. 1-9 against L1210 leukemia. Except for compound 7,the remaining analogues received at least one test in vivo: compounds 4,5,9, and 10 were not active or toxic when administered on day 1 (only) at 400 (21) Ehrlich, J.; Sloan, B. J.; Miller, F. A.; Machamer, I-?. E. Ann. N.Y. Acad. Sci. 1965, 130, 5-16. (22) Lee, H.; Vince, R. J. Pharm. Sci. 1980,69, 1019-1021. (23) W. M. Shannon and co-workers, unpublished results. (24) Geran, R. I.; Greenberg, N. H.; Macdonald, M. M.; Schumacher, A. M.; Abbott, B. J. Cancer Chemother. Rep., Part 3 1972, 3, 1-103.
672 Journal of Medicinal Chemistry, 1984, Vol. 27, No. 5
Shealy et al.
Table I. Evaluation of Carbocyclic Analogues of Ribofuranosides of 2-Aminopurines and 2-Amino-8-azapurines for Antiviral Activity in Vitroa compd
HSV-1, strain H F
substituent at position 6
MIC jo, mcg/mL
VRC
vaccinia VR
MIC jo, m c g / m l
Purines
c1
2 3
1.7
0 S
5 6
Od
NHCH, SCH,
8
1.2
20
0
4.6 1.3
2"
7
10
0
0.3 320
0.1
NT 3.8 NT NT
2.3
8-Azapurines 0 NT 9 c1 0.9 81 0.6 10 2 1.4 100 11 SCH, 0 NT ara-A (positive control) 2.0-2.2 2.5-10 Antiviral assays of these compounds were performed by using the H F strain of HSV-1 and the Lederle Chorioallantoic strain of vaccinia virus, both replicating in human epidermoid carcinoma no. 2 (I-I.Ep.-2) cells. The compounds tested against HSV-1 (HF) were also tested against influenza virus; none showed activity, Purine numbering system for 8azapurines. VR = virus rating; see the discussion. Strain 377 was used in evaluating compound 5 .
0
4
I _ _
Table 11. Tests of Compounds 2-11 for AntineoDlastic Activitv cytotoxicity compd 2
3
4 5
6
7 8 9 10 11
cells KB
H. Ep :2
H.Ep.-2 KB KB
KB KB H.Ep.-Z KB KB
L1210 in vivo4
ED,,, mcg/mL
>loo >loo
>loo 50 19
schedule day 1 q.d. 1-9 q.d. 1-9 q.d. 1-9 day 1 day 1 day 1 days 1, 5, 9 days 1, 5, 9 days 1, 5 , 9 q.d. 1-9 q.d. 1-9 q.d. 1-9 day 1 day 1 day 1 q.d. 1-9 q.d. 1-9 q.d. 1-9 q.d. 1-9
dose, mg/kg 400 200
100 50 300 150 75 75 38 19 38 19 9 400
TIC,b % 104
67t 85t 101
tC 755d 88
400 400 200 100 50 25
70t 92 94 71t 83t 101 109 99 t 67t 70t 89 102
100
108
400 400 100
111
18
>loo
8 > 100 > 100
q.d. 1-9 day 1 day 1 q.d. 1-9
97 94
Tested in accordance with the protocols of the National Cancer Institute.24 Mice were inoculated intraperitoneally with Solutions or suspensions of the compounds were administered intraperitoneally. T = treated mice; C = untreated, leukemic control mice. A dose is consideredz4to be toxic ( t ) if mortality by day 5 is greater than or equal t o three of six mice if TIC is
