Journal of Medicinal and Pharmaceut'ical Chemistry VOL. 3, No. 2 (1961)
A Coniparative Study of the Anticancer Activity of Some S-Substituted Derivatives of 6-Mercaptopurine and their Ribonucleosides* JOHN A. MONTGOMERY, THOMAS P. JOHNSTON, ANNEGALLAUHER, CARL R. STRINCFELLOW and FRANK M. SCHABEL, JR.,KetteringMeyer Laboratory,-f Southern Research Institute, Birminghum, Alubuwm
I. Introduction Because of the established anticancer activity of 6-mercaptopurine1 [purine-6(lH)-thione], we prepared the ribonucleoside of this purine some time ago in these laboratories.2 A careful comparative evaluation of the activity of this purine and its ribonucleoside against Carcinoma 755, a tumour unusually sensitive to purine antagonists, in BDF 1 mice showed the ribonucleoside t o have a greater therapeutic index in this test systems3 This initial result led us to synthesize and test a number of ribonucleosides of #-substituted derivatives of 6-mercaptopurine. The #-substituted 6-mercaptopurine derivatives themselves had been synthesized previously4 and found to have activity against Carcinoma 755.3 We have now made a study of the activity of this series of purines and their ribonucleosides against not only Carcinoma 755 but also against Sarcoma 180 and Leukemia L 1210.-f-f
* This work was supported by funds from the Cancer Chemotherapy National Service Center, National Cancer Institute, National Institutes of Health, Contract No. SA-43-ph-1740. t Affiliated with the Sloan-Kettering Institute. t i These are the three tumours employed in the primary screening programme of the Cancer chemotherapy National Service Center, U.S. Public Health Service. 265
266
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11. Discussion
A . Synthesis The facile conversion of inosine into 9-p-D-ribofuranosyl-9Hpurine-6(lH)-thione via the thiation of 9-(2,3,5-tri-O-benzoyl-/3-~ribof~ranosy1)hypoxanthine~ has made the thione a readily available starting material for the preparation of S-substituted derivatives." The direct methylation of 9-P-D-ribofuranosy1-9H-purine6(lH)-thione with iodoinethane in aqueous sodium hydroxide solution to give the 6-(methylthio) derivative has been described.5 Previously, the preparation of tlie 6-(methylthio) derivative had been achieved by the condensatioii of tlie chloromercuri derivative chloride, of 6-(methy1thio)purine with 2,3,5-tri-0-acetyl-~-ribosyl followed by removal of the acetyl groups.6 The synthesis of 6-(benzyltliio)-9-/3-~-ribofuranosyl-9H-pur~ne by the action of sodium a-toluenethiolate on 6-cldoro-9-~-ribofuranosyl-9H-purine has been reported. The 6-(alkylthio)-9-~-~-ribofuranosyl-9H-purines whose preparations are suniniarized in Table I were all prepared by the direct alkylation of 9-P-~-ribofuranosyl-9H-purine-6(1H)-thione either in dimethylformaniide with potassium carbonate as the acid acceptor (Method A) or in water with sodium hydroxide as the acid acceptor (Method B). Method A is essentially the method previously described for the preparation of a large number of 6-(alky1thio)purines in which dimetliylformamide was employed advantageously as a s ~ l v e n t . Dimethylformamide ~ proved to be a good medium for most of the alkylations that produced tlie ribonucleosides of Table I, but its great solvating power complicated the isolation of pure products in some cases. I n no instance in which dimethylformaniide was used was the presence of unchanged 9 -p-D-ribofuranosyl-9H-purine- 6 ( 1H)-thione de tected in the isolated product either by ultraviolet absorption spectroscopy or by paper chromatography. When the substituent was a methyl, cyanomethyl, or acetonyl group, method B was the preferred procedure ; when the substituent was a thenyl group, method A was preferred because of the competitive hydrolysis of 2-chloromethylthiophene in water. The products were obtained
* We wish to thank the Cancer Chemotherapy National Service Center for a generous supply of 0-P-n-ribof~iranofiyl-0H-purine-6( 1H)-thione.
MERCAPTOPURINE DERIVATIVES
267
J. A. MONTGOMERY ET AL.
268
in varying degrees of hydration, the water content depending on the solvent used for recrystallization and the drying conditions ; forced drying in vacuo a t temperatures above 80" was avoided because several of the compounds were observed t o be unstable when drying was attempted a t 100". Only one of the hydrated ribonucleosides [9-/3-~-ribofuranosyl-6-(2-thenylthio)-9H-purine] had a definite melting point. Examples of typical procedures employed are given in the Experimental Section. The ultraviolet absorption spectra of these ribonucleosides (Table 11)are, in general, quite similar to those of the corresponding B-(alkylthi~)purines.~ The greatest difference in the spectra Table 11. Ultraviolet absorption spectra of 8-substituted 9 -P-D-ribofuranosyl-9H -purine-6-thiolsa*
Methyl Ethyl Cyanomethyl Allyl Propyl Acetonyl Cyclopentyl 2-Thenyl Benzyl Cinnamyl
224 293 224 294 277 292 225-226 295 300
11.4 17.4 10.4 16.8 16.3 17.6 11.0 17.4 13.8
224 289 224 292 278 291 225-226 293 282
11.8 18.9 10.6 18.2 16.1 19.4 10.8 19.3 17.2
297 224 292 293d 254-255 295
18.5 15.8 18.0 18.9 19.7 23.3
295 224 293 292 255 295
20.2 15.8 18.6 20.6 19.5 24.7
224 289 224 292 278 291 225-226 293 255O 281 294-295 224 292 292 253-25ge 295
11.4 18.9 10.5 18.4 16.0 19.3 10.7 19.7
20.4
-
19.1 20.8 19.6 24.8
a Determined with either a Beckman Model DK-2 spectrophotometer (optical densities on a Beckmsn DU) or a Cary Model 1 4 spectrophotometer. b Shoulders not recorded but have patterns similar to those recorded for t h e corresponding 6 - ( a l k y l t h i o ) p ~ i n e s . ~0 Undergoes partial hydrolysis to inosine at p H 13. d cf. ref. 7. e Plateau.
occurs in the case of 6-(cyanomethy1thio)purine(289 mk) a t pH 13 wersus 6-(cyanomethylthio)-9-/3-~-ribofuranosyl-9H-pur~ne (278 mp). 6-(Acetonylthio)-9-~-~-ribofuranosyl-9H-pur~ne underwent
MERCAPTOPURINE DERIVATIVES
269
partial hydrolysis to inosine during the course of determining its spectrum a t p H 13. Paper chromatographic data and optical rotations of these compounds are recorded in Table 111.
B. Screening Results Carcinoma 755. The raw data obtained in screening these purines and their ribonucleosides against Carcinoma 755 in BDF 1 mice are presented in Table IV. With one exception, 6-(methylthio)-9-p-D-ribofuranosyl-9H-purine,* these materials exhibited a high degree of inhibitory action against this tumour. Prom these data the chemotherapeutic indices of the compounds were calculated. The results of these calculations are summarized in Table T7 in which the maximum tolerated dose (MTD); the minimum effective dose, which in this paper is defined as the lowest dose that will inhibit the growth of this tumour to 50 per cent of the untreated controls (ED5,)? ; the chemotherapeutic indices (MTD/ED,,); and the ratio of the chemotherapeutic index of the ribonucleoside to that of the purine from which it is derived (CIR/CIp)are given. The MTD’s and the ED5,’s were calculated in pmoles/kg/day t o give a reasonable comparison between these compounds that vary so widely in their niolecular weights. On this basis, two of the ribonucleosides, wix. B-(methylthio)-9-P-~ribofuranosyl-9H-purine and 9-P-~-ribofuranosyl-6-( 2-theny1thio)9H-purine, were more toxic than the parent purines, and these two compounds were the only ones more toxic than 6-mercaptopurine itself. The only compound to exhibit a smaller ED,, than 6-mercaptopurine was its ribonucleoside, and this was the only ribonucleoside that was significantly more effective against Carcinoma 755 than the parent purine, as can be seen from the ratios of the cheniotherapeutic indices. Five ribonucleosides were about as effective as the corresponding purines and four were considerably less effective, the ratios varying from 0 04 t o 0 4. * Sarcione and Stutzman have found that, in the rat, 6-(methy1thio)purine is metabolized to 6-mercaptopurine, and 6-mercaptopurine to 6-(methylthio)purine, but neither conversion takes place t o any great extent.O The significance of this observation with regard to the anticancer activity of 6-(methy1thio)purine or its ribonucleoside is not certain, nor is i t known whether other S-substituted derivatives of 6-mercaptopurine undergo the same type of cleavage i n vivo. i- The Ed,, values were obtained by an extrapolation procedure.*
270
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The effect of ribonucleosidation of this series of purines on their activity against Carcinoma 755 is then, with the one exception noted, not beneficial. Sarcoma 180 and Leukemia L1210. Because it does not necessarily follow that the results of screening these compounds against Sarcoma 180 and Leukemia L1210 would show the same pattern observed with Carcinoma 7 5 5 , these two test systems were also used in our evaluation and some variation in results was observed. 6-Nercaptopurine is less effective against Sarcoma 180 than it is against Carcinoma 7 5 5 , and its derivatives were also found to be less effective than they are against Cascinoma 7 5 5 . I n fact, the derivatives, with the exception of 6-mercaptopurine ribonuceloside, were all less effective than 6-mercaptopurine itself. 6-Mercaptopurine ribonucleoside, in these tests, inhibited Sarcoma 180 to a t least the same extent as 6-mercaptopurine. 6-Mercaptopurine and its ribonucleoside seem equally effective against Leukemia L1210, causing a significant increase in life-span of the leukemic mice. None of the other compounds tested caused any greater increase in life-span when employed under standardized conditions and, indeed, only one other ribonucleoside showed ‘plus’ activity. Table V I presents the Sarcoma 180 screening data and Table VI1 the Leukemia L1210 data. Three of the purines and the ribonucleosides of these same three purines caused a marked inhibition of Sarcoma 180, and three of the purines and two of the ribonucleosides produced a significant increase in life-span of the leukemic mice. A summary of these comparisons is given in Table V I I I in which the compounds are listed in decreasing order of their chemotherapeutic indices against Carcinoma 7 5 5 .
111. Conclusions This study of closely related derivatives of 6-mercaptopurine shows that no increase in anticancer activity is obtained from the ribonucleosidation of these purines. Only in the case of the parent compound, 6-mercaptopurine, was an increase observed and in that case only against Carcinoma 7 5 5 . A wide but not correlatable variation in host toxicity of the ribonucleosides was noticed.
J. A. MONTGOMERY ET AL.
282
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J. A. MONTGOMERY El' AL.
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Experimental
6-(Propylthio)-9-/3-~-ribofuranosyl-9H-purine monohydrate (iwethod A ) . To a stirred mixture of 9-/3-D-ribofuranosyl-gH-purine6(lH)-thione (1 00 g, 3 52 mmoles) and potassium carbonate (511 mg, 3.70 mmoles) iii dirnethylfornzamide (7 ml) was added 1-bromopropane (455 mg, 0.34 ml, 3.70 mmoles). When the exothermic reaction had ceased, the mixture was heated at 40-50" for 1 h. The mixture was then dissolved in 20 ml of water, and the solution, after being neutralized with 6 N hydrochloric acid, was evaporated to dryness under reduced pressure. The residue was extracted with dimethylformamide (3 x 5 ml), and the extract evaporated to dryness under reduced pressure a t BO", care being taken to remove all of the solvent. The residue was dissolved in 50 ml of hot water, and the solution was treated with Xorit, filtered and cooled. The white gel that formed was collected and dried a t 80" for 24 h in vacuo over phosphorus pentoxide; yield 667 mg (55 per cent) of analytically and chromatographically pure ribonucleoside monohydrate, m.p. indefinite with softening from 74" (Kofler Heizbank). An additional 343 1n.g (about 80 per cent pure by ultraviolet spectral analysis) was obtained as a second crop when the filtrate was evaporated under izitrogen to one-half volume; total yield 80 per cent. 9 -/3 - D - Ribofuranosyl- 6 - (2- thenylthio) - 9H -purine hemihydrate (Method A ) . 2-Chloromethylthiophene (245mg, 1- 85mmoles) was added dropwise to a stirred mixture of 9-/3-D-ribofuranosyl-9Hpurine-6( lH)-thione (500 ma, 1 76 mmoles), anhydrous potassium carbonate (256 mg, 1.85 mmoles), and diniethylformamide ( 5 ml). After the exothermic reaction had ceased, the mixture was stirred and heated a t 40-50" for 1 h. The mixture was then poured into 20 ml of water, and the pH of the resulting solution was adjusted from 9 to 7 with 6~ hydrochloric acid. The crude ribonucleoside precipitated as a gel when the solution was cooled. The mixture was evaporated to dryness under reduced pressure a t BO", the residue triturated in 5 ml of cold water, and the remaining white solid collected and dried for 40 h in vacuo over phosphorus pentoxide a t room temperature; yield 673 mg (shown by ultraviolet absorption spectra to be about 87 per cent pure). Two recrystallizations of a 550-mg sample of the crude product from methyl
286
J. -4.NONTGOMERY ET AL.
alcohol (Korit) gave 350 mg (corresponding to a 62 per cent yield) of analytically and chroinatographically pure 9-P-D-ribofuranosyl6-(2-thenylthio)-SH-purine heniihydrate (dried in vacuo over phosphorus pentoxide a t room temperature for 16 h and then a t 60" for 8 h), m.p. indefinite, with softening from 97" (Kofler Heizbank). From a larger run ( 10 6 nimoles of the thione) the yield of pure product was 7 1 per cent. 6-(Etlzylthio)-9-P-n-ribofuranosyl-9H-purine two-thirds hydrate (Method A ) . To a stirred mixture of 9-P-D-ribofuranosyl-9Hpurine-B(1H)-thione (8 20 g, 28 * 9 mnioles) and anhydrous potassiuni carbonate (4 * 20 g, 30 * 4 mmoles) in dimethylformamide ( 5 5 ml) was added dropwise bromoethane (3.32 g, 2 34 ml, 30 4 mmoles). The mixture was then heated a t 45-50" for 1 h with stirring and poured into 165 ml of water. The p H of the resulting solution was adjusted from 9 to 7 with 6 N hydrochloric acid, and the solution evaporated to dryness under reduced pressure. The residue was extracted with warm (60") dimethylformamide (4x 24 ml), and the extract evaporated to dryness a t about 60" under reduced pressure. The residue was then extracted with 50 nil of hot ethyl alcohol, a small amount of insoluble material being removed by filtration. Because crystallization of the ribonucleoside did not occur during prolonged refrigeration of the filtrate, the solvent was removed by evaporation in vacuo, and the residue was similarly treated with isopropyl alcohol. A small amount of hot isopropyl alcohol-insoluble material was removed, and the filtrate when cooled deposited a few crystals, which were collected and found to be impure ribonucleoside by paper chromatography. The filtrate was then evaporated to dryness in vacuo, and the residue dissolved in 20 ml of warm methyl alcohol, The niethanolic solution was drawn by suction through a capillary attached to a sintered glass filter into 1a 5 1. of ethyl ether. The resulting white precipitate was collected and dried in vacuo over phosphorus pentoxide for 7 h a t 80"; yield 4.86 g (54per cent, as the two-thirds hydrate). 6-(Cyanonzethylthio)-9-~-~-ribofurnno~yl-9H-pur~ne monohydrate (Method B ) . To a stirred solution of 9-P-n-ribofuranosyl-9Hpurine-6(lH)-thione (6 00 g, 21 ' 1mmoles) in approximately 0 a 4 N aqueous sodium hydroxide solution (54 ml) was added chloroaceto-
MERCAPTOPURINE DERIVATIVES
2af
-
nitrile (1 75 g, 21 1 mmoles). When the exothermic reaction had ceased, more chloroacetonitrile ( 0 . 1 g) was added. The mixture was then stirred a t room temperature for 30 min. The resulting solution, adjusted to p H 7 with 6 N hydrochloric acid, was chilled for about 5 h. The reddish-brown gel that formed was collected and dried in vacuo over phosphorus pentoxide first a t room temperature for 1 h, then a t 80" for 2 h. Recrystallization of the crude light-tan solid from isopropyl alcohol gave 4 . 4 5 g (62 per cent) of 6-(cyanomethylthio)-9-p-D-ribofuranosyl-9H-purine monohydrate (dried in vacuo over phosphorus pentoxide for 1 h at room temperature and then 4 h a t 80') as an off-white powder. Xcreening procedures. The animal assay procedures were performed according to the specifications established by the Cancer Chemotherapy National Service Center. Since these specifications have been set forth in detail,1° no description of the procedures will be included here. Summary. The ribonucleosides of ten S-substituted derivatives of 6-mercaptopurine have been prepared by the alkylation of 6-mercaptopurine ribonucleoside. The activity of these ribonucleosides against Carcinoma 755, Sarcoma 180, and Leukemia L1210 was compared with the activity of the parent purines against these same neoplasms. None of the ribonucleosides of these derivatives of 6-mercaptopurine showed any increase in anticancer activity over the parent. Acknowledgements. The authors wish to thank Dr. W. R. Laster, Jr., under whose direction the animal screening was carried out; Dr. H. E. Skipper for his helpful suggestions; Dr. W. J. Barrett, under whose direction the analytical determinations herein reported were carried out ; and Mrs. Sarah D. Clayton for technical assistance.
(Received 5 August, 1960) References Burchenal, J. H., Ellison, R. R., Murphy, M. L., Karnofsky, D. A., Sykes, M. P., Tau, T. C., Mermann, A. C., Yuceoglu, M., Meyers, W. P. L., Krakoff, I. and Alberstadt, N. Ann. N . Y . Acad. Sci., 60,359 (1954) Johnson, J. A., Jr., and Thomas, H. J. J . Amer. chem. Soc., 78, 3863 (1956) Skipper, H. E., Montgomery, J. A,, Thomson, J. R. and Schabel, F. If,, J r . Cancer Re8. 19, 425 (1959)
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Johnston, T. P., Holum, L. B. and Montgomery, J. A. J . Amer. chem. Soc., 80, 6265 (1958) Fox, J. J . , Wempen, I., Hampton, A. and Doerr, I. L. J . Amer. chem. SOC.,80, 1669 (1958) Hampton, A., Biesele, J. J., Moore, A. E. and Brown, G. B. J . Amer. chem. SOC.,78, 5695 (1956) Johnson, J. A., Jr., Thomas, H. J. and Schaeffer, H . J. J . Amer. chem. Soc., 80, 699 (1958) 8 Schabel, F. M., Jr., Skipper, H. E., Laster, R. W., Jr., andMontgomery, J. A. I n press Sarcione, E. J. and Stutzman, L. Proc. Amer. Ass. Cancer Res., 2, 342 (1958) Cancer Chemotherapy Reports, I, 42 (1959)
