Dec. 5, 1958
S-SUBSTITUTED
o-chlorobenzaldehyde, ca. 243--245". Based on the yield of semicarbazone obtained fraction I must have been >SO% aldehyde-this would represent a 10-15% recovery of the starting aldehyde. Fractions 11, I11 and IV represent the cinnamonitrile yield. Basing the cis-trans composition of each fraction bn infrared analyses and using the spectra of the pure isomers as reference, the composition of the three fractions was: 11, 510/0 trans and 42% cis; 111, 64% trans and 25% cis; IV, 83% trans and 7% cis. The total combined yield of 11, I11 and IV was 72 g. (45%) of the cinnamonitrile and this consisted of 56 g. (78%) trans and 16 g. (22%) cis. Fraction V, 16 g., probably consisted largely of p-(ochloropheny1)-glutaronitrile and was not further investigated. Amidines.-All were made essentially by the procedure of Lorz and Baltzlyl using dried benzene to increase the solubility of the reactants where necessary. Either the hydrochloride or a mixture of hydrochloride with (usually yellow) hydrobromide crystallized during the decomposition. A typical example is given below. N ,N-Dibutyl-o-chlorocinnamamidineHydrochloride .T o a Grignard reagent made from 3.09 g. (0.127 mole) of magnesium turnings, 14.2 g. (0.13 mole) of ethyl bromide and 120 ml. of absolute ether was added during 5 minutes 17.05 g. (0.132 mole) of di-n-butylamine dissolved in 50 ml. of anhydrous ether. The mixture was boiled for an additional half-hour, and a solution of 14.5 g. (0.089 mole) of trans-o-chlorocinnamonitrile of m.p. >40°, dissolved in 50 ml. of absolute ether, was added in a few minutes. The solution was boiled under reflux for 5 hours, and decomposed, after remaining overnight, by the addition of 50 ml. of 4 N HC1. The resulting solid was filtered, washed with 4 N HCl and then with ether. The residue was 27.7 g. of product, m.p. 210-222'. This was recrystallized from absolute ethanol-ethyl acetate giving a first crop of 15.6 g., m.p. 225-229". A second crop obtained by addition of ether weighed 8.2 g., m a p . 227-228'. N,N-Di-n-butyl-P-methylcinnamamidinehydrochloride was prepared from 59.7 g. of &methylcinnamonitrile and bromomagnesium dibutylamide, essentially by the procedure given above. Decomposition of the reaction mixture with aqueous hydrochloric acid led to the formation of three layers. The middle one, which was benzene soluble, and the aqueous layer, were treated with an excess of iced aqueous sodium hydroxide and the amidine base so liberated was taken up in ether, dried briefly over magnesium sulfate and distilled. A fore-run of solvent and of dibutylamine
[CONTRIBUTION FROM
THE
6-MERCAPTOPURINE
6265
(the latter removed in vacuo) was followed by 10.07 g. (12% of theory) of distillate, b.p. 120-126' (0.15 mm.). This was acidified in ethyl acetate-ether solution with gaseous hydrogen chloride, and treated with Skellysolve A to give an oil. This was redissolved and, after a week at -15O, a crystalline solid was formed. This was recroystallized from the same solvent mixture, m.p. 148.5-150.3 p-Cyclocitry1ideneacetamide.-To 15.7 g. (0.081 mole) of cyclocitrylideneacetic acid was added 48 g.. of thionyl chloride. After the acid had dissolved and the mixture had been heated for 40 minutes under reflux, the excess thionyl chloride was evaporated a t the water-pump up to bath temperature 42". The residual oil was taken up in 80 ml. of absolute ether and added dropwise to 200 ml. of absolute ether kept saturated with ammonia by passing in a rapid stream of gaseous ammonia throughout the addition and for 10 minutes more. The solution then was filtered to remove ammonium chloride, and the ethereal solution was washed with water, dried a2d concentrated. The resiThis was recrystallized due was 15.4 g., m.p. 143-146 from Skellysolve C to give 13 g. of white solid, m.p. 147148". Anal. Calcd. for CI~HIONO: N, 7.25. Found: N, 7.54. p-Cyclocitrylideneacetonitrile (11).-A mixture of 11.4 g. (0.059 mole) of p-cyclocitrylideneacetamide and 70 ml. of benzene (dried by azeotropic distillation) was heated under reflux while 7.1 g. (0.065 mole) of thionyl chloride was added dropwise over 10 minutes. An additional 20 ml. of benzene was added and the solution was boiled an additional 2 hours. It was then distilled in vacuo collecting 9.94 g. (96% of theory, if pure) of a yellow liquid, b.p. 132-136" (12 mm.). This was redistilled a t the same temperature [literature6b.p. 141' (17 mm.)]. N,N-Dibutyl-p-cyclocitrylideneacetamidine Hydrochloride .-The procedure previously given for cinnamamidines was followed with 9.94 g. of 0-cyclocitrylideneacetonitrile except that the reaction mixture was decomposed after 2.5 hours under reflux, by addition of aqueous acid. The precipitate was filtered off, and washed with water and ether to give 17.7 g. of solid, m.p. 195-200". This was dissolved in 500 ml. of boiling acetone, filtered from some inorganic impurity, and absolute ether was added to incipient turbidity (ca. 800 ml.), The first crop of felted needles had m.p. 192-198", and had a broad absorption maximum a t 289290 mp, e 8800. Anal. Calcd. for CeoH&-Ne: C1-, 10.42. Found: C1-, 10.65. N. Y . TUCKAHOE,
.
.
UTTERING-MEYER LABORATORY,' SOUTHERN RESEARCH INSTITUTE]
Synthetis of Potential Anticancer A.gents. XVI. S-Substituted Derivatives of 6Mercaptopurinela BY THOMAS P. JOHNSTON, LEE B. HOLUMAND JOHN A. MONTGOMERY RECEIVED JUNE 30, 1958 A number of S-substituted derivatives of 6-mercaptopurine have been prepared by a new procedure in which dimethylformamide was used as the reaction medium.
Because both 6-methylthiopurine and 6-benzylthiopurine have shown activity against Adenocarcinoma 755* and Sarcoma 1803comparable to that of 6-mercaptopurine, an extensive investigation of the anticancer activity of S-substituted derivatives ( 1 ) A5liated with Sloan-Kettering Institute. This work was supported by funds from the C. F . Kettering Foundation and the National Institutes of Health, Contract No. SA-43-ph-1740. Part XV. H. J. Schaeffer and H. J. Thomas, THISJOURNAL,80, 4896 (1958). ( l a ) Name in common usage; 6-purinethiol is used by "Chemical Abstracts." (2) H. E. Skipper, J. A. Montgomery, J. R . Thomson and P. M. Schabel, Jr., Proc. A m . Assoc. Cancer Reseavch, 2, 346 (1958). (3) D. A. Clarke, G. B. Elion, G. H. Hitchings and C. C . Stock, Cancer Research, 18, 445 (1958).
of 6-mercaptopurine is of great interest. For this study we have prepared the varied series of 6-alkyland 6-arylthiopurines summarized in Tables I and 11. Although the screening of these compounds has not yet reached the stage a t which a pattern of the structural effect on anticancer activity can be established, some interesting variations in activity and toxicity already have been noted. Speculation that the activity of the methyl and benzyl derivatives is related to an in vivo formation of 6-mercaptopurine by a biological cleavage of the thioether has been a d ~ a n c e d . ~ Such a mechanism is (4) G. B. Elion, G. H. Hitchings, D. A. Clarke and C. C . Stock, Proc. A m . Assoc. Cancer Research, 2 , 199 (1957).
TIIOAIAS P.JOHNSTON, LEE B. HoLuni
6266
AND JOHN
.I.~ I O X T G O V E R Y
1701.
so
TABLE I 6-ALKYL.THIOPURINES
R
X
Crude yield, Method
Kecrystn solvent'
M p.,b OC.
CarbcIn, % Calcd. Found
Hydrogen, R Calcd. Found
Nitrogen, ?'& Calcd. Found
I A-1 93" A 196d 46. 46.75 4.48' Ethyl 4.34 Br 95' B A-1 238 d. 50.51 50.53 3.18 3.19 29.45 29.37 2-~ropynyl' 9Sg A A-1 C1 258 d. 43.97 43.88 2.64 Cyanomethyl 2.66 36.63 36.99 A-1 A c1 176 49.98 49.98 4.19 Allyl 4.46 29.15 29.11 93O A Br 17gh A-1 49.75 5.19 49.46 5 . 4 4 28.84 28.86 Propyl I A 88' A- 1 239.5 49.46 49.59 5.19 5.15 28.84 28.98 Isopropyl Br '4-2 72 A' 42. 84 ca. 200 d. 42.76 4.11 4 . 2 5 28.55 28.59 2-Hydroxyethyl A- 1 c1 8.Y A 184.5 46.14 46.11 3 . 8 7 3.89 26.91 26.85 Acetonyl I 93.5' C, D A- 1 152' 51.90 51.89 5 . 8 1 5 . 8 9 26.90 26.99 Butyl c1 A- 1 87' A 40.18 264 dec. 40.60 3.37 3.54 33.47 33.62 Carbamoylmeth yl c1' A-2 9Bg*" A Chars >260 39.99 39.97 2.88 2 . 8 5 26.65 26.41 Carboxymethylk A-1 4Xg None 277-279 d." Br 39.16" 38.81 3.29 3.03 26.10 25.97 2-Chloroethyl A-2 E 228 9Sg Br 54.52 54.56 5.49 5 . 6 1 25.43 25.61 Cyclopentyl A- 1 93' F 115. 5p Br 54.02 53.06 6.35 6.39 25.20 25.22 Pentyl A- 1 80° E 169 d. 42.84 43.12 3.60 3 . 6 8 24.99 24.83 Methoxycarbonylmethyl Br 7307 A 2-Carboxyethyl C1' A-2 227 d.' 42. S i 42.58 3.60 3.59 24.99 24.66 2-Ethoxyethyl Br G, A 48.21 69" A- 1 143 47.86 5 . 3 3 5.33 24.99 24.73 2-Chloroallyl c1 A-1 90g A 42.38 .?2.56 3.11 3 . 2 2 24.72 24.57 173 86 A, H B 193' c1 4.24 Benzyl 59.50 59.57 4.16 A-1 2-Thenyl c1 99g I 48. 37 48. 62 3 . 2 5 3.36 22.56 22.51 186 I 4-1 60. 91 Piienethyl 9gg G 166.5 61.02 4.72 '4.70 21.86 21.86 B 161 :1.81 21.45 21.66 55.50 55.36 3.4G o-Fluorobenzyl c1 70 Xone B m-Fluorobenzvl c1 159 84 None 55.50 55.27 3 . 4 6 3 . 6 2 21.45 21.26 B p-Fluorobenzyl c1 3 . 7 7 21.45 21.68 229 86 None 55.50 55.49 3.4G A-1 J 87, 100" Octyi I 97O 59.05 59.11 7 . 6 3 7.66 21.19 21.40 '4-1 9gc I 210 Cinnamyl" c1 62.58 .4.51 4 . 5 3 20.89 20.63 62.68 c1 A- 1 102' E 76 57.58 3.7:: 170 Benzoylmethyl 3.70 20.73 20.34 2-Phenoxyethyl Br A-1 103' G 154 57.35 57,45 4.4.4 4..57 20.58 20.73 €3 78 Sone 202 52.20 52.02 3.2G c1 o-Chiorobenzyl 3 . 3 8 20.30 20.08 A-1 o-Chlorobenzyl c1 loog K 203 B 201.5 c1 60 C $2.20 52.15 3 . X 3.36 20.30 20.29 p-Chlorobenzyl A-1 B 94O p-Chlorobenzyl c1 20 1 50.1s B 262 50.32 3.16 2.98 21.38 24.27 p-Nitrobenzyl c1 76 None" Decyl I 61.60 61.64 8.27 8.11 19.16 18.68 9Sg J 95, 102" A-1 104O B 222 45.21 2.59 45.31 2.6,5 18.01 18.18 3,4-Dichlorobenzyl c1 A-l Dodecyl Br 63.70 63.49 8.S1 8.:',2 17.48 17.46 A-1 99O J 98 A, water; B, methanol; C, methanol-water; D, chlorobenzene; E, ethyl acetate: 17,carbon tetrachloride: G, benzene; 1-1, toluene; I, 2-butanone; J, Skellysolve C ; K, chloroform. * Taken on Iiofler Heizbank except where indicated. An Lit.5 m.p. 185'. .4nalyses give:i for product, m.p. exact 1: 1 molar ratio of K2C03 to 6-mercaptopurine hydrate used. 196". prepared by modificatioil of method B. f Sensitive to light. 0 A 1.12: 1.00 molar ratio of K2COato 6-mercaptopurine hydrate used. Lit.5 m.p. 163-165'. Crystallized from water as hydrated plates,and needles; water of crystallization Reported by removed by evaporating a methanolic solution to dryness in vacuo over PzOsa t 80". 1 Lit.5 m.g. 126-127". G. Huber, Angew. Chem., 68, 706 (1956). Sodium chloroacetate. Isolated a t PH 4. " Capillarj-, aluminum biock. Calcd.: C1, 16.9; found: C1, 16.7. p Lit.6 m.p. 78-79', Sodium P-chloropropir)rlate. ' Isolated at PI3 1. A liigl1er yield (%To)of less pure crude product was obtained from a reaction carried o u t in dimethyl sulfoxide. On Fisher- Johns Lit. block with moderate rate of heating. Fast heating on KofleroHeizbank gives decomposition point as high as 260'. ni.p. 178-180',6 193-194°.12 " Amorphous form melts at 87 , crystallizes and remelts a t looo, lit.6 m.p. 78-80". " V in ethylene; c j . W. Ziegenbein and W. Franke, Chem. Ber., 90, 2291 (1957)); No -CHC I I ~ . - 970 ~ : (trans-1,2-disubstituted Amorphous form melts CH2absorption at 995-955,915-905, 1420-1410. " Can be recrystallized from glacial acetic acid. a t 95", crystallizes and remelts at 102", lit.6 m.p. 84-85'. 0
not necessarily supported by, for example, the retentioii of activity by 6-phenylthiopurine and the loss of activity by 6-(methoxycarbonylmethylthio) purine. The DreDaration of a number of 6-alkylthiopu-
priate alkyl halides with G-mercaptopurine in aqueous alkaline solution, alcohol being added to increase the solubility of the less soluble halides. This procedure was patterned after that of Elion, Burgi and Hitchinas' for the methylation of 6 -
( 5 ) C. G . Skinner, W. Shive, R. G. Ham, D. C. Fitzgerald, Jr., and I
