January 1966
NOTES
Potentiometric titration of I I b (0.1668 g./100 ml. of 50% ethanol) with 0.0995 A' KaOH gave a pK, of 6.75. The starting reagent, I b (36.2 mg./lOO ml. of water, titrated with 0.025 AV XaOH), had pK,, = 4.36 and pK,, = 8.92. Anal. Calcd. for C12Hlo?\T103:C, 62.60; H, 4.38; N, 12.17; mol. wt., 230.2. Found: C, 62.34; H, 4.49; N, 12.17; mol. art., 232.8. The cyclization reaction was repeated as above except that 40 ml. of 1,1,2,2-tetrachloroethanewas used as the solvent and the refluxing time was reduced to 2 hr. This was the preferable method of preparation, as I I b was more soluble in the boiling qolvent and crystallized in 76% yield upon filtering and cooling. Cycli7ation was effected also in polyphosphoric acid, but the yield of I I b was low. 24Anilinomethyl)-7,7a-dihydro-7a-methyl-lH-pyrrolo[1,2-c] imidazole-l,3,5(2H,6H)-trione(IIc).-This compound was prepared by condensing IIa with equivalent quantities each of formaldehyde and aniline according to the procedure described previously3: yield 65%. ilfter recrystallization from ethanol, it melted at 152-152.5'; ""::A: 287, 240.5 mp ( E 1757, 11,410); CHSOH-HCI mp A,, 282, 239.5 mp ( C 796, 7871); X ~ ~ ~ o H 3 0 N - 286.5,238 KoH ( E 1782,12,708). Anal. Calcd. for C14H15N30J:C, 61.53; H, 5.53. Found: C, 61.73; H, 5.70. In a similar manner 24anilinomethyl)-7,7a-dihydro-7a-phenyl1H-pyrrolo[1,2-c]imidazole-1,3,5(2H,6H)-trione(IId) was prepared from I I b in 867, yield. After recrystallization from ethanol, it melted a t 150-151"; 287, 267, 240 mp ( e 1871, 1073, 13,414); A::ioH-HC' 281.5, 267, 263, 239.5 mp ( E 547, 471, 584,5165); AC,Sf,3°H-KoH 287,231 mp ( E 1841,21,228). Anal. Calcd. for C19HnN303: C, 68.05; H, 5.11. Found: C, 68.25; H, 5.10.
Since 6-hydroxylaminopurine was inhibitory to several mouse tumors and leukemias (Table I) and was toxic, y-p-i,-rihofuraiiosSrl-B-hydroxylaniiiiopuririe (11) was synthesized. It was found to induce a complete inhibition of mouse leukemia P815, but was ineffective on solid tumors. Mice survived the daily administration of 500 mg. of I1 for 2 weeks and a dose of 250 m g for 3 weeks.
Acknowledgment.-The authors wish to thank the National Science Foundation for an undergraduate research grant in support of this study. We also thank Jules Rrandes for his contribution.
Synthesis and Biological Activity of 9-~-D-Ribofuranosyl-6-hydroxplaminopurine LILLIIS J I E D R E K , A.~RON BEKDICH
ALFRED0 GISER-SOROLLA, AKD
Division of Biological Chemistry, Sloan-Kettering Institute for Cancer Research, and Sloan-Keftering Division, Graduate School of Xedzcal Sczences, Cornell L'nzaersity Medical College, S e w York-, A'ezc: York 10021 Received August 13, 1966
The simple derivative of adenine, 6-hydroxylaminopurine,Z has been found to induce mitotic inhibit>ionand nuclear degeneration of mouse Sarcoma 180 cells, but not normal embryo skin fibroblasts, over a concentration range of to lop4 When administered intraperitoneally to rats and mice at a single dose of 500 mg./kg., or at lower dosages over prolonged periods, 6-hydroxylaminopurine proved to be toxic (LDjofor a single dose, 470 mg./kg.).4 (1) This investigation was supported by funds from the National Cancer Institute, National Institutes of Health, Public Health Service (Grant C.1 03190-09) and T h e Atomic Energy Commission (Contract No. AT[30-11, 910) and aided by the G r a n t T-128F from the American Cancer Soriety and the First National City Bank Grant for Research from the American Cancer Society. Presented in p a r t a t the 150th National Meeting of the American Chemical Society, Atlantic City, N. J., Sept. 1965; Abstracts, p. 5P. A. B. is the recipient of a Public Health Service Research Career Award (3-K6-CA-22.533-0161) from the National Institutes of Health. (2) A. Giner-Sorolla and A. Bendich, J . A m . Chem. Soc., 80, 3932 (1958). (3) J. J. Biesele, unpublished results. (4) A. Giner-Sorolia, Ph.D. Thesis, Cornell Ziniversity, 1958; Dissertation Abstr., 20, 1148 (1959).
143
TABLE I EFFECT OF SOVEHYDROXYL ~ I I S O P L R I R E DERIV iTIVES OK ;\IOUSE TCMORS A N D LEI IiEMIAa Dose, mg / k g . / d w
Result
G-Hgdroxrlaminopurine Sarcoma 180 125 125 Mouse carcinoma E0771 Mouse carcinoma E0771 250 Rlouse carcinoma C1021 125 Ridgaay osteogenic sarcoma 125 Ridgwag ohteogenic sarcoma 250 Nouse leukemia P815 250 Mouse leukemia P815 reiistant t o 150 6-mercaptopurine Mouse leukemia P815 resistant to 40 6-mercaptopurine S-p-n-Ribofuranosy1-6-hydrosglnminopurine ( 11) 62, ,5 Sarcoma 180 A h u s e carcinoma E0771 125 Ridgway osteogenic snrcoma 125 1Zouse leukemia B8'2 125 Mouse leukemia B82 62. ;i Mouse leukemia P815 150 RIouse leukemia P815 resistant to 135 6-mercaptopurine RIouse leukemia P815 resistant, t o 90 &thioguanine Mouse leukemia P388 resistant to 90 6-thioguanine a The agents were administered intraperitoneally in physiological saline containing 0.5% carboxymethylcellulose for 2 weeks. -, no effect; &, slight inhibition; +, moderate complete inhibition. Data courtesy of Dr. inhibition; K. Sugiura and C. C. Stock of the Division of Experimental Cheniotherap y .
+++ + + +
++ +,
The synthesis of 9-p-~-ribofuranosyl-6-hydroxylaminopurine (11) was achieved in 90% yield by treatment of 9-p-~-ribofuranosyl-6-chloropurine (I) with an excess of hydroxylamine in ethanol. Compound I1 n7as catalytically hydrogenated to adenosine (111) in 93% yield (Scheme I). Attempts to prepare I1 from either SCHEME I
C'
bH
I
NHOH
OH
bH bH
dH b H
II
m
6-mercapto- or 6-methylmereaptopurine riboside upon interaction with hydroxylamine were unsuccessful; hypoxanthine or inosine were the reaction products.
Experimental Section;
9-fl-i~-Ribofuranosyl-6-hydroxylaminopurine (I1 ).--O-~-I)1~i1~of~rr:iiiosyl-6-chloropurine6 (I, 23:: g., 10 mnioles) was clissolved i n an ethanol solution of Iiyctrosylariiine 1350 nil., prcpared as indicated in ref. 2 ) , heated a t 50" for 6 hr., and then kept a t 25" overnight. The crystalline product which deposited WLS rollected and washed with a little cold water and then with ethanol to yield 2.58 g. (go?), m.p. 210-213'. T'pon recrystal: 21s'. [ e ] ?aii lization from methanol, colorless thin i i o t ~ i l ~ >1n.p. -57" (c 0.5, water), were obtained. :Incd. Calcd. for CloHlaNjOL:C', 42.40; H, 4.59: S , 24.73. Foruid: C', 12.10; 13, 4.86; ?;, 24.54. The product (TI) gave positive FeC,ls aiid p1iouplioniolyl)d:tle tests, both indicative of the hydrosylaniino functioii. I1 \\-a$ recovered iinchanged after 3 hr. boiling in water (10 nnd after heating the same soliltion at 125' iii an aut hr. The solubility of I1 was 6.9 g./l. of water a t 25 =t1 Ultraviolet Spectral Properties.-Compound I1 exhibited a t p l l 1.4, A,,,,x 265 m p ( E 17.7 X l o 3 ) ; a t pH 6.7 (phosphate buffer), A,, 265 nip ( e 14.3 X l o 3 ) ; a t pH 12.2, Xmar 252 mp,i shoulder a t 310 nip; a t pH 1.4, A,,,!, 232 nip ( B 3.31 X l o 3 ) ; a t pH 6.7, X,,i,, 230 l l l p 4.65 x 1 0 3 ) ; R I I :It ~ p~ 12.2, xmi,, 230 IIW; PK,, = :%.li 0.1, pK,,= 9.7 t 0 . 1 . x Hydrogenation of II.--!)-fl-~-r~il~oEiiraiiosyl-G-h~dro piiriiie (11, 40.0 nig., 0.14 mmole) XIS dissolved in 95' ethanol ( I t ) nil.), 5' platinum-ori-charcoal catalyst was added, and the suspension was hydrogenated at 1 atni. at 25". .ifter iiptalie of the theoretical volmne of Ht, the suspeiisioir was filtered, the catalyst was washed with a little ethanol, and tho combined filtrate3 were evaporated t o dryness under rediiced pressure. The residue was wished with 1 ml. of ethanol talline prodnct waq obtained (34.2 mg., WC,,), n1.p. 23223.1". The product, which no longer gave the Feel3 arid phosphoniolybdate tests, was identifie iosine (111)by its inisture nieltiiigpoiiit, ultraviolet hpectra at tfitfereiit pH valiies, : ~ i i d from ltf vdiies iti several .solvent
'.
(.>) Ultraviolet absorption spectra \rere determined with a Cary recordinc npectrolhotometer, ,\lode1 11. Paper chromatograms \\-ere run by I I I P ascending metlioil on Schleicher and S c h ~ i e l lXu. 1 paper in the follo\ring solvent s p t e m s : n n t e r saturated u itli I-butanol; 1-butanol saturated 7vitIi \rater (n ith or u i t h o u t 10:; ammonia) ; 1-butanol-formic acid\ratf'r ( i i :IO: 13. v.,'v.). .\Ielt,ing points \rere taken in a Thomas-€1007-er TTnimelt apparatus and were corrected. The microanalysis \vas carried (lilt by Spang .\Iicroanalytical Laboratory, Ann Arbor, ,\lich. (6) G. 1%. Brown and V, S. Keliky, .I. B i d . C h e m . , '204, l0lY ( 1 hupplied by Cj-clo Chemical Corp., Los . 1 2 .
IIId
N-Substituted Ureidobis(1-aziridiny1)phosphine Oxides
Although some K-wb3titutecl ureidobis(l-aziridiii~1)phosphine oxidevas obtained by Pesticides Research Grniiii, E. TI. Rquibir Co., Division of the Olin llathieson Chemical Corri. .'I c i m i i r e heneive publication covering these data will appear separately. (4) 11. RLtz and C . Grundmann, U. S. Patent 2,858,ROB (Oct. 28, 195X): R. Ratz, E. Koher, c'. Griindmann, and C , . Ottmann. I n o r y . Clism., 3, 7'57 t1064). ( 5 ) .\. T, I\irsanu\- a n d (1961
>.
SI. S, ,\larmt.t*, .I. Gcn, C h m . f . S S I : , 31, I 4 ! X
