1076 Journal ofMedicina1 Chemistry, 1974, Vol. 17, No. 10
Kanai, et a / .
Pyrimidine Nucleosides. 6. Syntheses and Anticancer Activities of N4-Substituted 2,2’-Anhydronucleosides Tadashi Kana$* Motonobu Ichino, Research Laboratories, Kohjin Company, Ltd., Komiyacho, Hachiouji City, Tokyo, Japan
Akio Hoshi, Fumihiko Kanzawa, a n d Kazuo Kuretani National Cancer Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo, Japan. Received October 16, 1973
Some derivatives of N4-substituted 2,2’-c-araC [N4-hydroxy- (2a), N4-methoxy- (2b), ,V4-methyl- (2c), and W-phenyl- (2d)I which are potential carcinostatic nucleosides were prepared by introducing the 2,Z’-anhydro linkage into the corresponding ribonucleosides in one step. Compound 2a was markedly active against leukemia L1210 in the mouse but showed only a weak inhibitory activity against L5178Y cells in culture. Compound 2a gave 110% ILS a t 300 mg/kg/day (5 days) but was toxic at 700 mglkgjday (5 days); 2b and 2d were inactive against leukemia L1210 in the mouse a t 100 mg/kg/day (5 days).
2,2’-cyc~o-l-~-~-~abinofuranosy~cytosine (abbreviated to c-araC) has been shown t o be effective in t h e t r e a t m e n t of mouse leukemia L12101-3 a n d other turn or^^,^ (C1498 leukemia, N F S sarcoma, adenocarcinoma 755, sarcoma 180, Ehrlich ascites carcinoma, lymphocytic sarcoma of Tokuzen Nakahara,S a n d N T F sarcoma’). T h e clinical usefulness of this compound h a s been also reported.8 5HalogenatedSJO a n d 3’,5’-di-O-acyl-c-araCS have been also found to be active against L1210 leukemia in mice. It is known t h a t c-araC is easily hydrolyzed in alkaline media to 1-P-D-arabinofuranosylcytosine;the latter undergoes rapid enzymatic deamination in m a n t o 1-P-Darabinofuranosyluracil (araU), a n inactive m e t a b ~ l i t e . ~ These findings prompted us t o prepare N4-substituted derivatives of c-araC which might be more stable under alkaline conditions. In view of t h e reported biological activity12 of N4-hydroxy-l-~-~-arabinofuranosylcytosine~~ (which was active in vitro against a line of P815 leukemia resistant t o 5-fluorouracil), N4-hydroxy- (2a), N4-methoxy- (2b), N4methyl- (2c), a n d N4-phenyl-c-araC (2d) were of interest as potential chemotherapeutic agents. This paper describes the preparation a n d preliminary biological testing of these compounds (2a-d). Chemistry. T h e direct introduction of hydroxyl or other groups into c-araC a t the N4 position is difficult because of t h e base lability of the 2,2‘-anhydro linkage. It occurred to us t h a t syntheses of 2a-d might be feasible by introduction of the 2,2‘-anhydro linkage into t h e preformed N4-hydroxy- (la),N4-methoxy- (lb), N4-methyl- ( I C ) ,a n d N4-phenylcytidine ( I d ) using the procedures reported previouslys 914 915 (Scheme I). Compounds la a n d l b were easily prepared by the amine exchange between cytidine and hydroxylamine or rnethoxyamine.l6 However, the reaction of araC with hydroxylamine under the same conditions employed for t h e preparation of la gave rise t o a r a U in a fair yield. This different behavior of araC with hydroxylamine might be due to intramolecular catalysis of the 2’-hydroxyl group as proposed by Notari a n d coworkers17 in t h e hydrolytic deamination. It was found t h a t the conversion of cytidine to IC a n d Id proceeded in good yields, using the modified procedure of Curran18 for t h e conversion of 2-methylthio-4-hydroxy6-aminopyrimidine t o 2,6-bis(methylamino-4-hydroxy)pyrimidine. Replacement of t h e 4-amino group of cytidine with methylamino or phenylamino group was achieved by fusing cytidine with methylammonium or phenylammonium acetate a t 160”. Cyclization was effected by heating l a - d with partially hydrolyzed phosphorus oxychloride in a small volume of ethyl acetate to give 2a-d in yields of 3 2 4 4 % .
Scheme I cytidine
--+
KHR
NR
“ O HO H Z COHd
I
HO
1
2
NHOH
+ araC
-HoH2cy( U H OH pH41
I
I
HO
HO 3
R=OH = OCH, c R=CH, d. R = C,H,
a.
b. R
T h e structural assignments for 2a-d rest upon their ultraviolet spectral properties a n d analyses. T h e 2,2‘-anhydro structure of 2 a and 2b was also confirmed by the downfield shift of the n m r signal due to H-2’. T h e nmr spectra of 2a a n d 2b deserve further comment. T h e signals due to H-5 of 2a appeared as a multiplet, a n d those due to H-5 of 2b appeared a t 6.19 a n d 6.17 p p m as a doublet of doublets. On the other hand, there appeared well-resolved doublets d u e to H-6 in t h e n m r , uiz., 8.34 a n d 8.09 ppm for 2 a a n d 7.28 a n d 7.13 p p m for 2b. These doublets are likely due to t h e presence of cis a n d trans isomers rather t h a n t o t h e coupling between H - 5 a n d H-6. T h e ratio of cis-trans isomers was estimated t o b e 3:2 based on the intensity of the H-6 proton for 2a or 2b in the nmr. As expected, compounds 2a a n d 2b were found to be more resistant t o alkaline hydrolysis t h a n c-araC. Nagyvary19 found t h a t large differences existed in the hydrolysis of c-araC 3’-phosphate catalyzed by bicarbonate a n d chloride ions, the bicarbonate ions being good catalysts for the hydrolysis of the 2,2’-anhydro linkage of c-araC. These authors used bicarbonate a n d borate buffer solutions to evaluate the stability of 2a to hydrolysis relative to c araC.
Journal of Medicinal Chemistry, 1974, Vol. 17, No. 10 1077
Pyrimidine Nucleosides
Table I. RfValues of Some Anhydronucleosides a n d Related Arabinonucleosides
Rr values in Solvent A Solvent B
Compd AraC AraU c-AraC 2a 3 2c
0.15 0.28 0.05 0.10 0.17 0.16 0.30 0.26 0.63
2C'a
2d
2d'"
0.65 0.81 0.64 0.72 0.63
d f
"Respective hydrolysate of 2 c a n d 2d with 2 N KOH for 1 hr at room temperature.
Table 11. Effect of Pyrimidine Anhydronucleosides against L1210 Leukemia a n d Cultured Cells (L5178Y)
Compd
% increase in Dose,a life span over mg/kg/day controls* (L1210 x5 leukemia)
c-AraC
100
3
100 30 100 300 500
2a
700 2b 2d
100 100
76 98 64 82 110 95
