J. Med. Chem. 1989,32,799-807
ported by the Auckland Division of the Cancer Society of New Zealand and by the Medical Research Council of New Zealand. Registry No. 5,59292-04-5; 6,30087-31-1; 7, 118537-75-0;8, 35614-21-2; 9, 88521-78-2; 10, 117570-56-6; 11, 117570-57-7; 12, 118537-76-1; 13, 117570-45-3; 14, 117570-46-4; 15, 117570-58-8; 16,118575-12-5; 17,118537-77-2; 18,118537-783; 19,117570-59-9; 20,118537-79-4;21,117570-47-5; 22,117570-48-6; 23,117570-49-7; 24,117570-64-6;25,117570-63-5; 26,117570-50-0; 27,117570-51-1; 28,117570-52-2;29,117570-70-4; 30,117570-71-5;31,117570-72-6; 32,117570-74-8; 33,117570-75-9; 34,117570-73-7;35,117570-76-0; 36,117570-77-1;37,117570-78-2; 38,117570-83-9;39,107517-48-6; 40,117571-26-3; 41, 117571-27-4; 42,118537-80-7; 43,43160-03-8; 44,117570-84-0; 45,117570-85-1; 46,117570-86-2;47,117570-89-5; 48,117570-90-8; 49,69200-01-7; 50,117571-23-0; 51,117570-91-9; 52, 69199-67-3; 53, 117570-92-0;54, 59292-09-0; 55, 59292-12-5; 56,54160-09-7; 57, 30087-35-5; 58,118537-81-8; 59, 118537-82-9; 60, 26539-21-9; 61, 54182-77-3; 62, 55950-72-6; 63, 117570-95-3; 64,117570-96-4;65,117571-25-2;66,117571-285; 67,117571-29-6; 68,43160-04-9; 69,43160-06-1; 70,117570-97-5; 71, 117570-98-6; 72,117570-99-7; 73,117571-00-3;74,117571-01-4; 75,117571-02-5; 76,117571-03-6; 77,117571-31-0; 78,117571-32-1; 79,15128-43-5; 80,117570-80-6;81,117570-81-7; 82,117570-82-8; 83,117571-12-7; 84,117571-13-8;85,117571-14-9;86,117571-15-0; 87,117571-16-1;
799
88,117571-17-2;VI (R2= 5’-CH3), 117571-38-7; VI (R2= 3’-CH3), 118537-92-1;VI (R2= H), 117571-22-9;VI (R = 4-CH3), 3869277-2; VI11 (R = 4’-OCHJ, 118537-90-9; VI11 (R = 4’-C1), 118537-91-0;XI1 (R = 4-CH3), 117571-33-2;XI11 (R = 4-CH3), 117571-34-3;XIV (R = 4-CH3), 117571-35-4;XV (R = 4-CH3), 117571-36-5; XVI (R = 4-cH3), 117571-37-6; sodium 2,4-dichlorobenzoate,38402-11-8; sodium 2-methylphenolate, 4549-72-8; sodium o-chlorobenzoate, 17264-74-3;sodium 2-chloro-3-methoxybenzoate, 118537-83-0; sodium 2,3-dichlorobenzoate, 118537-84-1;sodium 2-chloro-3-nitrobenzoate, 118537-852;sodium 2-chloro-4-methylbenzoate, 118537-86-3; sodium 2-chloro-4methoxybemoate, 118537-87-4;sodium 2-chloro-5-methylbenzoate, 118537-88-5; sodium 2-chloro-5-methoxybenzoate, 118537-89-6; sodium 2,5-dichlorobenzoate, 63891-98-5; sodium 2-chloro-5nitrobenzoate, 14667-59-5;sodium 5-chloro-2-methylphenoate, 40495-68-9; sodium 3-chloro-2-methylphenolate,118537-93-2; sodium o-methylphenolate, 4549-72-8; potassium 2-iodo-6methylbemate, 117571-21-8;2-chloro-4nitrobemic acid, 99-60-5; 5,5’-dinitrobiphenol-2,2’-dicarboxylic acid, 92159-34-7; potassium 2-chlorobenzoate, 16463-38-0; sodium 2-allyl-4-methylphenolate, 118537-943;2-(2-allyl-dmethylphenoxy)benzoicacid, 117571-24-1; 4-methylsalicylic acid, 50-85-1; benzyl bromide, 100-39-0; 6methylsalicylic acid, 567-61-3; &methyL2-(phenylmethoxy)benzoic acid, 118537-95-4;[6-methyl-2-(phenylmethoxy)phenyl]acetic acid, 118537-96-5; (2-hydroxy-6-methylpheny1)aceticacid, 38692-76-1.
2’-Fluorinated Isonucleosides. 1. Synthesis and Biological Activity of Some Methyl 2’-Deoxy-2’-fluoro-2’-pyrimidinyl-~-arabinopyranosides M. Bobek,*st S.-H. An,t D. Skrincosky,t E. De Clercq,* and R. J. Bernackit Grace Cancer Drug Center, Roswell Park Memorial Institute, New York Department of Health, Buffalo, New York 14263, and Rega Institute for Medical Research, Katholieke Universiteit, Leuven, B-3000, Leuven, Belgium. Received January 4, 1988
New reactions of methyl 2,2-difluoroglycosides are described that were utilized for synthesis of some novel nucleoside (2) derivatives. Thus, treatment of methyl 2-deoxy-2,2-difluoro-3,4-O-isopropylidene-a(~)-~-erythro-pyranoside with anhydrous HC1 resulted in selective displacement of one fluorine atom with chlorine to give a 2-deoxy-2chloro-2-fluoro glycoside 3. Reaction of 3 with silylated uracil in the presence of SnCL provided a 2-deoxy-2fluoro-2-uracil-substituted glycoside 4. 2-Fluoro-2-deoxy glycosides substituted with other pyrimidines at C-2 were prepared similarly by the reaction of acylated 2,2-difluoro or 2-fluoro-2-bromo derivatives (5 and 6, respectively) with silylated pyrimidines. The resulting 2’4uorinated isonucleosides were evaluated for their antitumor and antiviral activities. Compounds 7a,b, 8a,b, and 10a,b demonstrated 50% tumor cell growth inhibition in vitro (IC,) at laCIOd M. At similar concentrations no antiviral activity was observed in vitro. Therapeutic activity was obtained with 7a,b and 8a,b in DBA/2 mice with L1210 leukemia. Administration of 7a,b at 500 mg/kg, ip daily, for 5 consecutive days, resulted in a 55% increase in life span (% ILS) while administration of 8a,b in the same manner at 200 mg/kg caused a 29% ILS. Treatment with 7a,b to mice with drug-resistant L1210 sublines (5-FU and araC) resulted in 22 and 57% increases in life span, respectively. Lewis lung carcinoma and M5076 sarcoma in mice also responded to the administration of 7a,b with reductions in tumor growth for both tumors and significant increases in life span in mice with Lewis lung carcinoma. Although the mechanism of action of 7a,b is not known, it has been found to be a relatively fast-acting, cell-cycle nonspecific cytotoxic agent that decreases [3H]deoxyuridineincorporation, blocks L1210 cells at the G2phase of the cell cycle, and is not reversed by exogenous thymidine. These 2’4uorinated isonucleosides have demonstrated biological activity and may have potential as antitumor drugs.
As part of our investigation of gem-difluoro monosaccharides,’ we recently reported a selective nucleophilic displacement of fluorine in methyl 2,2-difluoro glycoeides.2J Various nucleophiles, including C-substituents and heterocyclic bases can be introduced at C-2 of methyl glycosides, the products of this reaction being novel carbohydrate derivatives substituted, at (3-2, with fluorine and the entering substituent. In this paper, we describe the synthesis and the results of initial antiviral and antitumor evaluation of some 2’-fluor0 analogues of methyl 2’-pyrimidinylarabinopyranosides. Chemistry. Methyl 3,4-O-isopropylidene-@-~-arabinopyranoside4t6was oxidized by a chromium trioxide-pyridine-acetic anhydride complexs to give methyl 3,4-o-isot
Roswell Park Memorial Institute. Katholieke Universiteit.
propylidene-~-~-erythro-pentopyranosid-2-ulose (1, Scheme I) in 84% yield. While compound 1 was previously prepared by similar oxidation using different the present method gave improved yields and was found better suitable for large-scale preparations. Fluorination (1) Sharma, R. A.; Kavai, I.; Fu, Y.-L.; Bobek, M. Tetrahedron Lett. 1977, 3433. ( 2 ) An, S.-H.; Bobek, M. Tetrahedron Lett. 1986, 27, 3219. (3) Bobek, M.; An, S.-H.; Skrincosky, D.; De Clercq, E.; Bernacki, R. Nucleic Acids Res., Symp. Ser. 1987, No. 18, 5. (4) Pratt. J. W.: Richtmver. - . N. K.: Hudson, C. S. J. Am. Chem. SOC.
ism, 74,2200.
(5) Rosenthal. A.: SDrinzl. M. Can. J. Chem. 1970. 48. 3253. (6) Garegg, PI J.; Skuelson, B. Carbohydr. Res. 1978, 67, 267. (7) Onodera, K.; Hirano, S.; Kashimura, N. J. Am. Chem. Soc.
1965,87, 4651.
(8) Sowa,W.; Thomas, G. H. S. Can. J. Chem. 1966, 44, 836.
0022-2623/89/ 1832-0799$01.50/0 0 1989 American Chemical Society
800 Journal of Medicinal Chemistry, 1989, Vol. 32, No. 4
Bobek et al.
qH3-
Scheme I
Scheme I1
-2
P O C H ,
f0
-
R'O O
O
C
H
;
-
R'O(-3OCH.
F
OR'
OR'
F
-2
-1
0
0
A)
0 Q0CH3
4 0
___c
I
F
0 0 OCH,
lo4 M, Table V) but demonstrated tumor cell growth inhibition against the L121OIBdUrd leukemia line and other tumor cell lines including FM3A/O, Raji/O, and Molt/4F. The appearance of growth-inhibitory activity for 9a,b, the thymine derivative, against the L1210/BdUrd line again suggests a mechanism of drug action for this class of growth-inhibitory agents not dependent on kinase activity. Exchanging the 0-acetyl groups with 0-hexanoyl (10a,b) resulted in altered antitumor activity; however, blocking the 3'- and 4'-positions on the sugar with the isopropylidine group (4) resulted in a complete loss of activity. The novel nucleoside derivatives (7a,b, 8a,b, lOa,b) containing esterified substituents on the sugar moiety are lipophilic compounds. This lipophilicity probably allows these compounds to freely enter tumor cells by passive diffusion. A study of the time dependence of growth inhibition demonstrated that 7a,b was growth inhibitory following a relatively short incubation period (less than 1 h). This is in contrast to the bioactivity of 5-FdUrd, which is a highly potent agent (ICN < M) when incubated with L1210 cells for 48 h, but requires long exposure times (>6 h) to demonstrate any growth-inhibitory effect on L1210 leukemia cells. There were other apparent differences noted in the mechanism of drug action between the 2'-fluoro nucleoside derivatives and 5-FdUrd. Inhibition of precursor incorporation by 8a,b and 7a,b was not similar to that by 5FdUrd. No differential sensitivity to 7a,b was observed between log-phase and plateau-phase L1210 cells while exponential-phase cells exhibited greater sensitivity to 5-FdUrd than plateau phase cells. Additionally, a G2 L1210 cell cycle block was observed with 7a,b while 5FdUrd caused an S-phase block. Exogenous thymidine reversed 5-FdUrd while it had little effect on 7a,b. The mechanism of action for these novel nucleosides is unknown at present and is under current investigation. It is of interest that G2 block is characteristic for DNA damaging agents which have similar cytotoxicity for exponential- and plateau-phase culture^.^"^^ G2arrest in
the cell cycle reflects DNA and chromosome damage by drugs.28 Antitumor activity for 7a,b in mice with tumors was modest. Administration of 100-500 mg/kg per day X 5 to mice with various tumors resulted in decreased tumor sizes and increased mouse life spans. These results suggest that these agents may have therapeutic potential as antitumor agents. Experimental Section Thin-layer chromatographywas performed on EM Science silica gel 60. The column chromatography was carried out with silica gel (230-400 mesh) from E. Merck Industries Co. All melting points were taken on a Mel-Temp capillary point block and are not corrected. The optical rotations were measured on a Perkin-Elmer polarimeter 241 MC. The nuclear magnetic resonance spectra were determined on a Varian XLlOO and Bruker (200 MHz) spectrometers using tetramethylsilane (TMS) or CFC13 as internal standards. Mass spectral fragmentation was carried out by a Finnigan 4000 GC-MS system 70 (eV) with intermediate resolution. Elemental analyses were carried out by Galbraith Laboratories, Inc., Knoxville, TN, and were within *0.4% of the calculated values. M e t h y l 3 , 4 - 0 -1sopropylidene-B-D-erythro -pentopyranosid-2-ulose (1). Cr03 (129.2 g, 1.29 mol) was added in small portions to a stirred mixture of methylene chloride (1.9 L) and pyridine (209.2 mL) followed by the dropwise addition of a solution of methyl 3,4-0-isopropylidene-~-~-arabinopyranoside~~ (66 g, 0.32 mol) in methylene chloride (500 mL) in a period of 30 min. Acetic anhydride (122.2 mL, 1.29 mol) was added to this mixture and the reaction was completed after stirring at room temperature for 2 h. The mixture was concentrated (-400 mL) and was poured into ethyl acetate (2 L). The slurry mixture was applied to a silica gel column (8 X 60 cm), which was eluted with ethyl acetate. The total effluent (4 L) was collected, evaporated, and dried in vacuo to a crystalline residue (54.7 g, 83.7%),which was recrystallized from ethyl acetate: Ir (KBr) 1740 (C=O), no OH (no hydrate form);5'H NMR (CDClJ 6 4.68 (1, s, H-l), 4.65 (1,d, J3,4 = 5 Hz, H-3), 4.52 (1,dd, 5 4 3 = 5 Hz, J4fi = 1.5 Hz, H-4), 4.23 (1,dd, J5,5, = 12 Hz, J5,4 = 1.5 Hz, H-5), 4.04 (1, d, J5,,5 = 12 Hz, H-5'), 3.49 (3, s, OCH3), 1.44 and 1.39 (6, 2 s, isopropyl); 13C NMR (CDC13+ TMS) S 198.5 (C-2), 110.3 (=CMe2), 100.8 (C-1),77.6 (C-3), 75.3 (C-4) 58.3 (C-5),55.6 (OCH3),27.1 and 26.1 (2 CHd. Methyl 2-Deoxy-2,2-difluoro-3,4-0 -isopropylidene-a(8)D-erythro-pentopyranoside(2). To an ice-cold solution of methyl 3,4-0-isopropylidene-~-~-erythro-pentopyranosid-2-~ose (1,45.16 g, 0.22 mol) in dry benzene (400 mL) was added dropwise a solution of (diethy1amido)sulfurtrifluoride (DAST)s (54 g, 0.33 mol) in dry benzene (100 mL) in a period of 30 min. The reaction mixture was stirred at room temperature for 18 h, diluted with ether (150 mL), and filtered through glass wool. Ice/water (200 mL) was slowly added to the cooled filtrate, and the separated organic layer was washed with saturated NaHC03 solution (200 mL X 3) and water (200 mL X 3) and dried ( N d O a ) . The organic solvent was removed by evaporation and the residue was distilled to give clear yellowish liquid bp 104-104.5 "C (0.2 mmHg); yield 38.5 g (78.1%);MS m/z = 209 (M' - Me); 'H NMR (CDC13) 6 5.2 (1, m, H-l), 4.84 (2, m, H-3, H-4), 4.23 (2, m, H-5,5'), 3.64 (d, JFw = 1.35 Hz, OCH,, a anomer), 3.62 (d, JFw = 1.31 Hz, OCHS, p anomer). Methyl 3,4-Di- 0 -acetyl-2-deoxy-2,2-difluoro-a(~)-~erythro-pentopyranoside (5). Compound 2 (4 mL, 0.021 mol) was shaken vigorously with 15 mL of 95% formic acid at room temperature (22 "C) for 8.5 min. The mixture was immediately evaporated to a syrup under reduced pressure (bath temperature
