J.Med. Chem. 1983,26, 1153-1158
1153
Synthesis and Biological Evaluation of Neutral Derivatives of 5-Fluoro-2/-deoxyuridine5/-Phosphate David Farquhar,*-t Nancy J. Kuttesch,? Michael G. Wilkerson,+ and Tammo Winkled Department of Developmental Therapeutics, The University of Texas M. D. Anderson Hospital and Tumor Institute a t Houston Texas Medical Center, Houston, Texas 77030,and Ciba-Geigy AG, CH4002 Basel, Switzerland. Received July 9, 1982
5-Fluoro-5~-(2-oxo-1,3,2-oxazaphosphorinan-2-yl)-2'-deoxyuridine (la) and 5-fluoro-5~-(2-oxo-1,3,2-dioxaphosphorinan-2-y1)-2'-deoxyuridine(lb) were prepared by reaction of 5-fluoro-2'-deoxyuridine(7a) and phosphoryl chloride with 3-amino-1-propanol and 1,3-propanediol, respectively. The thymidine analogues, IC and ld, were prepared similarly from thymidine. Compound l b was synthesized in better yield from 13a and trimethylene phosphate with triphenylphosphine/diethylazodicarboxylate as a condensing agent. Compounds l a 4 were resistant to degradation by 5/-nucleotidase, alkalinephosphatase, venom phosphodiesterase, and crude snake venom. None of these compounds were significantly biotransformed when incubated with mouse hepatic microsomal preparations in the presence of an NADPH-generating system. When administered intraperitoneally (ip) for 5 consecutive days, la was nearly as effective as 5-fluorouracil at prolonging the life spans of BDFl mice implanted intraperitoneally with leukemia P-388. However, much larger dosages of la were required for optimal activity. Compound l b administered similarly was only marginally effective. Neither la nor l b was active against a P-388 mutant resistant to 5-fluorouracil.
5-Fluorouracil (5-FU, NSC-19893) is used extensively, Scheme I often in combination with other drugs, in the palliative Cytochrome P 4 W mixed-function management of a number of human neoplasms, including x o oxidase carcinomas of the colon, breast, and In common with most other anticancer drugs, resistance to 5-FU NADPH / 02 constitutes a serious and vexing clincal problem. Thus, OH OH although 5-FU is the most effective single agent for the treatment of disseminated colon cancer, only 20% of pala, X = NH 2a, X NH b,X-0 b,X-0 tients with this disease achieve an objective r e s p ~ n s e . ~ ~ ~ Despite continued drug treatment, most, if not all, of these patients eventually relapse. Notwithstanding its widespread clinical use, the precise molecular mechanism of antitumor action of 5-FU has not been determined. However, it is generally accepted that the cytotoxicity of the drug is due to one or both of the following biochemical OH OH mechanisms: (1)biotransformation to 5-fluoro-2'-deoxy3s,X-NH 5 4a,X-NH b,X=O b,X-0 uridylic acid (5-FdUMP), which, in the presence of NSJV1°-methylenetetrahydrofolicacid binds covalently to 4-hydroxycyclophosphamide. This metabolite exists in thymidylate synthetase and inhibita de novo thymidylate equilibrium with aldophosphamide, its ring-open tautom(2) biosynthesis and, consequently, DNA transformation to 5-fluorouridine 5'-triphosphate (5-FUTP), which can be incorporated into mRNA, tRNA, or (1) C. Heidelberger in "Cancer Medicine", J. F. Holland and E. rRNA, with possible alteration of RNA structure and Frei 111, Eds., Lea and Febinger, Philadelphia, 1973, pp function."'6 Although the biochemical pharmacological 768-791. determinants of clinical antitumor response to 5-FU has (2) C. Heidelberger, Handb. Exp. Pharmacol., 38,193-231 (1974). (3) G.Bonadonna, A. Rossi, P. Valagussa, A. Banji, and U. Veronot been established, several impressive correlates have nesi, Cancer, 39,2904 (1977). been reported in experimental tumor system^.'^^^ A (4) C. G.Moertel in ref 1, pp 1597-1627. is that therapeutic response correlates frequent (5) C. G. Moertel, N . Engl. J. Med., 299, 1049 (1978). with the persistence of 5-FdUMP in tumor cells. Con(6) L. Bosch, E. Harbers, C. Heidelberger, Cancer Res., 25,977 versely, resistance appears to be associated with the rapid (1958). clearance of this metabolite from tumors. This resistance (7) R. J. Langenbach, P. V. Danenberg, and C. Heidelberger, mechanism cannot be overcome merely by administering Biophys. Res. Commun., 48,1565 (1972). (8) D. V. Santi, C. S. McHenry, and H. Sommer,Biochemistry, 13, 5-FdUMP. First,this metabolite is anionic at physiological pH and, therefore, is unlikely to penetrate into ~ e l l s . ~ ~ ~ 471 ~ ~(1974). (9) H. G.Mandel, Prog. Mol. Subcell Biol., 1, 82-135 (1969). Second, it is susceptible to rapid dephosphorylation by (10) D.S.Wilkinson, A. Cihals, and H. C. Pitot, J. Biol. Chem., 246, phosphohydrolases in plasma and in other tissues. To 6418 (1971). circumvent this problem, we have attempted to devise a (11) U. Stenram, A. Bengtsson, and R. Willen, Cytobios, 5, 125 strategy to generate 5-FdUMP directly within cells that (1972). (12) D. S.Wilkinson, T. D. Tisty, and R. J. Hanas, Cancer Res., 35, obviates the requirement for intracellular anabolism from 3014 (1975). 5-FU or the corresponding ribo- or deoxyribonucleoside. (13) R. I. Glaxer and A. L. Peale, Mol. Pharmacol., 15,270 (1979). Our approach is based upon the mechanism of action of (14) C. K.Carrico and R. I. Glazer, Biochem. Biophys. Res. Comcyclophosphamide. mun., 87,664 (1979). Cyclophosphamide is not independently c y t o t o ~ i c . ~ ~ (15) ~ ~ ~C. K.Carrico and R. I. Glazer, Cancer Res., 39,3694 (1979). It is biotransformed in vivo predominantly by hepatic (16) D. Kessel and T. C. Hall, Science, 145,911 (1966). (17) B.Ardalan, J. S. MacDonald, D. A. Cooney, and P. S. Schein, cytochrome P-450 dependent mixed-function oxidase32to
c"rocw
-
'
+HoustonTexas Medical Center. AG.
t Ciba-Geigy
Bull. Cancer, 66,55 (1979). (18) M. Chadwick and W. I. Rogers, Cancer Res., 32,1045 (1972). (19) C. E. Myers, R. C. Young, D. G. Johns, and B. A. Chabner, Cancer Res., 34,2682 (1974).
0022-2623/83/1826-1153$01.50/00 1983 American Chemical Society
1154 Journal of Medicinal Chemistry, 1983, Vol. 26, No. 8
Farquhar et al.
Scheme I1 H
---
la X b: X c, X d, X -
ea, x
b, X
N
L
-- -- - -
NH, R F, R’ = H 0, R F, R’ H NH, R - CH3, R‘ H 0, R -CH3 R‘ - H COCH 0, R F, 0, R CH3, R’ codH3
er.3334 Aldophosphamide is chemically unstable and spontaneously dissociates via an E2 elimination mechanism to give phosphorodiamidic mustard and acrolein. Substantial evidence has accumulated that phosphorodiamidic mustard is the metabolite responsible for the antitumor activity of c y c l ~ p h o s p h a m i d e . However, ~~~ 4-hydroxycyclophosphamide and aldophosphamide probably contribute significantly to the antitumor selectivity of the drug. Although the precise roles of these metabolites have not h e n established, several plausible hypotheses have ~~~~~
~
(20) C. E. Myers, R. C. Young, and B. A. Chabner, J. Clin. Znoest., 56, 1231 (1975). (21) M. Chadwick and C. Chang, Cancer Treat. Rep., 60, 845 (1976). (22) C. E.Myers, R. Diasio, H. H. Eliot, and B. A. Chabner, Cancer Treat. Rep., 3, 175 (1976). (23) B. Ardalan, M. D. Buscaglia, and P. S. Schein, Biochem. Pharmacol., 27,2009 (1977). (24) P. Klubes, K. Connelly, I. Cerna, and H. G. Mandel, Cancer Res., 38, 2325 (1978). (25) H. G. Mandel, P. Klubes, and D. J. Fernandes, Bull. Cancer, 66,49 (1979). (26) B.Ardalan, D. A. Cooney, H. N. Jayaram, C. K. Carrico, R. I. Glaser, J. MacDonald, and P. S. Schein, Cancer Res., 40,1431 (1980). (27) Y. M. Rustum, L. Danhauser, and G. Wang, Bull. Cancer, 66, 44 (1979). (28) K. C. Liebman and C. Heidelberger, J. Biol. Chem., 216,823 (1955). (29) P. M. Roll, H. Weinfeld, E. Carroll, and G. B. Brown, J. Biol. Chem., 220,439 (1955). (30) G. E.Foley, 0. M. Friedman, and B. P. Drolet, Cancer Res., 21,57 (1961). (31) N.Brock, and H.-J. Hohorst, Arzneim-Forsch., 13,1021 (1963). (32) D. L. Hill, W. R. Laster, and R. F. Struck, Cancer Res., 32,658 (1972). (33) A. Takamizawa, S.Matsumoto, T. Iwata, Y. Tochino, K. Katagiri, K. Yamaguchi, and 0. Shiratori, J.Med. Chem., 18,376 (1975). (34) C. Fenselau, M. N. Kan, S. Subba Rao, A. Myles, 0. M. Friedman, and M. Colvin, Cancer Res., 37,2538 (1977). (35) M. Colvin, C. A. Padgett, and C. Fenselau, Cancer Res., 33,915 (1973). (36) R. F. Struck, M. C. Kirk, M. H. Witt, and W. R. Laster, Biomed. Mass Spectrom., 2,46 (1975). (37) C. Fenselau, M. N. Kan, S. S. Rao, A. Myles, 0. M. Friedman, and M. Calvin, Cancer Res., 37,2538 (1977). (38) B. E.Domeyer and N. E. Sladek, Cancer Res., 40,174 (1980).
been One important role may be the transportion of the hydrophilic phosphordiamidic mustard across cell membra ne^.^^^^ If this postulate is correct, then the mechanism potentially can be exploited to transport other ionic organophosphates,such as 5-FdUMP, into cells (Scheme I). Thus, biooxidation of the cyclic 5’-phosphoramidate la in a manner similar to cyclophosphamide would give the hydroxy analogue 2a, which, conceivably, could penetrate cell membranes by passive diffusion or facilitated transport and then ring open to the acyclic analogue 3a. Elimination of acrolein from 3a would yield the phosphoramidate 4a, which could be converted to 5-FdUMP either by chemi~ a l or~ 9 ~ ~ hydrolysis. A similar oxidationelimination sequence beginning with the cyclic 5’phosphotriester l b would yield 5-FdUMP directly. A preliminary account of this work has appeared.48 Chemistry. Our approach to the synthesis of la and lb (Scheme 11)is based upon Yoshikawa’s method for the preparation of 5’-nucleotides from unsubstituted nucleoside~!~ Thus, 5-FUdR (7a) was reacted with partially hydrolyzed phosphoryl chloride in triethyl phosphate, and the product was treated in situ with a solution of 3-amino-lpropanol (6a) in chloroform in the presence of triethylamine. The crude reaction product was desalted by acetone precipitation and by ion exchange on DEAE-cellulose (bicarbonate form), and the neutral residue was chromatographed on silica to give unchanged 5-FUdR and a product that appeared homogeneous by TLC. However, analysis of the product by HPLC in the reverse-phase mode (p-Bondapak C18) showed that it consisted of a mixture of four components in the ratio 5:5:1:1. These components were separated by reverse-phase semipreparative chromatography. The UV spectrum of each component was almost identical with that of 5-FUdR. The mass spectra of the trimethylsilyl derivatives50 showed similar fragmentation patterns, each with a molecular ion at m / e 581 corresponding to the trisubstituted parent compounds. The ‘H NMR spectra at 360 MHz indicated that these compounds were diastereomeric pairs of positional isomers derived by substitution of the cyclic phosphoramidate moiety at the 5’- and 3’-hydroxyl groups. The diastereomers arise from the presence of a chiral phosphorus atom in these compounds. The spectrum of 5FUdR in Me2SO-d6shows the 3’-OH and the 5’-OH resonances as a one-proton doublet (JHOCH = 4 Hz)and a (39) N. E.Sladek, Cancer Res., 33,1150 (1973). (40) T.A. Connors, P. J. Cox, P. B. Farmer, A. B. Foster, and M. Jarman, Biochern. Pharmacol., 23, 115 (1974). (41) N.Brock, Cancer Treat. Rep., 60,301 (1976). (42) H.-J. Hohorst, U.Draeger, G. Peter, and G. Voelckler, Cancer Treat. Rep., 60,309 (1976). (43) T.Wagner, D. Heydrich, G. Voelcker, and H.-J. Hohorst, J. Cancer Res. Clin. Oncol., 96,79 (1980). 80,3752 (44) R.W. Chambers and J. G. Moffat, J. Am. Chem. SOC., (1958). (45) R. L. Letsinger and W. S. Mungall, J. Org. Chem., 35, 3800 (1970). (46) M. Smith, G.I. Brummond, and H. G. Khorana, J.Am. Chem. SOC., 83,698 (1961). (47) K. C. Tsou and K. F. Yip, Life Sci., 13,1505 (1973). (48) D. Farquhar, N. Klein, J. Liehr, and T. L. Loo, “Abstracts of Papers”, 177th National Meeting of the American Chemical Society, Honolulu, HI, Apr 1979,American Chemical Society, Washington, DC, 1979,Abstr MEDI 32. (49) M. Yoshikawa, T. Kato, and T. Takenishi, Bull. Chem. SOC. Jpn., 42, 3505 (1969). (50) R. G Smith and D. Farquhar, J. Heterocycl. Chem., 17,1659 (1980).
Derivatives of 5-Fluoro-2‘-deoryuridine 5’-Phosphate
one-proton triplet ( J H O C H = 4 Hz) a t 6 5.20 and 5.09, respectively. In the spectra of the two major isomers, which were closely similar, the 3’-OH doublet is present at 6 5.42 (JHpcH. = 4 Hz). However, the 5’-OH triplet is absent, indicatmg substitution at this position. This interpretation is supported by the downfield shift of the 5’-H resonances from 6 3.53-3.67 in 5-FUdR to 3.97-4.09 in the product. The chemical shift of the 3’-H resonances, by comparison, is virtually unchanged (6 4.22-4.27 in 5-FUdR vs. 4.17-4.33 in la). Bonding of the nitrogen atom to phosphorus is indicated by the presence of a multiplet a t 6 5.21 attributable to the NHP(0) proton. The methylene resonances of the cyclic phosphoramide moiety appear as complex two-proton multiplets a t 6 4.17-4.33 (OCH2CH2CH2NH), 1.51-1.67 (OCH,CH&HNH), and 2.96-3.16 (OCH2CH2CH2NH). The lH NMR spectra of the minor pair of diastereomers are generally similar to those of la, except that the 5’-OH resonance occurs as a one-proton triplet at 6 5.37, while the 3’-OH resonance is absent. In addition, the chemical shift of the 3’-H signal occurs approximately 0.5-ppm downfield from that of the corresponding protons of 5-FUdR. The position of the 5’-H resonance, however, is unchanged. Similar spectral analysis of the product derived from 7a and 1,3-propanediol(6b) showed that it consisted of a 6 1 mixture of the cyclic 5’- and the 3’-phosphate positional isomers. Stereoisomers are not possible for these compounds because the phosphorus atom is not chiral. These data provide further support for the observation of Dunlap et al.51 that the Yoshikawa reaction for the preparation of 5’-nucleotides from unsubstituted nucleosides is not regiospecific but gives substantial amounts of the 3‘-isomers. The thymidine analogues IC and Id were prepared similarly. Although HPLC analyses of these compounds indicated that they contained a mixture of 5’- and 3’-i~Ome1’~, detailed spectral analysis of each component was not attempted. Proof that the cyclic phosphate group in l b and Id was present in the 5‘-position of the molecule was obtained from chemical interconversion studies. Thus, the reaction of l b and ld with acetic anhydride in pyridine gave 9a and 9b, respectively; these products were spectrally and chromatographically identical with the products obtained from the reaction of 3‘-0-acetyl-5-fluoro-2-deoxyuridine (8a) and 3’-O-acetylthymidine (8b) with phosphoryl chloride and 1,3-propanediol (Scheme 11). This result precludes the possibility that the phosphoryl group in l b and Id is bonded to the 3‘-oxygen atom and, therefore, eliminates alternative structures such as the isomeric cyclic 3’,5’-nucleotides. Although the modified Yoshikawa synthesis provided a direct route to the desired cyclic 5’-nucleotides, the yields from these reactions were disappointingly low. However, considering the greater steric requirements of the cyclization reactions, this result is consistent with the report of Dunlap et al.6l that the Yoshikawa procedure gives only a 28% yield of 5-FdUMP from 5-FUdR. Since the amount of la formed by this method was insufficient for antitumor screening, an alternative synthesis of this compound was attempted from 5-FUdR and trimethylene phosphate (10) by using triphenylphosphine/ diethyl a~odicarboxylate~~ as the condensing agent (Scheme 111). Chromatography of the crude product on silica gel gave l b in 64% yield free ~
~~~
(51) W.H.Dawson, R. L. Cargill, and R. Bruce Dunlap, J . Carbohydr., Nucleosides Nucleotides, 4,363 (1977). (52) J. Kimura, Y. Fujisawa, T. Yoshizawa, K. Fukuda, and 0. Mitsunobu, Bull. Chem. SOC.Jpn., 52, 1191 (1979).
Journal of Medicinal Chemistry, 1983, Vol. 26, No. 8 1155
Scheme I11
OH
10
la
lb
of the 3’-isomer. Because of the comparatively good yield and high isomeric purity of the product and the ease of workup of the reaction, this is clearly the best method for the preparation of lb. A similar synthesis of la was not attempted because of the unavailability of the azo analogue of trimethylene phosphate. Because of the pronounced acid lability of phosphoramidates, it seems likely that this compound would undergo facile self-addition to yield a polymeric product. All of the cyclic 5’-phosphates were stable in neutral aqueous buffers for up to 24 h at room temperature. Although the corresponding phosphoramidates were also stable in neutral buffers, they were extremely susceptible to acid-induced decomposition. Therefore, care was taken to ensure neutral or slightly basic (pH 7-8) conditions during all chemical and biological manipulations. For the enzyme studies, the cyclic 5’-nucleotides were purified to homogeneity by reverse-phase semipreparative chromatography. However, since the separation of la into its component isomers in gram quantities was not possible, this compound was screened as a mixture of isomers. Enzyme Studies. Since it is important to the success of our strategy that the cyclic 5’-nucleotides are resistant to degradation by nucleotide-catabolizing enzymes, the stability of la-d was examined in the presence of 5’-nucleotidase (EC 3.1.3.5), alkaline phosphatase (EC 3.1.3.1), phosphodiesterase 1(EC 3.1.4.1), and crude snake venom. None of the compounds showed measurable degradation when incubated with these enzymes for 2 h at 37 “C. The susceptibility of these same compounds to oxidative biotransformation by cytochrome P-450 dependent mixed-function oxidases was examined by incubating them with hepatic microsomal preparations from phenobarbital-induced mice. All four compounds showed a 5 to 10% decrease in concentration over the 2-h incubation period. HPLC analysis of the supernatants from ethanol-quenched incubations showed a different chromatographic profile from NADPH-deficient or heat-deactivated controls, but no major new peaks were detected that were unambiguously derived from the 5’-cyclic compounds. Cyclophosphamide used as control was extensively metabolized under these conditions. Antitumor Screening. Compounds la and l b were screened against murine leukemia P-388 both sensitve and resistant to 5-FU (Table I). Administered intraperitoneally (ip) daily for 5 consecutive days at maximally tolerated dosages, la was only slightly less effective than 5-FU at prolonging the life spans of mice bearing the 5-FUsensitive tumor. However, much higher dosages were required for optimal activity. Compound l b , administered similarly, was only marginally active at the highest dose tested. Neither compound was effective against a P-388 mutant resistant to 5-FU. Discussion Our objectives in this study were to synthesize neutral analogues of 5-FdUMP which incorporate the structural features that contribute to the selectivity of cyclophosphamide. We hoped these analogues might penetrate into cells and overcome innate or acquired resistance to 5-FU due to the deletion of anabolic enzymes. The success of this strategy is critically dependent on the oxidative
1156 Journal of Medicinal Chemistry, 1983, Vol. 26, No. 8
Farquhar et al.
Table I. Effect of Neutral 5-FdUMP Analogues on the Survival of BDF, Micea Implanted Intraperitoneally with P-388 Leukemia Both Sensitive and Resistant to 5-FU P-38810 compd
dose, mg/kg, (qd, 1-51
saline 5-FU la
35 20 15 500 300 180 108 65 500 300 180 108
MST, days
P-388/5-FU % wte
%ILS~
change
9 8 19
-11 111
9.4 -21.8 -16.5
17 14 13 12
89 55 44 33
-17.9 -15.7 -13.9 -6.6
MST, days 10 9 11 11
9 10 10
-
% wte
% ILS
change
-10 10 10
2.1 -14.2 -3.9 -6.2
-10 0 0 -10 0 0 0
-5.0 -3.7 -3.4 1.5 0.6 1.2 4.1 6.3
9 12 33 10 -12.4 11 22 -8.8 10 11 10 10 -0.0 10 0 10 11 1.7 a Six mice per group; average weight 23 g. Median survival time 1 X 10' cells inoculated intraperitoneally on day 0. (MST) of 20 mice used as control was 9 days. Percentage increase in life span of treated animals (T) compared with saline-treated controls (C) was determined by the formula: ( T / C - 1)x 100. e The average percentage weight change on day 6 was taken as a measure of drug toxicity. lb
bioactivation of the analogues by cytochrome P-450 dependent mixed-function oxidases. Although the cyclic phosphoramidate la was almost as active as 5-FU against leukemia P-388, much higher dosages were required for optimal activity. This result is consistent with the minimal extent of biotransformation of la when incubated with mouse hepatic microsomes in the presence of an NADPH-generating system. The marginal activity of the cyclic phosphate lb, moreover, parallels the weak activity of the dioxo analogue of cycloph~sphamide.~~ However, it cannot be assumed that the antitumor activity of la at high dosages is due to oxidative bioactivation. Metabolites anticipated from this mode of biotransformation were not conclusively detected in microsomal incubates. Although la, in common with the other cyclic 5'-nucleotides, was not degraded in vitro by several different phosphohydrolases, conceivably it is catabolized in vivo to 5-FU or 5-FUdR by some other enzymes. This possibility is consistent with the inactivity of la against the 5-FU-resistant tumor. However, since the mechanism of drug resistance in this tumor has not been established, this interpretation is entirely speculative. Apart from the failure of the cells to anabolize 5-FU to 5-FdUMP, other resistance mechanisms are possible, such as increased thymidine salvage, altered thymidylate synthetase, or decreased drug incorporation into RNA. Subsequent to our initial report on the synthesis and biological evaluation of the cyclic nucleotides described herein, Hunston et al." reported the synthesis of a similar series of compounds. Compound la was marginally active against sarcoma 180 at a dose of 100 mg/kg but inactive against leukemia L1210 at 50 mg/kg (schedules not specified). Enzymes studies were not reported. In summary, it appears that the poor substrate affinity of the 5'-cyclic 5-FdUMP analogues for cytochrome P-450 mixed-function oxidase inherently limits their potential as latent precursors of the parent nucleotide. Currently, we are investigating approaches to overcome this problem. Experimental Section Proton nuclear magnetic resonance spectra ('H NMR) were recorded a t ambient temperature on a Varian Associates T-60A (53) . , H. Arnold. F. Boureaux. and N. Brock, Arzneim.-Forsch.,. 11,. 143 (1961j. (54) R. N. Hunston, M. Jehangir, A. S. Jones, and R. T. Walker, Tetrahedron, 36, 2337 (1980).
spectrometer or on a Bruker HX-360 spectrometer in the Fourier transform mode, in CDC&or Me2SO-d6,with tetramethylsilane as an internal standard. Mass spectra were obtained on a Finnegan Model 3300 quadrupole spectrometerin the electron-impact or chemical-ionization mode with a direct-inlet probe. Elemental analyses were performed by Galbraith Laboratories Inc., Knoxville, TN, and the results are within *0.4% of the theoretical values. All solvents were distilled immediately before use and were stored over freshly activated (300 "C/4 h) molecular sieves (type 4A). Nucleosides were dried over P205under vacuum at 100 "C for 24 h before use. All reactions were conducted in dry glassware and were protected from atmospheric moisture. Evaporations were carried out on a rotaty evaporator under high vacuum at a bath temperature of
