J. Med. Chem. 1990, 33, 2603-2610
2603
Synthesis and Evaluation of CY-[ [ (2-Haloethyl)amino]methyl]-2-nitro-1H-imidazole-l-ethanols as Prodrugs of CY-[ (l-Aziridinyl)methyl]-2-nitro-1H-imidazole-l-ethanol (RSU-1069) and Its Analogues Which Are Radiosensitizers and Bioreductively Activated Cytotoxins Terence C. Jenkins,' Matthew A. Naylor, Peter O'Neill,* Michael D. Threadgill,* Shirley Cole, Ian J. Stratford, Gerald E. Adams, E. Martin Fielden, Mark J. %to,* and Michael A. Stiers Medical Research Council Radiobiology Unit, Chilton, Didcot, Oxfordshire OX11 ORD, U.K., and Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105. Received January 16,1990
.
a-[(l-Aziridinyl)methyl]-2-nitro-1H-imidazole-l-ethanols, of general formula ImCH2CH(OH)CH2NCR1R2CR3R4, where Im = 2-nitroimidazole and R', R2, R3, R4 = H, Me, are radiosensitizers and selective bioreductively activated
cytotoxins toward hypoxic tumor cells in vitro and in vivo. Treatment of the aziridines with hydrogen halide in acetone or aqueous acetone gave the corresponding 2-haloethylamines of general formula ImCH2CH(OH)CH2+NH2CR1R2CR3R4XX-, where R', R2,R3, R4 = H, Me, and X = F, C1, Br, I. These 2-haloethylamines were evaluated as prodrugs of the parent aziridines. The rates of ring closure in aqueous solution at pH -6 were found to increase with increasing methyl substitution and to depend on the nature of the leaving group (I Br > C1>> F). A competing reaction of ImCH2CH(OH)CH2+NH2CHzCH2X X- (X = C1, Br) with aqueous HC03- ions gives 3-[2-hyroxy-3-(2nitro-1H-imidazol-l-yl)propyl]-2-oxazolidinone. The activities of these prodrugs as radiosensitizers or as bioreductively activated cytotoxins were consistent with the proportion converted to the parent aziridine during the course of the experiment. a-[[(2-Bromoethyl)amino]methyl]-2-nitro-1H-imidazole-l-ethanol (RB 6145, lo), the prodrug of a[ (l-aziridinyl)methyl]-2-nitro-l~-imidazole-l-ethanol(RSU-1069,3), is identified as the most useful compound in terms of biological activity and rate of ring closure under physiological conditions.
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Introduction It is generally known that many solid tumors contain areas of diminished oxygen supply. While the underlying reasons for this hypoxia can vary depending upon the type and environment of the tumor, this phenomenon provides a potential basis for selectivity of cancer chemotherapeutic agents towards tumors. Compounds which are anaerobically reduced to a cytotoxin should be more cytotoxic to hypoxic tumor cells than to oxygenated normal tissues.' Many nitroheterocyclic compounds, such as the 2-nitroimidazoles, owe their selective cytotoxicity toward hypoxic cells within tumors to their bioreductive propertiesS2 They are activated in vivo by anaerobic, enzymatic reduction to form metabolites which are considerably more cytotoxic than the parent compound from which they were derived. Nitroimidazoles also act as hypoxic cell radiosensitizers, acting primarily by fast free-radical mechanism^.^ The agents increase the sensitivity to radiation of the normally radiation-resistant hypoxic cells but have little or no effect on the response of well-oxygenated cells to radiation. The ability of these compounds to act as radio sensitizer^^ and hypoxic cell cytotoxins is related to their reduction potentials, although the mechanisms of the two effects are different. Misonidazole ( 1)5 has undergone extensive clinical evaluation as a radiosensitizer, but cumulative neurotoxicity severely limits the dosage which may be administered. An equally potent but less toxic analogue, etanidazole (2): is in phase I11 trials. An alternative approach has been to develop compounds with greater potency than 1 or 2 (Chart I). An example is the dual functional agent a[ (l-aziridinyl)methyl]-2-nitro-1H-imidazole-1-ethanol (3), which combines the 2-nitroimidazole ring with an alkylating moiety, resulting in a powerful hypoxic cell radiosensitizer and bioreductively activated cytotoxic agent both in vitro and in This aziridine 3 was selected from Present address: Cancer Research Campaign Biomolecular Structure Unit, Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK. Present address: School of Pharmacy, University of Bath, Bath BA2 7AY, UK. Warner-Lambert Co.
Chart I. Structures of the Hypoxic Cell Radiosensitizers Misonidazole (1) and Etanidazole (2)
I
I NO2
H
NO2 1
-
2
a series of compounds based upon its partition coefficient and activity in model tumors. The ability of 3 to sensitize hypoxic tumor cells to the lethal effects of radiation at 10-12 times less than the dose of 1 or 2 in experimental animal models led to its investigation clinically. Unfortunately, in early phase I trials, gastric toxicity (emesis) was observed.1° The maximum tolerated dose achievable in humans was considered to be too low to achieve a usefully high degree of radiosensitization. This led to a search for equally active but less toxic analogues. This study describes the synthesis, stability, and development of a series of [ (haloethyl)amino]propyl-2nitroimidazoles derived from 3 and its analogues. Preliminary biological studies of these compounds as radios(1) Sartorelli, A. C. Cancer Res. 1988, 48, 775. (2) Collected papers In Biochemical Pharmacology; Alexander, P., Gielen, J., Sartorelli, A. C., Eds.; Pergamon Press: Oxford,
U.K., 1986; Vol. 35, pp 1-122. (3) Adams, G. E.; Michael, B. D.; Asquith, J. C.; Shenoy, M. A.; Watts, M. E.; Whillans, D. W. Radiation Research: Biomedical, Chemical and Physical Perspectives; Academic Press: New York, 1975; pp 478-492. (4) Adams, G. E.; Flockhart, I. R.; Smithen, C. E.; Stratford, I. J.; Wardman, P.; Watts, M. E. Radiat. Res. 1976, 67, 9. (5) Asquith, J. C.; Watts, M. E.; Patel, K.; Smithen, C. E.; Adams, G. E. Radiation Res. 1974, 60, 108. (6) Brown, J. M.; Yu, N. Y.; Brown, D. M.; Lee, W. Znt. J . Radiat. Oncol. Biol. Phys. 1981, 7, 695. (7) Adams, G. E.; Ahmed, I.; Sheldon, P. W.; Stratford, I. J. Br. J . Cancer 1984, 49, 571. (8) O'Neill, P.; Jenkins, T. C.; Stratford, I. J.; Silver, A. R. J.; Ahmed, I.; McNeil, S. S.; Fielden, E. M.; Adams, G. E. AntiCancer Drug Des. 1987, I, 271. (9) Adams, G. E.; Ahmed, I.; Sheldon, P. W.; Stratford, I. J. Int. J . Radiat. Oncol. Biol. Phys. 1984, 10, 1653. (10) Horwich, A.; Holliday, S. B.; Deacon, J. M.; Peckham, M. J. Br. J. Radiology 1986, 59, 1238.
0022-2623/90/ 1833-2603$02.50/0 0 1990 American Chemical Society
Jenkins et al.
2604 Journal of Medicinal Chemistry, 1990, Vol. 33, No. 9
Scheme I. Synthesis of 2-Haloethylamines 9-18, 2-Hydroxyethylamines 19, 20, and 22, and Ethylamine 21
Scheme 11. Synthetic Approaches to 2,2,3,3-Tetramethylaziridine me
me
SNH.
T
I
NO,
NO:
7
12 19 21 22
I
NO,
R=H, X=F R-)le, X=OH
R=X=H R=H, X=OH
CF,CO; 20
ensitizers are also provided. Chemistry Compounds 3-6 were prepared as described previously by us8J1J2from oxirane 7 and the corresponding aziridine (Scheme I). The overall yield of 4 was increased considerably with stereochemicallypure cis-2,3-dimethylaziridine prepared from cis-2,3-epoxybutane by the method of Dickey et al.13 Alternative procedures for the synthesis of 2,2,3,3-tetramethylaziridine, used in the preparation of 6, were explored, in order to avoid the use of the expensive, noxious, and unreliable nitrosyl chloride.'*J5 The cyclization of 2-amino alcohols via the 0-sulfates fails for the more substituted examples. The insertion of [ (2,4-[dinitrophenyl)sulfenyl]nitrene,generated from 2,4dinitro benzenesulfonamide and lead tetraacetate,I6 into the double bond of 2,3-dimethylbut-2-ene gave 1-[ 2,4-dinitrophenyl)thio]-2,2,3,3-tetramethylaziridine(8) in high yield (Scheme 11). However, reductive cleavage of the S-N bond could not be achieved in preparatively useful procedures. Addition of iodine azide, formed in situ from sodium azide and iodine monochloride, across the double bond of 2,3-dimethylbut-2-ene, reduction of the resultant azide, and concomitant ring closure furnished the aziridine in acceptable yield" (Scheme I). The 2-haloethylamines 9-18 were obtained from the corresponding aziridines 3-6 by acid-catalyzed nucleophilic (11) Adams, G. E.; Ahmed, I.; Gibson, D.; Stratford, I. J. U.K. Patent GB 2123816A, Feb 8, 1984.
(12) Ahmed, I.; Jenkins, T. C.; Walling, J. M.; Stratford, I. J.; Sheldon, P. W.; Adams, G. E.; Fielden, E. M. Int. J . Radiat. Oncol. Bioi. Phys. 1986, 12, 1079. (13) Dickey, F. H.; Fickett, W.; Lucas, H. J. J . Am. Chem. SOC. 1952, 74, 944. (14) Closs, G. L.; Brois, S. J. J . Am. Chem. SOC.1960, 82, 6068. (15) Dornow, A.; Fust, K. J. Chem. Ber. 1957,90, 1769. (16) Atkinson, R. S.; Judkins, B. D. J. Chem. SOC.,Perkin Trans. 1 1981, 2615. (17) Hassner, A.; Matthews, G.; Fowler, F. J. Am. Chem. SOC.1969, 91, 5046.
ring opening with the appropriate hydrogen halide, as shown in Scheme 11. The yields were generally greater if anhydrous conditions were employed, thus avoiding the possibility of nucleophilic attack by water leading to the corresponding 2-hydroxyethylamines. Compounds 13 and 14 have the threo relative stereochemistry in the haloalkylamine moiety but were isolated as mixtures of diastereoisomers with respect to the chiral center adjacent to the hydroxy group. Proton NMR at 300 MHz and HPLC analysis revealed that 13 consisted of such diastereoisomers in the approximate 1:l ratio, indicating that the electrophilic centers in protonated 4, which are prochiral, are roughly equivalent in reactivity and/or steric crowding in the reacting conformation. HPLC analysis of 14 also indicated the presence of both the analogous diastereomers in similar quantities. Ring-closure of 13 and 14 in the presence of aqueous base gave exclusively cis2,3-dimethylaziridine 4, showing that both ring-opening and ring-closing reactions proceed with complete inversion at the reacting center. The correct regioisomeric structures of 15 and 16 as l,l-dimethyl-2-haloethylamines which are products of attack at CH,, rather than isomeric 2-halo-2-methylpropylamines resulting from alternative attack at CMe,, were also confirmed by two pieces of evidence from 'H NMR spectra. Firstly, the CMe2CH2Clprotons of 15, in solution in 10% deuterotrifluoroacetic acid in deuterium oxide, resonate as a sharp singlet at 6 3.90. In comparison, the spectrum of 19, in the same solvent, shows a corresponding singlet at 6 3.80 for the CMe,CH20D protons whereas that of isomeric alcohol 20, prepared in situ from 3 by hydrolytic ring opening in the NMR solvent, contains a singlet for the +ND2CH2CMe20Dprotons at 6 3.40. Secondly, when the spectra were recorded of solutions of these materials in anhydrous protiotrifluoroacetic acid, coupling was evident between the +NH2protons and the adjacent CH2CMe2 in 20 but was absent from the +NH,CMe,CH,OH signal of 19. The corresponding signal of 15 also showed no coupling to the +NH2protons. These observed regioselectivities of acid-catalyzed ring opening of 2,2-dimethylaziridineswith the weak nucleophilic water and the strong nucleophile chloride are consistent with the reportedI8 corresponding reactions of simple N-unsubstituted aziridines. The attack of chloride proceeds via a SN2-type mechanism involving the less hindered CH2 center; whereas water can only trap the more stable tertiary carbonium ion in a SN1-likeprocess. The diastereotropic nature of the NC(CH,), and C1C(CH,), groups of protons of 17 was revealed by proton NMR at 250 MHz in solution in (CDJ2S0. The A6 for the pairs of methyl groups were 0.02 and 0.01 ppm, respec(18)
Schatz, V. B.; Clapp, L. B. J . Am. Chem. SOC.1955,77, 5113.
Journal of Medicinal Chemistry, 1990, Vol. 33, No. 9 2605
Imidazoleethanols as Prodrugs
Scheme 111. 'One-Pot" Conversion of 2-Hydroxyethylamines 22 and 19 to 2-Bromoethylamines 10 and 16
Table I. Half-Life of the Agents in PBS Aqueous Solution at pH 5.5-6.0 Together with the Major Product Formed at 23 OC % methanol major substance half-life/min product for HPLC 9 >>300 ( 280)O 3 10 10 30 3 10 11 3 80 10 13.75 13 120 4 14 4 10 30b 15 76b 5 25
