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An activated carbamate, 2-nitrophenyl (2-fluoroethyl)nitrosocarbamate (3), was used to advantage in the synthesis of the water-soluble (2-fluoroethyl)nitrosoureas 6a--d from 2-aminoethanol, (1 alpha, 2 beta, 3 alpha)-2-amino-1,3-cyclohexanediol, cis-2-hydroxycyclohexanol, and 2-amino-2-deoxy-D-glucose. In a variation of this method, 2,4,5-trichlorophenyl (2-fluoroethyl)carbamate (4) was used to prepare the urea from which the essentially water-insoluble N-(2,6-dioxo-3-piperidinyl)-N-(2-fluoroethyl)-N-nitrosourea (6e) was derived. The anticancer activity of these nitrosoureas was determined against the murine tumors B16 melanoma and Lewis lung carcinoma and found to be significant and comparable to their chloroethyl counterparts. On the basis of results from both systems, the dihydroxycyclohexyl derivative 6b may be the most effective.

The high antiradiation activity and low toxicity of sodium 3-amino-2-hydroxypropyl hydrogen phosphorothioate (1) suggested the introduction of hydroxyl groups into other types of radioprotective phosphorothioates. A number of such compounds were synthesized, including S-3-(3-aminopropylamino)-2-hydroxypropyl dihydrogen phosphorothioate (11, n equals 3), S-2-(3-amino-2-hydroxypropylamino)ethyl dihydrogen phosphorothioate (20) and its propyl homolog 26, N,N'-(2-hydroxytrimethylene)bis(S-2-aminoethyl dihydrogen phosphorothioate) (40), S-2-[3-(2-hydroxyethylamino)propylaminoi1ethyl dihydrogen phosphorothioate (44), and sodium S-2-amino-2-(hydroxymethyl)-3-hydroxypropyl hydrogen phosphorothioate (49). Compounds 11 (n equals 3), 20, 26, and 49 were highly protective when administered intraperitoneally but were generally ineffective when given perorally, as were the other hydroxylated phosphorothioates prepared. The introduction of hydroxyl groups significantly enhanced the radioprotective properties of nonhydroxylated parent compounds, however, only in the case of intraperitoneally administered.

A reinvestigation of the aqueous decomposition of N-(2-chloroethyl)-N-nitrosoureas has shown that their mode of decomposition is dependent upon whether or not the solution is buffered at or near physiological pH. In distilled water, the 2-chloroethyl compounds decompose with the loss of 1 mol, or slightly less, of chloride ion per mole in nitrosourea and the formation of acetaldehyde and 3-4% of 2-chloroethanol. In buffer, the yield of 2-chloroethanol increases to 0.3-0.6 mol per mole of nitrosourea, the yield of chloride ion decreases to 0.5 mol per mole of nitrosourea, and the yield of acetaldehyde decreases to 0.1-0.4 mol per mole of nitrosourea. Evidence for the formation of the vinyl cation, a possible precursor of acetaldehyde, in these reactions is presented. In contrast to the results obtained with the N-(2-chloroethyl)-N-nitrosoureas, the decomposition of N,N'-bis(2-fluoroethy)-N-nitrosourea in distilled water gave almost 1 mol of 2-fluoroethanol per mole of nitrosourea and only 0.04 mol of acetaldehyde per mole of nitrosourea.

N-(2-chloroethyl)-N'-(cis-4-hydroxycylohexyl)-N-nitrosourea, a major metabolite of N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU), and its trans isomer were prepared from the corresponding 4-aminocyclohexanols. A convenient and stereospecific precursor was found in 2-oxa-3-azabicyclo[2.2.2]oct-5-ene hydrochloride, hydrogenation giving pure cis-4-aminocyclohexanol hydrochloride. The metabolites were, at nontoxic levels, at least as active as CCNU in tests against murine leukemia L1210 implanted both intraperitoneally and intracerebrally and, on a weight basis, were more active and more toxic. These observations and previously reported metabolic studies suggest that the anticancer activity of CCNU is due primarily to its metabolites.

The utility of the Lewis lung carcinoma as a secondary screen for the evaluation of nitrosoureas as anticancer agents has been assessed. The activity of this series of compounds was determined against both the early (before metastasis) and late (after metastasis) forms of the disease. Although some exceptions were noted, compounds most active against the early form of the disease were most active against the established tumor. A differentiation in activity based on the Lewis lung system was evident with nitrosoureas equally active against leukemia L1210, although the significance of this differentiation with respect to the human disease has not yet been established.

Evidence incicating that effective in vivo inhibition of hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) should produce antiprotozoal activity without significant toxic effects on mammalian hosts prompted syntheses of 1-substituted hypoxanthines bearing functionalized side chains whose groupings might interact with appropriate groupings of HGPRT to form covalent bonds or strong hydrophobic bonds. 3-(Fluorosulfonyl)benzoyl, 4-(fluorosulfonyl)benzoyl, 4-chlorobenzoyl, and bromacetyl derivatives of two parent amines, 1-(2-aminoethyl)-hypoxanthine and 1-(4-aminobenzyl)hypoxanthine, were synthesized for evaluation in this connection. None of these compounds extended the life span of Plasmodium berghei infected mice or showed significant in vitro inhibition of HGPRT from H.Ep.-2 cells, but 1-[2-(bromoacetamido)ethyl]hypoxanthine displayed in vivo activity against Trypanosoma rhodesiense.

The superior activity of N-(2-chloroethyl)-N'-(trans-4-methylcyclohexyl)-N-nitrosourea (MeCCNU) against advanced murine Lewis lung carcinoma in comparisons with the cis form and other nitrosoureas prompted the synthesis of a number of MeCCNU analogues, including several cis-trans pairs. The methyl group was replaced by a variety of substituents (CO2H, CH2CO2H, CO2Me, CH2OAc, CH2Cl, OMe); the trans-3-methylcyclohexyl, cis-2-methyl-1,3-dithian-5-yl, cis- and trans-2-methyl-1,3-dithian-5-yl-tetraoxide, and 1-methylhexyl (open-chain) analogues were also prepared. Preliminary tests against murine leukemia L1210 revealed therapeutic indices (ED50/LD10) ranging from 0.26 to 0.79; all but three analogues effected 50% cure rates at nontoxic doses, the open-chain analogue being one of the least active. In terms of therapeutic index, diequatorial (trans-4) isomers were, with one exception, as active as or, in four of the eight examples, somewhat more active than the corresponding axial-equatorial (cis-4) isomers. In this series, four of the five 2-fluoroethyl analogues prepared were clearly inferior to the corresponding 2-chloroethyl analogues.

cis-4-[[[(2-Chloroethyl)nitrosoamino]carbonyl]methylamino] cyclohexanecarboxylic acid (N-Me-cis-CCCNU) was synthesized in five steps from cis-4-aminocyclohexanecarboxylic acid via an N-tosylated intermediate. N-Me-cis-CCCNU, which is incapable of the facile decomposition that characterizes the clinically useful nitrosoureas, effected a significant cure rate of both early and established murine Lewis lung carcinoma, even though its in vitro half-life was approximately 5.5 times that of the unmethylated parent compound. This is the first observation of latent activity of a nitrosourea against an experimental solid tumor.

To enable further structure-activity comparisons among radioprotective phosphorothioates, S-2,omega-diaminoalkyl dihydrogen phosphorothioates were synthesized from L-2,4-diaminobutyric acid, L-ornithine, L-lysine, and DL-2,7-diaminoheptanoic acid as homologues of S-2,3-diaminopropyl dihydrogen phosphorothioate (4) and as isomeric analogues of S-2-[(omega-aminoalkyl)amino]ethyl dihydrogen phosphorothioates (e.g., 1). The preferred route that evolved from exploratory trials retained optical activity and involved the reduction of methyl 2,omega-bis(benzoylamino)alkanoates with lithium borohydride, debenzoylation-bromodehydroxylation, and reaction of the resulting 1-(bromomethyl)-1,omega-alkanediamine dihydrobromides with trisodium phosphorothioate. The products of an alternative route that involved the reduction of phthaloylated intermediates with sodium borohydride were racemic. Exploratory conversions of N-(omega-alkenyl)phthalimides failed to provide suitable precursors of the target compounds. In terms of a protective index, these homologues were significantly more radioprotective than the parent phosphorothioate 4 when administered intraperitoneally to mice prior to whole-body gamma irradiation. The homologues derived from L-lysine also showed good peroral activity. No apparent difference was observed in the protection afforded by optically active homologues and the corresponding racemates.