Intracellular polyamine pools are partially maintained by an active transport apparatus that is specific for and regulated by polyamines. Although mammalian transport activity has been characterized by kinetic studies, the actual protein itself has yet to be identified, purified, or cloned. As one approach to this problem, we attempted photoaffinity labeling of plasma membrane proteins using two specifically designed and synthesized polyamine conjugates as photoprobes. The first is a spermidine conjugate bearing the photoreactive moiety 4-azidosalicylic acid at the N 4 position via an alkyl linkage, and the second is a norspermine conjugate with 4-azidosalicylic acid at the N 1 position via an acyl linkage. Labeling of murine L1210 lymphocytic leukemia cells was carried out at 4°C to promote selective alkylation of cell surface proteins. Separation of plasma membrane proteins from cells cross-linked with the N 4 -spermidine conjugate by SDS-polyacrylamide gel electrophoresis revealed two heavily labeled proteins at ϳ118 and ϳ50 kDa (designated p118 and p50, respectively). Band p118 was more well defined and much more intensely labeled. Analogous proteins were also observed in human U937 lymphoma cells. Specificity of labeling was strongly suggested by competition with polyamines and analogs during labeling and further indicated by the nearly identical labeling of the same protein by the N 1 -norspermine photoprobe but not by the unconjugated photoreagent. Neuraminidase pretreatment of L1210 cells increased mobility of the p118, suggesting that it was glycosylated and, thus, of plasma membrane origin. In transport-deficient L1210 cells, p118 and p50 were found to have a slightly higher molecular mass and were accompanied by a less distinct protein band (ϳ100 kDa). These findings indicate the presence of a polyamine binding protein at the surface of murine and human leukemia cells, which could be directly or indirectly related to the polyamine transport apparatus.

In continuation of efforts to improve the antitumor selectivity of the 2,2-dimethylaziridine class of alkylating agents, a series of N-substituted bis(2,2-dimethyl-1-aziridinyl)phosphinic amides has been synthesized and evaluated. All of these compounds (3-15) were tested in vivo against leukemia P-388 in mice, where most of them caused significant increase of survival time at nontoxic dose levels. Some of the most active compounds were also tested against leukemia L1210, B16 melanoma, and colon 26 carcinoma; in the latter tests, the parent unsubstituted amide 3 appeared to show the highest antitumor activity. Since the dose-limiting toxicity of the clinically tested prototypes of this class of anticancer agents AB-132 (1) and AB-163 (2) had been found to be CNS toxicity attributable mainly to the inhibition of cholinesterase, the compounds were tested in vitro against the cholinesterases from horse serum, electric eel, and bovine erythrocytes, as well as in vivo for the inhibition of the cholinesterase present in the whole blood of mice. In all of these assays, the various members of the present series showed a wide range of anticholinesterase activities, ranging from almost zero (for 3) to even higher potency than that of the prototype 2. A similarly wide range of stability was observed toward hydrolytic ring opening of the 2,2-dimethylaziridine moieties. Several of the compounds, particularly 3, deserve further study.

With the aim of increasing the selectivity of the 2,2-dimethylphosphoraziridine type antitumor agents toward the intracellular site of DNA synthesis, a series of new compounds was synthesized in which the reactive bis(2,2-dimethyl-1-aziridinyl)phosphinyl (2,2-DMAP) group was linked through a carbamate or amide linkage to thymidine or cytosine nucleoside moieties. The 3'- and 5'-(2,2-DMAP)carbamates of thymidine (1 and 2) were found to be highly unstable, therefore the corresponding O-acetyl derivatives 5 and 6 were prepared by reacting 5'- and 3'-acetylthymidine, respectively, with dichloroisocyanatophosphine oxide followed by the addition of 2,2-dimethylaziridine and triethylamine. The 3'- and 5'-(2,2-DMAP)amides of thymidine 14 and 15 were prepared by reacting the appropriate thymidinylamines with bis(2,2-dimethyl-1-aziridinyl)phosphinyl chloride (17). The N4-(2,2-DMAP)amides of cytidine, 2'-deoxycytidine, and cytosine arabinoside (18, 19, and 20, respectively) were prepared by reacting the hydrochlorides of the O-peracetylated cytosine nucleosides with triethylamine and POCl3 and, subsequently, with 2,2-dimethylaziridine and triethylamine, to give the corresponding N4-(2,2-DMAP)cytosine nucleoside peracetates 21, 22, and 23, respectively, which were then deacetylated by aminolysis. However, the peacetate intermediates were found to be more stable and, probably for the same reason, also more active against P388 leukemia in mice than the deacetylated products. Particularly, 22 and 23 showed sufficient activity in this in vivo assay system to warrant further evaluation. The relationships between the antitumor activities, the chemical alkylating activities, and the cholinesterase inhibitory activities of these agents are discussed.

Two new drug candidates, in which a quinonoid moiety is linked to the reactive bis(2,2-dimethyl-1-aziridinyl)phosphinyl function, have been prepared and tested in vivo for antitumor activity and in vitro as potentiators of the cytotoxic effect of X-irradiation. Without irradiation only moderate effectiveness against leukemia P-388 in mice was exhibited by one of the quinonoid compounds that had sufficient water solubility to be used in the in vivo screening. However, both compounds were shown to potentiate the effect of X-irradiation in vitro by a colony-forming cell culture assay under hypoxic conditions.

(+/-)3-(3-(4,4-diphenylpiperidin-1-yl)propyl) 5-methyl 4-(3,4-dimethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate ((+/-)-DHP-014), is a new 4-aryl-1,4-dihydropyridine that can reverse multidrug resistance mediated by the ATP-binding cassette (ABC) transport proteins, P-glycoprotein, multidrug resistance-associated protein 1 and breast cancer resistance protein; it exhibits negligible calcium channel blocking activity. The objective of this work was to investigate the pharmacokinetics of this new compound in rats. Three intravenous (1, 2 and 5 mg/kg) and two oral (25 and 50 mg/kg) doses were administered to female Sprague-Dawley rats. A two-compartment model with nonlinear elimination best characterized the pharmacokinetic profiles after intravenous and oral administration in rats. The terminal half-life of (+/-)-DHP-014 increased and the systemic clearance significantly decreased at higher doses, indicating nonlinear elimination. The dose-dependent clearance is likely due to saturation of metabolism. The apparent volume of distribution of (+/-)-DHP-014 was 2.0 L/kg in rats and was unchanged with increasing intravenous doses of (+/-)-DHP-014. The estimated oral bioavailability of (+/-)-DHP-014 was 8.2%. The poor bioavailability is likely due to the poor solubility of the compound, as well as to substantial first-pass elimination.