Chemical Structure of Carbamoylating Groups and Their Relationship to Bone Marrow Toxicity and Antiglioma Activity of Bifunctionally Alkylating and Carbamoylating Nitrosoureas1

Although the antitumor effects of chloroethylnitrosoureas have been shown to be due primarily to DNA-DNA cross-linking by the alkylating moieties of these agents, the basis of the often accompanying bone marrow toxicity has been more controver sial. We report on the relative bone marrow toxicity of four model nitrosoureas with different alkylating and Carbamoylating activi ties: 1,3-bis(2-chloroethyl)-1-nitrosourea; 1,3-bis(frans-4-hydroxycyclohexyl)-1-nitrosourea; chlorozotozin, (2-[3-(2-chloroethyl)-3 -nitrosoureido]-2-deoxy-D-glucopyranose); and 1-(2-chloroethyl)-3-(|8-D-glucopyranosyl)-1-nitrosourea. Inhibitions of DNA, RNA, and protein synthesis in murine bone marrow cells and of colony growth of myeloid precursor cells (granulocyte-macrophage colony-forming units) were used as in vitro end points of myelotoxicity. Further, we determined the antiglioma activity of the four nitrosoureas on two human gliomas in a clonogenic tumor cell assay and studied the effect of the non-nitrosourea carbamoylators potassium cyanate, chloroethyl isocyanate, cyclohexyl isocyanate, ethyl isocyanate, and ethyl isothiocyanate on granulocyte-macrophage colony-forming units. The results show that, at equivalent drug exposures, clonogenic glioma cell kill was significant and comparative for 1,3-bis(2-chloroethyl)-1nitrosourea, 1-{2-chloroethyl)-3-{|8-D-glucopyranosyl)-1 -nitrosourea, and chlorozotocin; 1,3-bis(frans-4-hydroxycyclohexyl)-1-nitrosourea showed little activity. In contrast, granulocyte-macro phage colony-forming unit toxicity was low with chlorozotocin and 1-{2-chloroethyl)-3-{lo-D-glucopyranosyl)-1-nitrosourea and very high with 1,3-bis(2-chloroethyl)-1-nitrosourea and 1,3bis(frans-4-hydroxycyclohexyl)-1-nitrosourea. Of tr)e isocyanates, bone marrow toxicity was highest with chloroethyl isocyan ate and cyclohexyl isocyanate, intermediate with ethyl isocyan ate, and lowest with KOCN and ethyl isothiocyanate. Our results indicate that (a) bifunctional alkylation is essential for antiglioma activity of nitrosoureas and (b) myelosuppression is at least partly linked with carbamoylation but that structural entities in the Carbamoylating isocyanate rather than a quantitative degree of carbamoylation determine the degree of potential myelotoxicity. INTRODUCTION The CENUs3 have been proved clinically to be effective antitumor drugs and, because of their ability to cross the blood-brain barrier (1), have been especially useful in the chemotherapy of central nervous system cancers. However, some nitrosoureas, especially the non-sugar-containing ones, uniquely produce dose-limiting cumulative bone marrow toxicities (2, 3). Under physiological conditions, CENUs decompose to alkylating, crosslinking chloroethyl carbonium ions as well as reactive organic isocyanates (4, 5), and there is considerable evidence (6-11) to suggest that interstrand cross-linking of cellular DNA by the bifunctionally alkylating chloroethyl carbonium ions constitutes the major cytotoxic event and consequently is responsible for the antitumor effects. However, apart from their ability to inhibit DNA ligase and other cellular enzyme activity (12-17), there is less certainty about the effects of the Carbamoylating moieties of CENUs, although it has been speculated (3, 14, 18) and refuted (19-21) that the unwanted normal tissue toxicity of most CENUs might be associated with carbamoylation. The results of studies on the role of carbamoylation on bone marrow toxicity have, however, been inconclusive, because the carbamoylation models used have been either potassium cyanate or sugarcontaining nitrosoureas (19, 20). The myelotoxicity of Carbamo ylating, non-sugar, nonalkylating nitrosoureas have thus far not been studied in this context. In order to establish whether the chemical structure of a CENU or a Carbamoylating agent is related to its potential myelotoxicity, we investigated the effects of 4 structurally different nitrosoureas that differ in their alkylating and Carbamoylating activities. Inhibition of DNA, RNA, and pro tein synthesis in murine bone marrow cells and suppression of the colony growth of myeloid precursor cells (CFU-GM) were used as in vitro end points of myelotoxicity. In vitro antiglioma effects were determined in a clonogenic cell monolayer assay using a previously described method (22). The 4 model nitrosou reas investigated were BC NU(abifunctional alky lator and carbamcylator), BHCNU (a carbamoylator without alkylating activity), CHZ (a sugar-containing bifunctional alkylator with very low external Carbamoylating activity), and GANU (a sugar-containing bifunc1Supported in part by NIH Program Project Grant CA-13525 and NIH Grants CA-31882andCA-28529. 2Present address: Hippie Cancer Research Center and Wright State University School of Medicine, 4100 Kettering Boulevard, Dayton, OH 45439-2092. To whom requests for reprints should be addressed. Received 1/23/85; revised 4/29/85; accepted 5/2/85. 'The abbreviations used are: CENU, chloroethylnitrosoureas; BCNU, 1,3-bis(2chloroethylH -nitrosourea; BHCNU, 1,3-bis(frans-4-hydroxycyclohexyl)-1 -nitrosou rea; CHZ (chlorozotozin), 2-[3-(2-chloroethyl)-3-nitrosoureido]deoxy-D-glucopyranose; GANU, 1-(2-chloroethyl)-3-03-D-glucopyranosyl)-1-nitrosourea; CFU-GM, granulocyte-macrophage colony-forming units; CEIC, chloroethyl isocyanate; CyHIC, cyclohexyl isocyanate; EIC, ethyl isocyanate; ETIC, ethyl isothiocyanate; MEM, Eagle's minimum essential medium; EBS, Earle s balanced salts; CSF, colony-stimulating factor; i.e., intracerebral. CANCER RESEARCH VOL. 45 SEPTEMBER 1985