Inhibition of Microtubules and Cell Cycle Arrest by a New 1-Deaza-7 , 8-dihydropteridine Antitumor Drug , CI 980 , and by Its Chiral Isomer , NSC 6138631

CI 980 (NSC 613862; [S-(-)]) and NSC 613863 [R-(+)] are the two chiral isomers of ethyl 5-amino 1,2-dihydro-2-methyl-3.phenylpyrido[3,4b]pyrazin-7-ylcarbamate (NSC 370147), which is a mitotic inhibitor with in vivo and in vitro activity against murine multidrug-resistant sublines. We have characterized the inhibition of in vitro microtubule assembly by the S (CI 980) and R (NSC 613863) enantiomers, their actions on the cytoplasmic microtubule network of epithelial-like PtK2 cells, and on the cell cycle of different human and murine leukemias and PtK2 cells. Assembly of purified tubulin, or tubulin plus microtubule-associated proteins, into microtubules was substoichiometrically inhibited by both compounds, which also induced a slow depolymerization of preassembled microtubules. Half inhibitory concentrations were 0.4-0.7/~M and 1.6-2.1 /zM for the S and R isomers, respectively. Excess of both drugs induced polymerization of liganded tubulin into abnormal polymers similar to colchicine. The cytoplasmic microtubules of PtK2 cells were disrupted by both compounds in a concentrationand time-dependent manner, which was observed by indirect immunofluorescence microscopy and quantified by an enzyme-linked immunoassay of cytoskeletal tubulin. Half inhibitory concentrations were 6 nM S isomer, 100 nM R isomer, and 1 pM colchicine. Twenty ma S isomer or 500-700 nra R isomer gave nearly maximal effect. At these concentrations, half maximal microtubule depolymerization took place after 2 h of treatment. After drug removal, slow microtubule assembly and nearly complete reorganization of the cytoplasmic microtubules of PtK2 cells were observed (24 h). One nM S enantiomer or 25 nM R enantiomer induced mitotic arrest in 8 h in U937, HL60, and EIA leukemias. PtK2 cells also stopped in mitosis after a 24-h incubation with 50 nM R isomer or 5 nM S isomer. The inhibition of cell division was irreversible in the leukemic cells, while PtK2 cells partially resumed growth. Although the interactions of CI 980 with microtubules in vitro are not very different from other drugs, it is a most potent cellular microtubule and mitotic inhibitor. I N T R O D U C T I O N Several 1,2-dihydropyrido[3,4-b]pyrazines were prepared as intermediates for the synthesis of certain antifolates (1, 2); they did not function as analogues of folic acid but inhibited mitosis and caused cultured cells to accumulate in metaphase (3). Therefore, new compounds of this type have been synthesized and tested as inhibitors of cell proliferation and anticancer agents. Ethyl 5-amino-l,2-dihydro2-methyl-3-phenylpyrido[3,4-b]pyrazin-7-ylcarbamate, 2-hydroxyethanesulfonate, hydrate (NSC 370147) was very active in inhibiting proliferation of cultured L1210 cells and in increasing the mitotic index. It also inhibited pig brain microtubule polymerization and the Received 7/16/93; accepted 10/29/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Grant PB 87-0230 from the Direccion General de Investigacion Cientifica y Tecnica, French-Spanish grants, and grants from Association pour la Recherche sur le Cancer Federation de Centres de lutte Contre Cancer. 2 To whom requests for reprints should be addressed. binding of [3H]colchicine to purified tubulin and slightly enhanced the binding of [3H]vincristine to tubulin. NSC 370147 was synergistic with vincristine in killing cultured L1210 cells and increasing life spans of mice bearing P388 leukemia (4). Moreover, this compound had good antitumor activity against several tumors in culture and also in mice as well as against murine leukemic sublines that were resistant to most drugs used in cancer therapy (5, 6). NSC 370147 is a racemic compound. Its two chiral isomers, NSC 613862 [S-() ] (CI 980) and NSC 613863 [R-(+) ] (Fig. 1), have been prepared and present significant differences in inhibiting the proliferation of L1210 (7-10) and U937 cells (11). CI 980 is presently undergoing Phase I clinical trials (12). Frequently, one isomer of a drug may produce a desired effect while another may be less active, inactive, or even produce some undesired effect. Microtubule inhibitors are not an exception. Inversion of optical activity of the acetamide group of colchicine abolishes its activity (13), and isomerization of podophyllotoxin (c islactone) results in a 10-fold loss of activity (14). It has been shown that both isomers CI 980 [S-(-)] and NSC 613863 [R-(+) ] are able to bind to purified tubulin with equilibrium dissociation constants of approximately 0.2 ~M and 0.3 p,M, respectively, apparently overlapping the trimethoxybenzene locus of the colchicine binding site. Their different fluorescence properties upon binding to tubulin have suggested a different orientation for the isomers in the same locus (15). In order to gain insight into the mechanism of action of CI 980 and NSC 613863, we have studied their effect on in vitro microtubule polymerization and their action on the cytoplasmic microtubule network and cell division by using PtK2 cells as well as human and murine leukemias. MATERIALS AND M E T H O D S Chemicals, Culture Media, and Antibodies. The synthesis of NSC 613862 (CI 980) and NSC 613863 has been reported (8). Stock solutions were made in DMSO 3 and stored at -20~ Their concentrations were measured spectrophotometrically using extinction coefficients E374 nm 15100 and 15400 M -1 cm -1 for the S and R compounds, respectively (8). MTC was a gift from Dr. T. J. Fitzgerald (16). Its concentration was measured spectrophotometrically using the extinction coefficient •343 n m : " 17600 M-I"cm -1 (17). GTP, disodium salt, and colchicine were from Fluka. Podophyllotoxin was from Aldrich Chemical Co. Dulbecco's modified Eagle's medium, RPMI-1640, FCS, L-glutamine, nonessential aminoacids, and antibiotics were from Flow Laboratories (Irvine, United Kingdom). Propidium iodide, DM1A anti-tubulin monoclonal antibody, and goat-antimouse polyvalent immunoglobulins (fluoresceine-conjugated) were from Sigma Chemical Co. Rabbitantimouse immunoglobulins (peroxidase-conjugated) were from Dakopatts (Copenhagen, Denmark). 3 The abbreviations used are: DMSO, dimethyl sulfoxide; MTC, 2-methoxy-5-(2',3',4trimethoxyphenyl)-2,4,6-cycloheptatrien-l-one; MTP, microtubule protein; FCS, fetal calf serum; MES, 4-morpholineethanesulfonic acid; PEM, 100 mM piperazine-N,N'-bis(2ethanosulfonic acid), 1 mM EGTA, and 1 mM MgC12, pH 6.8; PBS, phosphate-buffered saline; BSA, bovine serum albumin.