[CANCER RESEARCH 44, 3245-3251, August 1984] Synthesis, Antitumor Activity, and DMA Binding Properties of a New Derivative of Amsacrine, A/-5-Dimethyl-9-[(2-methoxy-4- methylsulfonylamino)phenylamino]-4-acridinecarboxamide1'2 Bruce C. Baguley, William A. Denny, Graham J. Atwell, Graeme J. Finlay,3 Gordon W. Rewcastle, Simon J. Twigden, and William R. Wilson Cancer Research Laboratory and Department oÃ-Pathology, University of Auckland Medical School, Auckland, New Zealand ABSTRACT The 4-(W-methylcarboxamido)-5-methyl derivative of amsa- crine (NSC 249 992) has been synthesized as part of a program aimed at optimizing solid tumor activity in this series. Physico- chemical studies of this analogue ¡N-5-dimethyl-9-[(2-methoxy- 4-methylsulfonylamino)phenylamino]-4-acridinecarboxamide; NSC 343 499) indicate a slightly increased lipophilicity (estimated log p = 1.10), a decreased acridine base strength (pKa 6.40), and a 16-fold-higher association constant for double-stranded calf thymus DNA (K, 2.1 x 106 ivr1 at 0.01 ionic strength). Like amsacrine, the drug binds to DNA by intercalation. Inhibition of cell growth has been monitored by continuous drug exposure assays with a variety of rodent and human cell lines. The con centration for 50% inhibition varied from 6.7 nw (T-47D, a human breast carcinoma line) to 800 nM (P388/ADR, a murine cell line resistant to Adriamycin). A/-5-Dimethyl-9-[(2-methoxy-4-methyl- sulfonylamino)phenylamino]-4-acridinecarboxamide was cyto- toxic at growth-inhibitory concentrations and also induced cell cycle arrest in the G2 phase. It was active against P388 leukemia following administration by P.O., i.V., or i.p. routes, and it was superior to amsacrine, daunorubicin, and Adriamycin. It was curative towards i.v.-injected Lewis lung tumor in a proportion of animals when treatment was started on Day 1 or Day 5 after tumor inoculation. It also produced highly significant life exten sions against advanced tumors (treatment starting Day 9 after i.v. inoculation or on Day 8 after s.c. inoculation) and was comparable to cyclophosphamide in its effectiveness. It is a candidate drug for clinical trial. INTRODUCTION Amsacrine (NSC 249 992), a 9-anilinoacridine derivative, was first synthesized and shown to be active in a variety of experi mental tumors by Cain and Atwell (11). Clinical trials have dem onstrated useful activity against leukemia (1,28) and, to a lesser extent, against lymphoma (37). Activity in breast cancer is low but significant (27), whle activity in other solid tumors is consid ered insufficient to warrant further clinical trial (26). Studies in this laboratory have been aimed at the identification of analogues of amsacrine which might offer a broader clinical antitumor spectrum. Amsacrine binds to DNA by intercalation (36) and induces single-stranded and double-stranded breaks in the DNA of treated cells (42). It resembles the anthracycline antibiotic dau norubicin in its action but is less cardiotoxic. Since Adriamycin, differing from daunorubicin in only one substituent, has a mark edly broader antitumor spectrum (19), it may be hypothesized that appropriate substitution of amsacrine may increase its activ ity against clinical solid tumors. To this end, a large series of analogues of amsacrine have been synthesized, and their exper imental antitumor activity has been determined (2,6,11-13,15- 18). Many of these new analogues have now been evaluated to identify compounds with superior activity to amsacrine against solid tumors. One approach has involved the comparison of activity in vitro using a panel of human carcinoma cell lines. A second approach has been to investigate the activity of amsa crine analogues against the Lewis lung carcinoma in mice. This is the least responsive mouse tumor in the main NCI4 panel (23) and is resistant to Adriamycin, actinomycin D, ametantrone, amsacrine, daunorubicin, and mitoxantrone (7, 23). The results of these studies have been to a large extent convergent, and a disubstituted derivative of amsacrine (CI-921 ; NSC 343 499) has been identified as the most active derivative of amsacrine in the Lewis lung tumor system. The synthesis, DNA binding properties, in vitro cytotoxicity, and in vivo antitumor activity of this compound form the basis of the present study. A short report of some of these properties has been published previously (3). MATERIALS AND METHODS Synthesis of CI-921. The key intermediate for the preparation of CI- 921 , 5-methylacridone-4-carboxylic acid, was first synthesized from 7- methylisatin (31 ) (Chart 1, Method A), but this method proved impractical for large quantities. A better overall yield was obtained from 3-methylan- thranilic acid (Chart 1, Method B) (15), but this is an expensive starting material. We have recently developed a third route (Chart 1, Method C) from the readily obtainable 2-iodoisophthaiic acid.5 Conversion of this compound to 9-chloro-5-methylacridine-4-carbonyl chloride and subsequent selective reaction first with methylamine to provide A/-methyl-9-chloro-5-methylacridine-4-carboxamide followed by A/-(4-amino-3-methoxyphenyl)methanesulfonanilide gave the hydrochlo- ride salt of the desired compound as reported (15). The hydrochloride (10 g) was dissolved in 50% aqueous ethanol (500 ml) and treated with 1This work was supported by the Cancer Society of New Zealand, Inc., by its Auckland Division, and by the Medical Research Council of New Zealand. 2 Dedicated to Professor Bruce F. Cain, who directed the development of the amsacrine series until his death in January 1981. 3 Recipient of a Warner-Lambert Fellowship. Received January 24, 1984; accepted May 1,1984. 'The abbreviations used are: NCI, National Cancer Institute; CI-921, N-5- dimethyl-9-[(2-methoxy-4-methytsulfonylamino)phenylamino]-4-acridinecarboxam- ide (NSC 343 499):2-hydroxyethanesulfonate (1:1); ID»,drug concentration (nM) required to decrease the cell density at the end of cell culture by 50% relative to that of untreated control cultures; 0.01 AC buffer, 9.3 mm NaCI:100 ¡MEDTA: 2 HIM sodium acetate (pH 5.0; ionic strength, 0.01); ILS, increase in life span of treated tumor-bearing animals relative to that of untreated control mice; poly- (dA-dT), poly(deoxyadenylate-deoxythymidylate) (alternating); poly(dG-dC), poly(deoxyguanylate-deoxycy tidylate) (alternating). 5 Q. W. Rewcastle and W. A. Denny, manuscript submitted for publication. AUGUST 1984 3245