Synthesis, Antitumor Activity, and DMA Binding Properties of A

[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 oÃPathology, University of Auckland Medical School, Auckland, New Zealand

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

AUGUST 1984 3245

tumor size (measured in 2 dimensions with calipers) on Day 7. Mice were then ear-tagged and divided into groups of 8, tumors being measured thereafter 3 times weekly with vernier slide calipers. Drug treatment was initiated on Day 8. Tumor volume was calculated as 0.52 a'b. where a and b are the minor and major axes, respectively, of the tumor. The mean Â±S.E. was calculated for each on the basis of the logarithms of the tumor volumes. Cell Culture. L1210 and P388 cell lines grown in culture were the same cells as used for in vivo studies. The Adriamycin-resistant cell line P388/ADR was kindly provided by Mason Research, Inc. The mouse leukemia lines were passaged in DBA/2J carrier mice and adapted to cell culture for 3 weeks before use in growth inhibition assays. All 3 mouse leukemia lines were cultured in 24-well trays in RPMI 1640 medium (Grand Island Biological Co., Grand Island, NY) containing 10% (v/v) heat-inactivated fetal calf serum as described previously (9, 41). The remaining cell lines were cultured in 24-well trays in Â«-minimal CM COCÃ• essential medium containing 10% (v/v) heat-inactivated fetal calf serum without antibiotics and is described fully elsewhere (21, 41 ). Cells were judged Mycoplasme free by cytochemical staining (14). V-79-171 b cells Chart 1. Steps in the synthesis of CI-921. were from Dr. W. R. Inch, London, Ontario, Canada. A tissue culture- adapted Lewis lung cell line was provided by Dr. R. C. Jackson, Warner- excess aqueous KHCO3 to provide the free base. This was dried, Lambert Research Laboratories, Ann Arbor, Ml, and originated from the suspended in dry methanol (200 ml), and treated with methanesulfonic Southern Research Institute, Birmingham, AL (39). HCT-8 cells were acid (1.05 equivalents). The resulting solution was diluted slowly with originally established in culture by Tompkins ei a/. (35) and were also ethyl acetate at the boil until crystallization of the red methanesulfonate obtained from Dr. R. C. Jackson. Jurkat leukemia cells (30) were provided salt commenced. The yield was 11.1 g (94% from the crude hydrochlc- by Professor J. D. Watson, Department of Pathology, University of ride); m.p. 254-256Â°. Auckland Medical School. Descriptions of the other human tumor cell lines are given in the following references: HT-29 (22); LoVo (20); MCF- C2,H24N4O4S 7 (32); MDA-MB-231 (10); T-47D (25); and MM-96 (38). Found: C 52.11, H 5.2, N 9.8, S 10.9% The IDsoassay procedure involves the addition of drugs at the appro HCKCHÃ¼JzSOaHrequires:C, 52.8; H, 5.2; N, 9.5; S, 10.9%. priate dilution to exponentially growing cultures in 24-well trays and further growth of cells for 4 to 5 generations. Cells were counted at the Materials. Amsacrine was either synthesized in this laboratory as end of the incubation period using an electronic particle counter (Coulter). described previously (12) or obtained from Warner-Lambert as the ise- The clonogenicity of one of the cell lines (T-47D) was assessed by thionate. Cyclophosphamide (Bristol-Myers), 5-fluorouracil (Roche), Adri- trypsinizing (0.07% trypsin for 10 min), washing with growth medium, amycin (Farmitalia). and daunorubicin (May & Baker) were obtained as and replating in 60-mm Petri dishes containing 5 ml of medium. Colonies clinical formulations. Calf thymus DNA (type V) was from Calbiochem, of greater than 30 cells were counted 17 days later after staining with and diamidinophenylindole, ethidium bromide, poly(dA-dT), and poly(dG- 50% (v/v) aqueous ethanol containing 0.5% mÃ©thylÃ¨neblue. dC) were obtained from Sigma Chemical Co. Flow Cytometry. L1210 cell cultures (1 ml) were prepared for flow Mice. Breeding pairs of C57BL/6J and DBA/2J mice, obtained from cy tome try by addition of phosphate-buffered saline (1 ml) containing The Jackson Laboratory, Bar Harbor, ME, were used to establish a 0.4% Triton X and 10 /il of an ethandic solution of diamidinophenylindole breeding unit for the production of C57BL/6J x DBA/2J Fi (hereafter (0.2 mg/ml) according to the method of Taylor (34). Analysis was carried called B6D2F,) mice under conditions of constant temperature and out using an Ortho Instruments ICP 22A cytometer with a Model 2103 humidity, with sterile bedding, water, and food. multichannel analyzer. Proportions of cells in different phases of the cell Tumors. P388, L1210, and Lewis lung cells were kindly provided as cycle were estimated by fitting symmetrical curves to the Gr and G^ frozen stocks in 1977 by the Development Therapeutics Program, Divi phase peaks and measuring areas. sion of Cancer Treatment, NCI. Details of passaging procedures have DNA Binding. DNA binding isotherms were measured by equilibrium been described previously (7). P388 leukemia cells obtained by peritoneal dialysis using a Dianorm apparatus (Diachema A. G., Zurich, Switzerland) washing of carrier (DBA/2J) mice were inoculated i.p. (106 cells) in with two 1-ml compartments separated by Diachema membranes (M, phosphate-buffered saline [NaCI (8 g/liter):KCI (0.2 g/liter):KH2PO4 (0.2 cut-off, 10,000). Calf thymus DNA (1 to 15 Aseo units/ml) and CI-921 g/liter):Na2HPO4(1.15 g/liter):CaCI2(0.1 g/liter):MgCI2(0.1 g/liter)]. Lewis were dissolved in 0.01 AC buffer. The cells were rotated at 16 rpm for lung cells were prepared by passing minced s.c. tumors (from earner 15 to 20 hr at 25Â°(equilibrium was attained well before this time), and C57BL/6J mice) through 100-jtm nylon monofilament mesh. Nucleated the contents of each cell were then assayed spectrophotometrically. The cells were counted in 1% acetic acid with a hemocytometer, and the spectrum of the DNA-containing cell was measured using the DMA-free suspension was used for i.v. or s.c. inoculation of 108 cells/mouse. cell as a reference, and the bound drug concentration was calculated Life Extension Assays. Control groups included 20 to 30 mice, and from the extinction coefficient of DNA-bound drug (measured in a 1 mg/ experimental groups had 6 mice, all B6D2F, of either sex. Drug doses ml solution of DNA). were adjusted to pretreatment body weight in groups which were weight Viscometry. The extent of DNA unwinding induced by drug binding matched at intervals of 1 g body weight (18 to 19,19 to 20, 20 to 21, or was determined by measuring the viscosity of closed circular DNA at 21 to 22 g). Dose levels were set at 1.5-fold intervals, and the optimal 25Â°using methods published previously (36). The DNA was prepared dose was defined as the dose giving the highest ILS without causing from a mini-col E1 plasmid PML-21 (24), kindly provided by Dr. H. E. D. deaths from drug toxicity. Deaths were recorded daily, and the percent Lane, Department of Cell Biology, University of Auckland. Equilibrium ages of ILS were calculated at Day 50 (P388) or Day 60 (Lewis lung). sedimentation in ethidium bromide:cesium chloride was used for purifi Mice surviving at this time were excluded from the calculation and cation of the DNA. Small aliquots of drug solution (0.4 mg/ml in H2O) recorded separately. were added to an Ostwald viscometer containing 1.1 ml of the DNA Tumor Growth Delay Determinations. B6D2F, mice were inoculated solution (65 /jg/ml) in 0.01 AC buffer. The helix unwinding angle was s.c. (106 Lewis lung cells) on Day 0 and randomized with respect to calculated as 26Â°x r,/rd, where r, and r,, are the binding ratios at the

3246 CANCER RESEARCH VOL. 44

equivalence point for ethidium and CI-921, respectively. Binding ratios 28 r were calculated from the drug association constants determined by equilibrium dialysis using the McGhee and von Hippel equation (29).

RESULTS

Physicochemical Properties. The lipophilicity of CI-921 was determined using a thin-layer Chromatographie system to mea sure relative mobilities [Rm = log (1/flf - 1)], which are linearly related to the logarithms of the partition coefficients between n- octyl alcohol and water (log p values) (17). The Rm values determined for the cationic forms of CI-921 and amsacrine are, respectively, 0.34 and 0.18, corresponding to log p values of 1.10 and 0.60 (17,18) and indicating that the analogue is slightly more lipophilic (0.5 log p units) than amsacrine. Acridine pK<, values for amsacrine and analogues have been determined pre viously (2) in 20% aqueous dimethyl formamide. Under these conditions, amsacrine has a value of 7.43, while CI-921 is a 12L considerably weaker base, with a pKÂ«of6.40 (15). 0.02 0.04 0.06 0.08 0.10 DNA Binding Properties. Binding of CI-921 to double- D/P stranded calf thymus DNA in 0.01 AC buffer was accompanied Chart 3. Effects of CI-921 (O) and amsacrine (â€¢)on the reduced viscosity of by a bathochromic shift in the wavelength of maximal absorption closed circular duplex DNA in pH 5.0 acetate buffer. The molar ratio of drug to from 448 nm to 464 nm and a decrease in the extinction DNA phosphate (0/P) is shown on the abscissa. The reduced viscosity (r,) (units, dl/g) is shown on the ordinate. coefficient at the wavelength of maximal absorption from 12,740 to 8,360 cm~1 M~\ Equilibrium dialysis was carried out in this displacement method as described previously (5). Competition buffer using calf thymus DNA (Chart 2). The scatter of the data binding equations developed by McGhee and von Hippel (29) reflects the low concentrations of drug in the DNA-free compart were combined with a correction for quenching of DNA-bound ment. A theoretical curve was calculated using the equation ethidium fluorescence by bound drug (39). This quenching effect, derived by McGhee and von Hippel (29) for a model with neigh which may be due to an electron transfer phenomenon (8), was boring site exclusion and assuming a binding site size of n = 2 45% greater with CI-921 than with amsacrine at the same binding base pairs. The association constant obtained was 2.1 x 106 ivr1, 16-fold higher than that of 1.3 x 10s w~1 obtained (at pH ratio. Binding constants for poly(dA-dT) and poly(dG-dC) were, respectively, 1.15 x 106 NT1and 4.4 x 106 w~1, as compared to 6.0) with amsacrine at the same ionic strength (40). 3.7 x 10s M-1 and 4.5 x 105 KT1 for amsacrine (4). CI-921 The base sequence binding selectivity was examined using poly(dA-dT) and poly(dG-dC) in 0.01 AC buffer using an ethidium therefore demonstrated greater selectivity than did amsacrine for poly(dG-dC), as opposed to poly(dA-dT). The binding mode of CI-921 was investigated by viscometric 2.0 titration with closed circular duplex DNA (Chart 3). As the drug:DNA phosphate (D:P) ratio increased, the viscosity first increased and then decreased, indicative of intercalative binding. The unwinding angle of 18Â°(corrected for the proportion of unbound drug) is slightly less than that of amsacrine (20.5Â°)and 1.5 less than that of ethidium (26Â°). In Vitro Inhibition of Tumor Cell Growth. CI-921 was com pared to amsacrine in growth inhibition assays using a variety of rodent and human tumor cell lines (Table 1). CI-921 had a _L 1.0 potency similar to that of amsacrine in the mouse leukemia lines but was more active against the Lewis lung carcinoma and against all the human tumor cell lines tested. Adriamycin and daunorubicin, 2 other DNA binding drugs which differ only mini mally in structure but show different spectra of clinical activity, 0.5 were also compared (Table 1). It was of interest to determine whether differences in patterns of sensitivity between CI-921 and amsacrine were mirrored by differences between Adriamycin .1 and daunorubicin. The ratios of IDsovalues for each pair of drugs are therefore compared in Table 1 and indicate similar patterns. 0.1 0.2 0.3 0.4 0.5 Relationship of Growth Inhibition to Cytotoxicity. In order to determine whether growth inhibition was a result of cytotoxicity Chart 2. Scatchard plot for the interaction of CI-921 with double-stranded calf or merely cytostasis, T-47D cells, at the end of the ID.Â»assay, thymus DNA in pH 5.0 acetate buffer, r is the ratio of bound drug molecules to were plated to determine the number of clonogenic cells. The DNA base pairs, and D, is the free drug concentration. The line shows the predicted curve for intercalative binding, where each bound drug occludes 2 base pairs with relationship between total cell number and number of clonogenic an association constant of 2.1 x 10* M~\ cells is shown for various concentrations of amsacrine (3 to 50

AUGUST 1984 3247

Table 1 /DÂ»valuesof CI-921 and related agents ID,,:.(n-.iÂ¡Cell IDso(nM) CI-

cin:daunorubi Adriamy-crmecrine rubicinratio2118551815022083755673224838"Â¿\VAdriamy lineL1210P388P388/ADRV-79LewisHCT-8HT-29LoVoMCF-7MDA-231T-47DMM-96Jurkat100--ioin33 ratio cin1.40.782.641.10.200.290.300.170.230.270.100.130.24A leukemiaMouse 3014 1.2 leukemiaMouse 14550 0.86 leukemiaChinese 14516 1.5 fibroblastMousehamster 1927 0.59 carcinomaHumanlung 3070 0.47 carcinomaHumancolon 6372 0.36 carcinomaHumancolon 2547 0.28 carcinomaHumancolon 1379 0.26 carcinomaHumanbreast 1377 0.23 carcinomaHumanbreast 2028 0.40 carcinomaHumanbreast 2.150 0.24 melanomaHuman 6.312 0.32 T-cellleukemia.0oo 9.2100 0.30

r80

_i* 8Â°-M-; -iÃŸ60 â€¢â€¢o1 Â«o-UOÂ« -> UiWILs 40-Za0

20-2,20 v20 1160 - *Â»*j 100CI-92138128009.512.625201218316.7163.6CELLS40OriginMouse 80 V: / WELL %QO1-amsa-Amsa- JDauno Chart 4. Comparison of cell killing and growth inhibition by CI-921 (â€¢)and 100 r amsacrine(O), using cultured T-47D human mammarycarcinomacells. At the end of the standard IDÂ«assayprocedure, cells were counted to determine total cell densityor replatedat the appropriatedilutionto assess clonogenicity.Clonogenicity is expressed as a percentage of that of untreated cells. eo â€¢ nw) and CI-921 (0.5 to 10 nM) in Chart 4. The plating efficiency of treated cells decreased with increasing drug concentrations, eo indicating increasing cytotoxicity. The decrease was similar for u both drugs. 5s Cell Cycle Effects. The effects of CI-921 on the cell cycle ÃŽ 40 were investigated by flow cytofluorimetry using cultured L1210 *| J leukemia cells. The compound caused a dose- and time-depend 8 ent accumulation of cells in G2 phase. At a concentration of 100 20 nM, an increase in G2-phase cells was evident from 4 hr onwards. Exposure for 24 hr to a drug concentration of 6 nw produced a significant increase in G2-phase cells (27% as compared to the control value of 16%), and at higher concentrations, a high IO'Â» proportion of the total population could be trapped in G2 phase CHANNEL NUMBER (Chart 5). Charts. Flow cytofluorometric profiles of DMAcontent of cultured L1210 cells in logarithmic phase either before treatment (A) or 24 hr after addition of CI-921 Activity towards P388 Leukemia in Vivo. Although having a (100 nM)(B). |, position of pigeon erythrocytes which were used as an internal therapeutic range similar to that of amsacrine (active over a 10- marker. fold dose range), CI-921 provided a considerably higher life extension at the optimal dose (Chart 6). This superiority was manifested when the drug was given Â¡.p.,p.o., or i.v. (Chart 6; i.p. on Days 1, 5, and 9 after i.v. injection of tumor cells (Table Table 2). CI-921 was also therapeutically superior to Adriamycin 3). Adriamycin and daunorubicin were inactive with this route and daunorubicin, although its dose potency was lower (Table and schedule of administration. In 3 experiments, CI-921 pro 2). vided 14 of 17 sixty-day survivors and provided highly significant Activity towards the Lewis Lung Carcinoma in Vivo. Amsa life extensions in the remainder. crine had only marginal activity towards this tumor when injected In order to provide a larger number of target cells, the Lewis

3248 CANCER RESEARCH VOL. 44

u in

K CYCLO- 8 DOSE ma/kg PHOSPHAMIDE Â§ LO Chart 6. Dependence on dose of the percentage of ILS of animals inoculated i.p. on Day 0 with 10Â°P388 leukemia cells. CI-921 (O, A); amsacrine (â€¢,A).Drugs were administered on Days 1, 5, and 9 either i.p. (O, â€¢)orp.o. (A, A). 0.5 Table 2 10 15 20 Activity of CI-921 and other agents towards i.p.-inoculated P388 leukemia DAYS AFTER START OF TREATMENT of ILS dose at opti Chart?. Effects of CI-921 (A) and cyclophosphamide (D) on the growth of (mg/kg)a20 CompoundCI-921AmsacrineAdriamycinDaunorubicinCyclophosphamideRoutei.p.maldose200 (50day)3/6 advanced s.c.-implanted Lewis lung tumor. Drug treatment was initiated 8 days after s.c. inoculation of 10* tumor cells. CI-921 was administered 3 times daily i.p. every 4 days (20 mg/kg/dose), while cyclophosphamide was administered as a P.O.Â¡.V.Â¡.p. 50 164 1/6 single dose (220 mg/kg). Each group contained 8 animals. Growth of untreated 308.9 13678 1/60 tumors is also shown (â€¢).Bars,S.E. of tumor volume measurements. or 13.3 p.o.i.V.i.p.i.p.i.p.Optimal65303.92.7225% 62727960216Survivors0/6 0/61/60/65/6 (7), the solvent for i.p. dosage was 30% (v/v) aqueous ethanol. However, distilled water (optimal dose, 30 mg/kg; ILS, 203% with 5 of 6 sixty-day survivors) and 50% dextrose in water (optimal dose, 30 mg/kg; ILS, 161% with 4 of 6 survivors) were acceptable solvents, providing full dissolution of the agent. CI-

* Drug administered on Days 1, 5, and 9 after tumor inoculation, except for 921 thus showed activity comparable to cyclophosphamide (the NCI standard for this tumor) and superior to 5-fluorouracil. cyclophosphamide, which was administered on Day 1 only. Data (i.p.) for amsacrine represent an average of multiple determinations, and the optimal dose was either When treatment was initiated at Day 9, at a stage where tumor 8.9 or 13.3 mg/kg. nodules in the lung were 1 to 2 mm in diameter and the mice were approximately 7 days from death, a highly significant in TaUe3 Activity of CI-921 and other agents towards i.v.-inoculated Lewis lung carcinoma crease in life span was still achieved, although there were no long-term survivors. Advanced s.c. tumors were also responsive to treatment with CI-921 (Chart 7). On the basis of data from (day(s)of administra dose several experiments, control tumors grew in a fashion which CompoundCI-921AmsacrineAdriamycinDaunorubicinCyclophosphamideRoutei.p-i.p.Â¡-P-Â¡â€¢P-Â¡â€¢P-P.O.Â¡.V.i.p.Â¡â€¢P.P.O.Â¡.V.Â¡â€¢P-Â¡â€¢P-Â¡.p.Â¡-P-Scheduletion)11,5,955,9,139,(mg/kg)202020303050308.9(%)611683816786132127384238582512216185Survivors(60day)1/614/170/69/210/100/65/6000/61/60/60/65/65/6could be acceptably described by a Gompertzian growth equa tion (33), with an initial doubling time of 18 hr and a halving of the growth rate every 8 days. At the time of initiation of treatment, the tumor volume was estimated as 1 cu cm, and the doubling 13,175,9,135,9,131,5,95,9,135, time was 36 hr. The delay in tumor regrowth corresponded to an overall 6 Iog10cell kill (assuming no reversible growth inhibi

or13.3"8.9 tion) and was comparable to that of cyclophosphamide. Mea or13.3s75302.63.9225225ILS sured tumor volumes after 4 days were all significantly less than 9,135,9,135, those of the control tumors (p < 0.01). In contrast, amsacrine treatment at the highest tolerated dose resulted in no significant 9,135, reduction in tumor volume over the course of the experiment (data not shown). 9,1315Optimal DISCUSSION

* Average of multiple experiments; optimal dose was either 8.9 or 13.3 mg/kg. The primary aim of our studies with amsacrine analogues has been to develop compounds with activity against solid tumors. lung tumor is routinely grown in this laboratory for 5 days before This development can be regarded theoretically as an optimiza initiation of drug treatment (7). Under these conditions, CI-921 tion of pharmacokinetics and drug stability (to allow maximal still provided a proportion of 60-day survivors (Table 3). The exposure of tumor cells to drug) and optimization of intrinsic dose profile was quite similar to that of the P388 leukemia, and activity towards tumor cells (cell cycle selectivity, transport, the minimal effective dose (40% life extension) was 7.3 mg/kg. metabolism) as compared to normal cells. CI-921, developed as Although the i.p. route was the most effective, p.o. and i.v. a result of previous structure-activity studies (6, 18) which indi routes also provided high life extensions. As in previous studies cated the optimal physicochemical properties for this class of

AUGUST 1984 3249

B. C. Baguley et al. compound, has shown the highest levels of activity of any lively high water solubility of this compound. amsacrine derivative, particularly against the Lewis lung carci Acridine substitution of amsacrine has been shown previously noma. to influence the relative activity towards individual cell lines (41). The first physicochemical parameter to be investigated in the Table 1 represents an attempt to determine the extent of this 9-anilinoacridine series was lipophilicity, and a clear parabolic variation for CI-921 relative to amsacrine by presenting results dependence of antileukemic activity (as measured by increased with a number of different rodent and human cell lines in culture. life span) on lipophilicity was demonstrated (13). More recent Ratios of IDsovalues vary from 1.5 to 0.23 with the human lines studies have shown that measurements of in vitro and in vivo showing the greatest selectivity for CI-921. By comparison, the activity towards L1210 leukemia cells are related by a parabolic corresponding range for Adriamycin and daunorubicin is from term in lipophilicity (4), suggesting that this property plays an 2.6 to 0.1, and there is evidence of parallel effects with the 2 important role in the pharmacokinetics of drugs in this series. pairs of compounds. Although some of these effects may be Lipophilicity also appears to be of critical importance in determin artifacts of tissue culture techniques (e.g., differences in drug ing the activity of amsacrine derivatives towards the Lewis lung half-life in different culture media), the results suggest strongly carcinoma.6 CI-921, by combining a lipophilic (methyl) and hydro- that individual cell line sensitivity differences do exist and that philic (A/-methylcarboxamide) group, possesses near-optimal li CI-921 and Adriamycin show increased activity towards carci pophilicity as suggested by structure-activity relationships (15, noma cells as compared to amsacrine and daunorubicin. 18). These groups also have the additional advantage of confer In conclusion, CI-921 represents an analogue of amsacrine ring greater water solubility than is shown by amsacrine. which was selected from in vivo and in vitro screening of several Investigation of the other effects of acridine ring substituents hundred members (15,18) of the 9-anilinoacridine series. While of amsacrine was initially carried out by comparing the effect of it resembles amsacrine in its mode of action on cultured cells (G2 substitution on antileukemic activity with that expected on the arrest), its activity against P388 leukemia and Lewis lung carci basis of the changes in lipophilicity alone, the latter being ascer noma is superior to that of amsacrine and to all other derivatives tained with reference to homologous alkanesulfonamide series of amsacrine tested so far. A particular feature is its activity containing constant acridine substituent patterns (12, 13). A towards advanced Lewis lung carcinoma, growing either in the number of substituents showed increased activity over that lung or s.c., where it is similar to cyclophosphamide in its efficacy. expected from changes in lipophilicity alone (12, 16), and the 4- It is active against a number of other tumors including L1210 methyl (equivalent to the 5-methyl in CI-921 ) derivative (11) and leukemia, B16 melanoma, the CD8F1 mammary tumor, and the a variety of A/-substituted 4-carboxamide groups (16) showed colon 26 and 38 tumors.7 8 This activity, in combination with the large effects. Recently, both the 4-methyl and the 4-N-methyl selective activity towards human carcinoma cell lines in culture, carboxamide substituents have been identified as providing strongly suggests that it may be superior to amsacrine in the greatly increased activity towards the Lewis lung carcinoma in treatment of human carcinomas. It has now been selected as a mice (7). candidate drug for clinical trial. Substituents which result in such increases in activity may do so by altering a number of physicochemical properties simulta ACKNOWLEDGMENTS neously. A number of acridine substituents are known to increase DNA binding (6). Multivariate equations modelling dose potency The authors are grateful to Antoinette Kemohan, Rosalee Nash, Cherry Grim- wade, Julie Harris, and Susan Tapp for expert technical assistance and to Sally Hill indicate that both high DNA binding affinity and high acridine for preparing the manuscript. base strength (pKÂ«)areassociated with high dose potency. This may be rationalized in terms of increased retention in tumor cells REFERENCES (i.e., by drug binding to DNA) and decreased excretion rates 1. Ariin, Z. A., Sklaroff, R. B., Gee, T. S., Kempin, S. J., Howard, J., Clarkson, B. (since charged molecules are excreted at lower rates). However, D., and Young, C. W. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1984 American Association for Cancer Research. Synthesis, Antitumor Activity, and DNA Binding Properties of a New Derivative of Amsacrine, N -5-Dimethyl-9-[(2-methoxy-4-methylsulfonylamino)phenylamino] -4-acridinecarboxamide

Bruce C. Baguley, William A. Denny, Graham J. Atwell, et al.

Cancer Res 1984;44:3245-3251.

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