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(CANCER RESEARCH 56. 2094-2104.

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Pharmacological and Toxicological Aspects of New Imidazoacridinone Antitumor Agents1 Birgit Berger,2 Hans Marquardt, and Johannes Westendorf [)t'¡mrrmt'nt of TO.\ÕCOÕOX\. University of Htiinhurg Medicttl School, unti Fraunhofi'r Hdtnlntrx. Genntin\

liefHirltnent of Toxicology

tinti Eiivirontnenttil

Medicine.

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ABSTRACT

INTRODUCTION

Imidazoacridinones represent a new group of antitumor compounds developed by .1. Konopa and coworkers in Gdansk. Poland (YV. M. Cholody, J. Med. Chem., 33: 49-52, 199(1). The compounds exert activity

Several antitumor agents interact with DNA. either by the forma tion of covalent bonds or by noncovalent binding, i.e., intercalation (1). Polycyclic aromatic antineoplastic agents, such as doxorubicin (2), mitoxantrone (3), or anthrapyrazoles (4), belong to the group of DNA-intercalating agents. Modifications of the chemical structure of the anthracenediones led to the synthesis of a new group of promising antitumor agents, the imidazoacridinones (5). with a broad spectrum of antitumor activity in vitro (6). The biological activity of these antituinor agents is determined by a planar polycyclic ring system capable of binding to DNA and one or two polymethylenediamine side chains in a well-defined orientation to the ring system (5). The addition of a fourth ring to the basic tricyclic system increases the density of the TT-electron system and makes the chromophore proba bly more resistant to enzymatic reduction by radical species (7). An important aspect for the development of new antitumor agents is a selective cytotoxicity of these compounds against tumor cells. Because the development of resistance against a variety of antitumor agents by the overexpression of the P-glycoprotein is a common phenomenon during an antineoplastic chemotherapy (8-10). it is thus important to develop new agents that retain their activity in MDR3

against a broad spectrum of human tumors in the National Cancer Institute i/i vitro screening scheme. In this work, the in vitro cytotoxicity. cellular pharmacology, and genotoxic/transforming potential of Five se lected imidazoacridinones were studied. The compounds were highly cytotoxic (0.01-0.49 /IMI to dividing cells, such as Friend erythroleukemia cells (line F4-6), V79 Chinese hamster cells, and exponentially growing C3H/M2 mouse fihroblasts. In contrast, nondividing primary rat hepatocytes and C3H/M2 cells in confluency were less sensitive to the toxicity of the imidazoacridinones. Multidrug-resistant-overexpressing F4-6 cells, 200-fold resistant to doxorubicin. showed only partial resistance (4-10 fold) to the imidazoacridinones. The cellular transport of the fluorescent imidazoacridinones occurred rapidly, and most of the drug fluorescence was localized in the nucleus. Cellular accumulation and retention of two selected imidazoacridinones (C-1310 and C-1311 ) in sensitive as well as in resistant F4-6 cells were determined with laser-excited flow cytometry. After an incubation with C-1311 and C-1310 for 60 min at 37°C, the cellular accumulation of the less cytotoxic compound C-1310 was greater than that of C-1311. and for both compounds, the fluorescence in the resistant F4-6 cells was one-half of that in the sensitive F4-6 cells. Lowered temperature (4"C) reduced the cellular accumulation for both compounds comparable

in the sensitive and in the resistant F4-6 cells and was to the uptake in resistant F4-6 cells at 37°C.The resistant

F4-6 cells at 37 C showed, despite a lower loading level, a faster initial efflux compared to the sensitive F4-6 cells. The treatment of the resistant F4-6 cells with the multidrug-resistant modulator verapamil led to an enhanced accumulation of C-1310 and C-1311 by the cells. All five com pounds produced a dose-dependent inhibition of | '11|uridine and | "( '|th\ inuline incorporation

and, except for C-1336,

preferentially

inhibited

DNA synthesis. The affinity of the imidazoacridinones to DNA is also indicated by an increase of the DNA melting point by 9-11°C. The mutagenic potential of the imidazoacridinones was investigated in the hypoxanthine guunine phosphoribosyl transferase test,- the compounds C-1310 and C-1311 were additionally tested in the Salmonella typhi/nurium-microsome assay. Limited mutagenicity was detected in the hy poxanthine guanine phosphoribosyl transferase test, and in Salmonella typhimurium, mutagenicity was observed only in the strain TA1537. Fur thermore, no induction of DNA repair synthesis was observed after treat ment of primary hepatocytes with the five imidazoacridinones. The com pounds did not transform C3H/M2 fibroblasts. One derivative, C-1336, led to a significant induction of cell differentiation in Friend erythroleu kemia cells. The results of this study show that the imidazoacridinones display a strong cytotoxic effect in rapidly dividing cells and only a partial resistance toward multidrug resistant cells; in addition, they showed a limited mutagenic potential in Y79 fibroblasts and Salmonella typhi murium and no transforming potential in C3H/M2 cells. The imidazo acridinones are, therefore, an interesting group of new antitumor agents, and further//! vivo studies are warranted to explore the usefulness of these compounds for the treatment of human cancer.

tumor cells (11, 12). Additionally, it is important to develop new compounds with a reduced long-term toxicity, such as mutagenicity and carcinogenicity. because many of the clinically used cheinotherapeutic agents are genotoxic in cell cultures (13). laboratory animals (14, 15), and humans (16). Since the induction of cell differentiation is a new aspect of the therapeutic action of antileukemic agents ( 17, 18). it is important to investigate the capacity of antituinor agents to induce cell differentiation in relation to their cytotoxic potential. In the present investigation we, therefore, studied the pharmaco logical and toxicological properties of a variety of structurally related imidazoacridinones. MATERIALS

Chemicals. Imidazoacridinones were synthesized and kindly provided by Drs. W. M. Cholody (NCI. Frederick. MD) and J. Konopa (Technical Univer sity of Gdansk. Gdansk. Poland: Ref. 5). The five compounds that we inves tigated differ in their substitution at the imida/.o ring and the length of the side chain. The compounds C-1309, C-1310. and C-1336 have in common a methyl group at the imidazo ring. The compounds C-1311, C-1310. C-1309. and C-1335, C-1336 contain two or three carbons between the proximal and distal nitrogens in the side chain, respectively. All compounds contain two ethyl groups at the distal nitrogen except C-1309 (two methyl groups). Stock solutions of these compounds containing 1 mg/ml were prepared in distilled water. Dilutions were achieved by the addition of aqueous medium. The stability of the stock solution was checked by TLC on silica gel. Si-60. solvent: crtlorolbrni:methanol:acidic acid. IOO:2:/5. ['HJUridine (26 mCi/mmol) and l'4C]thymidine (52 mCi/mmol) were purchased from Amersham Buchler (Braunschweig.

Received 1/15/96; accepted .V4/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by the Hamburger Stiftung /ur Förderung der Krebs bekämpfung, e. V. 2 To whom requests for reprints should be addressed.

AND METHODS

Germany). a-MEM containing no nucleosides. Williams me

dium E. EBSS. trypan blue solution, and PCS were obtained from Life Technologies. Inc. (Karlsruhe. Germany). Neutral red was obtained from 'The abbreviations used are: MDR. multidrug resistance; CFE, colony-forming effi ciency; EBSS. Earle's balanced salt solution; UDS. unscheduled DNA synthesis: MCF. mean channel fluorescence.

2094

ASI'IXTS

()l: Nl:W IMIDA/.OACKIDINONI-S

LS6000IC).

Merck (Darmstadt. Germany): calf thymus DNA from Boehringer Mannheim (Mannheim. Germany): HEPES from Serva (Heidelberg. Germany): 6-thioguanine, (±) verapamil. l-methyl-3-nitro-nitrosoguanidine. and NP40

ED5l)s were calculated

from the dose-response

curves as the

concentration of drug required to reduce RNA/DNA synthesis to 50% of the untreated control. Determination of Drug Influx and Efflux. For fluorescent microscopy, HeLa cells were cultured on flying coverslips in test tubes according to Paul (29). The imida/oacridinones (0.1 nM) were added to the cells for IO min. Then the coverslips were washed in EBSS. mounted on glass slides, and fixed with liquid paraffin. Drug uptake was analyzed with a fluorescent microscope (ZEISS ICN Camera microscope) filter: excitation wavelength. 395-400 nm; emission wavelength. 460-470 nm: film. Kodak ektachrome 400 ASA. In the uptake experiment, sensitive and resistant F4-6 cells were exposed to C-1310 and C-1311 (2.3 and 4.6 /UM)at 4°Cand 37°C.After varying periods

from Sigma Chemical Co. (Deisenhofen. Germany). Determination of DNA Melting Points. To determine the DNA melting temperature. 0.01 M phosphate buffer containing 0.1 M EDTA at pH 7 was used. Drug and DNA (calf thymus DNA) concentrations were 0.01 M and 0.1 M. respectively. The absorbance was monitored at A = 260 nm with a Gilford System 2600 Microprocessor-controlled UV-VIS spectrometer. The cells were heated at a rate of l°C/min. and the absorbance was read every minute. 7"mwas obtained by plotting the absorbance against the temperature and taking the midpoint of the curve between the high and the low temperature-absorbance regions. A7"nl describes the difference in melting temperature between the drug-DNA complex and the uncomplexed DNA. Cell Cultures. Friend erythroleukemia cells, line F4-6. were kindly pro vided by Dr. W. Ostertag (Heinrich-Pette-Institut fürExperimentelle Virolo gie. Hamburg, Germany: Ref. 19). Cells were grown in a-MEM without nucleosides supplemented with 10% FCS. They differentiate along the erythroid pathway after treatment with DMSO (20) or a variety of other inducing agents (21-23). Doxorubicin-resistant cells, variant F4-6 Adriamycin 2R. were kindly provided by Dr. A. Schaefer (Fraunhofer Department of Toxicol ogy and Environmental Medicine. Hamburg. Germany). To ascertain the drug-resistant phenotype. the cells were periodically treated with doxorubicin

of incubation (0.5. 1, 2. 5, 10, 15. 30. and 60 min), the cells were centrifuged and washed with HBSS. The intracellular content was determined with a Becton Dickinson flow cytometer using a laser excitation of 4X8 nm and a logarithmic amplification. The histograms were gated so that only viable cells were used for analysis. The relative cellular content for a particular sample was obtained by subtracting the MCF of the treated cells from that of the untreated control cells. In efflux experiments, sensitive and resistant F4-6 cells were exposed to C-1310 and C-1311 (2.3 and 4.6 JAM)at 37°Cfor 15 min. The drugs were then removed, and the cells were washed twice with HBSS and resuspended in drug-free medium. After varying periods of incubation at 4°Cand 37°C(2. 10.

at 1 f¿g/mlfor 2 days (12). Before their use. the resistant cells were grown for several generations in drug-free medium (12). V79 cells were kindly provided by Dr. E. H. Y. Chu (Department of Human Genetics. University of Michigan. Ann Arbor. MI) and grown in DMEM supplemented with 10% calf serum. 2 mM glutamine. and 1% penicillin/ streptomycin. The C3H/M2 cells were established as a line by Marquardt et al. (24). according to the procedure by Chen and Heidelberger (25). Primary rat hepatocytes were prepared and cultured as described in "Induction of UDS." Salmonella typhimurium tester strains TA 98. TA 100. and TA 1537 were kindly provided by Dr. B. Ames (Department of Biochemistry, University of California. Berkley. CA) Cytotoxicity Studies. Exponentially growing sensitive and doxorubicinresistant F4-6 cells (1 X IO6 cells/ml) were treated with various concentra tions of the imida/.oacridinones for 48 h at 37°C. After 48 h, cells were

and 20 min), the cells were centrifuged and washed with HBSS; then the intracellular content was determined with a Becton Dickinson flow cytometer. To determine the cellular uptake of C-1310 and C-1311 in the presence of verapamil. the resistant F4-6 cells were incubated with the compounds (2.3 and 4.6 ¿¿M) for 30 min. To one part of the cells, the MDR modulator verapamil was added, and the incubation was continued for another 30 min. The cells were then centrifuged and washed with HBSS; then the intracellular drug content was determined with a Becton Dickinson flow cytometer. Mutagenicity in V79 Chinese Hamster Cells. The mutagenesis assay with mammalian cells was performed similar as described by Westendorf et til. (30). The cells were first cultivated for 5 days in hypoxanthine-aminopterinethymidine medium to eliminate spontaneous 6-thioguanine-resistant mutants (31). Thereafter. 2 X 10* cells were plated into 60-mm dishes containing 6-thioguanine-free

medium. After 18 h. aqueous solutions of the test com

collected, and the trypan blue excluding cells were counted (26). The drug effects were expressed as a percentage of survival compared to the untreated control cells, and the IC,,,s were determined graphically from the dose-

pounds were added for 24 h. Then the medium was replaced by fresh DMEM supplemented with 10% calf serum, and after 3 days, the cells were trypsinized and counted. For each concentration, five dishes, each containing I X IO2

response curves. The factor of resistance was determined by division of the 1C,,, of the resistant F4-6 cells by the 1C,,, of the sensitive F4-6 cells.

cells, were prepared for the Cytotoxicity assay with 6-thioguanine-free me dium, and another five dishes were plated with I X IO5 cells for mutagenicity

Cytotoxic effects of imidazoacridinones on V79 cells were analyzed by determining the CFE (27); cells were incubated with various concentrations of the imidazoacridinones for 24 h, and colonies were allowed to grow for 6-8 days. Cytotoxicity was expressed as the percentage of colonies in the treated dishes compared to the control. The IC,,,s were calculated from the dose-

studies with medium containing 6-thioguanine

(10 /¿g/ml).After 1 week, the

Cytotoxicity dishes were fixed and stained with Giemsa solution. The muta genicity dishes were refed every 2 days with medium containing 6-thiogua nine. After 10-15 days, these dishes were fixed and stained with Giemsa solution, and the number of 6-thioguanine-resistant colonies was counted. The

response curves. Cytotoxic effects of imidazoacridinones on primary hepatocytes were de termined with the neutral red assay (28). The primary hepatocytes were incubated for 24 h with various concentrations of the imidazoacridinones and thereafter washed with HBSS and incubated with Williams Medium E con taining neutral red for another 2 h. The neutral red was then extracted from the cells with 50% ethanol supplemented with 1% acetic acid. Cell viability was expressed as the percentage of untreated control. Cytotoxic effects of imidazoacridinones on exponentially growing and «influential cultures of C3H/M2 cells were determined with the neutral red assay (28) and the measurement of the CFE (27). Inhibition of DNA/RNA Synthesis. About l x K)6 F4-6 cells were

mutation frequency was calculated per Iff survivors. The cocultivation of V79 Chinese hamster cells with primary Wistar rat hepatocytes was performed as described by Langenbach et al. (32). Initially. 2 X IO5 V79 cells were plated into 25-cm tissue culture flasks; 24 h later, 5 x 10" freshly prepared primary rat hepatocytes suspended in Williams

incubated in a-MEM

monella t\pMmurium was performed as described by Ames et al. (33). Induction of UDS. This assay was performed in primary Wistar rat hepa tocytes according to the procedure described by Williams (34) and Butterworth et al. (35). Transformation Studies. The transformation assay with C3H/M2 cells was performed as described by Marquardt et al. (24) Induction of Differentiation. F4-6 cells were incubated for 4 days in a-MEM without nucleosides and supplemented with 10% FCS. The cell number at the start of the incubation was 2 X 105/ml. After the 4-day

with 10% FCS. 20 mM HEPES buffer, and different

concentrations of the imidazoacridinones on a shaking heater for 6 h. During the last hour, the cells were labeled with ['H]uridine (0.25 /xCi/ml) and [l4C]thymidine (0.05 /j.Ci/ml). The reaction was stopped with ice-cold 10% trichloroacetic acid (w/v) containing 2% PP¡(w/v). and the macromolecules were precipitated at 4°Cfor 1.5 h. The precipitate was centrifuged and washed twice with ice-cold 5% trichloroacetic acid (w/v) containing 2% PP¡(w/v). Then the pellet was dissolved for 15 min in 2 N NaOH at 60°C.Aliquots of this solution were added to a scintillation cocktail (Beckman Ready Safe II), and the radioactivity was counted in a liquid scintillation counter (Beckman Model

Medium E supplemented with 10% PCS were added. Two h later, medium was replaced with fresh Williams Medium E containing the test compounds. These cultures were incubated for 24 h. and then the medium was replaced by DMEM supplemented with 10% calf serum. After another 3 days, the cultures were trypsini/ed and handled as described above (V79 cells and primary hepato cytes can easily be distinguished by size and morphology). .SVi/i/io/icWd-Mii-rosdinc Assay. The bacterial mutagenicity assay with Sal-

2095

incubation period, the cells were collected, washed twice in PBS solution, and

ASI'iriS

01 Ni;\V IMIIlA/OACklDISONhS

between the amino moieties and no methyl group at the imidazo ring) showed the highest factor of resistance (13.3). Additionally, we examined the cytotoxic effect of the imidazo acridinones on primary rat hepatocytes (data not shown). In contrast to the fast-dividing F4-6 cells and V79 cells, the proliferation of

suspended in 1% NP40. The samples were sonicated twice for 10 s. Insoluble material was removed by centrifugaron, and the hemoglobin content was measured using the benzidine technique (36. 37)

RESULTS The chemical structures of the five ¡midazoacridinones we inves tigated are presented in Fig. 1. The compounds differ with regard to their substitution at the imidazo ring and the length of the side chain. DNA Melting Temperature. As illustrated in Table 1, the five investigated imidazoacridinones increased the melting temperature of calf thymus DNA by 8.7-11°C. indicating their capacity to stabilize the helical DNA structure to thermal denaturation. Compounds with out a methyl group on the imidazo ring (C-1311 and C-1335) showed slightly lower A7"ms as compounds with a methyl group on the

which was strongly inhibited by the investigated compounds (IC50s in the range of 0.01 to 0.1 JUM), we did not observe a significant cytotoxicity in primary, nondividing hepatocytes (38) up to concen trations of 7 /UM.To clarify whether the imidazoacridinones are rather cytostatic than cytotoxic, experiments with C3H/M2 cells, either in exponentially growing or nongrowing (confluential) cultures, were carried out. We investigated compound C-1311 as representative of

imidazo ring (C-1309, C-1310, and C-1336). Cytotoxicity Studies. The growth-inhibitory effects of the five selected compounds were comparatively investigated in different cell lines (Table 1). A characteristic for all compounds was a fairly steep dose-response curve. The evaluation of the !C50s for the imidazo acridinones in the sensitive F4-6 cells and the V79 cells resulted in comparable structure-activity relationships. Compounds having twocarbon spacers (C-1309. C-1310. and C-1311) between the proximal and distal nitrogens in the side chain showed a strong cytotoxic effect. An increase in the number of méthylène groups (C-1310 and C-1335) resulted in a decrease of biological activity. Compounds with a methyl group at the imidazo ring (C-1310 and C-1336) were less cytotoxic than their counterparts without a methyl group (C-1311 and C-1335). The resistant F4-6 cells, about 200-fold resistant to doxorubicin as compared to the sensitive F4-6 cells (12), showed a significantly smaller factor of resistance (4-13) towards the imidazoacridinones.

the group of imidazoacridinones. The cytotoxic effects of C-1311 on exponentially growing and confluent C3H/M2 cell cultures are shown in Fig. 2. The data dem onstrate considerable differences in the sensitivity of growing or nongrowing cells toward the imidazoacridinone. In C3H/M2 cells in a confluent phase, no toxicity was observed in the neutral red test and only marginal toxicity in the CFE when higher concentrations of C-1311 were used. In exponentially growing C3H/M2 cells again, no toxicity was observed in the neutral red assay but pronounced toxicity was observed in the CFE. Inhibition of DNA/RNA Synthesis. Table 1 shows the effect of the five imidazoacridinones on DNA and RNA synthesis in sensitive F4-6 cells determined by the incorporation of radioactive precursors. Compounds with two carbon spacers (C-1310 and C-1311) between the proximal and distal nitrogens in the side chain inhibited DNA synthesis to a greater extent than the derivatives with three carbon spacers in the side chain (C-1336 and C-1335). A methyl group at the imidazo ring (C-1310 and C-1336) decreased the potency to inhibit

For the cytotoxic effect of the imidazoacridinones in the resistant F4-6 cells, no structure-activity relationship was observed. The com pound C-1311 (with two carbons between the amino moieties and no methyl group at the imidazo ring) showed the smallest factor of resistance (3.8), while the compound C-1335 (with three carbons

DNA synthesis compared to derivatives without a methyl group at the imidazo ring (C-1311 and C-1335). For the inhibition of RNA syn thesis, no clear structure-activity relationship was observed. With the exception of C-1336, which inhibited DNA and RNA synthesis with equal potency, RNA synthesis was less sensitive to an inhibition by

C-1309

C- 1311

Fig. 1. Structure of the five investigated imidazoacridinones.

k 2096

ASPECTS OF NEW IMIDAZOACRIDINONES

Table I Cytiilimc effects tif the iinitlazimcridiiumes

im VT) cells and F4-6

WT/MDR

WT(JIM)0.0240.0360.0100.027 (°C)10.610.79.28.7 (UM)0.0440.0950.0300.111 CompoundC-1309C-1310C-1311C-1335

ofresistance8.510.03.813.3 1/iM)0.4040.6310.1810.435 (¿IM)0.7373.2330.5X90.826

C-1336''A7"m" >3.5Factor >7DNA 11.0V79 0.820CSDF4-6 0.490F4-6MDR(H.M)0.2040.3690.0380.358 2.384ED»'RNA 2.546DNA/RNAratio0.50.20.30.5 0.9 " The DNA melting temperature was determined with the imida/oacridinones (0.01 /AM)and calf thymus DNA (0.1 ¿¿M) in a 0.01 M phosphate buffer with 0.1 M EDTA (pH ~ 7). The heating rate was l°C/min. and the ahsorhance at À= 260 nm was read every minute. 7m were calculated from the absorbance/temperature curve. The A7nl were obtained by subtracting the 7"mfor the DNA from the 7m of the drug/DNA complex. Data represent the mean value of two separate experiments. * V79 cells were treated with different concentrations of the imidazoacridinones for 24 h. After 6-8 days, colonies were counted, and the 50'»inhibitory dose was determined from the dose-effect curves. The data represent the means of at least three separate experiments. F4-6 WT/MDR cells were treated with different concentrations of the ¡midaHiacridinones for 48 h. The percentage of trypan blue-negative cells was expressed compared to the untreated control, and the concentration of drug resulting in a 50% loss of cell viability was determined from the dose-response curve. Data represent mean values for at least three independent experiments. The factor of resistance is the quotient IC\M I;4-6/MDR/IC ones as novel anticancer agents: synthesis and biological evaluation. J. Med. Chem.. 27: 253-255, 1984. 5. Cholody. W. M., Martelli. S.. Paradziej-Lukowicz. J., and Konopa. J. 5-[(Aminoalkyl)amino]imidazo[4.5,1 -i/c]acridine-6-ones as a novel class of antineoplastic agents: synthesis and biological activity. J. Med. Chem.. 33: 49-52. 1990. 6. National Cancer Institute Developmental Therapeutics Program. In Vitro Screening Data Review Checklist. December 1991. 7. Showalter, H. D. H., Fry. D. W., Leopold, W. R.. Lown, J. W., Plambeck, J. A., and Reszka. K. 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33. Ames. B. N.. McCann. J.. and Yamasaki. E. Methods for detecting carcinogens and mutagens with the Sa/m