(CANCER RESEARCH 49, 5969-5978, November 1, 1989]

DNA Topoisomerase II-mediated Interaction of Doxorubicin and DaunorubicinCongeners with DNA1

Annette Bodley, Leroy F. Liu, Mervyn Israel, Ramakrishnan Seshadri, Yoshihiro Koseki, Fernando C. Giuliani,Stanley Kirschenbaum, Robert Silber, and Milan Potmesil2

Department of Biological Chemistry [A. B., L. F. L.J, The Johns Hopkins University School of Medicine, Baltimore, Maiyland 21205; Departments of Pharmacologyand Medicinal Chemistry, and Cancer Center [M. I., R. S., Y. K.j, University of Tennessee-Memphis, Memphis, Tennessee 38163; Farmitalia Carlo Erba, CentroRicerche [F. C. G.], Ñerviano, Italy; and Departments of Radiology fS. K., M. P.] and Medicine [R. SJ, New York University School of Medicine,New York, New York 10016

ABSTRACT

Three groups of doxorubicin and daunorubicin analogues, differing bytheir substituents on the chromophore and sugar moieties, were used inthis study. The 3'-A'-unsubstituted (Group I), 3'-/V-acyl (Group 2), and

.V-,V-alkyl (Group 3) analogues were tested for: (a) in vivo antitumoractivity and in vitro cytotoxicity; (b) cellular or tissue uptake and metabolic conversion; (c) strength of DNA intercalation; and (</) interactionwith DNA topoisomerase II (topo-II). Compounds of Group 1 werecytotoxic, were strongly intercalative, and, except for those with C-14side chain substitution, induced the formation of topo-II-DNA cleavablecomplexes. As shown previously, esterolysis of C-14-acyl substituentswas required to yield a metabolite which can interact with topo-II in thepurified system. The C-14-substituted compounds of Group 2 and theirC-14-unsubstituted metabolites were cytotoxic. These drugs were weakintercalators, and the C-14-unsubstituted congeners induced cleavablecomplex formation in the purified system, but with reduced potencyrelative to doxorubicin. The type of the 3 '-^Y-position substituent deter

mined whether Group 3 analogues were cytotoxic and strong intercalators, or less active and nonintercalating. Although C-14-unsubstitutedintercalators of Group 3 did not form cleavable complexes in the purifiedsystem, they were cytotoxic.

The study shows that DNA intercalation is required but not sufficientfor the activity by topo-II-targeted anthracyclines. In addition to theplanar chromophore which is involved in intercalation, two other domainsof the anthracycline molecule are important for the interaction with topo-II: (a) substitution of the ( -14 position totally inhibits drug activity inthe purified system, but enhances cytotoxicity by aiding drug uptake andpresumably acting on other cellular targets; and (b) substitutions on the3'-.-V position of the sugar ring can, depending on the nature of thesubstituent, inhibit intercalation and/or topo-II-targeting activity. Thesefindings may provide guidance for the synthesis and development of newactive analogues.

INTRODUCTION

Mammalian DNA topoisomerase II, a nuclear enzyme thatalters the topologica! state of DNA and is essential for cellreplication and viability (reviewed in Refs. 1-3), appears to bethe principal target for several groups of anticancer agents (3-6). A covalent complex between topoisomerase II and DNA isan obligatory intermediate in the catalysis of DNA topoisomer-ization. Stabilization of this complex by natural products ofmicrobial or plant origin and their analogues presents an initialevent which leads to cell death. The stabilization of the complexcan interfere with vital functions involving DNA replication.

Received 2/1/89; revised 6/29/89; accepted 8/3/89.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in part by USPHS Grants CA-37082, CA-37209, CA-11655, andCA-39662 from the National Cancer Institute, NIH, Department of Health andHuman Services; by Grant CH-348A from the American Cancer Society; andgrants from Farmitalia Carlo Erba and the Marcia Slater Society for Research inLeukemia.

2To whom requests for reprints should be addressed, at Department ofRadiology, Laboratory of Experimental Therapy, New York University Schoolof Medicine, 550 First Avenue, New York, NY 10016.

This interaction appears to play a substantial role in the cytotoxicity exerted by anthracyclines. The precise nature of theprocess, however, remains obscure.

It has been proposed that the formation of a ternary complexdrug-topoisomerase II-DNA could be a prerequisite for thestabilization of DNA cleavable complexes (7). It is unclearwhether the intercalative mode of drug-DNA binding or just anincreased concentration of drug molecules around topoisomerase II-DNA adducts is essential for this step. Our previousstudies (7) have shown that DXR3 analogues with undetectable

or low DNA binding stabilize the cleavable complex betweenthe enzyme and DNA; these results thus favor the formerpossibility. Understandably, even weak DNA binding, not detectable with the methods applied previously, could be important for the formation of drug-topoisomerase II-DNA ternarycomplexes.

In the present study, we have analyzed three groups of DXRand daunorubicin analogues, which differ by the substitutionpattern on the chromophore as well as on the sugar moiety.The drugs show a range of intercalative strengths from strongto undetectable. The study has identified some of the structuralproperties of the analogues which are connected with DNAintercalation and/or topoisomerase II inhibition. It also showsthat several biologically active intercalating agents apparentlydo not interact with this enzyme.

MATERIALS AND METHODS

Drugs and Drug Treatments. Table 1 lists the 24 DXR or daunorubicin analogues included in this study. Of the .V-A'-unsubstituted anthra

cyclines, all except ADI21 and AD268 were prepared in the laboratoriesof Farmitalia Carlo Erba as previously described (8-12). AD268 andS'-A'-acyl- or 3'-jV-alkylanthracyclines were synthesized as reported

earlier (13-17). The preparation of ADI 20 and ADI 21 will be describedelsewhere. All products were purified to homogeneity, the purity wastested by thin-layer and high-performance liquid chromatography, andeach compound was fully characterized by microchemical analysis aswell as by the IR, UV, and nuclear magnetic resonance spectral properties.

3The abbreviations used are: DXR. doxorubicin (Adriamycin); AD32. N-trifluoroacetyladriamycin-14-valerate; AD38. A'-acetyladriamycin: AD41. A'-tri-fluoroacetyladriamycin: AD92, A'-trifluoroadriamycinol; ADI 15. A'-pentafluo-ropropionyladriamycin; AD 120. iV-trifluoroacetyl-14-mcthoxydaunorubicin:AD121, 14-methoxydaunorubicin; AD133, A'-acetyladriamycin-14-valerate;AD143, A'-trifluoroacetyladriamycin-l4-0-hemiadipatc; AD194, /V-(n-bu-tyl)adriamycin-14-\alerate; ADI98. iV-benzyladriamycin-14-valerate; AD199,/V.jV-dimethyladriamycin-14-valerate: AD202. A',A'-di(n-butyl)adriamycin-14-val-erate: AD206, A;tiV-dibenzyladriamycin-14-valerate; AD268, adriamycin-14-thiovalerate: AD280. A'.A'-dimethyladriamycin: AD284. A'-(n-butyl)adriamycin:AD285. A',A'-di(fl-butyl)adriamycin: AD288. A'-benzyladriamycin; AD289, N,N-

dibenzyladriamycin; CE. cloning efficiency; DMSO, dimethyl sulfoxide:4'deoDXR. 4'-deoxydoxorubicin: 4'd4'IDXR. 4'-deoxy-4'-iododoxorubicin: 4-dmxDNR. 4-demethoxydaunorubicin; DTT. dithiothreitol: 4'epiDXR. 4'-epi-doxorubicin: HPLC, high-performance liquid chromatography; ¡.a.,intraarteri-ally. ID50. median inhibition dose; ILS, increase in life span; IR, infrared; LDgo.lethal dose at the 90°Vcell kill level: PAB. protein-associated DNA single-strandbreaks; SDS. sodium dodecyl sulfate: 14-thia DXR, 14-thiadoxorubicin; UV,ultraviolet.

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For in vitro study, the compounds were dissolved in saline (0.16 MNaCl), DMSO, or Diluent 12 (polyethoxylated castor oil:ethanol; Pharmaceutical Resources Branch, Division of Cancer Treatment, NationalCancer Institute) diluted with saline (1:5). Saline or Diluent 12 wasalso used for the formulation of drugs for in vivo application.

Murine Tumors. P388 lymphocytic leukemia cells were maintainedby serial i.p. passage in DBA/2 mice. For experimental purposes, IO6

cells were injected i.p. into each male BALB/c x DBA/2 F! (hereaftercalled CD2F,) or C57BL/6 x DBA/2 F, (hereafter called B6D2F,)mouse. The assay procedures were similar with the standard NationalCancer Institute protocols (18). Drug treatments began on Day 1 afterleukemia inoculation and, in some experiments, continued for 4 consecutive days. Drugs dissolved in saline were injected i.v., and thoseformulated in Diluent 12, i.p. The injected volume was adjusted to 0.1ml/10 g of body weight. In order to verify that tumor responsivenessdid not change from one series of experiments to another, DXR wasused in each series as a positive control. No differences in responseswere detected between the treatments with DXR formulated in Diluent12 as compared with saline. Treated mice were followed up to 90 days,and the treatment was evaluated either as cures or as the increase inlife span |% of ILS = 100 x (T/C - 1), where T is the median survivaltime of the drug-treated group in days, and C is median survival timein days for untreated controls].

DBA3S, a H cell lymphoma maintained by serial s.c. transplants inisogeneic mice of the DBA/2J inbred strain (19), was used for phar

macological studies on Day 7 after the tumor transfer.Tissue Culture Lines. P388 cells were obtained from inoculated DBA/

2 mice and maintained for two passages of transfer in 25-cm3 plastic

tissue culture flasks (Corning Glass Works, Corning, NY) in RPMI1964 medium (Grand Island Biological Co., Grand Island, NY), supplemented with 10% heat-inactivated fetal calf serum (Flow Laboratories, Inc., Rockville, MD), 20 ¿tM2-mercaptoethanol (Sigma ChemicalCo., St. Louis, MO), and antibiotics (100 Mg/ml of streptomycin andkanamycin, and 100 lU/ml of penicillin). The cultures were grown at37°Cin a humidified atmosphere of 95% air:5% CO2 (20-22).

Exponentially growing P388 or CCRF-CEM cells (human lympho-blastic leukemia line) were grown in multiwell tissue culture plates orin 25-cm3 tissue culture flasks (Corning Glass Works). P388 cells werecultured under conditions specified earlier, and 5x10* cells/ml were

exposed for 4 h to drugs dissolved in saline (for details of drug uptakestudies, see Table 3). CCRF-CEM cells were kept in Eagle's minimal

essential medium (Grand Island Biological Co.) supplemented with10% heat-inactivated fetal calf serum (Microbiological Associates, Be-

thesda, MD), 2 n\i glutamine, and 50 Mg/ml of gentamycin (23). Testcompounds were dissolved in saline or DMSO (1% DMSO finalconcentration in the assay) and added to the cultures (5 x IO5cells/ml)

in a final drug concentration ranging from 0.01 to 5.0 ¿tM,with half-log increments, and incubated for 48 h at 37'C in a humidified atmos

phere of 95% air:5% CO2. Experiments were repeated at least twice, ineach instance with saline or DMSO-exposed cultures serving as growth

Table 1 Doxorubicin and daunorubicin analogue (name, abbreviation/code number, and chemical formula)

D_0 OH

1

Type ofanalogue/name3'-/V-unsubstitutedanthracyclinesDoxorubicin

(Adriamycin)Daunorubicin(daunomycin)14-Methoxydaunorubicin"Adriamycin-14-thiovalerate*4-Demethoxydaunorubicin4'-Epidoxorubicin4'-Deoxydoxorubicin4'-Deoxy-4'-iododoxorubicinR?Abbreviationor

codeno.DXRDNRAD121AD2684-dmxDNR4'epiDXR4'deoDXR4'd4'IDXR0RIHHHHHOHHHnOHC'4-I/CÑ3

\R2Y_/A

'R3

R4R2OHOHOHOHOHHHIR3HHHHHHHHi

1'R4HHHHHHHHR5OHHOCH,SCO(CH2),CH,HOHOHOHMOOOoooooR7OCH,OCH,OCH,OCH,HOCH,OCH,OCH,Molecularwt543.57527.57557.6680.23497.54543.57527.57653.57

3 '-/V-Acylanthracyclines/V-Trifluoroacetyladriamycin-14-valerate'iV-Trifluoroacetyl-14-methoxydaunorubicin'/V-Acetyladriamycin-14-valerate'/V-Trifluoroacetyladriamycin-14-O-hemi-

adipate*/V-Acetyladriamycin/V-Trifluoroacetyladriamycin/V-TrifluoroacetyladriamycinolA'-Pcntafluoropropionyladriamycin

AD32AD120AD133AD143AD38AD41AD92AD115HHHHHHHHOHOHOHOHOHOHOHOHHHHHHHHH

COCF,COCF,COCH,COCF,COCH,COCF,COCF,COCF2CF,OCO(CH2),CH,OCH,OCO(CH2),CH,OCO(CH2)4COOHOHOHOHOHOoooo0H,OHOOCH,OCH,OCH,OCH,OCH,OCH,OCH,OCH,723.71653.61669.74767.72585.61639.58641.60689.59

3'-/V-Alkylanthracyclines/V-(n-Butyl)adriamycin-14-valerate'/V-Benzyladriamycin-

14-valerate*/V./V-Dimethyladriamycin-14-valerate"/V./V-Difn-butylladriamycin-

14-valerate'/V./V-Dibenzyladriamycin-14-valerate'/Vt/V-Dimethyladriamycin;V-(n-Butyl)adriamycinN,/V-Di(n-butyl)adriamycin,V-Benzyladriamycin/V.A'-DibenzyladriamycinAD

194AD198AD199AD202AD206AD280AD284AD285AD288AD289HHHHHHHHHHOHOHOHOHOHOHOHOHOHOHn-C4H,CH2C6H,CH,n-C4H,CH2C6HjCH,n-C4H,n-C4H,CH2C6H5CH2C6H5HHCH,n-C4H,CH2C6H5CH,Hn-C4H,HCH2C6H,OCO(CH2),CH,OCO(CH2),CH,OCO(CH2),CH,OCO(CH2),CH,OCO(CH2),CH,OHOHOHOHOHOOooooooo0OCH,OCH,OCH,OCH,OCH,OCH,OCH,OCH,OCH,OCH,683.82717.83655.77739.94807.96571.63599.69655.81633.69723.82

•¿�C-14position substituted (R5).

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controls, and DXR-treated cultures as quality controls. Cell countswere determined by hemocytometer and/or in a Model F CoulterCounter. The percentage of growth inhibition was determined usingdrug-exposed and control untreated cultures and expressed in /jmol as

the ID50(23).Mouse LI210 leukemia cells were grown as a suspension culture in

Fisher's medium supplemented with 10% heat-inactivated horse serumand 1% Pen-Strep (Grand Island Biological Co.). Suspensions of IO6cells/ml were exposed to various drug concentrations for l h at 37°C

in humidified 92% air:8% CO2. Cytotoxic effects of drugs were determined by cloning the cells in Fisher's medium with 15% heat-inacti

vated horse serum, 1% Pen-Strep, and 0.3% Noble agar (Difco Laboratories, Detroit, MI), as described previously (24). Cell colonies werescored under low-power magnification following 2 to 3 wk of incubationat 37°C.Each experimental point was done in quadruplicate andrepeated 2 to 5 times. Cell viability was determined from CE. "Survivingfraction" of a cell population was defined as the ratio of CE of drug-

treated cells to the CE of untreated (control) cells, with the LD90expressed in (¡mol.

Pharmacological Studies. For studies in hosts without tumors, groupsof male A/J mice or Sprague-Dawley rats were given injections i.v. ori.a. with a therapeutic dose equivalent of a test drug. Following drugadministration, blood samples were obtained at intervals ranging from0 to 24 h. Serum or plasma aliquots were extracted with chloro-form:propanol (3:1, v/v), evaporated to dryness, and stored at —¿�20°C

pending analysis.DBA/2J mice, bearing 1-wk-old DBA3S tumors, were given injec

tions i.p. of a single dose of the test drugs dissolved in saline (DXR) orDiluent 12 (AD32 or AD 143) and sacrificed l h after the injection.Upon removal, the tumor tissue was blotted dry and weighed, thenhomogenized in 9 volumes of Tris-HCI buffer, pH 8.5, with 3% SDS(w/v), and extracted 3 times with two volumes of ethyl acetate:propanol(9:1, v/v) (17).

For cellular pharmacology studies, P388, CEM, or LI210 cells inlog-phase growth were incubated with the test drug for various timeintervals. At the end of each incubation, cell pellets and media super-natants were obtained. The intracellular 3'-A'-unsubstituted drugs were

extracted with ethanol-saline, and their concentration, as well as theconcentration of metabolites, was measured spectrofluorometricallyagainst known standards at the excitation wavelength of 479 nm andemission of 593 nm. For the rest of the drugs, pelleted cells weresonicated and extracted with ethyl acetate:propanol (9:1, v/v), andsamples were processed as described above for in vivo studies. Detailsof the procedures have been published elsewhere (7).

HPLC Analyses. Thawed tissue and plasma samples were reconstituted in 1 ml of methanol and subjected to complementary reverse-phase and normal phase HPLC analysis (25, 26). Reverse-phase separation was done with a ¿j-Bondapak/phenyl column using a dual-pumpsolvent delivery system (Waters Associates, Milford, MA). The solventsystem consisted of ammonium formate buffer, 0.05 M, pH 4.0, andacetonitrile. Normal phase Chromatographie separation was achievedwith a Partisil-10 PAC column (30 cm x 3.9 mm; Whatman, Inc.,Clifton, NJ) and an eluting solvent system of chloroform versus chlo-roform:methanol:acetic acid:water mixture (85:15:5:1.5, v/v). Samplesderived from in vitro studies were analyzed only by reverse-phasechromatography; a phenyl-Radialpak lO-^m column (10 cm x 8 mm)mounted on a Z-Module radial compression unit was used in conjunction with the reverse-phase solvent system specified earlier. The flowrate conditions and the monitoring systems were described in recentreports (7, 27). Eluting peaks were identified by retention times relativeto those of authentic standards. Peaks were quantified by reference tostandard extraction curves for each drug or metabolite and correctedfor recovery of internal standards.

DNA Alkaline Elution. The methodology has been described in detailin previous publications (28-30). In all experiments, L1210 cells labeledwith [3H]thymidine (Amersham/Searle Corp., Arlington Heights, IL)and irradiated with 300 rads at 0°Cusing a 137Csradiator (Atomic

Energy of Canada, Ltd.) were used as internal standards in the elution(24). Cells un labeled with the isotope, 106/ml, were treated with thedrugs for l h at 37°Cin 92% air:8% CO2 atmosphere. Aliquots

containing IO6drug-treated cells were mixed with IO5cells serving asinternal standards and then applied to 2-^m-pore-sized polyvinyl chloride filters (Millipore, Bedford, MA). Cells were washed and lysed, andthe duplicate lysates were incubated at room temperature with 0.2 mg/ml of proteinase K. The elution proceeded as specified previously (30).SDS, 0.1%, was added to the eluting solution for proteinase K-treatedlysates. Fractions were collected at 3-h intervals over 15 h. Eluted DNAwas precipitated, and the precipitates were collected on Durapore filters(pore size, 0.22 ^m; Millipore), using a sample manifold, and dried.Each filter was incubated with 2 M 3,5-diaminobenzoic acid hydrochlo-ride (Sigma Chemical Co.) for 45 min at 60°C.The DNA-adduct

fluorescence was measured with an Aminco Bowman spectrofluo-rometer (xenon lamp, 415-nm excitation wavelength, 500-nm emissiondetection). Radioactivity of the samples was then also determined. Thedata were analyzed, and the frequency of PAB per IO6nucleotides was

calculated (31).DNA Unwinding Assay and Relative Strength of DNA Intercalation.

Reaction mixtures (60 >i\each) containing 50 mM Tris-HCI, pH 7.5,100 mM KC1, 10 miviMgCU, 0.5 mM DTT, 0.5 mM EDTA, 30 ¿ig/mlof bovine serum albumin, 1.5 pg of supercoiled pC15 DNA, 1.5 ^g ofrelaxed pC15 DNA, 45 ng of calf thymus topoisomerase I, and 0.1,0.5, 2.5, or 12.5 ¿¿g/mlof the test drug were incubated at 37°Cfor 30

min. Each reaction was stopped with the addition of 15 /il of aprewarmed solution (37°C)containing 2.5% SDS and 100 mM EDTA.

The reaction mixture was extracted once with phenol and once withchloroform and then precipitated with ethanol. Samples were electro-phoresed at 4°Cin a 1% agarose gel in 90 mM Tris, 90 mM boric acid,

and 5 mM MgCl2, pH 8.3, at 80 V for 20 h (32). A scale was constructedfor the evaluation of DNA intercalative strength of a drug relative toDXR intercalation. The strength of DXR was set, in arbitrary units,equal to 7. The number >7 indicates a greater, and <7 a lower, abilityof a drug to intercalate. It was assumed that the unwinding angle foreach compound is the same as that of DXR and that the extent ofunwinding is proportional to the intercalative binding constant of thedrug.

Spectrophotometric Determination of Drug-DNA Binding. Themethod applied is described in detail elsewhere (7). The absorbance ofanthracycline solutions was measured at 480 nm in the presence (.1)and absence of DNA (/40),using an ultraviolet/visible light spectropho-tometer (Model Lambda 3B; Perkin-Elmer, Norwalk, CT). The ratioA/AO was taken as a measure of the extent of DNA-drug binding, withvalues of 0.55 to 0.6 representing strong and those of 0.96 to 1.00 nodrug-DNA association.

DNA-Topoisomerase II. The enzyme as purified to homogeneity fromHeLa cells by modifications of the published procedure (33).

Assay for Topoisomerase II-mediated DNA Cleavage. The assay wasperformed as described previously (7, 34). DNA plasmid PMC41 waslinearized with EcoRl and end labeled at the 3' termini with the large

fragment of Escherichia coli DNA polymerase I in the presence of [a-32P]dATP and unlabeled dTTP. The test drugs were dissolved in DMSO

and diluted to desired concentrations with the incubation buffer. Cleavage reaction mixtures (20 n\ each) containing 50 mM Tris, pH 7.5, 100mM KC1, 10 mM MgCl2, 1 mM ATP, 0.5 HIMDTT, 0.5 mM EDTA, 30Mg/ml of bovine serum albumin, 20 ng of 3'-end labeled DNA, 5 ng of

purified human topoisomerase II, and various drug concentrations (5-fold dilution) were incubated for 20 min at 37°C.The reactions were

stopped with the addition of SDS to a final concentration of 1%. Eachreaction mixture was treated with proteinase K, 0.2 mg/ml, for 1 h at37°C.Reaction products were analyzed by electrophoresis using a 1%

agarose gel in a buffer containing 0.089 M Tris-borate and 0.002 MEDTA, pH 8.3. The cleavage pattern was visualized by autoradiogra-phy.

RESULTS

Drug Pharmacology and Antitumor Activity. Structural formulas of the test drugs are presented in Table 1. The compoundsare divided into three groups according to the substitutionpattern at the 3'-nitrogen of the daunosamine moiety (Table 1,

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Table 2 Doxorubicin and daunorubiàn analogues (in vivo and in vitro activity)

DrugP388"

in vivo

(cures or % ofILS4)CEM

invitro(ID50,MM*)L1210/nvitro (LO«,,

MM")3'-(V-unsubslituted

anthracyclinesDXRDNRAD121'AD268'4-dmxDNR4'epiDXR4'deoDXR4'd4'IDXR3

'-A/-Ac)lanthracyclinesAD32'AD120'AD13.VAD

143'AD38AD41AD92AD1I53'-iV-AlkylanthracvclinesAD

194'AD198'AD199'AD202'AD206'AD280AD284AD285AD288AD289320%

ILS100%ILS81%

ILS130%ILS161%ILS270%ILS300%ILS300%ILS100%

cures63%ILS81%

ILS100%cures27%

ILS481%ILS100%ILS372%

ILS>475%

ILS100%cures72%

ILS>770%ILS145%ILS164%ILS85%

ILS75%ILS0.050.030.320.450.0030.030.030.0090.33.572.50.261.20.211.030.440.110.110.022.1>5.30.30.080.40.5>5.00.310.92.00.230.420.320.188.315.28.87.012.37.210.44.42.94.63.37.1>49.55.19.837.010.2>55.4

°P388. i.p. mouse leukemia; CEM, tissue culture line of a human leukemia;

LI210. a tissue culture line of a mouse leukemia. Results confirmed in at leastone independent experiment for each test system.

* % of ILS. increase in life span, optimal drug doses qd 1 (3'-A/-unsubstituteddrugs except AD268) or qd 1-4 (the rest of the drugs).

' ID50,median inhibition dose, 48-h drug exposure; mean of 2 to 3 experimentswith a SD <10% of values shown.

'LO«,, a dose killing 90% of clonogen, 1-h drug exposure; mean of 2 to 5experiments with a SD < 20% of values shown.

'tl4 position-substituted analogues.

R3 and R4). Except for ADI21 and AD268, the anthracyclinemolecule of the 3'-./V-unsubstituted analogues is modified ateither the 4'-position of the daunosamine (Rl and R2) or the

C-4 position of the chromophore (R7). AD268 is the onlyanalogue of this group with a C-14 position bulky acyl sidechain (R5), whereas ADI21 contains a nonhydrolyzable 14-0-methyl ether function. A second group, the 3'-/V-acyl analogues,

is characterized by an acyl function attached to the nitrogen ofthe daunosamine (R4). Four of the analogues included in thisgroup also have C-14 position side chain substitution (R5). Theanalogues of the third group, 3'-./V-alkylanthracyclines, are

modified by mono- or dialkyl substitution at the sugar nitrogen(R3, or R3 and R4). In some of the compounds, there is also avalerate side chain substitution at C-14.

Drug effectiveness was tested in vivoand in vitro, using severalsystems (Table 2). A single dose of 3'-./V-unsubstituted ana

logues on Day 1 improved, to a varying degree (81 to 320% ofILS), the survival of mice implanted a day earlier with P388leukemia. The drugs were also effective against CEM in vitro.The in vivo treatment of P388 leukemia with the second groupof drugs, 3'-/V-acylanthracyclines, was based on an established

optimal treatment schedule (every day, Days 1 to 4) in terms ofcures or ILS. Both C-14 position substituted and unsubstitutedanalogues are represented by highly effective (AD32, AD 143,AD41, or AD 115) and less potent compounds (AD 120, AD 133,AD38, and AD92). The differential efficacy of the 3'-JV-acyl

analogues is further confirmed by the results of the in vitroCEM and LI210 screens. A comparable in vitro growth inhi

bition of CEM cells by the most active 3'-W-acyl analoguesrequires a 3.5-fold (AD41) to 7.3-fold (ADI 15) higher concentration than that of DXR. In the L1210 system, the differencein cell kill is approximately 13- to 28-fold.

Analogues of the third group, 3'-Ar-alkylanthracyclines, in

clude biologically effective as well as ineffective drugs. Amongthe C-14 position substituents, AD 194, AD 198, and AD202are highly effective in vivo against P388 leukemia and in vitroagainst CEM and L1210 cell lines. AD 199, despite modest invivo activity, shows marked activity in the in vitro assays. In theCEM growth inhibition assay, AD 199 is 3-fold more activethan DXR. AD280 and AD288, the respective C-14-unsubsti-tuted analogues of AD 199 and AD 198, are effective in vitro. Invivo, AD280 shows a modest enhancement in antitumor activitycompared with AD 199, while AD288 is significantly less effective than AD198. AD284, a C-14-unsubstituted analogue ofAD 194, is almost as effective against CEM leukemia as areDXR and AD 199. AD289 and its C-14-substituted congenerAD206 remain without major effectiveness against tumors invivo as well as in vitro. AD206 is metabolic-ally converted into

AD289 and AD288 in vivo, but not in vitro.The compounds of the first group are stable in various

biological systems, both in vivo and in vitro (Table 3). The rateof conversion into a reduced biologically active form (DXN, C-13-OH 4-dmxDNR, C-13-OH 4'deoDXR, or C-13-OH4'd4'IDXR) is relatively small. Of the drugs in the secondgroup, the C-14 position-substituted 3'-yV-acyl analogues AD32

and AD143 are largely converted into the C-14-unsubstitutedcongener AD41. This esterolysis has been documented in previous studies (7, 19, 25, 27). Similarly, the C-14-substituted 3'-yV-alkylanthracyclines AD 198 and AD 199 are partially transformed into the C-14-unsubstituted products AD288 or AD280,respectively. AD280 or AD288 appears stable, and no furtherbiotransformation has been detected in vitro.

Drug Interaction with DNA. The intercalative binding of eachanalogue to DNA was measured using an assay in which bothsupercoiled and relaxed DNAs are incubated with the drug inthe presence of DNA-topoisomerase I and allowed to come toan equilibrium. The inclusion of both topological forms of DNAensures that any inhibitory effect which the drugs may exert ontopoisomerase I will be detectable and could be compensatedfor in the assay conditions (32). Fig. 1 shows some of the gelswith assayed drug intercalation. Table 4, first data column,presents the results expressed as the relative strength of drugintercalation. The rating was, on a relative scale, from 0 (nointercalation) to 9 (strong intercalation). It appears that the testdrugs could be divided into three categories, strong, weak, andnonintercalating compounds. With the exception of AD202and its C-14-unsubstituted congener AD285, all biologicallyactive 3'-/V-unsubstituted analogues (first group) and 3'-N-

alkylanthracyclines (third group) are strong intercalators. Thetwo biologically inactive jV-alkyl derivatives AD206 and AD289did not show detectable intercalation. Analogues of the secondgroup (3'-/V-acylanthracyclines) are generally weak intercala

tors, with the relative strength <5. There was no evidence ofintercalation with AD32. Within the group, there is no apparentcorrelation between the strength of intercalation and antitumoractivity (compare active compounds AD32, AD 143, AD41, andAD115 with the less active AD133, AD38, or AD92).

Fig. 2 compares the relative strength of DNA intercalationwith the extent of DNA-drug binding, as has been determinedspectrophotometrically. It is apparent that, in general, bothmethods concur: 3'-yV-unsubstituted as well as 3'-TV-alkylanthracyclines are strong; and 3'-./V-acyl analogues are weak DNA

intercalators or binders. One exception to this generalization is5972

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ANTHRACYCLINES AND TOPOISOMERASE M-DNA INTERACTION

Table 3 Doxorubicin and daunorubkin analogues (metabolism in vivo and in vitro)

Biologically ineffective metabolites are not listed.

Drug.V-V-miMihsi

¡inteilanthracvclinesDXR4-dmxDNR4'epiDXR4'deoD\R4'd4'IDXR3'-W-AcylanthracvclinesAD32'AD

143'AD41AD923'-/V-AlkylanthracyclinesAD

198«AD199«AD280AD288Mouse/rat

strainDBA3S-B6DF/C3H'BALBA/B6D2F/A/J*DBA3S"Sprague-Dawley*DBA3S-Sprague-Dawley7In

vivo%

Unchanged91.979.099.099.085.016.726.753.96.515.0In

vitroMetabolitesDXN»

(8.1)'C-13-OH(21.0)C-13-OH(1.0)C-13-OH(1.0)C-13-OH(14.0)AD41

(81.1)AD92(I.3)AD41

(63.4)AD920.7)DXR

(4.5)AD41(42.3)AD920.9)DXR

(1.9)AD41(87.4)AD92(1.6)DXR

(2.0)AD288

(77.0)C-13-OH(8.0)Cell

lineP388"L1210'P388"P388'P388"P388'P388"CEM'L1210*L1210*CEM'CEM'L1210'L1210-L1210'L1210'%Unchanged100.0100.0100.096.0100.099.097.085.594.419.280.977.584.990.3100.0100.0MetabolitesC-13-OH

(4.0)C-13-OH(1.0)C-I3-OH(3.0)AD41

(11.7)AD92(0.5)DXR(0.4)AD41(5.6)AD41

(80.8)DXR

(7.2)AD92(0.6)DXN(6.0)AD288(15.1)AD280

(9.7)

" DBA3S lymphoma implanted s.c. into DBA/2J mice, levels of the drug and metabolites in the tumor tissue, 6 h after the i.p. injection of DXR ( 15 mg/kg), and

1 h after the injection of AD32 (80 mg/kg) or AD143 (64 mg/kg), 2 to 4 mice/point.* DXN, doxorubicinol (adriamycinol).' Numbers in parentheses, percentage.* P388, mouse leukemia line, 4-h exposure to 2.5 itg/mi, cell-associated drug uptake.'L1210. mouse leukemia line. 1-h exposure (MM)to DXR (0.88), AD32 (0.56), AD143 (1.0), AD198 (2.35), AD199 (4.1), and AD280 (2.38); cell-associated drug

and metabolite uptake.7Mice of various strains, levels of the drug, and metabolite in serum following an i.p. bolus (3 to 10 mg/kg).' C-14 position-substituted analogues.* A/J mice and Sprague-Dawley rats, levels of the drug, and metabolites at 3 h in serum following an i.v. bolus (5 mice), or in plasma following an ¡.a.bolus (4

rats); the bolus of AD143 was 50 mg/kg and of AD198, 5.0 mg/kg.' CEM. human leukemia line. 45-min exposure to 5.2 JIM(AD32) or 3-h to 10 MM(AD41 and AD92), cell-associated drug, and metabolite uptake. All tissue culture

experiments done in duplicates.

ABCDEFGHIJKLMNOPQRABCDEFGHIJKLMNO P Q R

ABC

Fig. 1. Drug intercalation. Form I pC15 DNA was relaxed using purified topoisomerase I in the presence of various drug concentrations. /, A. 1.5 ngof supercoiledand 1.5 ng of relaxed DNA; B, DNA plus 45 ng of topoisomerase I (controls); C to F, DNA plus topoisomerase I plus DXR (0.18, 0.92, 4.6, or 23.0 MM;3'-N-unsubstituted drug. Group 1); G to J, 4'deoDXR [0.19, 0.95, 4.75, or 23.75 MM(1)]; K to N, 4'd4'-IDXR [0.15, 0.76, 3.8, or 19.12 MM(1)]; O to A, 4'epiDXR [0.18,0.92,4.6, or 23.0 MM(1)]. 2, A and A, controls; CtoF, DXR(l); G to /, AD92 (0.16, 0.78, 3.9, or 19.5 MM;3'-/V-acylanthracycline, Group 2); K to A/, AD41 (0.16,

0.78, 3.9, or 19.5 MM(2)]; O to R, AD38 [0.17, 0.85, 4.27, or 21.37 MM(2)]. 3, A and B, controls; C to F, DXR (1); G to J, ADI 15 [0.14, 0.72, 3.62, or 18.12 MM(2)]; ATto A', ADI 33 [0.15. 0.74. 3.72. or 18.62 MM(2)]; O to K, AD206 (0.12, 0.62, 3.1, or 15.5 MM;3'-/V-alkylanthracycline, Group 3). 4. A and B. controls; C to F,DXR (1); G to J, AD198 [0.14, 0.7, 3.47, or 17.37 MM(3)]; K to N, ADI99 [0.15, 0.76, 3.8, or 19.0 MM(3)]; O to A, 4'dmxDNR [0.2, 1.0, 5.02, or 25.12 MM(1)].

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ANTHRACYCLINES AND TOPOISOMERASE ll-DNA INTERACTION

Table 4 Doxorubicin and daunorubicin analogues (DNA intercalation, topoisomerase ¡l-mediatedDNA cleavage, and inhibition of background cleavage)

Cell-free system

Topoisomerase II-mediatedcleavage

Drug.V-.V-misiilisiitiiU'uanthracyclinesDXRDNRAD121»AD268'4-dmxDNR4'epiDXR4'deoDXR4'd4'IDXR3

'-A'-AcylanthracyclinesAD32'AD

120*ADI33*AD143'AD38AD41AD92AD1153'-A/-AlkylanthracyclmesAD

194'AD198»AD

199»AD202'AD206*AD280AD284AD285AD288AD289DNAintercalation

(relativestrength)77679986031243226663076<170Range(fM)0.04-0.920.04-0.95ND<ND0.04-1.010.04-0.920.04-0.950.03-3.80NDNDNDND0.85-21.40.78-19.50.78-19.50.72-18.1NDNDNDNDNDNDND0.03-19.1NDNDMaximumeffect(flM)0.920.21.010.180.190.7721.4W19.519.518.1

w19.1Inhibition

of backgroundcleavage, maximum

effectOIM)>4.620.2aO.725.024.6>4.719.1ND18.2wND16.2

wX).7>0.720.15>0.7IS.Sw20.04>0.0720.0617.2L

1210 cells(PAB°induced

by 5 f¿\idrugconcentration)1.410.760.840.171.151.161.162.011.650.441.820.421.450.420.950.08"0.300.850.07'

* PAB, protein-associated DNA cleavage, frequency/10' nucleotides; average of 2 to 3 experiments.*C-14 position substituted.' ND, not detectable; w, weak.' Insignificant frequency of PAB.

10-

•¿�D

O0-Lû_

1 0.8 0.6SpectrophotometncAssay

(A/Ao)Fig. 2. Comparison of DNA-drug association measured by the unwinding and

spectrophotometric assays. For details, see "Materials and Methods" and Ref. 7.

AD 198, which, in the unwinding assay, shows strong intercalation, whereas little drug-DNA interaction was seen with thespectroscopic method (16, 35).

Topoisomerase II-mediated DNA Cleavage. Drug inductionof the topoisomerase II-mediated cleavage of plasmid DNAwas tested in cell-free systems. The results are presented inTable 4, second and third columns, as the range of drug concentrations at which DNA cleavage was observed and the optimal concentration at which maximal response was induced.Representative gels are shown in Fig. 3. The most strikingfeature was the inability of C-14 position-substituted analoguesto inhibit topoisomerase II and induce DNA cleavage. Tenanalogues with three types of C-14 substituents were tested.The removal of a thiovalerate, valerate, or O-hemiadipate

changes the 3'-Ar-unsubstituted and 3'-7V-acyl-inactive ana

logues into active inhibitors of topoisomerase II. Such biotransformation converts AD268 (first group) into 14-thiaDXR andits disulfide. For the compounds of the second group, themetabolic conversion of AD32 or AD143 results in AD41 andAD92, and of AD133 in ADI 15. There is a notable exceptionto this rule among the C-14-unsubstituted 3'-yV-alkylanthracy-

clines. Neither biologically active AD280 and AD288 nor inactive AD289 has shown topoisomerase II-mediated DNA cleavage. Finally, AD202 and its C-14-unsubstituted congenerAD285 are weak biologically active intercalators. AD285 is theonly drug among the tested 3'-./V-alkyl analogues which induces

topoisomerase II-mediated cleavage. In terms of DNA intercalation, both congeners AD202 and AD285 behave more likeanalogues of Group 2.

Incubation of the 3'-end-labeled DNA with purified topoi

somerase II in the absence of a drug results in slight DNAcleavage (Fig. 3, Lane B). This low level of activity has beentermed "background" cleavage (33). As has been shown in

previous studies (7, 34), DXR stimulates topoisomerase II-mediated cleavage of plasmid DNA at the concentration rangeof 0.04 to 0.92 ßM,and at a concentration of 4.6 Õ¿Mand higherthe cleavage reaction is either diminished or totally abolished(Fig. 3; Table 4). The rest of 3'-./V-unsubstituted DXR ana

logues, except AD268, stimulated the cleavage with comparableefficacy. The C-14 position-unsubstituted analogues of the 3'-

yV-acylgroup did not show the background inhibition. For sucheffects, substantially higher drug concentrations may be required than those used in the assay. AD268, a C-14-substitutedanalogue of the first group, and AD 198, AD 199, AD280, or

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ANTHRACYCLINES AND TOPOISOMERASE II-DNA INTERACTION

ABCOEFGHIJKLMNOPQ ABCDEFGHIJKLMNOPQ

Fig. 3. Drug-stimulated topoisomerase Il-me-diated DNA cleavage. /, A, 20 ng of PMC41 DNAend labeled with (a-32P]dATP; B, DNA plus 5 ngof human DNA-topoisomerase II (controls); C toG, DNA plus topoisomerase II plus DXR (0.04,0.18, 0.92, 4.6, or 23.0 ¿¡M;S'-yv-unsubstituteddrug. Group 1); H lo L, 4'epiDXR [0.04, 0.18,0.92, 4.6, or 23.0 UM (1)]; M to Q, 4-dmxDNR[0.04, 0.2, 1.0, 5.02, or 25.12 MM(1)]. 2, A and B,controls; C to G, DXR; H to L. 4'd4'IDXR [0.03,

0.15, 0.76, 3.8, or 19.12 MM (I)]; M to Q,4'deoDXR [0.04, 0.19, 0.95, 4.75, or 23.75 MM

(1)]. 3. A and B, controls; C to G, DXR (1); H toL, AD32 (0.03, 0.14, 0.69, 3.45, or 17.25 MM;3'-W-acylanthracycline, Group 2); M to Q. AD 143[0.03, 0.13, 0.65, 3.25, or 16.25 MM(2)]. 4, A andA, controls; C to G, DXR (l); H to L, AD41 [0.03,0.16, 0.78, 3.9, or 19.5 MM(2)]; M to Q, AD92[0.03, 0.16, 0.78, 3.9, or 19.5 MM(2)]. 5, A and B.controls; C to G, DXR (1); H to L, AD 198 (0.03,0.14, 0.7, 3.47, or 17.37 MM;3'-A'-alkylanthracyc-

lines, Group 3); M to Q, AD 199 [0.03, 0.15, 0.76,3.8, or 19.0 MM(3)). 6, A and B, controls; C to G,DXR (1); H to L, AD280 [0.04, 0.17, 0.87, 3.62,or 18.12 MM(3)].

1 2

ABCDEFGHIJKLMNOPQ ABCDEFGHIJKLMNOPQ

III

*.»

3 4

ABCDEFGHIJKLMNOPQ ABCDEFGHIJKL

AD288, biologically active drugs of the third group, effectivelyabolished the background cleavage reaction in the absence oftopoisomerase H-mediated DNA cleavage.

The drug effects on intact cells were tested using the LI210system. At a 5 ¿IMconcentration, the test drugs induced asignificant number of PAB (Table 4), except for the two biologically inactive 3'-jV-alkylanthracyclines AD206 and AD289.Among the 3'-./V-alkyl derivatives, both of these analogues are

least cytotoxic and show the lowest number of PAB. Detectionof PAB in cells treated with 3'-7V-acylanthracyclines provided

further evidence that, at cytotoxic concentrations, the prodrugs(AD32, AD120, AD133, and AD143) are intracellularly converted into topoisomerase H-interacting metabolites.

DISCUSSIONThe stabilization of the topoisomerase II-DNA cleavable

complex by anthracyclines appears to be a major cause of drug-

exerted cytotoxicity (4-6). The present study indicates that thisconclusion is valid for most but not all DXR analogues. Inaddition to the clinically important chemotherapeutic agentDXR, the test drugs included several potentially useful compounds (for review, see Ref. 36). Some of the analogues, suchas 4'epiDXR, dmxDNR, or AD32, have been investigated in

clinical trials; others are being screened in initial or advancedpreclinical tests. In the present study, several differences in thestructure-function relationship have been detected amongclosely related congeners. Summary of experimental findings ispresented in Table 5.

DNA Intercalation of Test Drugs. The extent of DNA intercalation seems to depend on the substitution of the daunosa-mine nitrogen (Table 1, R3 and R4). The 3'-yV-unsubstituted

analogues are strong intercalators. Analogues bearing one ortwo ./V-alkylsubstituents, such as A'-(n-butyl), TV-benzyl,or N,N-

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ANTHRACYCLINES AND TOPOISOMERASE II-DNA INTERACTION

Table 5 Summary of experimental findingsThe semiquantitative evaluation of various activities was as follows: +++. high; ++, intermediate; +, low; 0, absent.

Activity

Analogue (abbreviation) In vivo In vitro*DNA

intercalation"

TopoisomeraseII-mediated

cleavage"

3'-,V-unsubsli(uted anthracyclines

Doxorubicin (DXR)Daunorubicin (DNR)14-Methoxydaunorubicin' (ADI 21)Adriamycin-14-thiovalerate'(AD268)4-Demethoxydaunorubicin (4-dmxDNR)4'-Epidoxorubicin (4'epiDXR)4'-Deoxydoxorubicin (4'deoDXR)4'-Deoxy-4'-iododoxorubicin(4'd4'IDXR)

S'-A'-AcylanthracyclinesA-Trifluoroacetyladriamycin-14-valerate' (AD32)A-Trifluoroacetyladriamycin-14-methoxydaunorubicin' (AD 120)A-Acetyladriamycin-14-valerate' (ADI 33)A-Trifluoroacetyladriamycin-14-O-hemiadipate'(AD143)A-Acelyladriamycin (AD38)A'-Trifluoroacetyladriamycin (AD41 )A-Trifluoroacetyladriamycinol(AD92)A'-Pentafluoropropionyladriamycin (ADI 15)

3'-A'-AlkylanthracyclinesAr-(n-Butyl)adriamycin-14-valerate'(ADI94)A'-Benzyladriamycin-14-valerate' (AD 198)A',A'-Dimethyladriamycin-14-valerate'(AD199)A',A'-Di(fl-butyl)adriamycin-l 4-valerate'(AD202)A'.A'-Dibenzyladriamycin-l 4-valerate'(AD206)A'.A'-Dimethyladriamycin (AD280)

A-(n-Butyl)adriamycin (AD284)AT,A'-Di(n-butyl)adriamycin(AD285)A'-Benzyladriamycin (AD288)A'.A'-Dibenzyladriamycin (AD289)

•¿�+ See Table 4.•¿�SeeTable 2.

< 14 position-substituted analogues (Table 1).

dimethyl, also intercalate strongly. Their type of associationwith DNA could be at variance with that of DXR-DNA binding(37). W-Dibenzylation, as in AD206 or AD289, and W-dibu-tylation, as in AD202 or AD285, appear to significantly interfere with or preclude DNA interaction sterically.

The 3'-¿V-acylanthracyclines have either yV-acetyl, TV-trifluo-

roacetyl, or /V-pentafluoropropionyl substitution. All analoguesof this group, except for AD32, have shown a weak DNAintercalation. In this regard, the present results confirmed thelack of drug-DNA intercalation for AD32, as evidenced by avariety of other techniques (38-41). The limited sensitivity ofanthracycline-DNA absorbency assay used in previous work (7,42) has precluded the detection of low-level DNA intercalationby AD 143, AD41, and AD92. The data for AD32 suggest thatthe bulky C-14 substituent may further affect the weakenedDNA binding caused by the S'-A'-trifluoroacetyl substitution.

Drug Effects on Topoisomerase H-mediated DNA Cleavage.The side chain C-14 position substitution by a thiovalerate(AD268), valerate (AD32 and AD133), hemiadipate (AD143),or O-methyl ether (AD 120 and ADI21) prevents the analoguesfrom interacting with topoisomerase II (Table 4; Fig. 3). Themetabolic cleavage of a hydrolyzable, C-14 position-acyl substituent by plasma esterases, which has been extensively documented for AD32, AD 143, and related drugs in vivo and invitro (7, 43), converts AD32 or AD 143 into AD41 and AD92.Similarly, ADI33 is converted into AD38, while AD268 isdeesterified, presumably to 14-thiaDXR and its disulfide. AllC-14 position-unsubstituted 3'-./V-acylanthracyclines have beenshown to stimulate the topoisomerase 11-mediated cleavage.Thus, the study confirms previous observations (7) and lendsfurther support for the conclusion that metabolic activation of3'-ALacyl C-14 substituents precedes their interaction with to

poisomerase II. This also becomes applicable to the 3'-7V-alkylcompound AD202. For all active S'-W-unsubstituted and 3'-N-

acylanthracyclines (Groups 1 and 2), the strength of DNAcleavage paralleled the strength of drug intercalation. The three3'-jV-alkyl analogues (Group 3) AD280, AD284, and AD288are C-14-unsubstituted DNA intercalators which, however, donot stimulate the topoisomerase II mediated DNA cleavage.Although cytotoxic in vitro, they exhibit only moderate in vivoantitumor activity.

Inhibition of "Background" DNA Cleavage. Previous studies

(7, 34) have shown that high concentrations of DXR not onlyinhibit the drug-induced DNA cleavage but also the backgroundcleavage. It appears that, among S'-W-unsubstituted and 3'-N-

alkyl analogues, the inhibition of the background cleavage is afunction of the ability of a drug to intercalate into DNA. Thisapplies to both C-14-substituted and -unsubstituted analogues.However, AD280, AD284, and AD288, the C-14-unsubstituted3'-/V-alkylanthracyclines, do not cleave DNA in the presence

of topoisomerase II, yet the drugs are able to inhibit the background cleavage effectively. Ethidium bromide, a strong inter-calator with minimal cytotoxicity, is also known to inhibitbackground cleavage as well as DNA cleavage induced by otherdrugs (34). Unlike ethidium bromide, AD280 and AD288, aswell as their C-14-substituted congeners AD 199 and AD 198,induced PAB in cells and are cytotoxic in vitro or in vivo. Theobservations taken together suggest that, following the treatment with these 3'-jY-alkylanthracyclines, a different mode of

drug-topoisomerase II-DNA interaction or perhaps a new typeof topoisomerase II inhibition occurs. It is also possible thatthe analogues kill the cell by other mechanism(s), independentlyof topoisomerase II.

Drug Structure and Cytotoxicity. The side chain C-14 sulist i-

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ANTHRACYCLINES AND TOPOISOMERASE II-DNA INTERACTION

tution in AD32 and AD 143 is important for various biologicalproperties, including those connected with intracellular partitioning of the drug (reviewed in Ref. 7). The actual effectors oftopoisomerase II-related cytotoxicity are the C-14-unsubsti-tuted metabolites of these compounds, mainly AD41. Thetopoisomerase H-mediated DNA cleavage by 3'-W-acylanthra-

cyclines is comparable among the four C-14-unsubstituted analogues. However, AD38 and its C-14-substituted congener areonly marginally active in biological systems. While the 3'-N-

acetyl substitution of the two compounds has little effect on thetests conducted in cell-free systems, it may diminish in someway the cell kill efficacy. The C-13-OH forms of anthracyclinessuch as AD92 are generally credited with lower cytotoxicityrelative to their parent drugs. Some of the 3'-A'-unsubstituted(DXR, 4-dmxDNR) and the 3'-/V-alkyl analogues (for example,

AD 198, AD 199, AD280, and AD288) have shown a disparitybetween their in vivo and in vitro effectiveness. In either case,other topoisomerase II-unrelated mechanism(s) may participateto a various degree in vivo and in vitro and contribute to theoverall cytotoxicity.

In conclusion, the study of 24 analogues of DXR or dauno-mycin suggests that there are two domains of the anthracyclinemolecule which may determine some of the biological properties of the congeners. The first is localized at the C-14 positionof the chromophore Ring A. The side chain attached to thechromophore, be it acyl or nonhydrolyzable ether, may preventthe formation of a ternary complex drug-topoisomerase II-DNA (7). Consequently, topoisomerase II is not stabilized andthe cleavable complex is not formed. The second domain,important for anthracycline intercalation, is localized at the 3'-

nitrogen of the daunosamine moiety. The intercalative modeseems to be advantageous for the drug interaction with topoisomerase II-DNA complexes. While the daunosamine sugar ofthe drug molecule is positioned along the minor grove of DNA(44-46), the C-14 domain of the chromophore can be presentedfor further interaction with binding sites of the enzyme molecule. With the increasing number of the intercalated drugmolecules, the interaction may reach a saturation level andresult in the inhibition of background cleavage. Since the background inhibition is also present in C-14-substituted drugswhich do not stabilize the cleavable complex, these compoundsmay exert topoisomerase II inhibition which differs from thetrapping of the cleavable complex.

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1989;49:5969-5978. Cancer Res   Annette Bodley, Leroy F. Liu, Mervyn Israel, et al.   Daunorubicin Congeners with DNADNA Topoisomerase II-mediated Interaction of Doxorubicin and

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