Chem..Biol. Interactions, 44 (1983) 207-218 Elsevier Scientific Publishers Ireland .Ltd.

207

N E W A N T H R A C E N E D I O N E D E R I V A T I V E S : I N T E R A C T I O N W I T H D N A A N D B I O L O G I C A L E F F E C T S

M. PALUMBO', C. ANTONELLO b, I. VIANO', M. SANTIANO ~, O. GIA b, S. GASTALDP' and S. MARCIANI MAGN(Y'

"Institute of Organic Chemistry, Biopolymer Research Centre of CNR, Via Marsolo, 1, blnstitute of Pharmaceutical Chemistry, University of Padua, Via Marzolo, 5, 35100 Padua and Clnstitute of Pharmacology, University of Turin, Corso Raffaello, 30, 10125 Turin (Italy)

(Received May 12th, 1982) (Revision received September 26th, 1982) (Accepted October 4th, 1982)

SUMMARY

Two a n t h r a c e n e d i o n e de r iva t i ve s [1 . (w - d i e thy l aminop ropy lamido ) - 4 - hyd roxy - 9,10 - a n t h r a c e n e d i o n e hydrochlor ide (I) and 1 - (w - d i e thy lamino- p ropy lamido) - 2 - m e t h o x y - 4 - hydroxy - 9,10 - a n t h r a c e n e d i o n e hydrochlor ide (I])], h a v i n g an e lec t ron-r ich p l a n a r ch romophore and an amino - subs t i t u t ed side chain, h a v e been synthes ized . T h e i r b ind ing abi l i ty to D N A was in- ves t iga t ed by m e a n s of spectroscopic, equ i l i b r i um dia lys is and f luorescence m e a s u r e m e n t s . T h e i r inh ib i t ion efficiency on nucleic acid syn thes i s was also e v a l u a t e d both in mouse and h u m a n cells. O u r resu l t s indica te tha t , in compar i son wi th ad r i amyc in , compound I shows a s l ight ly w e a k e r com- p lexa t ion abi l i ty to DNA, whi le compound II i n t e rac t s wi th D N A a t a s u b s t a n t i a l l y lower level. T h e s e d a t a m a t c h qui te well wi th the biological r e sponse on the inh ib i t ion of D N A and R N A syn thes i s exh ib i ted by the above m e n t i o n e d compounds; in fac t compound I is s l ight ly less efficient t h a n a d r i a m y c i n and abou t ten t imes more efficient t h a n compound II. T h e close r e l a t i onsh ip be tween the r e su l t s of phys icochemica l and biological s tudies is discussed.

K e y w o r d s : D N A - Drug - In t e rac t ion - A n t h r a c e n e d i o n e - Binding

INTRODUCTION

T h e a n t i t u m o r effects of ce r t a in compounds of the a n t h r a c y c l i n e s t r u c t u r a l type h a v e been assoc ia ted wi th in t e rca la t ion of t he i r p l a n a r mo ie ty in to the D N A of r ap id ly p ro l i f e r a t i ng neoplas t ic cells a n d s u b s e q u e n t b lock ing of

0009-2797/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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RNA synthesis [1-5]. It is known that some antibiotic compounds of this group, like daunomycin and adriamycin, show a good clinical effectiveness not only against leukaemia, but also against solid tumors [6, 7], exhibiting, however, also undesired side effects, such as irreversible cardiotoxicity through the aglycone metabolite [8]. Several analogs of adriamycin have been studied in the literature, consisting essentially of an anthracenedione or a naphtacenequinone moiety, to which one or two aminoalkylamino side chains are bound [9-11]. For these compounds an intercalation mechanism into DNA has been proposed, based upon evidence obtained by means of several experimental techniques [10|.

With the aim of developing new analogs of adriamycin, two anthra- cenedione derivatives, namely l(w-diethylaminopropylamido)-4-hydroxy-9,10- anthracenedione (Fig. l(I)) and its 2-methoxy derivative (Fig. l(II)) have been synthesized and investigated. They meet the fundamental requirement for intercalation (i.e. the presence of an electron-rich planar chromophore) and present the additional possibility of an electrostatic interaction between the phosphate groups of DNA and their positively charged side-chains. In the present paper the binding ability of compounds I and II to DNA is studied, along with their inhibitory effects of nucleic acid synthesis, as a basic indication of their antiproliferative activity.

MATERIALS AND METHODS

Anthracenedione derivatives 1.Amino-4-hydroxy.9,10.anthracenedione and 1-amino-2-methoxy-4-hyd-

0 OH

0 N-C-CH2-CH2-N = (C2Hs)2 Ct T I II I ~" H 0 H

~ 0 ~ O-CH3 • ®

0 N-C-CH2-CH2I II -N=CC2H5)2C| 1"[ H O H

Fig. 1. Chemical structure of compounds I and II.

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roxy-9,10.anthracenedione (EGA-Chem. Co.) were purified by chromatho- graphy on silica gel columns.

1.(w-Chloropropylamido)-4-h ydroxy.9, l O.anthracenedione 1-Amino-4-hydroxy-9,10-anthracenedione (0.360g) was dissolved in ben-

zene (150 ml). A few drops of pyridine and I ml 3-chloropropionyl chloride were then added and the solution heated for 4 h at 70°C in a thermostatic bath. After removal of the solvent under reduced pressure, the residue was recrystallized from toluene/ligroin (1 : 2), to yield 0.430 g red-orange crystals; m.p. 206°C. The elemental analysis for C, H, N and C1 was in excellent agreement with the expected values.

~H-NMR(CDC13): 8 13.27(1H; s; 4-OH); 6 12.4(1H; very broad signal; NI-ICO); $ 9.11 (1H; d; J = 9.6; 2-H); B 8.31 (2H; m; 6-H and 9-H); 8 7.82 (2H; m; 7-H and 8-H); 8 7.36 (1H; d; J = 9.6; 3-H); 8 3.93 (2H; t; J = 6.8; --CH2C1) and $ 3.00(2H; t; J = 6.8; CO--CH~).

1 . (w - Dieth y lam inopropyla m ido) . 4 - h ydrox y - 9,10 - an th racenedione hydro- chloride (I)

1-(w-Chloropropylamido)-4-hydroxy-9,10-anthracenedione (0.400 g) was dissolved in absolute ethyl alcohol (200 ml). Diethylamine (4 ml) was then added and the solution heated for 4 h at 70°C in a thermostatic bath. After removal of the solvent under reduced pressure, a small amount of absolute ethanol was added to the solid residue along with 0.4ml concentrated hydrochloric acid and ethyl ether unless the solution became turbid. After cooling, the precipitate was separated and crystallized as a red-orange material from absolute ethanol/ligroin (2:1); m.p. 217°C. The elemental analysis for C, H, N and C1 was in excellent agreement with the expected values.

IH-NMR(D20): B 8.23(1H; d; J = 9.6: 2-H); 8 7.68(4H; m; 6-9H); 8 6.94(1H; d; J = 9.6; 3-H); 8 3.53(2H; t; J = 6.3; -CHr-N); 8 3.31(4H; q; J-- 7.3; N-CH r- CH3}; 8 2.95(2H; t; J = 6.3; CO-CH2-) and 8 1.37(6H; t; J = 7.3; N-CH2-CH3).

1-(oJ-Ch loropropylam ido)-2.methoxy.4,h ydroxy-9,10.anth racenedione 1-Amino-2-methoxy-4-hydroxy-9,10-anthracenedione (0.450g) was dis-

solved in benzene (150 ml). A few drops of pyridine and I ml 3-chloropro- pionyl chloride were then added and the solution heated for 4 h at 70°C in a thermostatic bath. The crude product, obtained after removal of the solvent under reduced pressure, was dissolved in benzene (30 ml) and purified on a silica gel column (3 × 40 cm) deactivated with 10% water. The product was finally crystallized from absolute ethanol/ligroin (1:1). Yield 0.400 g yellow needles; m.p. 226°C. The elemental analysis for C, H, N and C1 was in excellent agreement with the expected values.

IH-NMR(CDCls); 8 13.59(1H; s; 4-OH); B 11.63(1H; very broad signal; NI~ICO); 8 8.24(2H; m; 6-H and 9-H); 8 7.78(2H; m; 7-H and 8-H); 8 6.78(1H; s; 3-H); 8 3.96(3H; s; --OCH3); ~ 3.89(2H; t; J = 6.5; -CH2CI) and 5 2.97(2H; t; J = 6.5; CHrC1).

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1-(w-Dieth ylam inopropylam ido).2.methoxy-4-h ydroxy-9,10-anth racenedione (II) l(w - Chloropropylamido) - 2 - m e t hoxy - 4 - hydroxy - 9,10 - a n t h r a c e n e d i o n e

(0 .200g) was dissolved in abso lu te e thy l alcohol ( 1 0 0 m 1). D i e t h y l a m i n e (2 ml) was then added and the solut ion hea t ed for 4 h a t 70°C in a ther - mos ta t i c ba th . Af t e r so lvent r em ova l unde r reduced pressure , 10 ml of ab- solute e thano l were added to the solid res idue , a long wi th 0.3 ml concen t ra ted hydrochlor ic acid and e thy l e the r unless the solut ion became turbid . Af te r cooling, the p rec ip i t a t e was s e p a r a t e d and crys ta l l ized as a r ed -o range m a t e r i a l f rom abso lu te e thanol / l ig ro in (1:2); m.p. 235°C. T h e e l e m e n t a l ana ly s i s for C, H, N and C1 was in exce l len t a g r e e m e n t wi th the expected values .

tH-NMR(D20): ~ 7.50(4H; broad s ignal ; 6-9-H); ~ 6.42(1H; s; 3-H); 3.74(3H; s; 2-OCH3); 5 3.50(2H; t; J :: 6.7; -CHz-N) ; ~ 3.28(4H; q; J = 7.2; N-CHz-CH3); ~ 2.97(2H; t: J = 6.7; -CH,z-CO) and ~ 1.32(6H; t; J = 7.2; N-CH,z-CH3).

DNA Calf t h y m u s D N A was ob ta ined f rom S i g m a Chem. Co. St. Louis, MO

(U.S.A.) (Cat D 1501). I ts hypochromic i ty , d e t e r m i n e d according to M a r m u r and Doty [12] was g r e a t e r t h a n 40%.

Solutions Unless s ta ted otherwise , all physica l m e a s u r e m e n t s were car r ied out in

aqueous 0 . 0 1 M Tr i s buffer ( p H = 7.0), con ta in ing 2 - 1 0 3M NaC1 and 1 • 10 :* M E D T A a t 25°C. D N A and d rug concen t ra t ions were a l w a y s checked spec t ropho tomet r i ca l ly , us ing the fol lowing ext inc t ion coefficients: e = 6800 a t 260 n m for DNA; F = 6500 a t 460 n m for I; ~- -: 5700 a t 420 n m for II.

~ H.NMR measurements N M R spec t ra were recorded on a V a r i a n FT-80 A spec t romete r , and a re

g iven in p p m f rom TMS. Coupl ing cons tan t s a re g iven in Hz; all a s s i g n m e n t s a re in a g r e e m e n t wi th re la t ive peak a r ea s and were appl icable wi th decoupl ing expe r imen t s .

Spectrophotometric measurements Elect ronic absorp t ion spec t ra were ob ta ined on a double b e a m Perk in -

E lmer , Mod. 554 spec t ropho tomete r . F luo r ime t r i c t i t r a t i ons were car r ied out us ing a P e r k i n - E l m e r , Med. M P F 44 spec t rof luor imeter . T h e f luorescence in tens i t i es were d e t e r m i n e d by exci t ing all s a m p l e s a t w a v e l e n g t h s cor- r e spond ing to a p p r o x i m a t e l y cons tan t absorp t ions , to avoid abso rbance cor- rections.

Both i n s t r u m e n t s were equipped wi th a t h e r m o s t a t a b l e cell holder.

Equilibrium dialysis A dia lys ing m e m b r a n e (Visking Chem. Co., U.S.A.) was sandwiched be.

211

tween 2 plexiglas half-cells (dialysable volume --7 ml). In one part of each cell, the aqueous solution of derivatives I or II was introduced, while the other part contained a DNA solution at the same ionic strength and pH conditions. Equil ibrium was usually reached after standing 12h at 4°C, followed by 7 h shaking at 25°C in a thermostatically controlled bath. Known volumes from each compartment were then used for the quant i ta t ive evalua- tion of the equil ibrium distribution of the drug. This was determined directly on the aqueous solution in the compartment not containing complex, and af ter complex dissociation (by addition of an equal volume of 0.2 M LiCl in methanol) in the DNA compartment. The ability of the LiC1 solution to dissociate the DNA-complex was previously checked and a calibration curve for compounds I and II in the hydro-alcoholic medium was determined by absorption measurements.

Studies on DNA and R N A synthesis Nucleic acid synthesis was studied both on mouse and human cells. Cells

were routinely cultured at 37°C in Eagle's MEM (Wellcome, Beckenham, U.K.) in 75-cm 2 plastic flasks in a humidified CO2 incubator. Confluent cultures were trypsinized and the cell suspension was centrifuged. The pellet was suspended in medium and the resulting suspension was distributed in test tubes. Triplicate cultures were used for each experiment.

The incorporation of [SH]thymidine into DNA and [3H]uridine into RNA was assayed as previously described [13], with slight modifications.

The cell cultures containing the various concentrations of the compounds and the control cultures were incubated at 37°C for 24 h. After this period the cell cultures were washed twice into Earle's balanced saline solution. The medium was replaced with Eagle's medium containing 1 ~Ci/ml of tr i t iated thymidine (2 Ci/mmol Radiochemical Centre, Amersham) or 5 ~Ci/ml of tri- t iated uridine (41 mCi/mmol Radiochemical Centre, Amersham), respec-

t ive ly . The cultures were returned in incubator for 2 h. Then the radioactive medium was removed and the cultures were washed twice with 3 ml cold phosphate buffered saline solution, twice with 3 ml 10% TCA and once with 3 ml e thanol /e ther (3: 1, v/v). The dried fixed cells were then dissolved in 0.2 N NaOH and an aliquot used for the determination of the radioactivity in a LKB Wallac liquid scintillation counter.

Results were calculated on the basis of specific radioactivity incorporated into the nucleic acids and expressed as percentage of the radioactivity incorporated into the nucleic acid of control cells.

The data obtained were submitted to the probit analysis and expressed as the IDso concentration, i.e., the drug concentration which produces a 50% inhibition.

RESULTS AND DISCUSSION

Physicochemical studies on the DNA-binding process Spectrophotometric investigations. The electronic spectra of I and II exhi-

212

bi t absorp t ion m a x i m a in the visible, f a r a p a r t f r o m the absorp t ions due to the D N A chromophores . T h e l igand absorp t ion is subs t an t i a l l y affected upon addi t ion of DNA, as shown in Fig. 2. In both cases a red sh i f t occurs ( - 2 5 nm), a long wi th m a r k e d h y p o c h r o n i s m ( - 3 0 % decrease in ex t inc t ion coefficient). T h e s e changes in the p roper t i e s of the d r u g a re in favor of an in t e rca la t ion m e c h a n i s m for the e x a m i n e d compounds [9,14]. E x p e r i m e n t s on t h e r m a l d e n a t u r a t i o n , showing a s u b s t a n t i a l increase in Tm (>15°C) in the p resence of the drug, and f luorescence m e a s u r e m e n t s (see below) f u r t h e r suppor t the i n t e r ca l a t i ve mode of in te rac t ion . P r e l i m i n a r y d a t a on the aff ini ty t oward D N A can be ob ta ined by d e t e r m i n a t i o n of the lowest poly- nucleot ide concent ra t ion , which is able to induce the m a x i m u m spec t ra l change in the l igand s p e c t r u m (i.e. a t which prac t ica l ly all l igand molecules a re bound to DNA). For compound I the above m e n t i o n e d concen t ra t ion (about 5 t imes the l igand concen t ra t ion a t CL ~-- 10 5M) is s o m e w h a t lower t h a n for compound II u n d e r s i m i l a r e x p e r i m e n t a l condit ions. T h e s e f indings ind ica te a poorer in t e rac t ion wi th D N A for the la t t e r , as compa red to the former .

0: I 0

0.1

W (,..) 7

'~ O0

0

(1)

'~ 0.2

0.1

O0

1 I I I I 1

I I t I ~ ~ ,

b) 1

I

350 400 450 500 550 600

Fig. 2. (a) Absorption spectrum of I alone (1) and in the presence of a 6-fold excess of DNA (2). Cl = 9.5 - 10 -s M. (b) Absorption spectrum of II alone (1) and in the presence of a 10-fold excess of DNA (2). Cn = 1.0 • 10 -5 M.

213

Equilibrium dialysis measurements. For a quant i ta t ive evaluation of the binding properties of our anthracenedione derivatives to DNA, equilibrium dialysis measurements have been performed. The results, plotted in terms of Scatchard diagrams, are reported in Figs. 3a and 3b, respectively (r is the ratio of bound ligand to total DNA and m the free ligand concentration). For the evaluation of n (number of binding sites) and K (apparent binding constant) a l inear regression was used, according to the classical in- dependent-site model [15]. As the correlation coefficient was always quite satisfactory, the binding data were not analyzed according to more sophisti- cated theories [16]. Equil ibrium dialysis data indicate therefore the presence of one type of independent binding sites for both compounds I and II. Under our experimental conditions we obtained n = 0.50 -+ 0.03 and log K -- 5.0 -+ 0.1 for compound I and n = 0.26 +- 0.03 and log K = 4.8 -+ 0.1 for compound II. It

1.0

' 0 8 0

"T C)

E06 04

0.2

0.0

x

E

,)

1 6

1.4

1.2

1.0

0 8

06

04

0 2

O0 O0

D O0 01 0.2 0.3 04 05

r

b)

o

Oo o

° 0 % o

L I I I ,

01 02 03 04 1

O5 r

Fig. 3. Scatchard plot for system I-DNA (a) and system ILDNA (b) obtained from equilibrium dialysis measurements.

214

should accordingly be concluded that, whereas the binding sites exhibit quite similar affinities in both our substances, the number of sites available markedly differ in the two cases. Note however tha t the r range investigated with dialysis experiments is 0.06 + 0.2 for H and 0.2 + 0.4 for I.

Fluorescence measurements . It is well known that the interaction between fluorescent compounds and DNA often leads to substantial changes in the fluorescence quantum yield [17-19]. In both compounds I and II a dramatic quenching effect is observed in the presence of DNA, using an excitation wavelength at which complex and free ligand exhibit practically identical

Cf = ~f + 1 qf 1 (1) ~f - - ~ ~f-- Wb K n ~ f - ~b[DNA]

absorptions. If r ~ n, Eqn. (1) holds [19] where ~f and ~b are the fluorescence quantum yield of free and bound ligand respectively and ~ is the apparent quantum yield at a macromolecule concentration [DNA]. Unfortunately we were not able to observe appreciable fluorescence at DNA/ligand ratios high enough to verify the relationship r ~ n. However, since we can assume that the fluorescence intensity is linearly related to the ligand concentration in our experimental conditions, the following equation can be used [20]:

F0-F T

[Ld = F ~ : : D LT

where [Lb] is the bound ligand concentration, LT is the total ligand concen- tration, f is the fraction of fluorescence remaining when the ligand is completely bound to DNA, Fo and F are the fluorescence readings in the absence and in the presence of DNA, respectively. The Scatchard diagrams obtained from fluorescence measurements are reported in Fig. 4. Both exhibit a linear path as found in the equilibrium dialysis experiments. Using the independent-site model, we calculated n = 0.24 _+ 0.03 and log K = 5.8 + 0.1 for I and n = 0.25_+0.02 and l o g K = 4.7_+0.1 for H.

Whereas the latter set of data totally agree with those already given in this work for compound II, K- and n-values are completely different from the figures obtained by equilibrium dialysis for compound I. Interestingly, while for II the investigated r range is practically the same both in the dialysis and fluorescence experiments, this is not the case for I, as 0.2 < r < 0.4 in the dialysis and 0.05 < r < 0.24 in the fluorescence measurements. The conclusion follows tha t the r/m vs. r plot for compound I consists of two approximately linear paths, with different slopes. This behavior could be explained by (a) the presence of more than one non-interacting binding site, (b) a cooperative effect between interacting sites or (c) with the fact that, at any degree of binding saturation the number of free ligand binding sites depends not only on the number of ligands already bound, but also on the distribution of these along the lattice [16]. Explanation (c) should be apparently ruled out, since by

215

x

E

1.{

1.4

1.2

1.0

0.8

0 .6

0 4

0 2

0.0 ,, O 0

,)

o o

I v o 0 ~

0.1 0.2 l i

0.3

0

x

E

1.2 b)

1.0-

o

0 . 8 - o o o o

06 o o

0.2

i o.o i 1 I

0.0 0.1 0.2 0.3

0.4

Fig. 4. Sca t cha rd plot for t h e s y s t e m I -DNA (a) a n d s y s t e m I I -DNA (1)) ob ta ined f rom fluores- cence m e a s u r e m e n t s .

means of appropriate curve fitting programs, we could not find K- and n-values able to reproduce our experimental results satisfactorily enough. Of the remaining possibilities (a) appears more likely than (b), due to the almost abrupt slope variation at r = 0.2. Compound I should accordingly exhibit two kinds of non-interacting binding sites, with apparent constants differing by about one order of magnitude. Under the above assumptions the high K process is probably related to ligand intercalation into DNA, while the low K interaction could be due to (external) electrostatic binding between the phos- phate groups of the polynucleotide and the ionic tail of the anthracenedione

216

derivative. Studies at variable ionic strength are required in order to confirm our hypothesis.

As far as compound 11 is concerned, only one class of binding sites has been characterized. However, since no data at r > 0.2 are available, a second class of sites with a lower binding constant could be present. Prel iminary results of spectrophotometric t i tration experiments indicate that this is the case also for compound II.

Although the compounds we examined are structurally very similar, the apparent constants relative to the first binding process differ remarkably from each other. The presence of the -OCH3 group at position 2 should not introduce substantial electronic modifications in the planar anthracenedione moiety, as to strongly modify the 7r interaction between drug and DNA base pairs. In any case, as it produces a weak electron-donating effect, it should improve the above mentioned interaction. The lowering of the binding energy for derivative IT should be therefore principally related to the steric hin- drance originated by the methoxy group directly bound to the anthra- cenedione ring, which limits its intercalation ability.

It is finally worth comparing the parameters obtained in this work, with the values reported in the l i terature for similar compounds [9]. l(4'-Diethyl- amino- l ' .methylbutylamino)-9,10-anthracenedione hydrochloride (IV) exhibits n- and k-values rather close to the strongest binding in I (log K = 5.70, n = 0.2), whereas the corresponding 4-hydroxy derivative (V) shows a greater affinity toward DNA (log K = 6.50, n = 0.14). The chemical struc- ture of the above compounds would suggest a marked similarity between I and V, rather than I and IV. A possible explanation for the results, which indicate the opposite trend, could rest on the fact tha t the secondary amino group in compound V is replaced by an amido group in compound I. The electron-donating effect on the anthracenedione ring is therefore consider- ably reduced in the latter, leading to a less effective 7r interaction with DNA bases. On the other hand the presence of an equal number of substituent groups (i.e. -OH and -NHR), which exert similar electronic effects on the planar moiety, possibly leading also to hydrogen bending to the main-chain phosphate groups, could reasonably explain the comparable values of the thermodynamic parameters experimentally found for compounds I and IV.

Inhibition of DNA and RNA synthesis. The effectiveness of compounds I and II and adriamycin in reducing [3H]thymidine and [3H]uridine incor- poration in DNA and RNA is shown in Table I. Clearly compound I is much more effective than compound II in inhibiting DNA and RNA synthesis. Compound I, on the other hand, is about three times less active than adriamycin in inhibiting DNA synthesis and as active as adriamycin toward RNA synthesis. These effects are similar beth in mouse and human cells. In analogy to that already shown for daunomycin [21], it can be proposed that the compounds act chiefly on DNA, and DNA-dependent RNA synthesis, ra ther than on RNA.

It is now interesting to compare the thermodynamic parameters relative to compounds I, II (this work) and adriamycin [22, 23], with the biological

217

TABLE I

INHIBITION OF DNA AND RNA SYNTHESIS IN MOUSE FIBROBLAST (L929) AND HUMAN AMNIOTIC CELLS (WISH) AFTER ADDITION OF TESTED COMPOUNDS

IDs0 and limit8 of error (drug concentration (~rn/ml) which produces a 50% inhibition as compared to the control) values are reported. Time course 24 h.

DNA RNA

L929 WISH L929 WISH

Compound I 7 (5-9) 9.5 (6.3--14) 14 (11-17) 11 (9-14) Compound II 87 (50--160) 130 (110-150) 80 (60-.90) 130 (110-150) Adriamycin 2.5 (1-5) 3 (1--6) 9 (11-13) 10 (9-15)

effects due to the same substances. Binding studies indicate for these com. pounds very similar n-values, whereas the corresponding K-values differ considerably. In fact the constant relative to adriamycin under experimental conditions rather close to ours is about 3-4 times higher than for compound I and about 30-40 times higher than for compound 1I. These data agree very well with the reactivity ratios found in the inhibition measurements and give fur ther support to the proposed direct relationship between the molecular event of drug intercalation into DNA and the biological response. We there- fore conclude that joint physicochemical and biological studies can prove quite valuable in investigating the mechanism of action of potential drugs and in directing toward the synthesis of new more active compounds.

ACKNOWLEDGEMENT

The financial support of CNR (finalized project in Chemistry) is gratefully acknowledged.

REFERENCES

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