Synthesis and antineoplastic evaluation of N-enamine derivatives of 1,4-bis[(2-aminoethyl)amino]-9,10-anthracenedione

815

EurJMed Chem (1991) 26,815819 0 Elsevier, Paris

Synthesis and antineoplastic evaluation of N-enamine derivatives of 1,4-bis[(2-aminoethyl)amino]-9,lO-anthracenedione B Stefanskal, S Martelli*, J Paradziej-Lukowiczl, E Borowskil* ‘Department

of Pharmaceutical Technology and Biochemistry, 2Department of Chemical Sciences, University (Received

26 February

Technical University of Gdansk, 80-952 Gdansk, Poland; of Camerino, 62032 Camerino (MC), Italy

1990; accepted 6 May 1991)

Summary - Several N-enamine derivatives of 1,4-bis[(2-aminoethyl)amino]-9,1O-anthracenedione have been synthesized in the reaction of 1,4-bis[(2-aminoethyl)amino]-9,lO-anthracenedione with various P-dicarbonyl compounds. The obtained derivatives exhibited cytotoxicity in vitro comparable or lower to that of ametantrone but their in vivo activity against P388 murine leukemia was decreased. R&urn6 - SynthGse et Cvaluation antineoplastique de d&iv& N-Cnaminiques de la 1,4-bis[(2-aminoethyI)amino]-9,10-anthraGnedione. Plusieurs derive’s N-tnaminiques de la I ,4-bis[(2-aminoe’thyl)amino]-9,lO-anthracc?nedione ant e’te’ synthe’tise’s par la reaction de la I ,4-bis[(2-aminotthyl)amino]-9,10-anthracdnedione avec differents composes p-dicarbonyle’s. Les de’rivks obtenus montrent une cytotoxicitt? in vitro comparable ou infe’rieure a celle de l’ame’tantrone mais leur activite’ in vivo contre la leuce’mie murine P388 est diminue’e. anthracenedione

derivative

/ cytotoxic

activity

/ antitumor

activity

Introduction The high antineoplastic activity [ 141 and low cardiotoxicity [5-71 of ametantrone, 1,4-his{ [2-/(2hydroxyethyl)amino/ethyl]amino)anthracene-9,l Odione and of its 5,8-dihydroxy analog, mitoxantrone, has led to a structural modification study of 1,4bis[(aminoalkyl)amino]-9,10-anthracenediones. The results obtained allowed the determination of the structural requirements of the anthraquinone sidechains to exhibit antitumor activity by the compounds (so-called N-O-O triangulation pattern between the hetero atoms of the chromophore) [S--15]. Recently the synthesis and antineoplastic evaluation of a series of N-enamine derivatives of daunorubicin and doxorubicin have been reported [ 16, 171. It has been demonstrated, that one of these derivatives, N(1 -carboethoxypropen- 1 -yl-2)daunorubicin exhibits reduced subacute toxicity and diminished cardiotoxicity with retention of antileukemic activity [ 181. We thought it is of interest to examine whether the N*Correspondence

and reprints

enamine analogs of anthracenediones would exhibit improved properties, in a similar manner to such derivatives of anthracyclines. In this paper we describe the synthesis and antileukemic activity of several N-enamine derivatives of 1,4-bis[(2-aminoethyl)amino]-anthracene-9,lO-dione, in which the NW-substituents have different lipophilic properties as well as steric bulk. The obtained compounds fulfill the structural requirements for antitumor activity, namely a defined distance between both nitrogen atoms in the side chains and the oxygen atoms in the ring (‘triangular pattern’ [ 151). Chemistry To synthesize the above N-enamine derivatives (scheme 1, l-6), 1,4-bis[(2-aminoethyl)amino]-9,10anthracenedione was reacted with an excess of P-diketones, acetylenedicarboxylates and/or acetoacetic esters in mild conditions. The obtained compounds were purified by means of column chromatography and the yields were in the range of 50-70% with the exception of 6 which was obtained in 30% yield. The

816 Ph R-NH-C=CH-COPh

R - NH - C = CH - COR

a ii

MHC~-~~,NHR

I R = $=CHCOOCH,

2 R = y=CHCOCH,

.m3

2 R

q

CH3

y=CHCOOC2H5

2 R = F=CHCOC,H, CH3

CH3

,6 R = y=CHCOOCH,

_3 R = y=CHCOOC(CH3$

b

where: R’ - H, Me

R - residue of the sugar

lH-NMR spectrum of the enamine part of the condensation product of 1,4-bis[(2-aminoethyl)amino]-9, 1Oanthracenedione with 1-phenyl- 1,3-butanedione (compound 5) is similar to that of 2-deoxy-2-[2-(4oxo-4-phenyl-2-butenyl)amino]-D-glucose. It allowed us to propose the structure of compound 5, as in scheme 1.

COOCH,

CH3

Biological

Scheme 1.

structures of the new compounds l-6 were confirmed by their spectral data (IR and iH-NMR), MS determination (field desorption (FD) technique) and elemental analysis (see table I). It was stated that simple enamine derivatives of glycosylamines are intramolecularly bonded p-aminoa, P-unsaturated ketones [20-221 (scheme 2). Similar structures of intramolecularly bonded l-6 and also of N-enamine derivatives of daunorubicin were deduced from the lH-NMR spectra (the position of NH) signals at very low fields [8-lo]. R ti = CH /

\

\

/

b - R’

G - i

H...O

where: G - glycosyl R, R’ - alkyl or alkoxyl Scheme 2.

The reaction between a primary amine and an unsymmetrical P-dicarbonyl compound can, in principle, give rise to 2 isomeric enamines (structure a and b). It has been well established for condensates of benzoylacetaldehyde and/or 1-phenyl- 1,3-butanedione with 2-amino-2-deoxyglucose that these substances react with the carbonyl group of benzoylaldehyde and 1-phenyl- 1,3-butanedione which is further away from the phenyl group (structure a) [23].

activity

and discussion

The obtained derivatives, which constitute a novel group of Nw-substituted 1,4-bis[(aminoalkyl)amino]9, IO-anthracenediones, were tested for the growth inhibition of L1210 cells in vitro and against P388 murine leukemia in viva. The results are presented in table II. The evaluated compounds exhibit various cytotoxicities in vitro. Thus compounds 1, 2 and 3 exhibit EC,, comparable with that of ametantrone, and 4 and 6 of about 1 order of magnitude higher than ametantrone. Compound 5 was inactive. Compounds l-4 and 6 all show antitumor activity in vivo. The optimal doses for 1 and 2 are 2-fold lower than for ametantrone; however, their efficacy is decreased. It is probably due to their low solubility in water. As it was pointed out above, the corresponding N-enamine derivatives of daunorubicin obtained earlier exhibited improved biological properties [16-l 91. The optimal derivative - N-( 1-carboethoxypropen-1-yl-2)daunorubicin - has shown a marked decrease in cardiotoxicity with retainment of antitumor activity comparable with that of daunorubicin. Although the analogous anthraquinone derivatives, unlike the case of anthracyclines did not exhibit improved properties, it is of interest that the structureactivity relationship is similar for the obtained N-enamine derivatives of 1,4-bis[(2aminoethyl)amino]-9,lOanthracenedione as well as for the respective N-enamine derivatives of daunorubicin. Compounds 1,2 and 3 with carboalkoxy groups in the enamine part show better activity than with an acyl group (4). The presence of an additional carboalkoxy group (6) instead of the methyl group (1) decreases the potency (compound 6).

817 Table I. Physicochemical Comp No

properties

Formula

of l-6.

MP (“C, dec)

1H NMR (CDCl,) I ,4+ubstituents

IR (KBr) cm-1 (major peaks)

FD-MS mlz (relative intensity %)

1.95 (s, 6H, CH,), 3.6 (m, 14H, COOCH,, CH,N, ArNCHJ, 4.5 (s, 2H, CH=), 8.8 (t, 2H, NH), 10.75 (t, 2H, ArNH)

1280,1570, 1630

520 ([Ml+, loo), 521 ([M + l]+, 36)

2

110-11

1.25 (t, 6H, OCH,, CHs), 1.95 (s, 6H, CH,), 3.55 (t, 4H, CH,N), 4.08 (q, 8H, OCH,CH,, ArNCH& 4.5 (s, 2H, CH=), 8.8 (t, 2H, NH), 10.75 (t, 2H, ArNH)

1270,1580, 1610,1630, 1700

548 ([Ml+, loo), 549 ([M + l]+, 17)

3

166-7

1.45 (d, 18H, CWI,),, 1.92 6, 6H CH,), 3.55 (m, 8H, CH,N, ArNCH2), 4.48 (s, 2H, CH=), 8.7 (t. 2H, NH), 10.75 (t, 2H, ArNH)

1260, 1280, 1580,1610, 1630

604 ([Ml+, loo), 605 ([M + l]+, 29)

4

189-90

1.95 (s, 6H, CH,), 2.01 (s, 6H, COCH,), 3.62 (m, lOH, CH=, CH,N, ArNCH2), 10.7 (br s, H, NH), 11.0 (br s, 2H, ArNH)

1280,1580, 1610

488 ([Ml+, loo), 489 ([M + l]+, 15)

5

112-3

2.01 (s, 6H, CH,), 2.6 (m, lOH, CH=, NCH2, ArNCH,), 7.4 (m, 6H, Ar), 7.85 (m, 4H, Ar), 10.4 (br s, 2H, NH), 11.7 (br s, 2H, ArNH)

1280,1560, 1580,161O

612 ([Ml+, loo), 613 ([M + l]+, 36)

6

142-4

3.63 (t, 4H, CH,N), 3.66 and 3.75 (2s 12H, COOCHs), 3.71 (q, 4H, ArCHJ, 5.15 (s, 2H, CH=), 8.25 (t, 2H, NH), 10.75 (t, 2H, ArNH)

1270,1580, 1630, 1710

608 ([Ml+, loo), 609 ([M + l]‘, 71)

Experimental

protocols

Instrumental analysis Melting points were determined with a Boetius pHMK 05 apparatus and are uncorrected. Analyses indicated by elemental symbols were within f 0.4% of the theoretical values and were performed by the Laboratory of Elemental Microanalysis,

University of Camerino. IR spectra were recorded on UR 10 Zeiss spectrometer in KBr pellets; tH-NMR spectra were obtained with a Varian 90 MHz spectrometer using tetramethylsilane as internal standard. Molecular weights were determined by mass spectrometry (PD) on a Varian MAT 711 instrument. The instrumental conditions were the following: wire heating current S-20 mA, ion source temperature 70-lOO”C, accelerating voltage 4-6 kV.

818 Table II. Cytotoxic Comu

Amete

and antileukemic L1210 cells EC,,0 = SEA4 (nM) 84.7 k 33.2

1

48.4 f 11.3

2

70.3 Ik 20.2

3

4

5 6

68.5 f 12.3

720 = 295

25877 + 4221 660 f 195

activity

of 14 against L1210

leukemia

cells and P388 murine

leukemia.

P388 murine leukemia Dose 0.18 0.36 0.78 1.56 3.12 6.25 12.50 25.00 50.00 100.00 1.25 2.50 5.00 10.00 20.00 40.00 0.15 0.31 0.62 1.25 2.50 5.00 10.00 20.00 40.00 80.00 0.19 0.39 0.78 1.56 3.12 6.25 12.50 25.00 50.00 100.00 200.00 1.25 2.50 5.00 10.00 20.00 40.00 60.00 120.00

% TICh

ToxD SU?V~

117 125 150 160 200 250 300 200 70 60 122 144 167 167 167 156 100 110 120 156 156 189 189 178 167 175 125 140 150 160 160 150 190 170 210 250 30 100 111 122 133 133 156 127 117

Wt changed

7l7 717 7r7 717 717 717 ;:: 717 7l7 717 717 717 717 617 717 7l7 717 717 717 7P 7/7 ;; 717 717 717 717 717 ;; 7r7 7l7 717 717 i; 7/7 717 717 717 7i7

-

-

-

1.oo 1.oo 2.16 1.43 0.57 0.14 0.55 0.15 2.75 3.50 0.15 1.22 1.07 0.58 0.28 0.14 0.51 0.28 0.11 0.04 0.35 0.43 0.24 0.04 0.18 1.80 1.oo 0.16 1.50 0.80 0.11 0.22 0

- too - 2.16 1.oo 1.35 1.92 1.21 1.07 1.52

;:: 717

::t

ntf 1.25 2.50 5.00 10.00 20.00 40.00 80.00 160.00 240.00

125 140 140 150 160 170 220 158 158

717 717 T; 7l7 7/7 T:: 717

-

0.60 0.66 0.67 0.17 0.56 0.67 1.08 0.40 1.oo

aEC50 = SEM = concentration of compound required to inhibit the cellular protein bios nthesis by 50%. Each experiment was run at least 4 times and results are presented as average values = standard error of the mean (SEM). z % T/C = ratio of median survival time of treated to control mice expressed as a percentage. CTOXD SUN = survivor recorded on day 4 after first injection as a measure of drug toxicity. Wt change = weight change of mice between day 1 and 5 after the leukemic cell graft. eArnet = Ametantrone. Not tested.

819 Synthesis

Acknowledgments

General procedure for the synthesis of compounds l-4 A sample of 1 mmol of 1,4-bis[2-(aminoethyl)amino]-9,10anthracenedione in 50 ml of a mixture of chloroform-methanol (1O:l) was stirred with 8-10 equivalents of the appropriate fidicarbonyl reagent. The reaction was carried but at room temnerature or at 40°C during 3 h for comnound 6. 4-5 h for compounds 1,2,3 and 15 h for compounds 4 and 5. The course of the reaction was controlled by TLC using the solvent systems chloroform:methanol (lO:l), or toluene:acetone (2: 1). The reaction mixture was evaporated in vacua to a small volume and the crude product was isolated by precipitation with petroleum ether and purified on column chromatography using silica gel and the following solvent systems: chloroform: methanol (lO:l), or toluene:acetone (8:l) for 2, toluene: acetone (2:l) for 4, chlorofotmmethanol (15:l) for 1 and 5. Compounds 3 and 6 were purified using aluminum oxide in the solvent system chlorofonnmethanol (10: 1).

Financial support provided by the Institute of Immunology and Experimental Therapy of Polish Academy of Science (Project CPBR-11.5), of Ministry of National Education, Warsaw (grant NoP/01/161/90-2) and of the Italian Minister0 della Publica, Istruzione (Fondi 60%) is gratefully acknowledged. Thanks are also due to M Bontemps-Gracz for the evaluation of in vitro activity.

1

Biologic&

L

References

tests: in vitro cytoto-xicity evaluation

Tissue culture L1210, murine leukemia cells, were obtained from Roswell Park Memorial Institute (Buffalo, USA). The cells were maintained in RPM1 1640 medium supplemented with 5% fetal calf serum, penicillin G (100 units/ml) and streptomycin (100 pg/ ml) at 37°C in a controlled humid atmosphere of 5% CO? - 95% air. The cells were transplanted every 2-3 days. Cytotoxicity evaluation The cells in logarithmic growth were suspended in the medium to give a final density of 5 x 104 cells/ml. The tested compounds were dissolved in 50% ethanol to give 4 different concentrations. The cell suspension (4 ml) was distributed in test tubes to which 10 pl of the solutions of studied compounds were added. The test tubes were incubated at 37°C in a controlled humid atmosphere of 5% CO, - 95% air for 48 h. The cytotoxic activity (EC,, value) of the tested compounds was defined as their in vitro concentrations causing 50% cellular protein biosynthesis inhibition, measured by cell protein contents 1241. In vivo antileukemic

evaluation

Murine P388 leukemia was obtained from the Institute of Immunology and Experimental Therapy of Polish Academy of Science and injected ip in DBA/, mice according to standard protocols of the National Cancer Institute USA [25]. For test purposes mice (first generation hybrid (BALB/, x DBAA/,)F,) were given 106 P388 cells ip on day 0. Twenty-four h after tumor implantation, solutions of tested compounds in 2% methylcellulose in physiological saline were administered ip daily for 5 consecutive days. At higher doses, the compounds were given as a suspension, because of their limited solubility. The treated group consisted of seven animals and the control group of 18 animals. The median survival time (MST) of the treated (T) and control (C) groups was determined and the percentage T/C was calculated by the following formula [25]: % T/C = [(MST treated)/(MST control)] x 100.

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