J. Med. Chem. 1980,23, 1330-1335
1330
DNA Intercalating Compounds as Potential Antitumor Agents. 1. Preparation and Properties of 7H-Pyridocarbazoles Didier Pelaprat, Robert Oberlin, Ir6ne Le Guen, Bernard P. Roques,* DBpartement de Chimie Organique, ERA 613 (CNRS), UER des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire, 75006 Paris, France
and Jean-Bernard Le Pecq Laboratoire de Physicochimie MacromolBculaire, LA 147 (CNRS), U140 (INSERM), Institut Gustave Roussy, 94800 Villejuif, France. Received March 25, 1980 The DNA intercalating compounds derived from 6H-pyridocarbazole (ellipticines, olivacines) elicit high antitumor properties. In order to get information about the mechanism of action of these agents it is necessary to study structurally related analogues. For this purpose, various derivatives of the four isomeric 7"-pyridocarbazoles were synthesized by a single photochemical process on indolylpyridylethylenes. These derivatives are able to intercalate into DNA. The DNA binding affinities vary in the range of lo' to los M-l, depending mainly on the nature of the substituent, nitrogen quaternization being the most enhancing factor. The position of the pyridinic nitrogen does not markedly affect the DNA binding affinity. Three quaternized compounds elicit a significative but low antileukemic activity on L1210 mice leukemia. The properties of 7H-pyridocarbazoles are discussed and compared to those of 6Hpyridocarbazoles (ellipticines and olivacines).
Several antitumor drugs, s u c h as actinomycin D,' adriamycin,2 el lip ti cine^,^ and amin~acridines,~ interact Whereas strongly in vitro with DNA b y inter~alation.~,~ it is easy to understand why DNA-binding compounds exhibit cytotoxic properties, the selectivity of their action toward tumoral cells remains totally unexplained. This situation results from the lack of information o n the biochemical differences between normal and malignant cells. Nevertheless, the preparation of new derivatives able to intercalate into DNA remains an attractive way to look for new antitumor drugs. Furthermore, the s t u d y of the relation between the DNA binding process and the biological activity of a series of well-chosen compounds can lead to a better understanding of the mechanisms involved. In the series of 6H-pyridocarbazoles,several compounds, mainly derivatives of ellipticine ( 5 , l l - d i m e t h y l - 6 H pyrido[3,4-b]carbazole), exhibit antitumor proper tie^.^^',^ Activity on human tumors has been reported for two derivatives ( 9 - m e t h o ~ y e l l i p t i c i n eand ~ 2-methyl-9-hydroxyellipticinium acetate).l0 On the other hand, the size and the shape of the 6H-pyridocarbazole ring allow an almost perfect positioning in the intercalating site with a maximum overlap between the heterocyclic ring a n d the DNA base pairs.5 In addition, the presence of a positive charge on the nitrogen atom in these compounds helps the stabilization of the complex they form with Such W. Muller and D. M. Crothers, J. Mol. Biol., 35, 251 (1968). F. Dall'Acqua, M. Terbojevich, S. Marciani, B. Scarpinato, and L. Valentini, Biochim. Biophys. Acta, 103, 25 (1965). J. B. Le Pecq, Nguyen Dat Xuong, and C. Paoletti, R o c . Nutl. Acad. Sci. U.S.A., 71(12), 5078 (1974). B. F. Cain, G. J. Atwell, and W. A. Denny, J . M e d . Chem., 20(8), 987 (1977). S. C. Jain, K. K. Bhandary, and H. M. Sobell, J . Mol. Biol., 135, 813 (1979). T. R. Krugh and 3. W. Neely, Biochemistry, 12,1775 (1973). S. Goodwin, A. F. Smith, and E. C. Morning, J. Am. Chem. SOC.,81, 1903 (1959). J. B. Le Pecq, C. Gosse, Nguyen Dat Xuong, and C. Paoletti, C. R. Hebd. Seances Acud. Sci., Ser. D, 281, 1365 (1975). G. Math& M. Hayat, F. De Vassal, L. Schwartzenberg, M. Schneider, J. R. Schlumberger, C. Jasmin, and C. Rosenfeld, Reu. Eur. Etud. Clin. Biol., 15, 541 (1970). P. Juret, A. Tanguy, A. Girard, J. Y. LeTalaer, J. S. Abbatucci, Nguyen Dat Xuong, J. B. Le Pecq, and C. Paoletti, Eur. J. Cancer, 14, 205 (1978). J. B. Le Pecq and C. Paoletti, J . Mol. Biol., 27, 87 (1967). 0022-2623/80/l823-1330$01.00/0
Scheme I
1, R = COOH
7, 1 - N 8, 2-N 2, R = COCl 3, R = CON(CH,), 9, 3-N 4,R = CH,N(CH,), 5 , R = CH,N+(CH,),, 16, R = CH,P+Ph,, 1-
10, 1-N; pyrido-
[3,4-c] carbazole 11, 3-N; pyrido-
[3,2-c]carbazole 12, 2-N; pyrido[4,3-c]carbazole 13, 4-N; pyrido[ 2,3-c]carbazole
characteristic properties are also found in the series of 7Hand 11H-pyridocarbazoles. It is therefore surprising that I1
'
kH3' 5
5,1l-dimethyl-6H-pyrido-
[4,3-b]carbazole (ellipticine)
7
7H-pyridocarbazoles 1-N; pyrido[ 3,4-c]carbazole 2-N; pyrido[4,3-c]carbazole 3-N; pyrido[ 3,2-c]carbazole 4-N; pyrido[2,3-c]carbazole
so far only a few compounds of these series have been p r e ~ a r e d ' ~and - ~ that ~ the antitumor properties have been evaluated only for three20 of them (in the series of 7 H (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24)
M. Kulka and R. H. F. Manske, Can. J. Chem., 30,711 (1952). K. Agoramorthy and R. Robinson, Tetrahedron, 1,172 (1957). R. Huisgen, Justus Liebigs Ann. Chem., 559, 174 (1948). G. R. Clemo and D. G. I. Felton, J. Chem. SOC.,671 (1951). T. I. Tai, Yakugaku Zasshi, 76, 798 (1956). T. Yoshikawa, Yakugaku Zasshi, 81, 1317 (1961). H. V. Horlein, U.S.Patent 2 691 024. R. H. F. Manske and M. Kulka, J . Am. Chem. SOC.,72,4997 (1950). L. K. Dalton, S. Demerac, and T. Teitei, Aust. J . Chem., 22, 185 (1969). J. C. Perche, G . Saint Ruf, and N. P. Buu-Hoi, J . Chem. SOC. Perkin Trans. 1, 260 (1972). 0. De Silva and V. Snieckus, Synthesis, 5, 254 (1971). H. P. Husson, C. Thal, P. Potier, and E. Wenkert, J . Org. Chem., 35, 442 (1970). D. Rousselle, J. Gilvert, and C. Viel, C. R. Hebd. Seances Acad. Sci., Ser. C, 284, 377 (1977).
0 1980 American Chemical Society
Preparation and Properties of 7H-Pyridocarbazoles
Journal of Medicinal Chemistry, 1980, Vol. 23, No. 12 1331
pyrido[ 2,3-c]carbazoles). It thus seemed of great interest to prepare such derivatives and to study their biological activity. As a first approach, the 7H-pyridocarbazole series was investigated. For this purpose, a photochemical method able to provide the four isomeric compounds was selected. Synthesis, DNA binding properties, and antitumor activity of several variously substituted 7H-pyridocarbazole derivatives, including quaternary salts, are described. On the other hand, it has been shown that the dimerization of intercalating agents leads to a very large enThe 7H-pyridohancement of their DNA carbazole derivatives are very appropriate for the preparation and study of such dimeric agents, owing to the easy access to the four isomers. This allows the direct introduction of the chain linking the two rings on the nitrogen atom at different positions. Several such dimers were prepared and studied, some of them exhibiting marked antitumor properties, as reported in the following paper.% Chemistry. Three types of methods have been used in the synthesis of 7H-pyridocarbazoles: the Fischer-type cyclization of indoles,I2-l7the Doebner or Skraup synthesis of quinolines,18-21and photochemical synthesis, with ind ~ l y l p y r i d y l e t h y l e n e s as ~ ~the . ~ ~starting material. The photochemical synthesis proved to be the most appropriate method for our purpose. 7H-Pyridocarbazoles were prepared according to the Scheme I.22,24*29 5-Methoxyindole-2-carboxylic acid (1) was reacted with S0C12to give the acid chloride 2; 2 gave the amide 3 by reaction with dimethylamine. The amide 3 was reduced by LiAlH4to the amine 4, which was quaternized by methyl iodide. The ammonium salt 5 was transformed into phosphonium salt 6. Reaction of 6 with a pyridinealdehyde gave a mixture of the corresponding cis- and trans-indolylpyridylethylene7-9. The indolylpyridylethylenes were transformed into 7H-pyridocarbazoles, 10-13, by an oxidative photocyclization in ethanol in the presence of iodine. We modified the procedure described for the extraction of cis and trans isomers obtained in the Wittig reaction, in order to eliminate OPPh,. The cis and trans isomers were extracted from the reaction mixture by 1N HC1. The main quantity of OPPh3 remained in the organic phase. The isomers were recovered by simple neutralization of the acidic phase, in the presence of CH2C12or CHC1,. Chromatography was then much easier to carry out and gave better yields. Furthermore, the successive steps from 1 to the phosphonium iodide 6 gave nearly quantitative yields. The photocyclizations led to good yields (Table I), except in the case of cis- and trans-7, which gave only 25% of 13 (7H-pyrido[2,3-c]carbazole).In this case, secondary reactions were also taking place. The pK, of compounds 10 to 19 have been measured by UV spectroscopy and are reported in Table 11. The pK, values are influenced by the position of the pyridinic nitrogen in the heterocycle: the greater the steric hindrance around the nitrogen, the lower the pK. Besides, in the 7H-pyrido[3,2-c]carbazolesfavorable mesomeric forms
Table I. Conditions of the Photocyclization of Indolylpyridylethylene Derivatives t o 7H-Pyridocarbazoles
(25) J. B. Le Pecq, M. Le Bret, J. Barbet, and B. P. Roques, Proc. Natl. Acad. Sci. U.S.A. 72(8), 2915 (1975). (26) E. S. Canellakis, R. M. Fico, A. H. Sarris, and Y. H. Shaw, Biochem. Pharmacol., 25, 321 (1976). (27) B. Gaugain, J. Barbet, R. Oberlin, B. P. Roques, and J. B. Le Pecq, Biochemistry, 17, 5071 (1978). (28) D. Pelaprat, A. Delbarre, I. Le Guen, J. B. Le Pecq, and B. P. Roques, J. Med. Chem., following paper in this issue. (29) J. A. Eenkhorn, S. 0. De Silva, and V. Snieckus, Can. J. Chem., 51, 792 (1973).
H
no.
X
10
1-N
11 __
3-N -~
yield of time of photoirradiation, cyclization, h 1mpa %
24 1 92b 2-N 90 2, 3 25 4-N 320 3 75 1 = 100-W medium-pressure Hanovia photoreactor; 2 = Rayonet photoreactor (3500 A ) ; 3 = Hanau TQ 150 highpressure photoreactor. Total yield for compounds 1 0 and 11. 12 13
resulting from the delocalization of the lone electronic pair of the pyrrolic nitrogen increase the pK values. This feature is also present in compound 10, but the steric hindrance prevents the quaternization even with a methyl group. In relation with the basicity of these componds, a W study showed the occurrence of an anhydronium base form at basic pH (14) in the case of compound 23. We checked that no change in the spectrum occurred for compound 24, which has a methylated pyrrolic nitrogen. As demonstrated by their low pK, values (pK, < 7), 7H-pyridocarbazoles are not completely protonated at physiological pH. The quaternization of the pyridinic nitrogen is thus necessary to obtain fully charged drugs at this pH, allowing strong interactions with DNA. Quaternization of pyridinic nitrogens was achieved with the corresponding alkyl halides in DMF.23 Demethylation of the methoxy group was performed in 48% HBr. Methylation of the pyrrolic nitrogen was obtained by reaction of the pyridocarbazole with NaH and ICH, in DMF.30 For this series, the various compounds synthesized are collected in Tables I1 and 111. Interaction with DNA. In the first step, the DNA binding constants of 7H-pyridocarbazoles were measured by competition with ethidium bromide." The required presence of a positive charge in the molecule for a strong DNA binding is clearly demonstrated by the increase in the DNA affinity constants (Kap)as a function of the pH (Table 11). Consequently, due to their low pK, the 7Hpyridocarbazoles were quaternized in order to obtain permanently, positively charged derivatives at physiological pH. As expected, such quaternization markedly increased the K , at pH 7.4 (Tables I1 and 111). The b N A affinities vary only slightly with the position of the nitrogen into the ring of the 7H-pyridocarbazoles, the largest values being obtained for the series of 7Hpyrido[4,3-c]carbazole(compounds 20-22,25,27, and 28). This indicates that the subsequent change in the conjugation does not play a crucial role in the formation of the intercalating complex. The influence of the nature of the quaternizing substituent is clearly shown by the values of Kapobtained for compounds 20 (X= 2-N+CH3),21 (X= 2-N+(CH2)zOH),and 22 (X = 2-N+(CH2)2-l-piperidyl);in this series, the introduction of a more hydrophylic chain lowers the DNA affinity, at pH 7.4. In addition, we demonstrated that the presence of a positively charged nitrogen (30) R. W. Guthrie, A. Brossi, F. A. Mennona, J. G. Mullin, R. W. Kierstead, and E. Grunberg, J.Med. Chem., 18(7), 755 (1975).
1332 Journal of Medicinal Chemistry, 1980, Vol. 23, No. 12
Pelaprat et al.
Table 11. Physical Properties and DNA Affinity Constants of Nonquaternized Pyridocarbazoles
no. 10
R, X mp, "C formulaa pH 7.4 anal. PK PH 5 PH 6 H 1-N 169-170 C16H12N20 C , H , N 5.25 0.18 0.32 1 11 H 3-N C, H, N 6.4 0.77 1.1 1.5 254 c 16 H12N20 12 H 2-N C,H,N 5.75 0.35 0.44 1.7 288 C,,H,2N*O 13 H 4-N C, H, N 5.6 0.16 0.40 1.2 230 C16H,2N20 14 H 1-N C, H, N 5.35 1.15 0.16 0.3 183 I 5H l o N ' 2 15 H 2-N 6.4 C, H, N 0.42 1.15 3 > 290 Cl,HlON,O 16 H 3-N C,H,N 6.75 0.19 0.82 2.4 >290 Cl,HlON,O 17 1.57 C,H,N 5.35 0.23 CH, 1-N 0.37 110 17 14NZ0 18 C, H, N 5.9 1.46 5.3 2.5 CH, 2-N 127 Cl,Hl,N,O 19 C, H, N 6.6 1.23 CH, 3-N 11 4.67 156 C,,H,4N,O a Analyses for the indicated elements were within 0.4% of the theoretical values for the formulas given. The affinity constants (K,) of the drugs for calf thymus DNA were measured o n a Zeiss PMQ I1 apparatus by competition with ethidium bromide, in 0.2 M buffer, Tris-HC1, pH 7.4, cacodylate, p H 6.0, or acetate, pH 5.0. Scatchard plots were calculated with a 9810 A Hewlett Packard calculator.
OCH, OCH, OCH, OCH, OH OH OH OCH, OCH, OCH,
Table 111. DNA Binding Constants and Anti-L1 210-Activity of Quaternary Salts
:& lR
R2
K, x
no. R, 20 OCH, 21 OCH, 22e OCH,
H H H
23 24 25 26 27 28
OCH, OCH, OH OH OCH, OCH,
H CH, H H CH, CH,
3-N+CH,, 13-N+CH3,12-N'CH,, 13-N'CH3, 12-N'CH,, 1-
2gf
H
H
4-N'CH,, 1-
R,
X 2-N+CH3,12-N+(CH,),OH, 12-N'(CH2),N-l-piperidyl,
c1-
FC P
9
> 290 >290 >290 > 290 > 290 > 290 290
> 290
2-N+(CH2),N-l-piperidyl, > 2 9 0
c1-
280
OD MTD,fracmg/ tion formula anal. a M-' kg MTD C,,H,,N,OI c, H, N, 1 2.9 50 0.5 C,,H,,N,O,I~1.5H,O C, H, N, I 1.1 100 0.5 C2,H2,N,0C1~1.5H,0~C, H, N, 0, C1 1.2 1 0 1" 1HCI C,,H,,N201 c, H, N, I 2 5 0 0.5 Cl,Hl,N201 c, H, N, I 3.1 150 0.5 C,,H,,N,01~0.3H20 C, H, N, I 1.8 25 1 C16Hl,N,01~1H20 c, H, N, 1 0.67 5 0 0.5 C,,H17N,01 c, H, N, 1 6.95 2 5 0.7 C,,H,,N,OCl~lH,O~ C, H, N, 0, C1 3 10 0.5 1HC1 C,,H,,N,I c , H, N, 1 0.6 50 0.5
%
T/ Cc
pd
ns 116 290 "C. Anal. Anal. (C15H10NZO) (Ci5HioN20) C, H, N. 7-Methyl- 10-methoxy-7H-pyridocarbazoles 17-19. Sodium hydride (150 mg, 3.18 mmol) was added to a solution of 250 mg (1.4 mmol) of pyridocarbazole in 3 mL of dry DMF. The mixture was stirred for 1.5 h under nitrogen gas. A solution (0.11 mL) of methyl iodide in 3 mL of dry DMF was then added dropwise. The reaction was kept stirring for one night at 25 "C, and ether (1mL) was added to facilitate crystallization of the 7-methyll0-methoxy-7H-pyridocarbazoles.17: 60% yield; mp 170 "C. Anal. (C17H14N20) C, H, N. 18: 62% yield; mp 127 "C. Anal. (CI7Hl4N20)C, H, N. 19: 60% yield; mp 156 "C. Anal. (C17Hi4N20) C, H , N. Quaternization of 7H-Pyridocarbazoles by Methyl Iodide (20,23-27 a n d 29). The corresponding 7H-pyidocarbazole (0.8 mmol) was dissolved into 3 mL of DMF a t 50 "C and 2.5 mL (4.2 mmol) of ICH3 was added. The mixture was stirred for 3 h a t 50-55 "C and concentrated in vacuo. Recrystallization of the precipitate in methanol gave about 70% of the desired 7Hpyridocarbazolium salts. 20: 73% yield; mp >290 "C. Anal. (C17H15N201)C, H, N, I. 23: 68% yield; mp >290 "C. Anal. (C17H16N201)C, H, N, I. 24: 68 yield; mp >290 "C. Anal. (C18H17N201)C, H, N, I. 25: 73% yield; mp >290 "C. Anal. (C16H&01*0.3H20) C, H, N, I. 2 6 60% yield; mp 290 "C. Anal. (C16H13N201)C, H, N, I. 27: 78% yield; mp >290 "C. Anal. (ClBHl7N20I)C, H, N, I. 29: 66% yield; mp 280 "C. Anal. (Ci6Hi3NzI) C, H, N, 1. 2-(2-Hydroxyethyl)-lO-methoxy-7 H-pyrido[4,3-c]carbazolium Chloride (21). Chloroethanol(2.5 mL) was added to a solution of 300 mg (1.2 mmol) of 12 in 10 mL of DMF, and the mixture was stirred overnight a t 65 "C. DMF was evaporated to half its amount in vacuo, and methanol was added (3 mL); 21 crystallized 58% yield; mp >290 "C. Anal. (C18H1~201-1.5H20) C, H, N, I. Quaternization of 12 and 18 by Chloroethylpiperidine (22 a n d 28). A solution of 0.8 mmol of chloroethylpiperidine hydrochloride in 10 mL of water was neutralized by 59 mg of Na2C03, and the base was extracted with 5 mL of chloroform. The organic phase was dried over calcium chloride and poured into a solution of 0.5 mmol of 12 or 18 in 3 mL of DMF. The mixture was kept overnight a t 80 "C. The precipitate was washed with ether and dried in vacuo. This precipitate was then dissolved in 15 mL of methanol and 0.1 mL of water, 0.2 mL of 2.2 N HC1 in methanol was added, the methnaol was evaporated, and the resulting precipitate was thoroughly washed with ether to remove the excess of HCl. This solid was then dried in vacuo, over calcium chloride, a t 80 "C. 22: 50% yield; mp >290 "C. Anal. (C23Hz4N30C1. 0.5H2O.lHCl) C, H, N, 0, C1. 28: 50% yield; mp >290 OC. Anal. (C24Hz8N30Cl*lH20*1HCl) C, H, N, 0, C1. Determination of K,, Values. A Zeiss PMQ-2 spectrophotometer with a ZFM-4 fluorescence attachment was used a t the maximum sensitivity. Excitation of the buffer solution (3 mL in 1-cm path-length glass cells) was achieved using a high-pressure mercury lamp and a 546-nm (M-466) filter. The wavelength of emission was 590 nm and a slit of 0.2 mm was used at the entrance
of the monochromator. The temperature was 25 "C in all the experiments. The buffers were 0.2 M Tris-HC1, pH 7.4, sodium cacodylate, pH 6, or sodium acetate, pH 5. Four cells were used simultaneously for each experiment. The first cell contained the buffer, the second one DNA (4 ,ug/mL), and the third and fourth ones contained the agent plus DNA. The compounds were used a t a concentration of M. Microliter portions of EthBr (ethidium bromide) (100 pg/mL in buffer) were added. Scatchard plots of competition were calculated with a 9810-1 Hewlett Packard calculator. Viscosimetry. Viscosimetric measurements were performed a t 25 "C in a semimicro dilution capillary viscosimeter with a suspended level (Cannon Instrument Co, State College, Pa.) mounted in a highly accurate thermoshted water bath. Flow times were measured to f0.1 ms by the combined use of photoelectric sensors and an electronic timer.31 The unwinding angle of the DNA helix caused by the binding of the different derivatives was measured with covalently closed circular DNA from PM2 phage.31 The reduced viscosities were calculated in units of dL/g, and 7 red is plotted vs. r, the number of bound compounds per nucleotide of DNA, as described by Saucier et a1.33 To measure the length increase of short DNA segments, the intrinsic viscosity of sonicated calf thymus DNA in the presence of an increasing concentration of agents was measured. Log [q]/[7l0 was plotted as a function of log (1+ 2r) where [7] and [710 are the intrinsic viscosities of the DNA measured in the presence and in the absence of bound agent, respectively, and r is the number of bound agent per nucleotide of DNA. These two procedures have been previously described by Saucier et al.33 Biological Testing. L1210 cells (lo5)were inoculated intraperitoneally into DBA2 mice. There were 20 to 30 animals per group. Twenty-four hours later, one group was treated with an intraperitoneal inoculum of the studied drug and another group (control group) received the same volume of the solvent used to dissolve the compound under study. Deaths were recorded every day a t the same hour. Animals which survived for more than 45 days were considered cured. The mean s w i v a l time of treated animals (T) was compared to that of control animals (C). The increases in life span (ILS) were calculated as T / C X 100. The acute toxicity was determined by the usual procedure. The highest dose which could be administered without causing animal death (MTD) was taken as unity, and dosages were expressed in fraction of MTD to appreciate the chemotherapeutic indexes. The statistical significance of the results was determined using Student's t test.
Acknowledgment. This work has been done under the auspices of EORTC Screening and Pharmacology Group. The authors are very grateful to T. M. Fasy for helpful discussions and A. Bouju for typing the manuscript. The technical assistance of J. Couprie and J. Morizet is gratefully acknowledged. This research was supported by grants from the Institut National de la Sant6 et de la Recherche Mgdicde, the Centre National de la Recherche Scientifique, the D616gation Gdn6rale ti la Recherche Scientifique et Technique, The UniversitG Ren6 Descartes (Paris V), and the Universit6 Pierre et Marie Curie (Paris VI).
