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Synthesis and Biological Evaluation of 11-Substituted 6-Aminobenzo[c]phenanthridine Derivatives, a New Class of Antitumor Agents Ilka Kock, Dieter Heber, Matthias Weide, Ulrich Wolschendorf, and Bernd Clement* Department of Pharmaceutical Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstraβe 76, D-24118 Kiel, Germany Received November 11, 2004
The synthesis of 11-substituted 6-amino-11,12-dihydrobenzo[c]phenanthridines and 11substituted 6-aminobenzo[c]phenanthridines through an efficient method is described. The antiproliferative activity of selected compounds against a wide panel of tumor cell lines was tested in the in vitro anticancer screening and the in vivo hollow fiber assay of the National Cancer Institute. Several compounds turned out to exhibit considerable cytotoxicity for tumor cells. For the study of structure-activity relationships different substituents were introduced in the 11-position. Compounds with methoxyphenyl substituents tended to show the highest potency. Several compounds exhibited noteworthy antitumor activity with GI50 values across all cell lines 0.60 is considered significant. COMPARE analysis indicated that the most growth-inhibitory compounds 33-35, 37 and also compounds 8, 13, 17 shared a response pattern with antimitotic agents, particularly an interaction with tubulin, whereas compounds 10 and 39 did not show correlations with any agents of the NCI database. Indeed, the derivatives 33 and 34 could be assigned clearly to the group of agents interacting with tubulin while the remaining compounds 8, 13, 17, 35, 37 displayed no such strong correlations as the abovementioned derivatives. We will try to relate derivatives 33 and 34 to structures that were found in an earlier study toward identification of antimitotic agents from the NCI database.15 Among the series of compounds evaluated in the in vitro screen, derivatives 10, 13, 33-35, 37, 39 were selected for further in vivo trials. In Vivo Antitumor Activity. The hollow fiber assay is used as the initial in vivo experience for compounds found to have reproducible activity in the in vitro anticancer drug screen and provides quantitative indices of drug efficacy.16 The results of the antiproliferative activity for all tested compounds are listed in Table 3. Compounds with a combined ip + sc score g20, a sc score g8 or a net cell kill of one or more cell lines were considered significantly active. The derivatives 10 and 34 exhibited a combined ip + sc score g20, while all compounds except derivative 37 displayed a sc score g8. Indeed, the 3,4,5-trimethoxyphenyl-substituted dehydro derivative 34 is the most potent compound representing the highest score on the
11-Substituted 6-Aminobenzo[c]phenanthridines
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Table 4. Yields, Melting Points, and Systematic Names of All Synthesized Compounds compd
name
yield [%]
mp [°C]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
6-amino-11,12-dihydro-11-phenylbenzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(2-methoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(3-methoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(4-methoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(2,3-dimethoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(2,4-dimethoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(2,5-dimethoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(3,4-dimethoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(3,5-dimethoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(2,3,4-trimethoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(2,4,5-trimethoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(2,4,6-trimethoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(3,4,5-trimethoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(2-pyridyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(3-pyridyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(4-pyridyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(2-furyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(2-thienyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(3-bromophenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(4-chlorophenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(4-fluorophenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(2-ethoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(4-ethoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(4-methylphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-[4-(2-propyl)phenyl]benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(1-naphthyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(3-hydroxy-4-methoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(4-hydroxy-3-methoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(3-hydroxy-2,4-dimethoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(4-hydroxy-3,5-dimethoxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11,12-dihydro-11-(3,4-methylenedioxyphenyl)benzo[c]phenanthridinium chloride 6-amino-11-phenylbenzo[c]phenanthridinium perchlorate 6-amino-11-(2,4-dimethoxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(3,4,5-trimethoxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(2,3,4-trimethoxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(2,3-dimethoxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(3,4-dimethoxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(3-methoxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(2-furyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(2-thienyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(1-naphthyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(4-methylphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(4-chlorophenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(4-fluorophenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(3-bromophenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(2,4,5-trimethoxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(2-ethoxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(3,4-methylenedioxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(2,5-dimethoxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(4-ethoxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(3,5-dimethoxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(2,4,6-trimethoxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(2-methoxyphenyl)benzo[c]phenanthridinium perchlorate 6-amino-11-(4-methoxyphenyl)benzo[c]phenanthridinium perchlorate
52 81 60 66 49 53 56 57 36 64 29 27 60 30 36 38 62 38 66 43 70 62 53 49 81 38 28 26 23 40 89 50 45 44 51 42 38 48 35 52 41 47 41 57 58 56 42 45 48 13 15 18 4 14
355 339 288 286 210 205 318 249 308 283 298 246 272 238-240 248 (dec) 248 248 232 271 (dec) 263 312 325 273 230 278 265 212 324 327 225 (dec) 293 326 336 296 289 (dec) 287 290 305 274 297 332 293 338 330 286 (dec) 306 304 319 291 305 334 316 341 328
in vivo assay. Compounds with a trimethoxyphenyl group in the 11-position (10, 13, 34, 35) contributed to high antiproliferative activity, whereas compounds bearing a dimethoxyphenyl substituent (33, 37) generally showed less potency. Therefore, trimethoxyphenyl substituents in the 11-position seem to be an important feature leading to higher antitumor potencies. In contrast to the in vitro results, the dihydro derivatives 10 and 13 displayed an enhanced activity in this in vivo study compared with most of the dehydro derivatives. Planarity of the ring system is apparently not required to ensure significant cytotoxicity in vivo. Due to these results the DNA intercalation as a mechanism of action is rather improbable. In this case it had to be expected that the dehydro derivatives show stronger effects related to their planar ring system. The deviating antitumor activities could be explained by an antimitotic
mechanism for most of the compounds as shown in COMPARE analysis. Moreover, corresponding to our derivatives the antimitotic agents colchicin and podophyllotoxin contain trimethoxyphenyl groups. These trimethoxyphenyl groups interact with a specific region of tubulin and are able to inhibit polymerization of tubulin.17 As compounds with a trimethoxyphenyl substituent were the most potent ones in the in vivo hollow fiber assay, an antimitotic mechanism of action could be postulated. Conclusions A method for the synthesis of novel benzo[c]phenanthridine derivatives was developed. In contrast to other more complicated methods the compounds were prepared in a simple one step synthesis by the reaction of 2-methylbenzonitrile and aromatic aldehydes. By this
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procedure a series of 6-amino-11,12-dihydrobenzo[c]phenanthridines 1-31 was synthesized, showing the broad applicability of this synthetic method. These compounds could easily be converted into the corresponding dehydro derivatives 32-54. Furthermore, the synthesized compounds exhibited remarkable antitumor activities in in vitro and in vivo assays. These derivatives are therefore under development as potential antitumor agents. Structure-activity relationships within the dehydro series revealed that methoxyphenyl substituents in the 11-position lead to derivatives with enhanced activity. By COMPARE analysis an interaction with tubulin as antimitotic mechanism could be postulated. The mechanism of the antitumor activity is the subject of further investigations.
bonate and several times with water. The organic layer was dried over sodium sulfate. After evaporation of the solvent, perchloric acid (70%) was added with vigorous stirring to obtain the 11-substituted 6-aminobenzo[c]phenanthridinium perchlorates. The precipitate was collected, washed with diethyl ether, and crystallized from methanol. Yields, melting points and systematic names of all synthesized compounds are listed in Table 4.
Acknowledgment. The authors thank the Developmental Therapeutics Program of the National Cancer Institute, Bethesda, MD, for providing the in vitro and in vivo antitumor screening data. Special thanks are due to Dr. Dan Zaharevitz for helpful discussions. We acknowledge the excellent experimental assistance of M. Ko¨nig. We also thank Dr. U. Girreser for NMR and MS spectral experiments. The financial support of the Fonds der chemischen Industrie is greatly appreciated.
Experimental Section Melting points were determined with a Bu¨chi 510 melting point apparatus and on a Thermowar microhotstage and are reported uncorrected. 1H NMR spectra were obtained on a Bruker ARX 300 spectrometer at 300 K. Chemical shifts (δ values) are reported in ppm using TMS as internal standard. All coupling constants (J values) are quoted in Hz. The following NMR abbreviations are used: br (broad), s (singlet), d (doublet), t (triplet), m (unresolved multiplet), Ar (aromatic proton). IR spectra (as KBr disks) were determined on a Perkin-Elmer FT-IR 16 PC spectrometer and are expressed in cm-1. Electron impact mass spectra (70 eV) were recorded on a Hewlett-Packard 5989 A mass spectrometer. The relative intensities of the peaks are listed in parentheses. Elemental analyses were performed by the Microanalytical laboratory of Ilse Beetz, Kronach, Germany, and were within (0.4% of the theoretical values unless indicated otherwise. All reagents were purchased from commercial sources and used without further purification. General Method for the Preparation of 11-Substituted 6-Amino-11,12-dihydrobenzo[c]phenanthridines 1-31. A solution of 2-methylbenzonitrile (80 mmol) and the relevant aldehyde (40 mmol) in DMPU (40 mL) was added dropwise to a stirred solution of potassium tert-butylate (88 mmol) in DMPU (90 mL) at 35 °C under an atmosphere of nitrogen. The resulting mixture was stirred for 3-4 h at 35-40 °C and then decomposed with ice water (400 mL) containing ammonium chloride (80 mmol). The aqueous layer was extracted with methylene chloride. After evaporation of the solvent, 5 N hydrochloric acid was added with vigorous stirring to obtain the 11-substituted 6-amino-11,12-dihydrobenzo[c]phenanthridine hydrochlorides. The precipitate was collected, washed with methylene chloride, and crystallized from methanol/water (3:1), if not stated otherwise, or purified by sublimation under high vacuum conditions. Analytical HPLC. Chromatographic separations of the racemic 6-amino-11,12-dihydrobenzo[c]phenanthridines 1, 3-6, 8, 10, 13-21, 24-26 were performed using a Waters 510 pump, a 250 × 4.6 mm CHIRACEL-OD column at room temperature, and a Waters 484 tunable absorbance detector set at 254 nm. The mobile phase consisted of n-hexane and i-propanol (85/15 v/v or 70/30 v/v). A flow rate of 1 mL/min was employed. In each case 5 µL was analyzed by HPLC (concentration of the analytes: 1 mg/mL). General Method for the Preparation of 11-Substituted 6-Aminobenzo[c]phenanthridines 31-54. A solution of DDQ in 1,4-dioxane was added to a stirred solution of the 11substituted 6-amino-11,12-dihydrobenzo[c]phenanthridine (base) in 1,4-dioxane. The reaction mixture was stirred and refluxed for 12 h. Then, the mixture was cooled, poured into 500 mL of a saturated solution of sodium hydrogencarbonate, and stirred vigorously. The aqueous layer was extracted several times with diethyl ether. The combined organic extracts were washed once with 300 mL of a saturated solution of sodium hydrogencar-
Supporting Information Available: Spectroscopic data (1H NMR, 13C NMR, IR, MS) and elemental analysis results. This material is available free of charge via the Internet at http://pubs.acs.org.
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