6-Alkyl-12-formyl-5,6-dihydroindolo[2,1-alpha]isoquinolines have been shown to inhibit the growth of human mammary carcinoma cells by an unknown mode of action. One of the possible molecular targets is the tubulin system which is involved in cell division. A number of 5,6-dihydroindolo[2,1-alpha]isoquinolines with methoxy or hydroxy groups in positions 3, 9, and/or 10 and various functional groups such as formyl, acetyl, cyano, alkylimino, and alkylamino in position 12 were synthesized and evaluated for both inhibition of tubulin polymerization and cytostatic activity in MDA-MB 231 and MCF-7 human breast cancer cells. In the tubulin polymerization assay, only hydroxy derivatives were active, whereas both the hydroxy derivatives and some of the methoxy compounds inhibited cell growth. In order to establish a correlation between the inhibition of tubulin polymerization and cytostatic activity in the hydroxy series, two of the most active racemates were separated into the enantiomers. In both assays, the relative potencies of the hydroxy derivatives were in a similar order. Highest activity was found for the (+)-isomers of 6-propyl- (6b) and 6-butyl-12-formyl-5,6-hydro-3,9-dihydroxyindolo[2,1-alpha]isoquino line (6c) with IC50 values of 11 +/- 0.4 and 3.1 +/- 0.4 microM, respectively, for the polymerization of tubulin at 37 degrees C (colchicine: 2.1 +/- 0.1 microM). The active hydroxy derivatives displaced 40-70% of [3H]colchicine from its binding site in the tubulin at concentrations 10-fold higher than that of colchicine. The data suggest that hydroxy-substituted indolo[2,1-alpha]isoquinolines bind to the colchicine-binding site and inhibit the polymerization of tubulin. This action can be assumed to be responsible for the cytostatic activity of the hydroxy derivatives and might also contribute to the antitumor effect of the corresponding methyl ethers.

A number of 6-alkyl-12-formyl-5,6-dihydroindolol[2,1-a]isoquinolines were synthesized by the Bischler-Napieralski reaction from the respective 1-alkyl-2-(3-methoxyphenyl)ethylamines and bromo-substituted (methoxyphenyl)acetic acid chlorides followed by a second ring closure reaction involving a base-generated benzyne intermediate. The methoxy functions in positions 3 and 9 or 10 were cleaved with BBr3 and the free hydroxy groups converted into the acetates. The enantiomers of the most potent derivatives were separated by liquid chromatography on triacetylcellulose. All of the compounds tested bind to the calf uterine estrogen receptor. The relative binding affinities (RBA) ranged from 0.5 to 3.9 (17 beta-estradiol: RBA = 100) and were dependent on the position of the oxygen function in the indole moiety. The 3,10-diacetoxy derivatives showed higher RBA values than the corresponding 3,9-substituted tetracycles. There was no major difference in binding affinity between (+)- and (-)-enantiomers. Computer-assisted molecular modeling studies showed that the chiral carbon atom 6 of the indoloisoquinoline is likely to mimic the C-11 atom of estradiol. In the mouse uterine weight test, only the 3,10-diacetoxy series exhibited weak estrogenic activity at higher doses. The antiestrogenic effects found with all the compounds varied considerably. Maximum inhibition of estrone-stimulated uterine growth was found for the ethyl derivative 7d (80% with 250 micrograms/animal per day). All derivatives strongly inhibited the growth of human breast cancer cells in vitro. There was no significant difference between hormone-sensitive MCF-7 cells and hormone-independent MDA-MB 231 cells. Cytostatic activity was higher for the 3,9-substituted indoloisoquinolines than for the 3,10-analogues and dependent on the length of the alkyl group at C-6. The maximum effect was found with the butyl derivative 7g. When the enantiomers of the ethyl (7c), propyl (7e), and butyl derivative were studied, a strong difference in activity was observed between the stereoisomers. The IC50 values of the (+)-forms were usually only a tenth of those of the levorotatory isomers. Maximum cytostatic activity was found with (+)-7g: 85% inhibition at 1 x 10(-7) M in MCF-7 cells and 94% inhibition at 2.5 x 10(-7) M in MDA-MB 231 cells. This stereospecificity indicates a selective action on a biochemical target. Since no interaction with DNA was observed, the precise mode of action still remains to be elucidated.

Inhibition of Tubulin Polymerization by 5,6-Dihydroindolo[2,1a]isoquinoline Derivatives. -3-Hydroxy-substituted indoloisoquinolines, e.g. (I), as well as their corresponding methyl ethers, both series exhibiting cytostatic activity, are evaluated for inhibition of tubulin polymerization. Only in case of the hydroxy series correlation between inhibition of tubulin polymerization and cytostatic activity is established, whereas the methyl ether series act certainly via an alternate mechanism. The (+)-enantiomers of compounds (Ia) and (Ib) show the highest activity as inhibitors of tubulin polymerization. -(GOLDBRUNNER, M.; LOIDL, G.; POLOSSEK, T.; MANNSCHRECK, A.; VON ANGERER, E.; J. Med. Chem. 40 (1997) 22, 3524-3533; Inst. Pharm., Univ. Regensburg, D-93053 Regensburg, Germany; EN)

6-Alkyl-12-formylindolo(2,1-a)isoquinolines.Syntheses, Estrogen Receptor Binding Affinities, and Stereospecific Cytostatic Activity.-Bischler-Napieralski ring closure reaction of the amides (III), obtained from the amines (I) and the acid chlorides (II) yields the benzylisoquinolines (IV) which undergo a second ring closure reaction on treatment with sodium (methylsulfinyl) methanide, forming the dihydroindoloisoquinolines (V). Vilsmeier-Haack formylation of (V) gives the compounds (VII) which are converted to the desired 6-alkyl-12-formylindolo(2,1-a)isoquinolines, e.g. (±)-(IX). After separation, the two enantiomers of (IX) are tested for their cytostatic activity which depends on the kind of stereoisomer. Further pharmacological investigations are described in the original paper. -(POLOSSEK, T.; AMBROS, R.; VON ANGERER, S.; BRANDL, G.; MANNSCHRECK, A.; VON ANGERER, E.; J.