Indanocine, a microtubule-binding indanone and a selective inducer of apoptosis in multidrug-resistant cancer cells.

BACKGROUND: Certain antimitotic drugs have antitumor activities that apparently result from interactions with nontubulin components involved in cell growth and/or apoptotic cell death. Indanocine is a synthetic indanone that has been identified by the National Cancer Institute's Developmental Therapeutics Program as having antiproliferative activity. In this study, we characterized the activity of this new antimitotic drug toward malignant cells. METHODS: We tested antiproliferative activity with an MTT [i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, mitochondrial damage and cell cycle perturbations with flow cytometry, caspase-3 activation with fluorometry, alterations of the cytoskeletal components with immunofluorescence, and antimicrotubule activity with a tubulin polymerization assay. RESULTS/CONCLUSIONS: Indanocine is a cytostatic and cytotoxic indanone that blocks tubulin polymerization but, unlike other antimitotic agents, induces apoptotic cell death in stationary-phase multidrug-resistant cancer cells at concentrations that do not impair the viability of normal nonproliferating cells. Of the seven multidrug-resistant cell lines tested, three (i.e., MCF-7/ADR, MES-SA/DX5, and HL-60/ADR) were more sensitive to growth inhibition by indanocine than were their corresponding parental cells. Confluent multidrug-resistant cells (MCF-7/ADR), but not drug-sensitive cancer cells (MCF-7) or normal peripheral blood lymphocytes, underwent apoptotic cell death 8-24 hours after exposure to indanocine, as measured by sequential changes in mitochondrial membrane potential, caspase activity, and DNA fragmentation. Indanocine interacts with tubulin at the colchicine-binding site, potently inhibits tubulin polymerization in vitro, and disrupts the mitotic apparatus in dividing cells. IMPLICATIONS: The sensitivity of stationary multidrug-resistant cancer cells to indanocine suggests that indanocine and related indanones be considered as lead compounds for the development of chemotherapeutic strategies for drug-resistant malignancies.

Regulation of ornithine decarboxylase induction by deguelin, a natural product cancer chemopreventive agent.

Deguelin, a plant-derived rotenoid, mediates potent chemopreventive responses through transcriptional regulation of phorbol ester-induced ornithine decarboxylase (ODC) activity. To explore the mechanism of this effect, the activity of this compound was evaluated with a number of model systems. Using cultured mouse epidermal 308 cells, the steady-state levels of both 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ODC mRNA and c-fos were decreased by treatment with deguelin. ODC activity was also inhibited by bullatacin and various antimitotic agents (podophyllotoxin, vinblastine, and colchicine), but only deguelin and bullatacin were active as inhibitors of ODC levels in a TPA-independent c-Myc-mediated induction system using cultured BALB/c c-MycER cells. These results suggest that antimicrotubule effects, as mediated by rotenone, for example, are not responsible for inhibitory activity facilitated by deguelin. This was confirmed by use of an in vitro model of tubulin polymerization in which deguelin and a variety of other rotenoids were investigated and found to be inactive. As anticipated, however, NADH dehydrogenase was inhibited by these rotenoids. Moreover, inhibition of this enzyme correlated with a rapid depletion of ATP levels and potential to inhibit either TPA- or c-Myc-induced ODC activity. It therefore seems that deguelin-mediated interference with transient requirements for elevated energy can inhibit the induction of ODC activity and thereby yield a cancer chemopreventive response.

The effects of chelidonine on tubulin polymerisation, cell cycle progression and selected signal transmission pathways.

Chelidonine is a tertiary benzophenanthridine alkaloid known to cause mitotic arrest and to interact weakly with tubulin. Our interest in chelidonine began when we found it to be a major contaminant of Ukrain, which is a compound reported to be selectively toxic to malignant cells. The effects of chelidonine in two normal (monkey kidney and Hs27), two transformed (Vero and Graham 293) and two malignant (WHCO5 and HeLa) cell lines, were examined. Chelidonine proved to be a weak inhibitor of cell growth, but no evidence for selective cytotoxicity was found in this study. It was confirmed that chelidonine inhibits tubulin polymerisation (IC50 = 24 microM), explaining its ability to disrupt microtubular structure in cells. A G2/M arrest results, which is characterised by abnormal metaphase morphology, increased levels of cyclin B1 and enhanced cdc2 kinase activity. Exposure of all cell lines examined to chelidonine leads to activation of the stress-activated protein kinase/jun kinase pathway (SAPK/JNK).

Antitubulin effects of derivatives of 3-demethylthiocolchicine, methylthio ethers of natural colchicinoids, and thioketones derived from thiocolchicine. Comparison with colchicinoids.

Esterification of the phenolic group in 3-demethylthiocolchicine and exchange of the N-acetyl group with other N-acyl groups or a N-carbalkoxy group afforded many compounds which showed superior activity over the parent drug as inhibitors of tubulin polymerization and of the growth of L1210 murine leukemia cells in culture. A comparison of naturally occurring Colchicum alkaloids with thio isosters, obtained by replacing the OMe group at C(10) with a SCH3 group, showed the thio ethers to be invariably more potent in these assays. The comparison included 3-demethylthiodemecolcine prepared from 3-demethylthiocolchicine by partial synthesis. Thiation of thiocolchicine with Lawesson's reagent afforded novel thiotropolones which exhibited high antitubulin activity. Their structures are fully secured by spectral data. Colchicine and several of its analogues show good antitumor effect in mice infected with P388 lymphocytic leukemia, and all of them show high affinity for tubulin and inhibit tubulin polymerization at low concentration. Consequently, antitubulin assays with this class of compounds can serve as valuable prescreens for the initial evaluation of potential antitumor drugs.

Synthesis, antitubulin and antimitotic activity, and cytotoxicity of analogs of 2-methoxyestradiol, an endogenous mammalian metabolite of estradiol that inhibits tubulin polymerization by binding to the colchicine binding site.

In order to define the structural parameters associated with the antitubulin activity and cytotoxicity of 2-methoxyestradiol, a mammalian metabolite of estradiol, an array of analogs was synthesized and evaluated. The potencies of the new congeners as inhibitors of tubulin polymerization and colchicine binding were determined using tubulin purified from bovine brain, and the cytotoxicities of the new compounds were studied in a variety of cancer cell cultures. Maximum antitubulin activity was observed in estradiols having unbranched chain substituents at the 2-position with three non-hydrogen atoms. 2-Ethoxyestradiol and 2-((E)-1-propenyl)-estradiol were substantially more potent than 2-methoxyestradiol itself. The tubulin polymerization inhibitors in this series displayed significantly higher cytotoxicities in the MDA-MB-435 breast cancer cell line than in the other cell lines studied. The potencies of the analogs as cytotoxic and antimitotic agents in cancer cell cultures correlated with their potencies as inhibitors of tubulin polymerization, supporting the hypothesis that inhibition of tubulin polymerization is the mechanism of the cytotoxic action of 2-methoxyestradiol and its congeners. Several of the more potent analogs were tested in an estrogen receptor binding assay, and their affinities relative to estradiol were found to be very low.

Synthesis and evaluation of stilbene and dihydrostilbene derivatives as potential anticancer agents that inhibit tubulin polymerization.

An array of cis-, trans-, and dihydrostilbenes and some N-arylbenzylamines were synthesized and evaluated for their cytotoxicity in the five cancer cell cultures A-549 lung carcinoma, MCF-7 breast carcinoma, HT-29 colon adenocarcinoma, SKMEL-5 melanoma, and MLM melanoma. Several cis-stilbenes, structurally similar to combretastatins, were highly cytotoxic in all five cell lines and these were also found to be active as inhibitors of tubulin polymerization. The most active compounds also inhibited the binding of colchicine to tubulin. The most potent of the new compounds, both as a tubulin polymerization inhibitor and as a cytotoxic agent, was (Z)-1-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)ethene (5a). This substance was almost as potent as combretastatin A-4 (1a), the most active of the combretastatins, as a tubulin polymerization inhibitor. Compound 5a was found to be approximately 140 times more cytotoxic against HT-29 colon adenocarcinoma cells and about 10 times more cytotoxic against MCF-7 breast carcinoma cells than combretastatin A-4. However, 5a was found to be about 20 times less cytotoxic against A-549 lung carcinoma cells, 30 times less cytotoxic against SKMEL-5 melanoma cells, and 7 times less cytotoxic against MLM melanoma cells than combretastatin A-4. The relative potencies 5a greater than 8a greater than 6a for the cis, dihydro, and trans compounds, respectively, as inhibitors of tubulin polymerization are in agreement with the relative potencies previously observed for combretastatin A-4 (1a), dihydrocombretastatin A-4 (1c), and trans-combretastatin A-4 (1b). The relative potencies 5a greater than 8a greater than 6a were also reflected in the results of the cytotoxicity assays. Structure-activity relationships of this group of compounds are also discussed.

Synthesis and biological evaluation of dihydrobenzofuran lignans and related compounds as potential antitumor agents that inhibit tubulin polymerization.

A series of 19 related dihydrobenzofuran lignans and benzofurans was obtained by a biomimetic reaction sequence involving oxidative dimerization of p-coumaric, caffeic, or ferulic acid methyl esters, followed by derivatization reactions. All compounds were evaluated for potential anticancer activity in an in vitro human disease-oriented tumor cell line screening panel that consisted of 60 human tumor cell lines arranged in nine subpanels, representing diverse histologies. Leukemia and breast cancer cell lines were relatively more sensitive to these agents than were the other cell lines. Compounds 2c and 2d, but especially 2b (methyl (E)-3-¿2-(3, 4-dihydroxyphenyl)-7-hydroxy-3-methoxycarbonyl-2, 3-dihydro-1-benzofuran-5-ylprop-2-enoate), the dimerization product of caffeic acid methyl ester, containing a 3',4'-dihydroxyphenyl moiety and a hydroxyl group in position 7 of the dihydrobenzofuran ring, showed promising activity. The average GI(50) value (the molar drug concentration required for 50% growth inhibition) of 2b was 0.3 microM. Against three breast cancer cell lines, 2b had a GI(50) value of <10 nM. Methylation, reduction of the double bond of the C(3)-side chain, reduction of the methoxycarbonyl functionalities to primary alcohols, or oxidation of the dihydrobenzofuran ring to a benzofuran system resulted in a decrease or loss of cytotoxic activity. Compound 2b inhibited mitosis at micromolar concentrations in cell culture through a relatively weak interaction at the colchicine binding site of tubulin. In vitro it inhibited tubulin polymerization by 50% at a concentration of 13 +/- 1 microM. The 2R, 3R-enantiomer of 2b was twice as active as the racemic mixture, while the 2S,3S-enantiomer had minimal activity as an inhibitor of tubulin polymerization. These dihydrobenzofuran lignans (2-phenyl-dihydrobenzofuran derivatives) constitute a new group of antimitotic and potential antitumor agents that inhibit tubulin polymerization.

Antineoplastic agents. 443. Synthesis of the cancer cell growth inhibitor hydroxyphenstatin and its sodium diphosphate prodrug.

A structure-activity relationship (SAR) study of the South African willow tree (Combretum caffrum) antineoplastic constituent combretastatin A-4 (3b) led to the discovery of a potent cancer cell growth inhibitor designated phenstatin (5a). This benzophenone derivative of combretastatin A-4 showed remarkable antineoplastic activity, and the benzophenone derivative of combretastatin A-1 was therefore synthesized. The benzophenone, designated hydroxyphenstatin (6a), was synthesized by coupling of a protected bromobenzene and a benzaldehyde to give the benzhydrol with subsequent oxidation to the ketone. Hydroxyphenstatin was converted to the sodium phosphate prodrug (6e) by a dibenzyl phosphite phosphorylation and subsequent benzyl cleavage (6a --> 6d --> 6e). While hydroxyphenstatin (6a) was a potent inhibitor of tubulin polymerization with activity comparable to that of combretastatin A-1 (3a), the phosphorylated derivative (6e) was inactive.

Antitumor agents. 150. 2',3',4',5',5,6,7-substituted 2-phenyl-4-quinolones and related compounds: their synthesis, cytotoxicity, and inhibition of tubulin polymerization.

As part of our continuing search for potential anticancer drug candidates in the 2-phenyl-4-quinolone series, we have synthesized a series of 6,7-methylenedioxy-substituted and unsubstituted 2-phenyl-4-quinolones, as well as related compounds. Their in vitro inhibition of human tumor cell lines and tubulin polymerization is reported. In general, a good correlation was found between cytotoxicity and inhibition of tubulin polymerization. Compounds 7, 9, 13, 16, 22, 23, 36, and 37 showed potent inhibitory effects in both assays. All rigid analogs (47-49) and trimethoxy-substituted compounds showed little or no activity. Substitution at the 4'-position also resulted in compounds with little or no activity, except for hydroxyl or methyl groups at this position. Further investigation is underway to determine if substitution at the 3'-position will result in compounds with increased activity.

Synthesis of analogs of 2-methoxyestradiol with enhanced inhibitory effects on tubulin polymerization and cancer cell growth.

A new series of estradiol analogs was synthesized in an attempt to improve on the anticancer activity of 2-methoxyestradiol, a naturally occurring mammalian tubulin polymerization inhibitor. The compounds were evaluated as inhibitors of tubulin polymerization and the binding of [3H]colchicine to tubulin, as well as for in vitro cytotoxicity in human cancer cell cultures. Overall, the most potent of the new compounds were 2-(2',2',2'-trifluoroethoxy)-6-oximinoestradiol, 2-ethoxy-6-oximinoestradiol, and 2-ethoxy-6-methoximinoestradiol. These agents lacked significant affinity for the estrogen receptor. The cytotoxicities of the compounds correlated in general with their abilities to inhibit tubulin polymerization, thus supporting inhibition of tubulin polymerization as the primary mechanism causing inhibition of cell growth.

Antitumor agents. 155. Synthesis and biological evaluation of 3',6,7-substituted 2-phenyl-4-quinolones as antimicrotubule agents.

A series of 3',6,7-substituted 2-phenyl-4-quinolones were designed and synthesized as antimitotic antitumor agents. All compounds showed cytotoxic effects (log GI50 < or = -4.0; log drug molar concentration required to cause 50% inhibition) against the growth of a variety of human tumor cell lines, including those derived from solid tumors such as non-small cell lung, colon, central nervous system, ovary, prostate, and breast cancers, when evaluated in the National Cancer Institute's 60 human tumor cell line in vitro screen. The most potent compound (26) demonstrated strong cytotoxic effects with GI50 values in the nanomolar or subnanomolar range in almost all the tumor cell lines. Compound 26 was also a potent inhibitor of tubulin polymerization and radiolabeled colchicine binding to tubulin, with activity comparable to those of the potent antimitotic natural products colchicine, podophyllotoxin, and combretastatin A-4.

Antineoplastic agents. 410. Asymmetric hydroxylation of trans-combretastatin A-4.

The South African willow tree Combretum caffrum has yielded a number of potent cancer cell growth inhibitors. The present SAR studies of the antineoplastic agent combretastatin A-4 (1c) were focused mainly on the olefinic bridge to determine the effects on cancer cell growth and, potentially, to better define the combretastatin A-4 binding site on tubulin. The geometric trans-isomer 3a of combretastatin A-4 was converted to the (1S,2S)- and (1R,2R)-vicinal diols 4c and 4d, respectively, under Sharpless' asymmetric dihydroxylation conditions. Cancer cell line testing showed the (1S, 2S)-diol 4c to be more potent than its enantiomer 4d. Diol 4c weakly inhibited tubulin polymerization (IC50 = 22 microM, versus 1.2 microM for combretastatin A-4), while 4d was inactive (IC50 > 40 microM). Esterification of either stereoisomer at the diol and/or phenolic positions resulted in elimination of inhibitory activity.

Antitumor agents. 196. Substituted 2-thienyl-1,8-naphthyridin-4-ones: their synthesis, cytotoxicity, and inhibition of tubulin polymerization.

As part of our continuing search for potential anticancer drug candidates in the 2-aryl-1,8-naphthyridin-4-one series, we have synthesized a series of substituted 2-thienyl-1, 8-naphthyridin-4-ones. Most compounds showed significant cytotoxic effects (log GI(50) < -4.0; log molar drug concentration required to cause 50% growth inhibition) against a variety of human tumor cell lines in the National Cancer Institute's in vitro screen, including cells derived from solid tumors such as non-small-cell lung, colon, central nervous system, melanoma, ovarian, prostate, and breast cancers. The most active compounds (31-33,40) demonstrated strong cytotoxic effects with ED(50) values in the micromolar or submicromolar range in most of the tumor cell lines. The most cytotoxic compounds inhibited tubulin polymerization at concentrations substoichiometric to the tubulin concentration. The most potent inhibitors of polymerization (40,42,43) had effects comparable to those of the potent antimitotic natural products podophyllotoxin and combretastatin A-4 and to that of NSC 664171, a particularly potent, structurally related analogue. Only compound 40 was a potent inhibitor of the binding of radiolabeled colchicine to tubulin, and it was both the most cytotoxic agent and the most effective inhibitor of polymerization among the newly synthesized compounds.

Structure-activity requirements for flavone cytotoxicity and binding to tubulin.

A series of 79 flavones related to centaureidin (3,6,4'-trimethoxy-5, 7,3'-trihydroxyflavone, 1) was screened for cytotoxicity in the NCI in vitro 60-cell line human tumor screen. The resulting cytotoxicity profiles of these flavones were compared for degree of similarity to the profile of 1. Selected compounds were further evaluated with in vitro assays of tubulin polymerization and [3H]colchicine binding to tubulin. Maximum potencies for tubulin interaction and production of differential cytotoxicity profiles characteristic of 1 were observed only with compounds containing hydroxyl substituents at C-3' and C-5 and methoxyl groups at C-3 and C-4'.

6-Alkylamino- and 2,3-dihydro-3'-methoxy-2-phenyl-4-quinazolinones and related compounds: their synthesis, cytotoxicity, and inhibition of tubulin polymerization.

As part of our continuing search for potential anticancer candidates among 2-phenyl-4-quinolones and 2-phenyl-4-quinazolinones, two series of 6,7,2',3',4',5'-substituted 2-phenyl-4-quinazolinones and 6,2',3',4',5'-substituted 2,3-dihydro-2-phenyl-4-quinazolinones were synthesized and evaluated for cytotoxicity and as inhibitors of tubulin polymerization. In general, a good correlation was found between the two activities. Five of the 6-substituted heterocyclic 2-phenyl-4-quinozolinones (37-51) showed significant cytotoxicity against a panel of human tumor cell lines with EC(50) values in the low micromolar to nanomolar concentration ranges. Compound 38 was the most potent of these compounds, as well as the most potent inhibitor of tubulin polymerization in this series. The activity of 38 was in the same range as those of the antimitotic natural products, colchicine, podophyllotoxin, and combretastatin A-4. Substituted 2-phenyl-4-quinazolinones and 2, 3-dihydro-2-phenyl-4-quinazolinones also displayed highly selective cytotoxicity against the ovarian cancer 1A9 and P-gp resistant KB-VIN cell lines.

Antitumor Agents. 211. Fluorinated 2-phenyl-4-quinolone derivatives as antimitotic antitumor agents.

Fluorinated 2-phenyl-4-quinolone derivatives were synthesized and evaluated in National Cancer Institute's 60 human tumor cell line in vitro screen. From the results, the ketone moiety plays an essential role in activity. Among the compounds tested, 2'-fluoro-6-pyrrol-2-phenyl-4-quinolone (13) exhibited the most potent cytotoxic activities (log GI(50) < -8.00) against renal and melanoma tumor cell lines. Compound 13 was also a potent inhibitor of tubulin polymerization (IC(50) = 0.46 microM) and of radiolabeled colchicine binding to tubulin, with activities comparable to those of the potent antimitotic natural products colchicine, podophyllotoxin, and combretastatin A-4.

Synthesis and biological evaluation of 2-acyl analogues of paclitaxel (Taxol).

The anticancer drug paclitaxel (Taxol) has been converted to a large number of 2-debenzoyl-2-aroyl derivatives by three different methods. The bioactivities of the resulting analogues were determined in both tubulin polymerization and cytotoxicity assays, and several analogues with enhanced activity as compared with paclitaxel were discovered. Correlation of cytotoxicity in three cell lines with tubulin polymerization activity showed reasonable agreement. Among the cell lines examined, the closest correlation with antitubulin activity was observed with a human ovarian carcinoma cell line.

Morpholino derivatives of benzyl-benzodioxole, a study of structural requirements for drug interactions at the colchicine/podophyllotoxin binding site of tubulin.

We performed a structure-activity evaluation of the effects of methoxy substituents in the benzyl moiety of a series of morpholinyl Mannich base derivatives of 6-benzyl-1,3-benzodioxol-5-ol ("morpholino compounds") on the ability of these compounds to inhibit tubulin polymerization in vitro. Structurally these agents are most similar to the natural product podophyllotoxin and, like podophyllotoxin, they inhibited in vitro tubulin polymerization, tubulin-dependent GTP hydrolysis, and the binding of colchicine to tubulin. The benzyl ring (C ring) of these compounds appeared to be analogous to the trimethoxybenzene ring (E ring) of podophyllotoxin (with its methoxy substituents at the 3', 4' and 5' positions), but the morpholino compound superficially most similar to podophyllotoxin (with 3', 4' and 5' methoxy substituents) was the least active in the series. The most potent methoxy-substituted morpholino compounds bear these substituents either at the 2' and 4' positions (NSC 370277) or at the 2', 4' and 6' positions (NSC 381577). NSC 370277 and NSC 381577 were essentially identical in their inhibitory effects on tubulin polymerization, but the latter compound was considerably more effective as an inhibitor of the binding of colchicine to tubulin. The most active of the monomethoxy substituted compounds bore this group at position 4'. A number of compounds with alternative substituents at this position (in particular, alkyl-substituted amines) also had significant in vitro inhibitory effects on tubulin polymerization. Although the morpholino compounds appear to possess only limited cytotoxicity, these findings suggest possible modifications of the antimitotic benzyl-benzodioxole compounds described previously [Batra et al., Molec. Pharmac. 27, 94 (1985)] to enhance their antineoplastic activity.

Partial synthesis and antitubulin activity of minor colchicum alkaloids: N-acetoacetyl-deacetylcolchicine and 2-demethylspeciosine (speciocolchine).

Two minor Colchicum alkaloids, N-acetoacetyl-deacetylcolchicine [1] and 2-demethylspeciosine [7], were synthesized. The diacetate 8 of 2-demethylspeciosine was also prepared. The antitubulin activity of these compounds, in comparison to colchicine, was measured. N-Acetoacetyl-deacetylcolchicine [1] has in vitro activity similar to that of colchicine. Both 2-demethylspeciosine [7] and the diacetate 8 were considerably less potent inhibitors of tubulin polymerization.

Activities of the microtubule-stabilizing agents epothilones A and B with purified tubulin and in cells resistant to paclitaxel (Taxol(R)).

Epothilones A and B, natural products with minimal structural analogy to taxoids, have effects similar to those of paclitaxel (Taxol(R)) in cultured cells and on microtubule protein, but differ from paclitaxel in retaining activity in multidrug-resistant cells. We examined interactions of the epothilones with purified tubulin and additional cell lines, including a paclitaxel-resistant ovarian carcinoma line with an altered beta-tubulin. The epothilones, like paclitaxel, induced tubulin to form microtubules at low temperatures and without GTP and/or microtubule-associated proteins. The epothilones are competitive inhibitors of the binding of [3H]paclitaxel to tubulin polymers. The apparent Ki values for epothilones A and B were 1.4 and 0.7 microM by Hanes analysis and 0.6 and 0.4 microM by Dixon analysis. In the paclitaxel-sensitive human cell lines we examined, epothilone B had greater antiproliferative activity than epothilone A or paclitaxel, while epothilone A was usually less active than paclitaxel. A multidrug-resistant colon carcinoma line and the paclitaxel-resistant ovarian line retained sensitivity to the epothilones. With Potorous tridactylis kidney epithelial (PtK2) cells examined by indirect immunofluorescence, microtubule bundles appeared more rapidly following epothilone B treatment, and there were different proportions of various mitotic aberrations following treatment with different drugs.