Design, synthesis and biological evaluation of dihydronaphthalene and benzosuberene analogs of the combretastatins as inhibitors of tubulin polymerization in cancer chemotherapy

A novel series of dihydronaphthalene and benzosuberene analogs bearing structural similarity to the combretastatins in terms of 1,2-diarylethene, trimethoxyphenyl, and biaryl functionality has been synthesized. The compounds have been evaluated in regard to their ability to inhibit tubulin assembly and for their cytotoxicity against selected human cancer cell lines. From this series of compounds, benzosuberene analogs 2 and 4 inhibited tubulin assembly at concentrations comparable to that of combretastatin A-4 (CA4) and combretastatin A-1 (CA1). Furthermore, analog 4 demonstrated remarkable cytotoxicity against the three human cancer cell lines evaluated (for example GI(50)=0.0000032 microM against DU-145 prostate carcinoma).     

Structure-activity relationships of 2-, 4-, or 6-substituted estrogens as aromatase inhibitors

Aromatase, which is responsible for the conversion of androgens to estrogens, is a potential therapeutic target for the selective lowering of estrogen levels in patients with estrogen-dependent breast cancer. To develop a novel class of aromatase inhibitors, we tested series of 2- and 4-substituted (halogeno, methyl, formyl, methoxy, nitro, and amino) estrones (7 and 9), as well as series of 6alpha- and 6beta-substituted (alkyl, phenalkyl, and alkoxy) estrones (13 and 14), and their estradiol analogs (8, 10, 11, and 12) as aromatase inhibitors. All of the inhibitors examined blocked the androstenedione aromatization in a competitive manner. Introduction of halogeno and methyl functions at C-2 of estrone as well as that of a phenalkyl or methyl function at the C-6alpha or C-6beta position markedly increased affinity to aromatase (apparent K(i) value=0.10-0.66 microM for the inhibitors versus 2.5 microM for estrone). 6alpha-Phenylestrone (13c) was the most powerful inhibitor among the estrogens studied, and its affinity was comparable to that of the androgen substrate androstenedione. Estradiol analogs were much weaker inhibitors than the corresponding estrone compounds in each series, indicating that the 17-carbonyl group plays a critical role in the formation of a thermodynamically stable enzyme-inhibitor complex.     

Hybrid Pharmacophore Design,Molecular Docking,Synthesis, and Biological Evaluation of Novel Aldimine‐Type Schiff Base Derivatives as Tubulin Polymerization Inhibitor

A series of hybrid aldimine‐type Schiff base derivatives including trimethoxyphenyl ring and 1,2,4‐triazole‐3‐thiol/thione were designed as tubulin inhibitors. The molecular docking simulations on tubulin complex (PDB: 1SA0) revealed that derivatives with nitro and/or chloro or dimethylamino substitutes (4‐nitro, 2‐nitro, 3‐nitro, 4‐Cl‐3‐nitro, and 4‐Me₂N) on the aldehyde ring were the best compounds with remarkable binding energies (?9.09, ?9.07, ?8.63, ?8.11, and ?8.07 kcal mol^?1, respectively) compared to colchicine (?8.12 kcal mol^?1). These compounds were also showed remarkable binding energies from ?10.66 to ?9.79 and ?10.12 to ?8.95 kcal mol^?1 on human (PDB: 1PD8) and Candida albicans (PDB: 3QLS) DHFR, respectively. The obtained results of cytotoxic activities against HT1080, HepG2, HT29, MCF‐7, and A549 cancer cell lines indicated that 4‐nitro and 2‐nitro substituted compounds were the most effective agents by mean IC₅₀ values of 11.84 ± 1.01 and 19.92 ± 1.36 μm , respectively. 4‐Nitro substituted compound (5 μm ) and 2‐nitro substituted compound (30 μm ) were able to strongly inhibit the tubulin polymerization compared to colchicine (5 μm ) and 4‐nitro substituted compound displayed IC₅₀ values of 0.16 ± 0.01 μm compared to that of colchicine (0.19 ± 0.01 μm ). This compound also showed the lowest MIC values on all tested microbial strains including three Gram‐positive, four Gram‐negative, and three yeast pathogens.     

Synthesis and biological evaluation of B-ring modified colchicine and isocolchicine analogs

A series of modified colchicine and isocolchicine analogs (C-7 substituent) were synthesized and evaluated in vitro against a PC3 cancer cell line and for inhibition of microtubule polymerization. The colchicine analogs all displayed strong inhibition of tubulin polymerization, while compounds 6 and 20 also possessed an increased cytotoxic activity as compared to colchicine. More importantly, isocolchicine analogs 7, 15, and 17 showed inhibition of microtubule polymerization with IC(50) values ranging from 58 to 68muM. In addition, 7 displayed strong cytotoxic activity with an IC(50)=93nM which was more potent than colchicine analog 12.     

Synthesis, nicotinic acetylcholine receptor binding, and pharmacological properties of 3'-(substituted phenyl)deschloroepibatidine analogs

A series of 3'-(substituted phenyl)deschloroepibatidine analogs (5a-j) were synthesized. The alpha4beta2( *) and alpha7 nicotinic acetylcholine receptor (nAChR) binding properties and functional activity in the tail-flick, hot-plate, locomotor, and body temperature tests in mice of 5a-j were compared to those of the nAChR agonist, nicotine (1), epibatidine (4), and deschloroepibatidine (13), the partial agonist, varenicline (3), and the antagonist 2'-fluoro-3'-(substituted phenyl)deschloroepibatidine analogs (7a-j). Unlike epibatidine and deschloroepibatidine, which are potent agonists in the tail-flick test, 5a-k show no or very low antinociceptive activity in the tail-flick or hot-plate test. However, they are potent antagonists in nicotine-induced antinociception in the tail-flick test, but weaker than the corresponding 2'-fluoro-3'-(substituted phenyl)deschloroepibatidines.     

The B-ring substituent at C-7 of colchicine and the alpha-C-terminus of tubulin communicate through the "tail-body" interaction

The carboxy terminals of alphabeta-tubulins are flexible regions rich in acidic amino acid residues that play an inhibitory role in the polymerization of tubulin to microtubules. We have shown that the binding of colchicine and its B-ring analogs (with C-7 substituents) to tubulin are pH sensitive and have high activation energies. Under identical conditions, the binding of analogs without C-7 substituents is pH independent and has lower activation energy. Beta-C-terminus-truncated tubulin (alphabeta(s)) shows similar pH sensitivity and activation energy to native tubulin (alphabeta). Removal of the C-termini of both subunits of tubulin (alpha(s)beta(s)) or the binding of a basic peptide P2 to the negatively charged alpha-C-terminus of tubulin causes a colchicine-tubulin interaction independent of pH with a low activation energy. Tubulin dimer structure shows that the C-terminal alpha-tail is too far from the colchicine binding site to interact directly with the bound colchicine. Therefore, it is likely that the interaction of the alpha-C-terminus with the main body of tubulin indirectly affects the colchicine-tubulin interaction via conformational changes in the main body. We therefore conclude that in the presence of tail-body interaction, a B-ring substituent makes contact with the alpha-tubulin and induces significant conformational changes in alpha-tubulin.     

Substituted 2-(3′,4′,5′-trimethoxybenzoyl)-benzo[b]thiophene derivatives as potent tubulin polymerization inhibitors

The central role of microtubules in cell division and mitosis makes them a particularly important target for anticancer agents. On our early publication, we found that a series of 2-(3′,4′,5′-trimethoxybenzoyl)-3-aminobenzo[b]thiophenes exhibited strong antiproliferative activity in the submicromolar range and significantly arrested cells in the G2–M phase of the cell cycle and induced apoptosis.In order to investigate the importance of the amino group at the 3-position of the benzo[b]thiophene skeleton, the corresponding 3-unsubstituted and methyl derivatives were prepared. A novel series of inhibitors of tubulin polymerization, based on the 2-(3,4,5-trimethoxybenzoyl)-benzo[b]thiophene molecular skeleton with a methoxy substituent at the C-4, C-5, C-6 or C-7 position on the benzene ring, was evaluated for antiproliferative activity against a panel of five cancer cell lines, for inhibition of tubulin polymerization and for cell cycle effects. Replacing the methyl group at the C-3 position resulted in increased activity compared with the corresponding 3-unsubstituted counterpart. The structure–activity relationship established that the best activities were obtained with the methoxy group placed at the C-4, C-6 or C-7 position. Most of these compounds exhibited good growth inhibition activity and arrest K562 cells in the G2–M phase via microtubule depolymerization.     

Stereochemistry of sodium borohydride reduction of tryptophan synthase of Escherichia coli and its amino acid Schiff's bases

Tryptophan synthase alpha 2 beta 2 complex containing [4'-3H]pyridoxal phosphate was reduced with sodium borohydride in the presence of various substrates and analogs in an attempt to trap reaction intermediates. Reduction in the presence of L-serine gave noncovalently bound radioactive material which was identified as phosphopyridoxylalanine, presumably resulting from reduction of the intermediate Schiff's base formed between pyridoxal phosphate and alpha-aminoacrylate. The tritium in this compound was located in the pro-R position at C-4', indicating that reduction of the Schiff's base double bond had occurred on the Si face at C-4'. On the other hand, analysis of phosphopyridoxyllysine obtained by hydrolysis of the reduced [3H]pyridoxal-P-alpha 2 beta 2 protein showed that the internal Schiff's base had been reduced on the C-4' Re face, suggesting a cofactor reorientation upon substrate binding. Analysis of phosphopyridoxylalanine from a reduction of unlabeled alpha 2 beta 2 complex in the presence of (2S,3R)-[2,3-2H2]serine with tritiated sodium borohydride demonstrated the presence of tritium at C-4' (50%), C-2 (20%), and C-3 (30%). According to the configuration at C-3, reduction of the phosphopyridoxal-alpha-aminoacrylate Schiff's base has occurred from the same side of the molecule at C-4' and C-3.     

Synthesis of mono- and dideoxygenated alpha,alpha-trehalose analogs

In this work, we describe the synthesis and NMR characterization of four mono- and four dideoxygenated analogs of alpha,alpha-D-trehalose. The symmetrical (2,2'-, 3,3'-, 4,4'- and 6,6'-) dideoxy analogs were obtained via selective protection and subsequent radical deoxygenation of the desired hydroxyl group set. The unsymmetrical (2'-, 3'-, 4'- and 6'-) monodeoxy analogs were synthesized by desymmetrization of alpha,alpha-trehalose and subsequent deoxygenation under radical conditions. Complete assignment of all (1)H and (13)C resonances in the spectra of these deoxytrehaloses was achieved through the extensive use of 2D [(1)H,(1)H] and [(1)H,(13)C] correlation NMR experiments. The synthesis of these trehalose analogs sets the stage for future biochemical and NMR-based studies to probe the substrate interactions of trehalose with the recently identified mycobacterial sulfotransferase Stf0.     

Relationship between mutagenic potency in Salmonella typhimurium and chemical structure of amino- and nitro-substituted biphenyls

All positional isomers of mononitro- and monoaminobiphenyls and those of dinitro-, diamino- and aminonitrobiphenyls, which have one substituent on each benzene ring, were assayed for mutagenicity in Salmonella typhimurium by the Ames method. The results suggest that the structural requirements favoring mutagenic activity are the presence of substituents at the 4-position and their absence at the 2'-position. The introduction of an amino group to the 3'- or 4'-position of 4-nitrobiphenyl or a nitro group to 3'- or 4'-position of 4-aminobiphenyl enhanced the mutagenicity. Among the mutagenic compounds, 4-nitro analogues were mutagenic in strains TA98 and TA100 in the absence of a microsomal metabolic activation system. Strain TA98NR was not reverted by the direct-acting mutagens, whereas strain TA98/1,8-DNP6 was as revertible as strain TA98; these results suggest that the direct-acting mutagenicity involves the reduction of the nitro group by bacterial nitroreductase but does not involve specific esterification enzymes.     

Synthesis and biological evaluation of a novel class of isatin analogs as dual inhibitors of tubulin polymerization and Akt pathway

A novel series of 5,7-dibromoisatin analogs were synthesized and evaluated for their cytotoxicities against four human cancer cell lines including colon HT29, breast MCF-7, lung A549 and melanoma UACC903. Analogs 6, 11 and 13 displayed good in vitro anticancer activity on the HT29 human colon cancer cell line in the 1 μM range. Analogs 5, 9 and 12, containing a selenocyanate group in the alkyl chain were the most promising compounds on the breast cancer MCF-7 cell line. Biological assays relating to apoptosis were performed to understand the mechanism of action of these analogs. Compounds 5 and 6 were found to inhibit tubulin polymerization to the same extent as the anticancer drug vinblastine sulfate, but compounds 11 and 13 inhibited significantly better than vinblastine. Further western blot analysis suggested that compound 6 at 2 μM reduced both levels and phosphorylation state of Akt. Compounds 11 and 13 at 1 μM caused reduced Akt protein levels and strongly suppressed the phosphorylation of Akt. Therefore, 11 and 13 were demonstrated as efficient dual inhibitors of both tubulin polymerization and the Akt pathway and good candidates for further study. More importantly, the strategy of microtubule and Akt dual inhibitors might be a promising direction for developing novel drugs for cancer.     

Electrochemical and electron spin resonance studies of actinomycin D and other phenoxazones

Electrochemical studies on actinomycin D (1) and two analogs, 2-amino-3-phenoxazone (2) and 1,2,4-trichloro-7-nitrophenoxazone (3) were analyzed by polarography and ESR spectroscopy. The polarograms of the three compounds in acetonitrile all show two reduction waves. ESR experiments confirm that the first reduction wave corresponds to a one-electron transfer process which produces a phenoxazone free radical anion and the second wave corresponds to a subsequent one-electron transfer producing a diamagnetic dianion. Substitution with electron-withdrawing groups such as NO2 (at C-7) and chloro (at C-1, C-2 and C-4)3 facilitated the reduction of the phenoxazone ring system to a free radical (i.e., half-wave potentials; 1, -0.815 V; 2, -0.920 V; 3, -0.135 V). It was found, by computer simulation of the ESR spectra, that the spin density in the electrochemically generated free radicals from 1, 2 and 3 was preferentially located in the benzenoid ring and at the N-10 nitrogen. For radicals obtained from 1 and 2, only a small residual spin density could be detected in the quinoid ring. Since 1 can be metabolized to a free radical in cells, these free radical forms of 1 and its analogs may represent reactive forms of the phenoxazone nucleus.     

Comparison of ivermectin, doramectin, selamectin, and eleven intermediates in a nematode larval development assay.

Chemical substitutions at pharmacologically relevant sites such as C-5, C-13, C-22,23, and C-25 were examined in ivermectin, doramectin, selamectin, and a series of 11 other intermediates using a larval development assay with Haemonchus contortus. A range of activities spanning 5 orders of magnitude were manifest with small changes in the substituents to the 14 avermectins. Within this compound series, there was no major potency advantage or disadvantage to a disaccharide over a monosaccharide substituent at C-13. Ivermectin and doramectin were each fully effective at a concentration of 0.001 microg/ml, and both were similar to their respective monosaccharide homologs. Specific patterns emerged among the analogs with substituents at C-5. Analogs possessing hydroxyl groups at C-5 were superior in activity by several orders of magnitude over those with oxo substituents. Replacement of the oxo with an oxime (NOH) restored activity to some degree but did not restore it to the level of those possessing the hydroxyl substituent. Consequently, ivermectin and doramectin that possess hydroxyl moieties at C-5 were superior against H. contortus to those like selamectin that have oxime substituents. There was no advantage for analogs with a single or double bond at C-22,23 within the cyclohexyl series, and these analogs had equivalent activity as those with a single bond at C-22,23 in the sec-butyl/isopropyl series. However, there was superior activity for the analog series that possessed the combination of a double-bond at C-22,23 and a sec-butyl/isopropyl substituent at C-25. As a result, the most potent compound in this test was not any of the 3 commercialized avermectins but was a monosaccharide with a double bond at C-22,23, an hydroxyl at C-5, and a sec-butyl/isopropyl moiety at C-25.     

Synthesis and structure-activity relationships of cephalosporins, 2-isocephems, and 2-oxaisocephems with C-3′ or C-7 catechol or related aromatics

A series of cephalosporins, 2-isocephems, and 2-oxaisocephems with C-3′ catechol-containing (pyridinium-4-thio)methyl groups and 2-isocephems with C-7 catechol related aromatics have been prepared and evaluated for antimicrobial activity. It turns out that these compounds have highly potent activity against Gram-negative bacteria, especially resistant pathogens such as Pseudomonas aeruginosa. The most active compound of the series was (6S,7S)-7-[2-(2-aminothiazol-4-yl)-2-[(Z)-[(1,5-dihydroxy-4-pyridon-2-yl)methoxy] imino]acetamido]-3-[[[(4-methyl-5-carboxymethyl)thiazol-2-yl]thio]methyl]-8-oxo-1-aza-4-thiabicyclo [4.2.0] oct-2-ene-2-carboxylic acid which exhibited potent in vitro activity against clinically isolated P. aeruginosa and Acinetobacter baumanii which is also resistant to many anti-infectives, and good in vivo efficacy against clinically isolated P. aeruginosa.

A series of cephalosporins, 2-isocephems, and 2-oxaisocephems and C-3′ or C-7 catechol or related aromatics have been prepared and evaluated for antibacterial activity.     

Synthesis,cytotoxic evaluation and molecular docking study of 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazoles as tubulin polymerization inhibitors

A series of cis-restricted 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazole analogues of combretastatin A-4 were synthesized and investigated for inhibition of cell proliferation against three cancer cell lines, HT-29, MCF-7, and AGS, and a normal mouse fibroblastic cell line, NIH-3T3, using an MTT assay. The biological study showed that 2-(methylthio) substituted compounds showed little cytotoxic activity against the four cell lines. In contrast, the presence of the 2-(benzylthio) group on the thiazole ring resulted in a significant improvement in cytotoxic activity relative to the 2-(methylthio) substituted derivatives. Furthermore, the inhibition of tubulin polymerization by some potent compounds was evaluated. All the compounds studied were moderate tubulin polymerization inhibitors. The flow cytometry analysis confirmed that the synthesized compounds led to cell cycle arrest at the G₂/M phase. Docking simulation was performed to insert these compounds into the crystal structure of tubulin at the colchicine binding site to determine a probable binding model.     

Synthesis and evaluation of diaryl sulfides and diaryl selenide compounds for antitubulin and cytotoxic activity

We have devised a procedure for the synthesis of analogs of combretastatin A-4 (CA-4) containing sulfur and selenium atoms as spacer groups between the aromatic rings. CA-4 is well known for its potent activity as an inhibitor of tubulin polymerization, and its prodrugs combretastatin A-4 phosphate (CA-4P) and combretastatin A-1 phosphate (CA-1P) are being investigated as antitumor agents that cause tumor vascular collapse in addition to their activity as cytotoxic compounds. Here we report the preparation of two sulfur analogs and one selenium analog of CA-4. All synthesized compounds, as well as several synthetic intermediates, were evaluated for inhibition of tubulin polymerization and for cytotoxic activity in human cancer cells. Compounds 3 and 4 were active at nM concentration against MCF-7 breast cancer cells. As inhibitors of tubulin polymerization, both 3 and 4 were more active than CA-4 itself. In addition, 4 was the most active of these agents against 786, HT-29 and PC-3 cancer cells. Molecular modeling binding studies are also reported for compounds 1, 3, 4 and CA-4 to tubulin within the colchicine site.     

Novel side chain analogs of 1alpha,25-dihydroxyvitamin D(3): design and synthesis of the 21,24-methano derivatives

The syntheses of the new 21,24-methano derivatives of 1alpha,25-dihydroxyvitamin D(3) [viz. 1(S),3(R)-dihydroxy-17(R)-(1',4'-cis-(4'-(1'-hydroxy-1'-methylethyl)-cyclo-hexyl))-9,10-seco-androsta-5(Z),7(E),10(19)-triene (MC 2108) and its (1',4'-trans)-isomer (MC 2110)] are described. The key step is the establishment, by Diels-Alder reaction on a CD-ring side chain diene intermediate prepared from vitamin D(2), of a 1,4-disubstituted cyclohexene moiety in the side chain. Hydrogenation to a 1:1 mixture of cis and trans cyclohexane derivatives and separation of the two series at a stage prior to the standard Horner-Wittig coupling with the (Hoffmann-La Roche) ring-A building block were other important steps in the syntheses of the target analogs. The relative configurations of intermediates were assigned by NMR spectroscopy. MC 2108 and MC 2110 are of interest as conformationally locked side chain derivatives to probe the receptor interactions of not only the parent vitamin D hormone but also its biologically active symmetrical 'double side chain' analog [21-(3'-hydroxy-3'-methylbutyl)-9,10-seco-cholesta-5(Z),7(E),10(19)-triene-1(S),3(R),25-triol (MC 2100)], 'both' side chains of which can formally be traced out in the new analogs. The preferred conformations, inferred from an analysis of (13)C-NMR characteristics, notably the chemical shift of C-17 in a series of analogs, to have the tertiary alcohol (1'-hydroxy-1'-methylethyl) substituent equatorial on the cyclohexane chair, are confirmed by molecular modeling.     

Structural determinants of blockade of eosinophil activation, adhesion and secretion by synthetic analogs of phomactin

We examined the structural determinants of phomactin analogs to assess their efficacy as antagonist of PAF. Six analogs of phomactin were synthesized to determine their inhibitory effects on adhesion, superoxide release, leukotriene C4 (LTC4) synthesis and [3H]PAF binding in human eosinophils. Phomactin analogs inhibited both PAF- and IL-5-induced eosinophil adhesion. Analog A, which bears an alkene moiety between C-1 and C-14, a ketone at the C-2 position, and an alkyne moiety between C-3 and C-4, had the greatest anti-adhesive effect. Change of the alkene between C-1 and C-14 to an alkane (analog I) decreased the anti-adhesive effect by 2.5-4 fold, while substitution of ketone by hydroxyl (analog G) at the C-2 position caused an 11-fold decrease in the anti-adhesive effect. Substitution of the alkyne moiety between C-3 and C-4 by an alkene (B and E) or alkane (D) blocked completely the anti-adhesive effect. Analogs A and I completely blocked superoxide release from eosinophils caused by phorbol-12-myristate-13-acetate or PAF and LTC4-release caused by fMLP plus cytochalasin B. Change of the alkyne moiety between C-3 and C-4 to an alkene (B and E) or alkane (D) blocked completely these inhibitory effects of phomactin. Analog A decreased the maximal binding of [3H]PAF binding to eosinophils without change of the apparent dissociation constant. We conclude that phomactin analogs are specific non-competitive PAF antagonists and have exceptional efficacy in inhibiting adhesion, metabolic activity and leukotriene secretion in human eosinophils. We further define the structural alterations in the phomactin molecule that regulate its inhibitory functions.     

Specificity of acceptor binding to Leuconostoc mesenteroides B-512F dextransucrase: binding and acceptor-product structure of alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 by inversion of the hydroxyl and by replacement of the hydroxyl with hydrogen

The specificity of acceptor binding to the active site of dextransucrase was studied by using alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 positions by (a) inversion of the hydroxyl group and (b) replacement of the hydroxyl group with hydrogen. 2-Deoxy-alpha-methyl-D-glucopyranoside was synthesized from 2-deoxyglucose; 3- and 4-deoxy-alpha-methyl-D-glucopyranosides were synthesized from alpha-methyl-D-glucopyranoside; and alpha-methyl-D-allopyranoside was synthesized from D-glucose. The analogs were incubated with [14C]sucrose and dextransucrase, and the products were separated by thin-layer chromatography and quantitated by liquid scintillation spectrometry. Structures of the acceptor products were determined by methylation analyses and optical rotation. The relative effectiveness of the acceptor analogs in decreasing order were 2-deoxy, 2-inverted, 3-deoxy, 3-inverted, 4-inverted, and 4-deoxy. The enzyme transfers D-glucopyranose to the C-6 hydroxyl of analogs modified at C-2 and C-3, to the C-4 hydroxyl of 4-inverted, and to the C-3 hydroxyl of 4-deoxy analogs of alpha-methyl-D-glucopyranoside. The data indicate that the hydroxyl group at C-2 is not as important for acceptor binding as the hydroxyl groups at C-3 and C-4. The hydroxyl group at C-4 is particularly important as it determines the binding orientation of the alpha-methyl-D-glucopyranoside ring.