Search

As part of our continuing search for potent inhibitors of tubulin polymerization, two novel series of 42 10-(4-phenylpiperazine-1-carbonyl)acridin-9(10H)-ones and N-benzoylated acridones were synthesized on the basis of a retrosynthetic approach. All newly synthesized compounds were tested for antiproliferative activity and interaction with tubulin. Several analogs potently inhibited tumor cell growth. Among the compounds tested, 10-(4-(3-methoxyphenyl)piperazine-1-carbonyl)acridin-9(10H)-one (17c) exhibited excellent growth inhibitory effects on 93 tumor cell lines, with an average GI50 value of 5.4 nM. We were able to show that the strong cytotoxic effects are caused by disruption of tubulin polymerization, as supported by the EBI (N,N'-Ethylenebis(iodoacetamide)) assay and the fact that the most potent inhibitors of cancer cell growth turned out to be the most efficacious tubulin polymerization inhibitors. Potencies were nearly comparable or superior to those of the antimitotic reference compounds. Closely related to this, the most active analogs inhibited cell cycling at the G2/M phase at concentrations down to 30 nM and induced apoptosis in K562 leukemia cells. We believe that our work not only proves the excellent suitability of the acridone scaffold for the design of potent tubulin polymerization inhibitors but also enables synthetic access to further potentially interesting N-acylated acridones.

A novel series of 1,5- and 1,8-disubstituted 10-benzylidene-10H-anthracen-9-ones and 10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-ones was synthesized to assess the substituent effects on biological activity. The 3-hydroxy-2,4-dimethoxy-benzylidene analogue 16 h displayed strong antiproliferative activity against several tumor cell lines, including multi-drug resistant phenotypes. Flow cytometric studies showed that KB/HeLa cells treated by elected compounds were arrested in the G2/M phases of the cell cycle. Among the compounds tested for inhibition of tubulin polymerization, 14 compounds proved to be exceptionally active with IC(50) values1 microM. In the 1,5-dichloro-derived series of benzylideneanthracenones, E/Z isomers were separated and biological effects were monitored. We found that the olefinic geometry had no significant effect on biological activity. Furthermore, the E isomeric 1,5-dichloro-substituted phenacylidenes entirely proved to be more potent inhibitors of tubulin polymerization than the recently described 10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-ones. In conclusion, the present study improves understanding of the action of anthracenone-based tubulin polymerization inhibitors and contributes to the design of further potent anti-tubulin drugs.

Tubulin-binding 9-benzylidene-naphtho[2,3-b]thiophen-4-ones 1a and 1b and benzylidene-9(10H)-anthracenone 2 were evaluated for their ability to induce cell death. We examined the effect of the molecules on cell cycle progression, organization of microtubule networks, and apoptosis induction. As determined by flow cytometry, cancer cells were predominantly arrested in metaphase with 4N DNA before cell death occurred. By using indirect immunofluorescence techniques we visualized microtubule depolymerization recognizable by short microtubule fragments scattered around the nucleus. The incubation with 1a and 2 resulted in chromatin condensation, nuclear fragmentation, and cell shrinkage, which are, among others, typical features of apoptotic cell death. Furthermore, time- and dose-dependent induction of apoptosis in SH-SY5Y cells was detected via cleavage of Ac-DEVD-AMC, a fluorigenic substrate for caspase-3. We observed a lower apoptotic activity in neuroblastoma cells overexpressing Bcl-xL, suggesting activation of the mitochondrial apoptosis pathway. Western blot analysis demonstrated that caspase-3, an apoptosis mediator, was activated in a time-dependent manner after exposure of SH-SY5Y cells to drugs 1a and 2. Taken together, the agents investigated in the present study display strong apoptosis-inducing activity and therefore show promise for the development of novel chemotherapeutics.

Benzenesulfonate derivatives of naphtho[2,3-b]thiophen-4(9H)-one and 9(10H)-anthracenone were prepared and found to inhibit microtubule formation by an in vitro tubulin polymerization assay. Several analogues showed potent cytotoxic activity in an assay based on K562 leukemia cells with IC50 values of

A novel series of 9-benzylidene-naphtho[2,3-b]thiophen-4-ones and structurally related compounds were synthesized and evaluated for their ability to inhibit tubulin polymerization. The 4-hydroxy-3,5-dimethoxy-benzylidene analogue 15d was identified as a potent cytotoxic agent in an assay based on K562 leukemia cells. Antiproliferative activity of 15d and the 2,4-dimethoxy-3-hydroxy-benzylidene analogue 15e was additionally evaluated against a panel of 12 tumor cell lines, including multidrug resistant phenotypes. All resistant cell lines were sensitive to these compounds. Concentration-dependent flow cytometric studies showed that K562 cells as well as KB/HeLa cells treated by 15d were arrested in the G2/M phases of the cell cycle. Moreover, four compounds strongly inhibited tubulin polymerization with activities higher or comparable to those of the reference compounds. In competition experiments, the most active compounds strongly displaced radiolabeled colchicine from its binding site in the tubulin, showing IC50 values virtually 3- to 4-fold lower than that of colchicine.

A series of 2-arylmethyl-substituted anthracenones were synthesized and tested as inhibitors of three types of 12-lipoxygenase isoforms in epidermal homogenate of mice, bovine platelets and porcine leucocytes. Their inhibitory activities were compared with those to inhibit the 5-lipoxygenase enzyme in bovine leucocytes. Structure–activity relationships are described with particular emphasis on modifications of the terminal aryl nucleus. The ability of the compounds to selectively inhibit the 12-lipoxygenase enzymes was dependent on a high overall lipophilicity of the inhibitor, whereas compounds with decreased lipophilicity were also inhibitors of the 5-LO enzyme. Among the more lipophilic inhibitors, the unsubstituted 2-phenylmethyl analogue 6a as well as the carboxylic acid ester 6q appeared to be selective inhibitors of platelet-type 12-LO isoform.

The synthesis and biological properties of 10-benzoyl-1,8-dihydroxy-9(10H)-anthracenones are described. The compounds were evaluated for their ability to inhibit the growth of the human keratinocyte cell line HaCaT and the 5-lipoxygenase enzyme in bovine polymorphonuclear leukocytes. In addition, generation of hydroxyl radicals was determined as measured by deoxyribose degradation. The results obtained with the new compounds show that substitution with a 4-hydroxy (4f) or a 4-benzyloxy group (4i) at the phenyl ring of the C-10 substituent resulted in potent antiproliferative agents. No correlation was found between hydroxyl-radical formation and the 5-LO inhibitory or antiproliferative action of the compounds.

Summary A novel series of 10-aminomethylene substituted 1,8-dihydroxy-9(10H)-anthracenones was synthesized and evaluated both in the bovine polymorphonuclear leukocyte 5-lipoxygenase (5-LO) and in the HaCaT keratinocyte proliferation assay for their enzyme inhibitory and antiproliferative activity, respectively. The synthesis required readily available formanilides as starting materials and a modified Vilsmeier type reaction with the parent anthracenone. The most potent inhibitor of 5-LO was a 4-hydroxyphenyl analog, whereas a 4-nitrophenyl substituent was essential for potent antiproliferative activity. The results of this study indicate that an activated double bond at C-10 of phenylalkylidene-substituted anthracenones is required for potency.

A novel series of carboxylic and hydroxamic acids based on 1,8-dihydroxy-9(10H)-anthracenone were synthesized from 8-hydroxy-1-methoxy-9,10-anthracenedione as the key intermediate and evaluated both in the bovine polymorphonuclear leukocyte 5-lipoxygenase (5-LO) assay and in the HaCaT keratinocyte proliferation assay for their enzyme inhibitory and antiproliferative activity, respectively. The most potent inhibitors in both assays were the N-methylated hydroxamic acids 5d-8d with straight chain alkyl spacers. Incorporation of these structural features on the anthracenone pharmacophore resulted in increased inhibitory activity against 5-LO while the antiproliferative activity was retained. In addition, prooxidant properties as measured by deoxyribose degradation and cytotoxicity as assessed by LDH release were largely reduced as compared with the antipsoriatic anthralin. Contrary to anthralin, antioxidant properties were observed as documented by the reactivity of the novel compounds against free radicals and inhibition of lipid peroxidation in model membranes.

A total of 53 N-benzoylated phenoxazines and phenothiazines, including their S-oxidized analogues, were synthesized and evaluated for antiproliferative activity, interaction with tubulin, and cell cycle effects. Potent inhibitors of multiple cancer cell lines emerged with the 10-(4-methoxybenzoyl)-10H-phenoxazine-3-carbonitrile (33b, IC(50) values in the range of 2-15 nM) and the isovanillic analogue 33c. Seventeen compounds strongly inhibited tubulin polymerization with activities higher than or comparable to those of the reference compounds such as colchicine. Concentration-dependent flow cytometric studies revealed that inhibition of K562 cell growth was associated with an arrest in the G2/M phases of the cell cycle, indicative of mitotic blockade. Structure-activity relationship studies showed that best potencies were obtained with agents bearing a methoxy group placed para at the terminal phenyl ring and a 3-cyano group in the phenoxazine. A series of analogues highlight not only the phenoxazine but also the phenothiazine structural scaffold as valuable pharmacophores for potent tubulin polymerization inhibitors, worthy of further investigation.

A series of anthracenone-based oxime ethers and -esters were synthesized in order to evaluate their antiproliferative activity. Several investigated compounds displayed strong antiproliferative activity against K562 leukemia cells and proved to be strong inhibitors of tubulin polymerization. In this context, anthracenone-based oxime ethers and -esters are considered to contribute to the development of novel antiproliferative drugs, based on tubulin interaction.

A series of 10-(2-oxo-2-phenylethylidene)-10H-anthracen-9-ones were synthesized and evaluated for interactions with tubulin and for antiproliferative activity against a panel of human and rodent tumor cell lines. The 4-methoxy analogue 17b was most potent, displaying IC(50) values ranging from 40 to 80 nM, including multidrug resistant phenotypes, and had excellent activity as an inhibitor of tubulin polymerization (IC(50) = 0.52 microM). Concentration-dependent flow cytometric studies showed that KB/HeLa cells treated with 17b were arrested in the G2/M phases of the cell cycle (EC(50) = 90 nM). In competition experiments, 17b strongly displaced [(3)H]-colchicine from its binding site in the tubulin. The results obtained demonstrate that the antiproliferative activity is related to the inhibition of tubulin polymerization.

A novel series of 10-benzylidene-9(10H)-anthracenones and 10-(phenylmethyl)-9(10H)-anthracenones were synthesized and evaluated for antiproliferative activity in an assay based on K562 leukemia cells. The 3-hydroxy-4-methoxybenzylidene analogue 9h was found to be the most active compound (IC(50) K562: 20 nM). Structure-activity relationships are also considered. The highly active compound 9h and the 2,4-dimethoxy-3-hydroxybenzylidene analogue 9l were tested against five tumor cell lines using the XTT assay, including multidrug resistant phenotypes. Induction of cell death in a variety of tumor cell lines was determined in a monolayer assay using propidium iodide. Noteworthy, all compounds within the series induced elongations in K562 cells similar to vinblastine-treated cells. The effect of the lead compound 9h on K562 cell growth was associated with cell cycle arrest in G2/M. Concentrations for 50% KB/HeLa cells arrested in G2/M after treatment with 9h and 9l were determined and found to be in the range of 0.2 microM. Additionally, we monitored the dose dependent caspase-3-like protease activity in K562 cells and MCF-7/Casp-3 cells treated with 9h, indicating induction of apoptosis. Western blotting analysis demonstrated that 9h caused a shift in tubulin concentration from the polymerized state found in the cell pellet to the unpolymerized state found in the cell supernatant. Seven compounds strongly inhibited tubulin polymerization with activities higher or comparable to those of the reference compounds such as colchicine, podophyllotoxin, and nocodazole. In general, the antiproliferative activity correlated with inhibition of tubulin polymerization. The most active compounds strongly displaced [(3)H]colchicine from its binding site in the tubulin, yielding IC(50) values 3- to 4-fold lower than that of colchicine. The novel benzylidene-9(10H)-anthracenones described in the present study constitute an interesting group of highly active and easily accessible antimitotic agents that inhibit tubulin polymerization.

In recent years, enormous progress has been made in the field of tubulin targeting agents. Several companies and academic laboratories have entered this field and competition has become very strong. Nevertheless, clinically promising compounds often face substantial limitations, such as high systemic toxicity, poor water solubility and bioavailability, as well as complex synthesis and isolation procedures. As a drawback of established drugs, like paclitaxel or the vinca alkaloids, the outcome of cancer chemotherapy is often affected by the emergence of the multidrug resistance phenotype. Among the recently disclosed tubulin polymerization inhibitors, there are several interesting low molecular weight compounds with improved oral bioavailability and demonstrated activity against multi-drug resistance positive phenotypes. As documented by the imidazole-based combretastatin analogs, to name just one example, chemical optimization of a lead structure resulted in compounds with potent in vitro and in vivo activity along with appropriate pharmacodynamic and pharmacokinetic requirements for a potential therapeutic candidate. Currently, several compounds are undergoing Phase I or Phase II clinical trials, among them orally bioavailable sulfonamides or dolastatin 10. Several other compounds are close to entering Phase I trials. The purpose of this review is to focus on the most recent advances in tubulin polymerization inhibitors from a survey of the published patent literature and related publications between late 1999 and April 2002. However, biological data, especially for the inhibition of tubulin polymerization, obtained from different laboratories cannot easily be compared.

A series of 2-arylalkyl-substituted anthracenones were tested as inhibitors of three types of 12-lipoxygenase isoforms in epidermal homogenate of mice, bovine platelets and porcine leukocytes. Their inhibitory activities were compared with those to inhibit the 5-lipoxygenase enzyme in bovine leukocytes. The compounds were synthesised by Marschalk, Wittig or Horner–Emmons reaction at the anthracenedione stage and then reduced to the anthracenones. Structure–activity relationship for the chain linking the anthracenone nucleus and the phenyl ring terminus was investigated. The 2-phenylethyl analogues were among the most potent inhibitors, and 3,4-dimethoxy-substituted 10f was identified as a selective inhibitor of the 12-LO enzymes over 5-LO. Selectivity for 12-LO isoforms was observed with an increase in the overall lipophilicity of the inhibitors. However, none of the linker chains of the 2-substituted anthracenones provided inhibitors that were able to discriminate between the 12-LO isoforms.

The synthesis of 2-substituted anthracenonyl acetic acid (2-AA) derivatives is described. The key step is the Marschalk reaction of 1-hydroxy-8-methoxy-anthracenedione with glycolic acid. After protection of the resulting 2-anthracenonyl acetic acid derivative, the 2-monoalkylated derivatives are selectively obtained by direct alkylation. The methodology proves quite general and allows for the introduction of various substituents onto the 2-position of the carboxylic side chain. Reduction of the anthracenediones proceeds with concomitant protecting group removal and provides final 2-AA products in good yields. The results of initial biological studies demonstrate enhanced 5-lipoxygenase inhibition compared to anthralin.