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The marine sponge-derived polyketide discodermolide is a potent antimitotic agent that represents a promising natural product lead structure in the treatment of cancer. Discodermolide shares the same microtubule-stabilising mech- anism of action as Taxol ® , inhibits the growth of solid tumours in animal models and shows synergy with Taxol. The pronounced cytotoxicity of discodermolide, which is maintained against cancer cell lines that display resistance to Taxol and other drugs, combined with its scarce availability from its natural source, has fuelled significant academic and industrial interest in devising a practical total syn- thesis as a means of ensuring a sustainable supply for drug development. This chapter surveys the various total syntheses of discodermolide that have been completed over the period 1993-2007, focusing on the strategies employed for introduction of the multiple stereocentres and achieving control over the alkene geometry, along with the various methods used for realising the pivotal fragment couplings to assemble progressively the full carbon skeleton. This dedicated synthetic effort has triumphed in removing the supply problem for discodermolide, providing suf- ficient material for extensive biological studies and enabling its early stage clinical development, as well as facilitating SAR studies for lead optimisation.

A series of N-aryl-N'-(2-chloroethyl)ureas (CEUs) and derivatives were synthesized and evaluated for antiproliferative activity against a wide panel of tumor cell lines. Systematic structure--activity relationship (SAR) studies indicated that: (i) a branched alkyl chain or a halogen at the 4-position of the phenyl ring or a fluorenyl/indanyl group, (ii) an exocyclic urea function, and (iii) a N'-2-chloroethyl moiety were required to ensure significant cytotoxicity. Biological experiments, such as immunofluorescence microscopy, confirmed that these promising compounds alter the cytoskeleton by inducing microtubule depolymerization via selective alkylation of beta-tubulin. Subsequent evaluations demonstrated that potent CEUs were weak alkylators, were non-DNA-damaging agents, and did not interact with the thiol function of either glutathione or glutathione reductase. Therefore, CEUs are part of a new class of antimitotic agents. Finally, among the series of CEUs evaluated, compounds 12, 15, 16, and 27 were selected for further in vivo trials.

The cryptophycins are a unique family of 16-membered macrolide antimitotic agents isolated from the cyanobacteria Nostoc sp. Their molecular target is tubulin protein wherein they are the most potent known stabilizers of microtubule dynamics and depolymerize microtubules at higher concentrations. They also deactivate the Bcl2 protein and produce apoptotic response much more quickly and at considerably lower concentrations than clinically utilized compounds. The presence of several amide and ester linkages within the cryptophycin core provides access to very convergent total synthetic approaches. Likewise, the modularity of the structure renders their synthesis amenable to structure-activity studies in several regions of the molecule. The in vivo hydrolytic instability of the C5 ester was a key obstacle to the successful identification of a clinical candidate. This problem was ameliorated by increased substitution at C6 as in the presence of gem-dimethyl substitution in the clinical candidate, cryptophycin-52. © 2002 John Wiley & Sons, Inc. Med Res Rev, 22, No. 2, 85–101, 2002; DOI 10.1002/med.10002

Compound 8H is a phenylcinnamide that induces G2/M-phase cell cycle arrest and cell death in cancer cell lines. Here we show that 8H exerts its cytotoxic activity through disruption of microtubule dynamics in vitro and in cell culture. A series of cinnamide derivatives were synthesized and evaluated, and several new compounds were identified that improve on the activity of the parent compound, with IC(50) values for induction of cell death ranging from 1 to 10 microM. Notably, these compounds retain potency in the HL-60/VCR leukemia cell line, which is resistant to antimitotic cancer drugs vincrisitine and paclitaxel through up-regulation of P-glycoprotein drug efflux pumps. As P-glycoprotein expression is often responsible for drug resistance in cancer and the exclusion of compounds from the central nervous system, 8H and its derivatives merit further examination as potential antimitotic therapeutics, specifically for brain cancers and cancers that are resistant to standard antimitotic agents.

Disorazoles are macrocyclic polyketides first isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. Both the major fermentation product disorazole A1 and its much rarer companion disorazole C1 exhibit potent cytotoxic activity against many human tumor cells. Furthermore, the disorazoles appear to bind tubulin uniquely among known antimitotic agents, promoting apoptosis or premature senescence. It is uncertain what conveys tumor cell sensitivity to these complex natural products. Therefore, we generated and characterized human tumor cells resistant to disorazole C1. Resistant cells proved exceedingly difficult to generate and required single step mutagenesis with chronic stepwise exposure to increasing concentrations of disorazole C1. Compared with wild-type HeLa cells, disorazole C1-resistant HeLa/DZR cells were 34- and 8-fold resistant to disorazole C1 and disorazole A1 growth inhibition, respectively. HeLa/DZR cells were also remarkably cross-resistant to vinblastine (280-fold), paclitaxel (2400-fold), and doxorubicin (47-fold) but not cisplatin, suggesting a multidrug-resistant phenotype. Supporting this hypothesis, MCF7/MDR cells were 10-fold cross-resistant to disorazole C1. HeLa/DZR disorazole resistance was not durable in the absence of chronic compound exposure. Verapamil reversed HeLa/DZR resistance to disorazole C1 and disorazole A1. Moreover, HeLa/DZR cells expressed elevated levels of the drug resistance ATP-binding cassette ABCB1 transporter. Loss of ABCB1 by incubation with short interfering RNA restored sensitivity to the disorazoles. Thus, the multidrug resistance transporter ABCB1 can affect the cytotoxicity of both disorazole C1 and A1. Disorazole C1, however, retained activity against cells resistant against the clinically used microtubule-stabilizing agent epothilone B.

Cryo-EM is ideally suited for the study of cytoskeleton polymers and their interaction with cellular partners. Our understanding of microtubule (MT) structure and interactions has benefited tremendously from the application of different EM techniques, from the use of electron crystallography to determine the first high-resolution structure of tubulin, to electron tomographic reconstructions of unique MT-based organelles; from molecular details governing the regulated interaction of MTs with kinesin motors, to an atomic description of how antimitotic agents bind to tubulin and affect MT stability. In this chapter, we review these structural findings with an emphasis on how cryo-EM enables our studies.

The synthesis of ring analogues and derivatives of the S isomer of ethyl [5-amino-1,2-dihydro-2-methyl-3-phenylpyrido[3,4-b]pyrazin-7 - yl[carbamate, (S)-1, a potent antimitotic agent with anticancer activity, was directed toward the determination of the contribution of several structural features of this compound to biological activity. Replacement of the 5-amino with a 5(6H)-oxo group and either transposing the 6-ring nitrogen to or incorporation of a ring nitrogen at the 8-position caused a significant decrease in in vitro activity and destroyed in vivo activity. Although in vivo cytotoxicity was reduced, in vitro activity at higher doses relative to (S)-1 was retained by replacement of the 5-amino group with hydrogen and by expansion of the 1,2-dihydropyrazine to give a dihydro-1,4-diazepine ring.

The binding of four potent antimitotic agents, rhizoxin (RZX), phomopsin A (PMS-A), ansamitocin P-3 (ASMP-3), and vinblastine (VLB), to tubulins from RZX-sensitive and -resistant strains of Aspergillus nidulans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae was investigated. Mycelial extracts to which RZX could bind contained β-tubulin with Asn as the 100th amino acid residue (Asn-100) in all cases, and those without affinity for RZX contained β-tubulins with either Ile-100 or Val-100. Though PMS-A shares the same binding site as RZX and ASMP-3 on porcine brain tubulin (Asn-100), only ASMP-3 bound Asn-100 fungal tubulins in a competitive manner with respect to RZX. PMS-A and VLB, which strongly bind to porcine brain tubulin, did not bind to any of the fungal mycelial extracts examined. The results indicate differential interactions of these antimitotic agents with brain and fungal tubulins.

The antimitotic agent taxol was tested for toxicity towards fungi from different taxonomic groups and found to be particularly active against oomycete fungi. In germinating zoospore cysts of the oomycete Phytophthora capsici the mechanism of action of taxol was shown to involve inhibition of mitosis, presumably resulting from an effect on microtubules. Various taxol analogues with deleted A-ring C-13 side chain substituents were tested for toxicity towards P. capsici and Aphanomyces cochlioides to provide insight into structural features required for activity. The importance of the side chain was shown by the much lower activity as compared to taxol of analogues lacking all or part of the side chain. The effect of stereochemistry at the C-2' position on fungitoxicity towards oomycetes was similar to that reported previously on mammalian microtubule assembly.

Full details of a study to a synthesis of the optically active C13-C26 lower subunit of rhizoxin including the side-chain chromophore characteristic of the full class of antimitotic agents are described. A key element of the synthesis is the stereoselective introduction of the C18-C19 trisubstituted olefin through use of a Wadsworth-Horner-Emmons condensation of 3 with β-keto phosphonate 38 bearing resident functionality suitable for the diastereoselective introduction of C15-C17 employing a hydroxyl-directed reduction of the resultant β-hydroxyl ketone

The 3,4,5-trimethoxyphenyl and 3-hydroxy-4-methoxyphenyl rings of combretastatin A-4 are deemed optimal for its activity as antimitotic agent. The replacement of either one by a naphthalene ring results in compounds with a potency comparable to that of the parent compound. These results show that the naphthalene ring is a good surrogate for the 3,4,5-trimethoxyphenyl or the 3-hydroxy-4-methoxyphenyl rings of combretastatin A-4 and that neither of them is essential for the antitumor activity., Financial support came from Spanish DGICYT (SAF98-0103). R.P.L.C. thanks the Spanish Ministerio de Educación y Ciencia (MEC) for his ‘contrato de reincorporación’. Biological evaluation was supplied by Instituto Biomar S.L. (León, Spain).

An intramol. SNAr reaction was applied to the synthesis of a 13-membered meta-cyclophane that is the structural core of phomopsin and ustiloxin-type antimitotics. Two types of facile beta -elimination processes are also presented. [on SciFinder (R)]

This Highlight summarises synthetic efforts towards the marine sponge-derived antimitotic agent spirastrellolide A and its congeners, including the first total synthesis by Paterson and co-workers.

Mitotic abnormalities are a common feature of human cancer cells, and recent studies have provided evidence that such abnormalities may play a causative, rather than merely incidental role, in tumorigenesis. One such abnormality is prolonged activation of the mitotic checkpoint, which can be provoked by a number of the gene changes that drive tumor formation. At the same time, antimitotic chemotherapeutics exert their clinical efficacy through the large-scale induction of prolonged mitotic checkpoint activation, indicating that mitotic arrest is influential in both the formation and treatment of human cancer. However, how this influence occurs is not well understood. In this perspective, we will discuss the current evidence in support of the potential mechanisms by which prolonged activation of the mitotic checkpoint affects both tumorigenesis and antimitotic chemotherapy.

The effect of colchicine and other antimitotic drugs was studied in cultures of 11-day chick embryo breast muscle. Exposure of such cultures to 10-6 M colchicine results in fragmentation of the elongate myotubes into rounded, cytoplasmic sacs (myosacs) containing various numbers of nuclei. Comparison of the dose-response relation between myotube fragmentation and metaphase arrest suggests that the underlying mechanism may be similar in both cases. Low temperature does not duplicate the effects of colchicine. Glycerinated myotubes are not affected by the mitotic inhibitors. The effect of colchicine on myotubes is reversible. Myosacs elongate within several days after removal from colchicine. However, the regenerated myotubes fail to incorporate additional mononucleated cells. Colchicine does not interfere with the process of fusion itself, but the metaphase block prevents cells from entering that phase of the cell cycle during which fusion can occur. Cells arrested in mitosis by colchicine do not recover when incubated in normal medium. Colcemid-induced arrest is reversible and does not prevent subsequent fusion of the cells.

International audience; Several small weight indole derivatives (D-64131, D-24851, BPR0L075, BLF 61-3, and ATI derivatives) are potent tubulin polymerization inhibitors and show nanomolar antiproliferative activity. Among them, indolobenzazepin-7-ones were recently disclosed as potent antimitotic agents. In an effort to improve this structure, we prepared new derivatives in order to evaluate their antiproliferative activity. 5,6,7,9-Tetrahydro-8H-indolo[2,3-e][3]benzazocin-8-one (1m) was found to be the most potent derivative inhibiting the cell growth of several cancer cell lines in the lower nanomolar range.

This Highlight summarises synthetic efforts towards the marine sponge-derived antimitotic agent spirastrellolide A and its congeners, including the first total synthesis by Paterson and co-workers.

The aim of this study was to investigate the mechanism of inhibition of Eg5 (kinesin spindle protein), a mitotic kinesin that plays an essential role in establishing mitotic spindle bipolarity, by the novel small molecule inhibitor K858. K858 was selected in a phenotype-based forward chemical genetics screen as an antimitotic agent, and subsequently characterized as an inhibitor of Eg5. K858 blocked centrosome separation, activated the spindle checkpoint, and induced mitotic arrest in cells accompanied by the formation of monopolar spindles. Long-term continuous treatment of cancer cells with K858 resulted in antiproliferative effects through the induction of mitotic cell death, and polyploidization followed by senescence. In contrast, treatment of nontransformed cells with K858 resulted in mitotic slippage without cell death, and cell cycle arrest in G1 phase in a tetraploid state. In contrast to paclitaxel, K858 did not induce the formation of micronuclei in either cancer or nontransformed cells, suggesting that K858 has minimal effects on abnormalities in the number and structure of chromosomes. K858 exhibited potent antitumor activity in xenograft models of cancer, and induced the accumulation of mitotic cells with monopolar spindles in tumor tissues. Importantly, K858, unlike antimicrotubule agents, had no effect on microtubule polymerization in cell-free and cell-based assays, and was not neurotoxic in a motor coordination test in mice. Taken together, the Eg5 inhibitor K858 represents an important compound for further investigation as a novel anticancer therapeutic. [Cancer Res 2009;69(9):3901–9]

Progression through mitosis requires a balance of active microtubule-interacting proteins (MIPs) that stabilize and destabilize microtubules in mitotic spindles. Molecular interactions between MIPs and tubulin or microtubules are important for cell cycle progression. Insights into these interactions can contribute to understanding the mechanisms underlying cell cycle interference by antimitotic agents that halt cell cycle progression in mitosis. We examined the direct interaction of the antimitotic agent, vinblastine with tubulin and stathmin using AUC, in order to understand how changes in stathmin levels during the cell cycle might affect the cellular drug response. Vinblastine acts during G2/M phase of the cell cycle and reduces microtubule dynamics. At high doses it destabilizes microtubules in mitotic spindles. We found in vitro that stathmin reduces the potency of vinblastine. Vinblastine was found to compete for tubulin-stathmin oligomers, at the same time as it induced tubulin spiral formation. To extend these data to a cellular context, we investigated changes in intracelluar MIP levels in response to paclitaxel, an antimitotic agent known to stabilize microtubules at high concentrations. Using qRT-PCR we found that paclitaxel treatment of human breast cancer MCF7 cells leads to a significant reduction in MAP4 and stathmin mRNA levels. Interestingly, the levels return to pre-paclitaxel treatment levels after a 4-day drug washout, suggesting that paclitaxel alters transcript levels. We found that the ratios of MAP4/stathmin increased after or during drug treatment. These data suggest that changes in MIPs levels alter the cellular response to drugs. These results also suggest that disruption of the cell cycle by antimitotic agents can alter the relative amounts of MIPs and thus affect the balance needed for normal progression through the cell cycle. These results must be taken into account when modeling the cellular response to antimitotic drugs.

1-(2,4-dimethoxyphenyl)-3-(1-methylindolyl) propenone, namely IPP51, was identified by screening a library of 3-indolyl-1-phenylpropenones. IPP51 was investigated for its ability to inhibit proliferation and/or to induce apoptosis of human bladder cancer cell lines and to assess its potential use in bladder carcinoma treatment. After treating the cells with IPP51 for 24 h, the title compound induced a predominant and reversible G2+M accumulation at the prometaphase stage of mitosis. However, when used for a longer period, it leads to cell apoptosis. These results suggest that the compound has potential anticancer activities, which could be useful in bladder cancer treatment.

Much of our knowledge about microtubules has come from detailed morphological, biochemical, and cell biological studies. As more is learned about these organelles, questions regarding the in vivo regulation of their expression and function become increasingly important. Genetics provides an approach to address these more subtle questions in the living cell. Mammalian mutants with microtubule alterations have been isolated using selections for resistance to the cytotoxic effects of a number of antimitotic drugs. A subset of these mutants have clearly defined alterations in α- or in β-tubulin, and these have been used to explore the mechanisms by which mammalian cells acquire resistance to this class of drugs. In addition, the mutants are providing valuable insights into how tubulin expression is regulated, into what factors determine the extent of microtubule assembly in living cells, into the domains of tubulin that are involved in assembly, and into the role of microtubules in essential cellular processes.

The interaction of antimitotic drugs with guanine nucleotides in the tubulin-microtubule system is reviewed. Antimitotic agent-tubulin interactions can be covalent, entropic, allosteric or coupled to other equilibria (such as divalent cation binding, alternate polymer formation, or the stabilization of native tubulin structure). Antimitotics bind to tubulin at a few common sites and alter the ability of tubulin to form microtubules. Colchicine and podophyllotoxin compete for a common overlapping binding site but only colchicine induces GTPase activity and large conformational changes in the tubulin heterodimer. The vinca alkaloids, vinblastine and vincristine, the macrocyclic ansa macrolides, maytansine and ansamitocin P-3, and the fungal antimitotic, rhizoxin, share and compete for a different binding site near the exchangeable nucleotide binding site. The macrocyclic heptapeptide, phomopsin A, and the depsipeptide, dolastatin 10, bind to a site adjacent to the vinca alkaloid and nucleotide sites. Colchicine, vinca alkaloids, dolastatin 10 and phomopsin A induce alternate polymer formation (sheets for colchicine, spirals for vinblastine and vincristine and rings for dolastatin 10 and phomopsin A). Maytansine, ansamitocin P-3 and rhizoxin inhibit vinblastine-induced spiral formation. Taxol stoichiometrically induces microtubule formation and, in the presence of GTP, assembly-associated GTP hydrolysis. Analogs of guanine nucleotides also alter polymer morphology. Thus, sites on tubulin for drugs and nucleotides communicate allosterically with the interfaces that form longitudinal and lateral contacts within a microtubule. Microtubule associated proteins (MAPs), divalent cations, and buffer components can alter the surface interactions of tubulin and thus modulate the interactions between antimitotic drugs and guanine nucleotides.

Chromosome doubling of microspore-derived plantlets and calli is a critical step in haploid breeding programs. For anther culture experiments, colchicine, (S)-N-(5,6,7,9-tetrahydro-1,2,3,10-tetrame-thoxy-9-oxobenzo[a]heptalen-7-yl-acetamide, is the most commonly used compound in doubling the chromosomes of seedlings derived from such experiments. Colchicine, however, is carcinogenic and expensive. We evaluated three antimitotic agent (colchicine ; trifluroryzolin, 2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)benzenamine (...)

One of the main secondary toxic side effects of antimitotic agents used to treat cancer patients is intestinal mucositis. This one is characterized by compromised digestive and absorptive functions, barrier integrity, and immune competence. At the same time, food intake is decreased, which may induce intestinal damages per se. The aim of the study was to characterize which alterations are specific to methotrexate, independently of the anorexic effect of the drug. Male Sprague-Dawley rats received subcutaneously saline solution as control group or 2.5 mg/kg of methotrexate during 3 days (D0-D2). Methotrexate-treated rats were compared with ad libitum and pair-fed controls. Histological examinations and specific markers of the immune and nonimmune gut barrier function were assessed at D4 or D7. Compared with ad libitum and pair-fed controls, methotrexate induced at D4 villus atrophy associated with epithelial necrosis. Mucosal protein synthesis rate and mucin contents of methotrexate treated rats were reduced. At the same time, cathepsin D proteolytic activity was increased compared with ad libitum and pair-fed controls, whereas calpain activity was increased when compared with the only pair-fed controls. These intestinal lesions were associated with various metabolic disturbances such as increased TNF-α level and inflammation score in the jejunum but also disturbances of amino acid concentrations in the duodenum and plasma. At D7, these alterations were partially or completely normalized. In addition to the consequences of a low food intake, methotrexate further impairs different biological processes leading to a dramatic loss of gut homeostasis. Targeted nutritional management of chemotherapy receiving patients should be set up to prevent or limit such alterations.

Haspin/Gsg2 is a kinase that phosphorylates histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin, therefore, is a novel target for development of antimitotic agents. We report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for haspin. Histone H3 peptide was used as a substrate, and a europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at K(m) concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC( 50) determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of 2 unrelated kinases. EC( 50) values in cells were determined using a cell-based ELISA of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of haspin that will have clear utility in basic research and possible use as starting points for development of antimitotic anticancer therapeutics.

Herein, we describe the structure–activity relationship study of a new 1-(arylalkyl)-11 H -benzo[ f ]-1,2-dihydropyrido[3,2, c ][1,2,5]oxathiazepine 5,5-dioxide series of antimitotic agents. The pharmacological results obtained from previous works allowed us to identify compound 1 as a new cytotoxic agent inhibiting tubulin polymerization. We have undertaken the synthesis of its non-methylated analogue 7 and have extended our investigations to a novel, structurally related benzopyridooxathiazepine dioxide series. Among all analogues synthesized in this study, compound 10b was the most promising, being 12-fold more potent than compound 1 . Its activity over a panel of five tumoral cell lines was in the nanomolar range for all of the histological types tested and flow cytometric studies performed on L1210 cells showed an accumulation of the cells in the G2/M phases of the cell cycle with a significant percentage of tetraploid cells (8N DNA content). This interesting pharmacological profile, resulting from inhibition of tubulin polymerization, encouraged us to perform preliminary in vivo studies.

We present a 2D agent-based stochastic solid tumor model which incorporates cellular response to a chemotherapeutic drug such as paclitaxel (Taxol). First, we show that our model not only reproduces the findings of past mathematical models but also agrees qualitatively with previously observed experimental findings in vitro. Then, the model is used to study two contrasting methods of chemotherapeutic dosing: (1) maximum tolerated dose (MTD) and (2) metronomic dosing. Results of our simulations confirm the typical clinical observation that a single front-loaded dosing to MTD leads to a period of remission followed by recurrence, while metronomic dosing maintains or reduces the tumor size. These results suggest patient treatment strategies should favor the more recently proposed metronomic dosing paradigm over the traditionally used MTD therapy. The model proposed here is adaptable to different solid tumor types as well as different chemotherapeutic drug pharmacokinetic and pharmacodynamic properties, making it ideal for similar studies on different clinical problems.

Forty-six new compounds were synthesized on the basis of our knowledge of the 3-haloacylamino benzoylurea (HBU) series. Structure-activity relationship (SAR) analysis indicates that (i) the configuration of the chiral center in 1 (JIMB01) is not indispensable for the activity, (ii) the phenyl ring is essential, and (iii) a substitution at the 6-position of the phenyl ring with a halogen enhances the activity. Among the analogues, 11e and 14b bearing 6-fluoro substitution showed potent activities against nine human tumor cell lines, including CEM (leukemia), Daudi (lymphoma), MCF-7 (breast cancer), Bel-7402 (hepatoma), DU-145 (prostate cancer), PC-3 (prostate cancer), DND-1A(melanoma), LOVO (colon cancer), and MIA Paca (pancreatic cancer) with IC 50 values between 0.01 and 0.30 microM. 14b inhibited human hepatocarcinoma by 86% in volume in nude mice. The mechanism of 14b is to inhibit microtubule assembly, followed by the M-phase arrest, bcl-2 inactivation, and then apoptosis. We consider 14b promising for further anticancer investigation.

International audience; The extracellular polysaccharide hyaluronan (HA) controls cell migration, differentiation and proliferation, and contributes to the invasiveness of human cancers. In order to investigate the sensitivity of cancer cells to antimitotic agents, we developed a cross-linked HA hydrogel, a three-dimensional matrix in which cells can invade and grow. We have studied three cell lines (SA87, NCI-H460 and H460M), from primary tumors and metastases, that migrated into the HA hydrogel and proliferated giving rise to clusters and colonies. Concurrently, we studied the growth of these cell lines in a usual monolayer culture system. In these two models, increasing concentrations of doxorubicin and 5-fluorouracil were evaluated for their ability to inhibit tumor cell growth and colony formation. Taken together, our data suggest that the cancer cells were more resistant in the three-dimensional model than in monolayer cell systems. The antimitotic drugs were efficient after 24 h of treatment in the monolayer cultures, whereas they were significantly efficient only after one week of incubation in the HA hydrogels. Herein, we show that this cross-linked matrix provides a three-dimensional model particularly appropriate for investigating mechanisms involved in cancer cell line sensitivity to antimitotic drugs.

The 2-(3,4,5-trimethoxybenzoyl)-2-aminoindole nucleus was used as the fundamental structure for the synthesis of compounds modified with respect to positions C-4 to C-7 with different moieties (chloro, methyl, or methoxy). Additional structural variations concerned the indole nitrogen, which was alkylated with small alkyl groups such as methyl or ethyl. We have identified 1-methyl-2-(3,4,5-trimethoxybenzoyl)-3-amino-7-methoxyindole as a new highly potent antiproliferative agent that targets tubulin at the colchicine binding site and leads to apoptotic cell death.

Microtubules are among the most successful targets of compounds potentially useful for cancer therapy. A new series of inhibitors of tubulin polymerization based on the 2-amino-3-(3,4,5-trimethoxybenzoyl)-4,5,6,7-tetrahydrothieno[b]pyridine molecular skeleton was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization, and cell cycle effects. The most promising compound in this series was 2-amino-3-(3,4,5-trimethoxybenzoyl)-6-methoxycarbonyl-4,5,6,7-tetrahydrothieno[b]pyridine, which inhibits cancer cell growth with IC(50)-values ranging from 25 to 90 nM against a panel of four cancer cell lines, and interacts strongly with tubulin by binding to the colchicine site. In this series of N(6)-carbamate derivatives, any further increase in the length and in the size of the alkyl chain resulted in reduced activity.

Tubulin targeting agents constitute an important class of anticancer drugs. By acting either as microtubule stabilizers or destabilizers, they disrupt microtubule dynamics, thus inducing mitotic arrest and, ultimately, cell death by apoptosis. Three different binding sites, whose exact location on tubulin has been experimentally detected, have been identified so far for antimitotic compound targeting microtubules, namely the taxoid, the colchicine and the vinka alkaloid binding site. A number of ligand- and structure-based molecular modeling studies in this field has been reported over the years, aimed at elucidating the binding modes of both stabilizing and destabilizing agent, as well as the molecular features responsible for their efficacious interaction with tubulin. Such studies are described in this review, focusing on information provided by different modeling approaches on the structural determinants of antitubulin agents and the interactions with the binding pockets on tubulin emerged as fundamental for antitumor activity.To describe molecular modeling approaches applied to date to molecules known to bind microtubules, this paper has been divided into two main parts: microtubule destabilizing (Part 1) and stabilizing (Part 2) agents. The first part includes structure-based and ligand-based approaches to study molecules targeting colchicine (1.1) and vinca alkaloid (1.2) binding sites, respectively. In the second part, the studies performed on microtubule-stabilizing antimitotic agents (MSAA) are described. Starting from the first representative compound of this class, paclitaxel, molecular modeling studies (quantitative structure-activity relationships – QSAR – and structure-based approaches), performed on natural compounds acting with the same mechanism of action and temptative common pharmacophoric hypotheses for all of these compounds, are reported.

The marine sponge-derived polyketide discodermolide is a potent antimitotic agent that represents a promising natural product lead structure in the treatment of cancer. Discodermolide shares the same microtubule-stabilising mech- anism of action as Taxol ® , inhibits the growth of solid tumours in animal models and shows synergy with Taxol. The pronounced cytotoxicity of discodermolide, which is maintained against cancer cell lines that display resistance to Taxol and other drugs, combined with its scarce availability from its natural source, has fuelled significant academic and industrial interest in devising a practical total syn- thesis as a means of ensuring a sustainable supply for drug development. This chapter surveys the various total syntheses of discodermolide that have been completed over the period 1993-2007, focusing on the strategies employed for introduction of the multiple stereocentres and achieving control over the alkene geometry, along with the various methods used for realising the pivotal fragment couplings to assemble progressively the full carbon skeleton. This dedicated synthetic effort has triumphed in removing the supply problem for discodermolide, providing suf- ficient material for extensive biological studies and enabling its early stage clinical development, as well as facilitating SAR studies for lead optimisation.

Dolastatin 10, a potent antimitotic peptide from a marine animal, strongly inhibits microtubule assembly, tubulin-dependent GTP hydrolysis, and the binding of vinca alkaloids to tubulin. In studies of the binding of [3H]vincristine to the protein, with vinblastine as a control for competitive inhibition (Ki, 6.6 microM), we found that the macrolide antimitotic agents maytansine and rhizoxin were also competitive inhibitors (Ki values, 3.1 and 12 microM). Dolastatin 10 and an unrelated peptide antimitotic, phomopsin A, were more potent but noncompetitive inhibitors (Ki values, 1.4 and 2.8 microM). Since maytansine and, to a much lesser extent, vinblastine interfere with nucleotide exchange on tubulin, all drugs were examined for effects on nucleotide interactions at the exchangeable GTP site. Rhizoxin had effects intermediate between those of vinblastine and maytansine. Both peptides inhibited binding of radiolabeled GTP to tubulin even more strongly than did maytansine, but no drug displaced nucleotide from tubulin. The drugs were evaluated for stabilizing effects on the colchicine binding activity of tubulin. The peptides prevented loss of this activity, and vinblastine provided partial protection, while rhizoxin and maytansine did not stabilize tubulin. A tripeptide segment of dolastatin 10 also effectively inhibits tubulin polymerization and GTP hydrolysis. The tripeptide did not significantly inhibit either vincristine binding or nucleotide exchange, nor did it stabilize colchicine binding. These findings are rationalized in terms of a model with two distinct drug binding sites in close physical proximity to each other and to the exchangeable GTP site on beta-tubulin.

A series of chalcones was evaluated as antimitotic agents. One of these, (E)-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2-pr open- 1-one) (73), was found to be an effective antimitotic agent at a concentration of 4 nM in an in vitro HeLa cell test system. When evaluated in experimental tumor models in vivo, this compound exhibited antitumor activity against L1210 leukemia and B16 melanoma.

Curacin D is a novel brine shrimp toxic metabolite isolated from a Virgin Islands collection of the marine cyanobacterium Lyngbya majuscula. Structure elucidation of curacin D was accomplished through multidimensional NMR, GC/MS, and comparisons with curacin A. Curacin D provides new insights into structure-activity relationships in this natural product class as well as some aspects of the likely biosynthetic pathway of the curacins.

The inv(16)(p13q22) is found in de novo AML and is closely associated with the FAB subtype M4eo. The inv(16) is rarely reported in therapy-related AML (t-AML) patients. Herein, we report a case of t-AML with inv(16) after combination chemotherapy using antimitotic agent and alkylating agent (cis-platin-paclitaxel) for ovarian serous cystadenocarcinoma.

Antimitotic agents that target the dynamic equilibrium between the microtubule polymer and tubulin heterodimers are key components of chemotherapeutic regimens for various solid tumors. These agents can be divided into two major classes based on their effect on microtubule polymerization and the mass of microtubule polymers: those that inhibit polymerization, such as the vinca alkaloids and those that stabilize microtubules, such as the taxanes and epothilones. The taxanes paclitaxel (Taxol) and docetaxel (Taxotere) were the first antimicrotubule agents approved for use in solid tumors, but their usefulness is often limited by development of drug resistance. The epothilones are distinguished from the taxanes structurally and functionally and have been shown in vitro and in preclinical models to have superior potency to the taxanes. The epothilones are not susceptible to P-glycoprotein-mediated efflux and have shown activity against taxane-resistant tumors. Other natural-product microtubule-stabilizing agents also have promising pharmacologic profiles. This article discusses mechanisms of drug resistance and summarizes scientific and clinical data supporting the potential of novel microtubule-stabilizing agents for achieving broad antitumor efficacy without the emergence of drug resistance. The ability to reduce the development of resistance with the epothilones and other microtubule-stabilizing agents may provide additional treatment options at the time of presentation and in the setting of taxane resistance.

Several 2-amino-pyrimido[4,5-c]quinolin-1(2H)-ones variously substituted at positions 3, 5, and 9 were prepared from their corresponding lactones. The target compounds were investigated for in vitro cytotoxic activity against a panel of human cancer cell lines, namely, lung fibrosarcoma HT-1080, colon adenocarcinoma HT-29, and breast carcinoma MDA-MB-231. Analysis of data revealed that the presence of chloro at position 9 has a major positive impact on cytotoxic activity. Additional halogen substitution at the para position of the 3-phenyl group further enhances activity. Furthermore, compound (25) was found to dose-dependently inhibit tubulin polymerization. In accordance, flow cytometric analysis of the most potent compounds (23-26) indicated that the tested compounds induce cell cycle arrest in the G(2)/M phase. The obtained results introduce the rarely described pyrimido[4,5-c]quinolin-1(2H)-one ring system as a new scaffold for promising antimitotic agents.

Two new series of inhibitors of tubulin polymerization based on the 2-amino-3-(3,4,5-trimethoxybenzoyl)benzo[b]thiophene molecular skeleton and its 3-amino positional isomer were synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization, and cell cycle effects. Although many more 3-amino derivatives have been synthesized so far, the most promising compound in this series was 2-amino-6-methyl-3-(3,4,5-trimethoxybenzoyl)benzo[b]thiophene, which inhibits cancer cell growth at subnanomolar concentrations and interacts strongly with tubulin by binding to the colchicine site.

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:22:27Z No. of bitstreams: 0Bitstream added on 2014-05-27T14:43:54Z : No. of bitstreams: 1 2-s2.0-34248579599.pdf: 604626 bytes, checksum: 1957b4bfbf6c652cb41fa86d4a6aa46d (MD5) Made available in DSpace on 2014-05-27T11:22:27Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-04-01 • Aim: To observe the effects of topical application of 5-fluorouracil (5-FU) on the rabbits corneal limbus. • Methods: 5-FU was applied topically to the corneoscleral region of rabbits eyes. The animals were sacrificed immediately or 7, 15, 30 and 60 days later, and tissues were submitted to histological examination. • Results: All animals presented corneoscleral deepithelization close to the site of application during the immediate post-operative period, whereas on the 4th postoperative day ulceration was no longer present. Histological examination showed absence of epithelium and slight edema. After one week (G2), 2 animals presented epithelial defects, thickened epithelium with larger basal cells and loose chromatin, and slight subepithelial edema. The remaining groups showed no alterations. • Conclusion: The 5-FU topically on the corneoscleral limbus postpone the epitelization. Department of Ophthalmology Faculdade de Medicina de Botucatu, 18618-000-Botucatu-Sao, Paulo State Department of Pathology Faculdade de Medicina de Botucatu, 18618-000-Botucatu-Sao, Paulo State Department of Ophthalmology Faculdade de Medicina de Botucatu, 18618-000-Botucatu-Sao, Paulo State Department of Pathology Faculdade de Medicina de Botucatu, 18618-000-Botucatu-Sao, Paulo State

Microtubule inhibitors such as Vinka alkaloids (e.g., vinblastine and vincristine) and Taxans (e.g., taxol/paclitaxel and docetaxel) are potent therapeutic drugs for cancer treatment. The cellular consequences of taxol/paclitaxel treatments over a wide range of doses (1 nmol/L–100 μmol/L) have

ABT-751 is a novel antimitotic agent that binds tubulin at the colchicine binding site. ABT-751 is undergoing Phase I trials in children, but has not been evaluated against a range of pediatric tumor models in vivo.ABT-751 was evaluated against 27 subcutaneously implanted xenograft models of childhood cancer including neuroblastoma [4], osteosarcoma [4], Ewing sarcoma [2] rhabdomyosarcoma [8], medulloblastoma [1] and eight kidney cancer lines (six Wilms tumors, two rhabdoid). ABT-751 was administered at 100 mg/kg P.O. on a schedule of 5 days on, 5 days off, 5 days on, repeating the cycle at 21 days. Tumor diameters were measured at 7 day intervals for a period of 12 weeks. Three measures of antitumor activity were used: (1) clinical response criteria [e.g., partial response (PR), complete response (CR), etc.]; (2) treated to control (T/C) tumor volume at day 21; and (3) a time to event measure based on the median event free survival (EFS) of treated and control lines.ABT-751 induced regression in 4 of 25 models (16%) including models of neuroblastoma that are refractory to vincristine and paclitaxel. Other regressions occurred in rhabdomyosarcoma and Wilms tumor models. ABT-751 significantly increased event free survival (EFS2.0) in eight models (33%) in addition to those with objective responses.ABT-751 demonstrated intermediate activity against this tumor panel. Neuroblastoma models appear somewhat more sensitive to this agent, with objective regressions also in rhabdomyosarcoma and Wilms tumor. ABT-751 was also active in several tumor lines intrinsically refractory to vincristine or paclitaxel.

A novel series of 2-amino and 2'-aminocombretastatin derivatives were synthesized and evaluated for antitumor activity. Several compounds had excellent antiproliferative activity as inhibitors of tubulin polymerization. Compounds 11, 20, and 21 with IC(50) values of 1.6, 1.7, and 1.8 microM, respectively, exhibited more potent inhibition of tubulin polymerization than colchicine and approximately as active as combretastatin A-4. They also displayed antiproliferative activity with an IC(50) values ranging from 11 to 44 nM in a variety of human cell lines from different organs. Structure activity relationship information suggests that the NH(2) substituent at the 2-position of either ring A or ring B in combretastatin molecular skeleton may play an important role in the bioactivity of this series of compounds.

Some of the most significant therapeutic leads and agents used for the treatment of cancer target microtubule dynamics. Paclitaxel is an exceptional example that is currently used for treating a wide range of tumors. New, non-taxane microtubule stabilizers, including several epothilones, are advancing through clinical trials. Laulimalide is a potent microtubule stabilizer that binds to tubulin at a site that does not overlap the taxane-binding site. It is active against paclitaxel-resistant cancer cells. Notwithstanding its therapeutic potential, laulimalide is relatively unstable, rearranging to a more stable but less active isomer. The goal of this study was to evaluate the ability of laulimalide and two designed laulimalide analogues, C16-C17-des-epoxy laulimalide (LA1) and C20-methoxy laulimalide (LA2), to inhibit cell proliferation in combination with other tubulin-binding and non-tubulin-binding antiproliferative antimitotic agents. The synthetic laulimalide analogues retain the mechanism of action of the natural compound but do not share its instability. We studied the ability of the laulimalides to act synergistically with paclitaxel, 2-methoxyestradiol, and monastrol, an Eg5 kinesin inhibitor. The results show that all three of the laulimalides acted synergistically with paclitaxel and 2-methoxyestradiol to inhibit proliferation with the analogues exhibiting significantly larger synergistic effects. The combination of laulimalide and monastrol was not synergistic and provided only additive effects. The laulimalide analogues LA1 and LA2 had a greater degree of synergy with both paclitaxel and 2-methoxyestradiol than was observed with laulimalide. Our results show that the laulimalides together with other tubulin-binding antimitotic agents provide synergistic antiproliferative actions. The data are consistent with the previously reported ability of laulimalide and paclitaxel to act synergistically to polymerize tubulin in vitro. These important findings suggest that specific combinations of microtubule-targeting agents should be considered for clinical utilities as they have excellent potential to improve clinical response.