Synthesis and evaluation of antiproliferative microtubule-destabilising combretastatin A-4 piperazine conjugates.

Microtubules are a validated clinical target for the treatment of many cancers. We describe the design, synthesis, biochemical evaluation, and molecular modelling studies of a series of analogues of the microtubule-destabilising agent, combretastatin A-4 (CA-4). Our series of 33 novel compounds contain the CA-4 core structure with modifications to the stilbene linking group, and are predominantly piperazine derivatives. Synthesis was achieved in a two-step process by firstly obtaining the acrylic acid via a Perkin reaction using microwave enhanced synthesis, followed by coupling using either DCC or Mukaiyama's reagent. All target compounds were screened for antiproliferative activity in MCF-7 breast cancer cells. Hydroxyl derivative (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-phenylpiperazin-1-yl)-2-(3,4,5-trimethoxyphenyl) propenone (4m) displayed potent antiproliferative activity (IC50 = 190 nM). Two amino-containing derivatives, (E)-3-(3-amino-4-methoxyphenyl)-1-(4-phenylpiperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4q) and (E)-3-(3-amino-4-methoxyphenyl)-1-(4-(p-tolyl)piperazin-1-yl)-2-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4x), were the most potent with IC50 values of 130 nM and 83 nM respectively. Representative compounds were shown to depolymerise tubulin, induce G2/M arrest and apoptosis in MCF-7 cells but not peripheral blood mononuclear cells, and induce cleavage of the DNA repair enzyme poly ADP ribose polymerase (PARP) in MCF-7 cells. Modelling studies predict that the compounds bind to tubulin within the colchicine-binding site. These compounds are a valuable addition to the library of CA-4 analogues and 4m, 4q and 4x will be developed further as novel, water-soluble molecules targeting microtubules.

Involvement of NF-κB in mediating the anti-tumour effects of combretastatins in T cells.

Purpose The combretastatins (CAs) are known to exhibit anti-tumour activity but the underlying mechanism remains to be fully elucidated. Inflammation plays a critical role in altering the function of cancer cells and evasion of cell death and increased proliferation are characteristics of transformed malignancies. Many of the proteins involved in these pathways are regulated by the transcription factor NF-κB which can be activated by tumour necrosis factor (TNF-α), a pro-inflammatory cytokine released by both malignant and immune cells within the tumour microenvironment. In this study, we examined the ability of combretastatin A-4 (CA-4) and its novel, cis-restricted analogue CA-432 to target the NF-κB signalling pathway in T cells. Methods Effects of the CAs on the viability of DND-41 leukaemia and Jurkat lymphoma T-cell lines was assessed by the alamar blue assay. Induction of apoptosis and effects on expression levels of key apoptotic proteins was established though flow cytometry and western blotting. Modulation of the NF-κB signalling pathway was determined through western blotting and through assessment of NF-κB reporter gene activity. Results CA-4 and CA-432 reduced cell viability and induced apoptosis in DND-41 and Jurkat T cells and sensitised the cells to TNF-α-induced apoptosis through inhibition of the NF-κB signalling pathway. Suppression of the NF-κB pathway downregulated NF-κB-dependent gene products involved in cell survival (IAPs, Bcl-2 and Mcl-1), proliferation (cyclin D1) and inflammation (COX-2). Furthermore, both CA-4 and CA-432 inhibited TNF-α-induced NF-κB activation through the inhibition of IκBα degradation and p65 nuclear translocation and decreased NF-κB reporter gene activity. Conclusions Our data indicate that the anti-cancer properties of comebretastatins may be mediated in part through targeting the NF-κB pathway. This study provides new insights into the molecular mechanisms of CA compounds and a potential application of combretastatins for inflammatory diseases such as cancers, which are associated with abnormal NF-κB activation.

Antiproliferative and Tubulin-Destabilising Effects of 3-(Prop-1-en-2-yl)azetidin-2-Ones and Related Compounds in MCF-7 and MDA-MB-231 Breast Cancer Cells.

A series of novel 3-(prop-1-en-2-yl)azetidin-2-one, 3-allylazetidin-2-one and 3-(buta-1,3-dien-1-yl)azetidin-2-one analogues of combretastatin A-4 (CA-4) were designed and synthesised as colchicine-binding site inhibitors (CBSI) in which the ethylene bridge of CA-4 was replaced with a ß-lactam (2-azetidinone) scaffold. These compounds, together with related prodrugs, were evaluated for their antiproliferative activity, cell cycle effects and ability to inhibit tubulin assembly. The compounds demonstrated significant in vitro antiproliferative activities in MCF-7 breast cancer cells, particularly for compounds 9h, 9q, 9r, 10p, 10r and 11h, with IC50 values in the range 10-33 nM. These compounds were also potent in the triple-negative breast cancer (TBNC) cell line MDA-MB-231, with IC50 values in the range 23-33 nM, and were comparable with the activity of CA-4. The compounds inhibited the polymerisation of tubulin in vitro, with significant reduction in tubulin polymerization, and were shown to interact at the colchicine-binding site on tubulin. Flow cytometry demonstrated that compound 9q arrested MCF-7 cells in the G2/M phase and resulted in cellular apoptosis. The antimitotic properties of 9q in MCF-7 human breast cancer cells were also evaluated, and the effect on the organization of microtubules in the cells after treatment with compound 9q was observed using confocal microscopy. The immunofluorescence results confirm that ß-lactam 9q is targeting tubulin and resulted in mitotic catastrophe in MCF-7 cells. In silico molecular docking supports the hypothesis that the compounds interact with the colchicine-binding domain of tubulin. Compound 9q is a novel potent microtubule-destabilising agent with potential as a promising lead compound for the development of new antitumour agents.

Design and synthesis of multifunctional microtubule targeting agents endowed with dual pro-apoptotic and anti-autophagic efficacy.

Autophagy is a lysosome dependent cell survival mechanism and is central to the maintenance of organismal homeostasis in both physiological and pathological situations. Targeting autophagy in cancer therapy attracted considerable attention in the past as stress-induced autophagy has been demonstrated to contribute to both drug resistance and malignant progression and recently interest in this area has re-emerged. Unlocking the therapeutic potential of autophagy modulation could be a valuable strategy for designing innovative tools for cancer treatment. Microtubule-targeting agents (MTAs) are some of the most successful anti-cancer drugs used in the clinic to date. Scaling up our efforts to develop new anti-cancer agents, we rationally designed multifunctional agents 5a-l with improved potency and safety that combine tubulin depolymerising efficacy with autophagic flux inhibitory activity. Through a combination of computational, biological, biochemical, pharmacokinetic-safety, metabolic studies and SAR analyses we identified the hits 5i,k. These MTAs were characterised as potent pro-apoptotic agents and also demonstrated autophagy inhibition efficacy. To measure their efficacy at inhibiting autophagy, we investigated their effects on basal and starvation-mediated autophagic flux by quantifying the expression of LC3II/LC3I and p62 proteins in oral squamous cell carcinoma and human leukaemia through western blotting and by immunofluorescence study of LC3 and LAMP1 in a cervical carcinoma cell line. Analogues 5i and 5k, endowed with pro-apoptotic activity on a range of hematological cancer cells (including ex-vivo chronic lymphocytic leukaemia (CLL) cells) and several solid tumor cell lines, also behaved as late-stage autophagy inhibitors by impairing autophagosome-lysosome fusion.

Synthesis and Antiproliferative Evaluation of 3-Chloroazetidin-2-ones with Antimitotic Activity: Heterocyclic Bridged Analogues of Combretastatin A-4.

Antimitotic drugs that target tubulin are among the most widely used chemotherapeutic agents; however, the development of multidrug resistance has limited their clinical activity. We report the synthesis and biological properties of a series of novel 3-chloro-ß-lactams and 3,3-dichloro-ß-lactams (2-azetidinones) that are structurally related to the tubulin polymerisation inhibitor and vascular targeting agent, Combretastatin A-4. These compounds were evaluated as potential tubulin polymerisation inhibitors and for their antiproliferative effects in breast cancer cells. A number of the compounds showed potent activity in MCF-7 breast cancer cells, e.g., compound 10n (3-chloro-4-(3-hydroxy-4-methoxy-phenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one) and compound 11n (3,3-dichloro-4-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-azetidin-2-one), with IC50 values of 17 and 31 nM, respectively, and displayed comparable cellular effects to those of Combretastatin A-4. Compound 10n demonstrated minimal cytotoxicity against non-tumorigenic HEK-293T cells and inhibited the in vitro polymerisation of tubulin with significant G2/M phase cell cycle arrest. Immunofluorescence staining of MCF-7 cells confirmed that ß-lactam 10n caused a mitotic catastrophe by targeting tubulin. In addition, compound 10n promoted apoptosis by regulating the expression of pro-apoptotic protein BAX and anti-apoptotic proteins Bcl-2 and Mcl-1. Molecular docking was used to explore the potential molecular interactions between novel 3-chloro-ß-lactams and the amino acid residues of the colchicine binding active site cavity of ß-tubulin. Collectively, these results suggest that 3-chloro-2-azetidinones, such as compound 10n, could be promising lead compounds for further clinical anti-cancer drug development.

Synthesis and Biological Evaluation of 1-(Diarylmethyl)-1H-1,2,4-triazoles and 1-(Diarylmethyl)-1H-imidazoles as a Novel Class of Anti-Mitotic Agent for Activity in Breast Cancer.

We report the synthesis and biochemical evaluation of compounds that are designed as hybrids of the microtubule targeting benzophenone phenstatin and the aromatase inhibitor letrozole. A preliminary screening in estrogen receptor (ER)-positive MCF-7 breast cancer cells identified 5-((2H-1,2,3-triazol-1-yl)(3,4,5-trimethoxyphenyl)methyl)-2-methoxyphenol 24 as a potent antiproliferative compound with an IC50 value of 52 nM in MCF-7 breast cancer cells (ER+/PR+) and 74 nM in triple-negative MDA-MB-231 breast cancer cells. The compounds demonstrated significant G2/M phase cell cycle arrest and induction of apoptosis in the MCF-7 cell line, inhibited tubulin polymerisation, and were selective for cancer cells when evaluated in non-tumorigenic MCF-10A breast cells. The immunofluorescence staining of MCF-7 cells confirmed that the compounds targeted tubulin and induced multinucleation, which is a recognised sign of mitotic catastrophe. Computational docking studies of compounds 19e, 21l, and 24 in the colchicine binding site of tubulin indicated potential binding conformations for the compounds. Compounds 19e and 21l were also shown to selectively inhibit aromatase. These compounds are promising candidates for development as antiproliferative, aromatase inhibitory, and microtubule-disrupting agents for breast cancer.

The vascular targeting agent combretastatin-A4 and a novel cis-Restricted {beta}-Lactam Analogue, CA-432, induce apoptosis in human chronic myeloid leukemia cells and ex vivo patient samples including those displaying multidrug resistance.

Combretastatin-A4 (CA-4) is a natural derivative of the African willow tree Combretum caffrum. CA-4 is one of the most potent antimitotic components of natural origin, but it is, however, intrinsically unstable. A novel series of CA-4 analogs incorporating a 3,4-diaryl-2-azetidinone (ß-lactam) ring were designed and synthesized with the objective to prevent cis -trans isomerization and improve the intrinsic stability without altering the biological activity of CA-4. Evaluation of selected ß-lactam CA-4 analogs demonstrated potent antitubulin, antiproliferative, and antimitotic effects in human leukemia cells. A lead ß-lactam analog, CA-432, displayed comparable antiproliferative activities with CA-4. CA-432 induced rapid apoptosis in HL-60 acute myeloid leukemia cells, which was accompanied by depolymerization of the microtubular network, poly(ADP-ribose) polymerase cleavage, caspase-3 activation, and Bcl-2 cleavage. A prolonged G(2)M cell cycle arrest accompanied by a sustained phosphorylation of mitotic spindle checkpoint protein, BubR1, and the antiapoptotic proteins Bcl-2 and Bcl-x(L) preceded apoptotic events in K562 chronic myeloid leukemia (CML) cells. Molecular docking studies in conjunction with comprehensive cell line data rule out CA-4 and ß-lactam derivatives as P-glycoprotein substrates. Furthermore, both CA-4 and CA-432 induced significantly more apoptosis compared with imatinib mesylate in ex vivo samples from patients with CML, including those positive for the T315I mutation displaying resistance to imatinib mesylate and dasatinib. In summary, synthetic intrinsically stable analogs of CA-4 that display significant clinical potential as antileukemic agents have been designed and synthesized.

ß-Lactams with antiproliferative and antiapoptotic activity in breast and chemoresistant colon cancer cells.

A series of novel 1,4-diaryl-2-azetidinone analogues of combretastatin A-4 (CA-4) have been designed, synthesised and evaluated in vitro for antiproliferative activity, antiapoptotic activity and inhibition of tubulin polymerisation. Glucuronidation of CA-4 by uridine 5-diphosphoglucuronosyl transferase enzymes (UGTs) has been identified as a mechanism of resistance in cancer cells. Potential sites of ring B glucuronate conjugation are removed by replacing the B ring meta-hydroxy substituent of selected series of ß-lactams with alternative substituents e.g. F, Cl, Br, I, CH3. The 3-phenyl-ß-lactam 11 and 3-hydroxy-ß-lactam 46 demonstrate improved activity over CA-4 in CA-4 resistant HT-29 colon cancer cells (IC50 = 9 nM and 3 nM respectively compared with IC50 = 4.16 µM for CA-4), while retaining potency in MCF-7 breast cancer cells (IC50 = 17 nM and 22 nM respectively compared with IC50 = for 4 nM for CA-4). Compound 46 binds at the colchicine site of tubulin, and strongly inhibits tubulin assembly at micromolar concentrations comparable to CA-4. In addition, compound 46 induced mitotic arrest at low concentration in both cell lines MCF-7 and HT-29 together with downregulation of expression of antiapoptotic proteins Mcl-1, Bcl-2 and survivin in MCF-7 cells. These novel antiproliferative and antiapoptotic ß-lactams are potentially useful scaffolds in the development of tubulin-targeting agents for the treatment of breast cancers and chemoresistant colon cancers.

3-Vinylazetidin-2-Ones: Synthesis, Antiproliferative and Tubulin Destabilizing Activity in MCF-7 and MDA-MB-231 Breast Cancer Cells.

Microtubule-targeted drugs are essential chemotherapeutic agents for various types of cancer. A series of 3-vinyl-ß-lactams (2-azetidinones) were designed, synthesized and evaluated as potential tubulin polymerization inhibitors, and for their antiproliferative effects in breast cancer cells. These compounds showed potent activity in MCF-7 breast cancer cells with an IC50 value of 8 nM for compound 7s 4-[3-Hydroxy-4-methoxyphenyl]-1-(3,4,5-trimethoxyphenyl)-3-vinylazetidin-2-one) which was comparable to the activity of Combretastatin A-4. Compound 7s had minimal cytotoxicity against both non-tumorigenic HEK-293T cells and murine mammary epithelial cells. The compounds inhibited the polymerisation of tubulin in vitro with an 8.7-fold reduction in tubulin polymerization at 10 M for compound 7s and were shown to interact at the colchicine-binding site on tubulin, resulting in significant G2/M phase cell cycle arrest. Immunofluorescence staining of MCF-7 cells confirmed that ß-lactam 7s is targeting tubulin and resulted in mitotic catastrophe. A docking simulation indicated potential binding conformations for the 3-vinyl-ß-lactam 7s in the colchicine domain of tubulin. These compounds are promising candidates for development as antiproiferative microtubule-disrupting agents.

Piperlongumine (piplartine) and analogues: Antiproliferative microtubule-destabilising agents.

Piperlongumine (piplartine, 1) is a small molecule alkaloid that is receiving intense interest due to its antiproliferative and anticancer activities. We investigated the effects of 1 on tubulin and microtubules. Using both an isolated tubulin assay, and a combination of sedimentation and western blotting, we demonstrated that 1 is a tubulin-destabilising agent. This result was confirmed by immunofluorescence and confocal microscopy, which showed that microtubules in MCF-7 breast cancer cells were depolymerized when treated with 1. We synthesised a number of analogues of 1 to explore structure-activity relationships. Compound 13 had the best cytotoxic profile of this series, showing potent effects in human breast carcinoma MCF-7 cells whilst being relatively non-toxic to non-tumorigenic MCF-10a cells. These compounds will be further developed as potential clinical candidates for the treatment of breast cancer.

ß-Lactam analogues of combretastatin A-4 prevent metabolic inactivation by glucuronidation in chemoresistant HT-29 colon cancer cells.

Glucuronidation by uridine 5-diphosphoglucuronosyl transferase enzymes (UGTs) is a cause of intrinsic drug resistance in cancer cells. Glucuronidation of combretastatin A-4 (CA-4) was previously identified as a mechanism of resistance in hepatocellular cancer cells. Herein, we propose chemical manipulation of ß-lactam bridged analogues of Combretastatin A-4 as a novel means of overcoming drug resistance associated with glucuronidation due to the expression of UGTs in the CA-4 resistant human colon cancer HT-29 cells. The alkene bridge of CA-4 is replaced with a ß-lactam ring to circumvent potential isomerisation while the potential sites of glucuronate conjugation are deleted in the novel 3-substituted-1,4-diaryl-2-azetidinone analogues of CA-4. We hypothesise that glucuronidation of CA-4 is the mechanism of drug resistance in HT-29 cells. Ring B thioether containing 2-azetidinone analogues of CA-4 such as 4-(4-(methylthio)phenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (27) and 3-hydroxy-4-(4-(methylthio)phenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (45) were identified as the most potent inhibitors of tumour cell growth, independent of UGT status, displaying antiproliferative activity in the low nanomolar range. These compounds also disrupted the microtubular structure in MCF-7 and HT-29 cells, and caused G2/M arrest and apoptosis. Taken together, these findings highlight the potential of chemical manipulation as a means of overcoming glucuronidation attributed drug resistance in CA-4 resistant human colon cancer HT-29 cells, allowing the development of therapeutically superior analogues.

Inhibition of γ-secretase activity synergistically enhances tumour necrosis factor-related apoptosis-inducing ligand induced apoptosis in T-cell acute lymphoblastic leukemia cells via upregulation of death receptor 5.

T-cell acute lymphoblastic leukemia (T-ALL) is a rare and aggressive hematopoietic malignancy prone to relapse and drug resistance. Half of all T-ALL patients exhibit mutations in Notch1, which leads to aberrant Notch1 associated signaling cascades. Notch1 activation is mediated by the γ-secretase cleavage of the Notch1 receptor into the active intracellular domain of Notch1 (NCID). Clinical trials of γ-secretase small molecule inhibitors (GSIs) as single agents for the treatment of T-ALL have been unsuccessful. The present study demonstrated, using immunofluorescence and western blotting, that blocking γ-secretase activity in T-ALL cells with N-[(3,5-difluorophenyl) acetyl]-L-alanyl-2-phenyl] glycine-1,1-dimethylethyl ester (DAPT) downregulated NCID and upregulated the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5). Upregulation of DR5 restored the sensitivity of T-ALL cells to TRAIL. Combination index revealed that the combined treatment of DAPT and TRAIL synergistically enhanced apoptosis compared with treatment with either drug alone. TRAIL combined with the clinically evaluated γ-secretase inhibitor 3-[(1r, 4s)-4-(4-chlorophenylsulfonyl)-4-(2, 5-difluorophenyl) cyclohexyl] propanoic acid (MK-0752) also significantly enhanced TRAIL-induced cell death compared with either drug alone. DAPT/TRAIL apoptotic synergy was dependent on the extrinsic apoptotic pathway and was associated with a decrease in BH3 interacting-domain death agonist and x-linked inhibitor of apoptosis. In conclusion, γ-secretase inhibition represents a potential therapeutic strategy to overcome TRAIL resistance for the treatment of T-ALL.

Isouronium and N-hydroxyguanidinium derivatives as Cell growth inhibitors: A comparative study.

Based on the results obtained from a computational study on the suitability of the isouronium and N-hydroxyguanidinium cations as hydrogen bond donors/acceptors, the DNA binding of a series of isouronium derivatives was assessed by DNA thermal denaturation experiments and compared to related N-hydroxyguanidines. Due to the poor DNA binding observed, the nature of the diaromatic linker was explored by preparing the corresponding amide-linked bis-isouronium derivative and measuring its DNA affinity. Next, the inhibitory effects of the isouronium derivatives on cell viability were evaluated in two different cancer cell lines providing IC50 values in the range of 36.9-57.4 µM (HL-60, leukemia), and 17.3-33.9 µM (Kelly, neuroblastoma). These values are comparable to those previously found for the N-hydroxyguanidine series. Compounds with the -S- linker (3, 6, and 10) proved to be considerably active in the HL-60 cells and even more active in the Kelly cell line. No correlation was found between DNA minor groove binding and cell growth inhibition; hence, activity may depend on different modes of action. Further studies into the apoptotic potential of these compounds indicated that, besides inhibiting cell viability and proliferation, derivatives 9 and 10, are significant apoptosis-inducers in both cell lines. Results obtained with HL-60 cells suggest that G2/M arrest and subsequent apoptosis induced by compound 10 are associated with microtubular depolymerisation, loss of mitochondrial membrane potential and activation of the caspase cascade. Moreover, the effects of compound 10 on cell viability and apoptosis in two non-cancereous cell lines (NIH3T3 and MCF-10A) indicate none or minimal toxicity.

The pyrrolo-1,5-benzoxazepine, PBOX-15, enhances TRAIL­induced apoptosis by upregulation of DR5 and downregulation of core cell survival proteins in acute lymphoblastic leukaemia cells.

Apoptotic defects are frequently associated with poor outcome in pediatric acute lymphoblastic leukaemia (ALL) hence there is an ongoing demand for novel strategies that counteract apoptotic resistance. The death ligand TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) and its selective tumour receptor system has attracted exceptional clinical interest. However, many malignancies including ALL are resistant to TRAIL monotherapy. Tumour resistance can be overcome by drug combination therapy. TRAIL and its agonist antibodies are currently undergoing phase II clinical trials with established chemotherapeutics. Herein, we present promising therapeutic benefits in combining TRAIL with the selective anti-leukaemic agents, the pyrrolo-1,5-benzoxazepines (PBOXs) for the treatment of ALL. PBOX-15 synergistically enhanced apoptosis induced by TRAIL and a DR5-selective TRAIL variant in ALL-derived cells. PBOX-15 enhanced TRAIL-induced apoptosis by dual activation of extrinsic and intrinsic apoptotic pathways. The specific caspase-8 inhibitor, Z-IETD-FMK, identified the extrinsic pathway as the principal mode of apoptosis. We demonstrate that PBOX-15 can enhance TRAIL-induced apoptosis by upregulation of DR5, reduction of cellular mitochondrial potential, activation of the caspase cascade and downregulation of PI3K/Akt, c-FLIP, Mcl-1 and IAP survival pathways. Of note, the PI3K pathway inhibitor LY-294002 significantly enhanced the apoptotic potential of TRAIL and PBOX-15 validating the importance of Akt downregulation in the TRAIL/PBOX-15 synergistic combination. Considering the lack of cytotoxicity to normal cells and ability to downregulate several survival pathways, PBOX-15 may represent an effective agent for use in combination with TRAIL for the treatment of ALL.

Synthesis and Biochemical Evaluation of 3-Phenoxy-1,4-diarylazetidin-2-ones as Tubulin-Targeting Antitumor Agents.

Structure-activity relationships for a series of 3-phenoxy-1,4-diarylazetidin-2-ones were investigated, leading to the discovery of a number of potent antiproliferative compounds, including trans-4-(3-hydroxy-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (78b) and trans-4-(3-amino-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (90b). X-ray crystallography studies indicate the potential importance of the torsional angle between the 1-phenyl "A" ring and 4-phenyl "B" ring for potent antiproliferative activity and that a trans configuration between the 3-phenoxy and 4-phenyl rings is generally optimal. These compounds displayed IC50 values of 38 and 19 nM, respectively, in MCF-7 breast cancer cells, inhibited the polymerization of isolated tubulin in vitro, disrupted the microtubular structure in MCF-7 cells as visualized by confocal microscopy, and caused G2/M arrest and apoptosis. Compound 90b possessed a mean GI50 value of 22 nM in the NCI60 cell line screen, displayed minimal cytotoxicity, and was shown to interact at the colchicine-binding site on ß-tubulin. Phosphate and amino acid prodrugs of both 78b and 90b were synthesized, of which the alanine amide 102b retained potency and is a promising candidate for further clinical development.

Antitumor effect of pyrrolo-1,5-benzoxazepine-15 and its synergistic effect with Oxaliplatin and 5-FU in colorectal cancer cells.

Some compounds of a series of novel pyrrolo-1,5-benzoxa(thia)zepine, a well-known group of tubulin targeting agents, display anti-tumor effects mainly inducing cell cycle arrest and apoptosis in several human cancer models. A member of this family, pyrrolo-1,5-benzoxazepine-15 (PBOX-15), has previously shown potent pro-apoptotic activity in a variety of human tumor cell types, with minimal toxicity toward normal blood and bone marrow cells. In this study, we evaluated the PBOX-15-mediated effects in human colorectal cancer cell (CRC) lines, DLD-1 and HT-29. The compound, used at concentrations equal to or greater than 1 µM, inhibited the proliferation of human CRC cells, inducing a significant cell cycle arrest in the G2/M phase. In DLD-1 cells, treatments prolonged over 48 h triggered a strong activation of the intrinsic apoptotic pathway as indicated by activation of caspase-9, caspase-3 and PARP cleavage. Moreover, nanomolar concentrations of PBOX-15, significantly improved the oxaliplatin and 5-fluouracil-induced anti-proliferative effects in DLD1 cell line. The observed synergistic interaction of both PBOX-15/Oxaliplatin and PBOX-15/5FU may involve activation of p38 MAPK and JNK pathway, which in turn significantly increased caspase-3 cleavage in DLD-1 cells, treated with PBOX-5/Oxaliplatin but not with PBOX-15/5FU. Moreover, PBOX-15/5FU-treated cells showed an increase in expression of the pro-apoptotic protein Bax. Taken together, these results show that PBOX-15 could represent a promising compound for the treatment of human CRC and a strong candidate for novel therapeutic options.

Development of novel cyclic peptides as pro-apoptotic agents.

Our recent finding that paclitaxel behaves as a peptidomimetic of the endogenous protein Nur77 inspired the design of two peptides (PEP1 and PEP2) reproducing the effects of paclitaxel on Bcl-2 and tubulin, proving the peptidomimetic nature of paclitaxel. Starting from these peptide-hits, we herein describe the synthesis and the biological investigation of linear and cyclic peptides structurally related to PEP2. While linear peptides (2a,b, 3a,b, 4, 6a-f) were found inactive in cell-based assays, biological analysis revealed a pro-apoptotic effect for most of the cyclic peptides (5a-g). Cellular permeability of 5a (and also of 2a,b) on HL60 cells was assessed through confocal microscopy analysis. Further cellular studies on a panel of leukemic cell lines (HL60, Jurkat, MEC, EBVB) and solid tumor cell lines (breast cancer MCF-7 cells, human melanoma A375 and 501Mel cells, and murine melanoma B16F1 cells) confirmed the pro-apoptotic effect of the cyclic peptides. Cell cycle analysis revealed that treatment with 5a, 5c, 5d or 5f resulted in an increase in the number of cells in the sub-G0/G1 peak. Direct interaction with tubulin (turbidimetric assay) and with microtubules (immunostaining experiments) was assessed in vitro for the most promising compounds.

ß-Lactam estrogen receptor antagonists and a dual-targeting estrogen receptor/tubulin ligand.

Twelve novel ß-lactams were synthesized and their antiproliferative effects and binding affinity for the predominant isoforms of the estrogen receptor (ER), ERα and ERß, were determined. ß-Lactams 23 and 26 had the strongest binding affinities for ERα (IC50 values: 40 and 8 nM, respectively) and ERß (IC50 values: 19 and 15 nM). ß-Lactam 26 was the most potent in antiproliferative assays using MCF-7 breast cancer cells, and further biochemical analysis showed that it caused accumulation of cells in G2/M phase (mitotic blockade) and depolymerization of tubulin in MCF-7 cells. Compound 26 also induced apoptosis and downregulation of the expression of pro-survival proteins Bcl-2 and Mcl-1. Computational modeling predicted binding preferences for the dual ER/tubulin ligand 26. This series is an important addition to the known pool of ER antagonists and ß-lactam 26 is the first reported compound that has dual-targeting properties for both the ER and tubulin.

Aromatic bis-N-hydroxyguanidinium derivatives: synthesis, biophysical, and biochemical evaluations.

In this paper we report the synthesis of a new family of hydroxyguanidinium aromatic derivatives (4a-g) as potential minor groove binders and cytotoxic agents. Their DNA affinity was evaluated by thermal denaturation experiments using salmon sperm DNA. The antiproliferative effects of derivatives 4a, 4d, and 4f were evaluated in human promyelocytic HL-60, breast carcinoma MCF-7, and neuroblastoma Kelly cell lines using the AlamarBlue viability assay, and IC(50) values were obtained. All three compounds were active in the HL-60 cell line. In particular, 4b exhibits antiproliferative effects in all three cell lines while 4d reduced HL-60 and Kelly viability. Both 4b and 4d produced considerable antiproliferative activity in the Kelly cell line. Derivative 4d was chosen for further cell cycle and apoptosis studies using flow cytometric analysis of cellular DNA content.

Novel cis-restricted ß-lactam combretastatin A-4 analogues display anti-vascular and anti-metastatic properties in vitro.

Combretastatin A-4 (CA-4) is a naturally occurring microtubular-destabilising agent that possesses potent anti-tumour and anti-vascular properties both in vitro and in vivo. Clinical trials to date indicate that its water-soluble prodrug, combretastatin A-4 phosphate (CA-4P), is well tolerated at therapeutically useful doses. However, the stilbenoid structure of CA-4, consisting of two phenyl rings linked by an ethylene bridge, renders the compound readily susceptible to isomerisation from its biologically active cis-conformation to its more thermodynamically stable but inactive trans-isomer. To circumvent this problem, we synthesised a series of cis-restricted CA-4 analogues. Replacement of the ethylene bridge with a 1,4-diaryl-2-azetidinone (ß-lactam) ring provided a rigid scaffold thus preventing cis-trans isomerisation. We previously documented that these tubulin-depolymerising ß-lactam compounds potently induced cell cycle arrest and apoptosis in a variety of cancerous cell lines (including those displaying multidrug resistance) and ex vivo patient samples, whilst exerting only minimal toxicity to normal cells. The purpose of this study was to elucidate the effect of the ß-lactam compounds on both tumour vascularisation and tumour cell migration, two critical elements that occur during the growth and metastatic progression of tumours. We established that two representative ß-lactam compounds, CA-104 and CA-432, exerted both anti-endothelial effects [G2/M arrest and apoptosis of primary human umbilical vein endothelial cells (HUVECs)] and anti-angiogenic effects [inhibition of HUVEC migration and differentiation and reduced vascular endothelial growth factor (VEGF) release from MDA-MB-231 breast adenocarcinoma cells]. In addition, we established that lead analogue, CA-432, abrogated the migration of MDA-MB-231 cells indicating an anti-metastatic function for these compounds. In summary, our results to date collectively indicate that these cis-restricted ß-lactam CA-4 analogues may prove to be useful alternatives to CA-4 in the treatment of cancer but with the added advantage of improved stability of the cis-isomer.