Metabolism of Flavone-8-acetic Acid in Mice.

Flavone-8-acetic acid (FAA) is a potent antivascular agent in mice but not in humans. Assuming that FAA was bioactivated in mice, we previously demonstrated that 6-OH-FAA was formed from FAA by mouse microsomes but not by human microsomes; its antivascular activity was 2.1- to 15.9-fold stronger than that of FAA, and its antivascular activity was mediated through the Ras homolog gene family (Rho) protein kinase A (RhoA) pathway. The present work aimed to study FAA metabolism in order to verify if 6-OH-FAA is formed in mice. Using synthesized standards and high-performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection and mass spectrometry (MS) analysis, we herein demonstrated, for the first time, that in vitro FAA and its monohydroxylated derivatives could directly undergo phase II metabolism forming glucuronides, and two FAA epoxides were mostly scavenged by NAC and GSH forming corresponding adducts. FAA was metabolized in mice. Several metabolites were formed, in particular 6-OHFAA. The antitumor activity of 6-OH-FAA in vivo is worthy of investigation.

Not flavone-8-acetic acid (FAA) but its murine metabolite 6-OH-FAA exhibits remarkable antivascular activities in vitro.

Flavone-8-acetic acid (FAA) has been proved to be a potent vascular-disrupting agent in mice. Unfortunately, FAA did not produce any anticancer activity in clinical trials. Previously, we had reported that FAA is metabolized by mouse microsomes into six metabolites, whereas it was poorly metabolized by human microsomes, with fewer metabolites formed in lesser amounts. Especially, 6-OH-FAA was not formed by human microsomes. In this work, two major available metabolites, 4'-OH-FAA and 6-OH-FAA, were tested and compared with the parent compound FAA for their potential antivascular activities in vitro. The ability of the products to induce morphological changes, disrupt preformed capillaries of EA.hy926 endothelial cells and inhibit tubulin polymerization in vitro was assessed. The action mechanism was determined using the RhoA and Rac1 inhibitors. At 25 µg/ml, 6-OH-FAA induced morphological changes and membrane blebbing, whereas 300 µg/ml of FAA and 4'-OH-FAA slightly changed the morphology without inducing membrane blebbing. At 300 µg/ml, 6-OH-FAA produced morphological changes that were 2.1-6.9-fold greater than that produced by FAA and 4'-OH-FAA, an effect that was consistent with its much greater inhibitory effect on tubulin polymerization compared with FAA and 4'-OH-FAA. 6-OH-FAA significantly disrupted the EA.hy926 cell capillaries. 6-OH-FAA activities were prevented in EA.hy926 cells pretreated with RhoA, but not Rac1, inhibitor. In this short communication we report for the first time that, in vitro, 6-OH-FAA, a mouse-specific FAA metabolite, exhibits significantly stronger antivascular activities compared with FAA and 4'-OH-FAA, which are mediated through the RhoA kinase pathway.

Antivascular and anti-parasite activities of natural and hemisynthetic flavonoids from New Caledonian Gardenia species (Rubiaceae).

A series of 16 flavonoids were isolated and prepared from bud exudate of Gardenia urvillei and Gardenia oudiepe, endemic to New Caledonia. Most of them are rare polymethoxylated flavones. Some of these compounds showed noticeable activity against Leishmania (Leishmania) amazonensis, Plasmodium falciparum and Trypanosoma brucei gambiense, in addition to tubulin polymerization inhibition at low micromolar concentration. We also provide a full set of NMR data as some of the flavones were incompletely described.

Bioavailability of polyphenol liposomes: a challenge ahead.

Dietary polyphenols, including flavonoids, have long been recognized as a source of important molecules involved in the prevention of several diseases, including cancer. However, because of their poor bioavailability, polyphenols remain difficult to be employed clinically. Over the past few years, a renewed interest has been devoted to the use of liposomes as carriers aimed at increasing the bioavailability and, hence, the therapeutic benefits of polyphenols. In this paper, we review the causes of the poor bioavailability of polyphenols and concentrate on their liposomal formulations, which offer a means of improving their pharmacokinetics and pharmacodynamics. The problems linked to their development and their potential therapeutic advantages are reviewed. Future directions for liposomal polyphenol development are suggested.

Evaluation of Nonradiative Clinical Imaging Techniques for the Longitudinal Assessment of Tumour Growth in Murine CT26 Colon Carcinoma.

Background and Objectives. To determine the most appropriate technique for tumour followup in experimental therapeutics, we compared ultrasound (US) and magnetic resonance imaging (MRI) to characterize ectopic and orthotopic colon carcinoma models. Methods. CT26 tumours were implanted subcutaneously (s.c.) in Balb/c mice for the ectopic model or into the caecum for the orthotopic model. Tumours were evaluated by histology, spectrofluorescence, MRI, and US. Results. Histology of CT26 tumour showed homogeneously dispersed cancer cells and blood vessels. The visualization of the vascular network using labelled albumin showed that CT26 tumours were highly vascularized and disorganized. MRI allowed high-resolution and accurate 3D tumour measurements and provided additional anatomical and functional information. Noninvasive US imaging allowed good delineation of tumours despite an hypoechogenic signal. Monitoring of tumour growth with US could be accomplished as early as 5 days after implantation with a shorter acquisition time (<5 min) compared to MRI. Conclusion. MRI and US afforded excellent noninvasive imaging techniques to accurately follow tumour growth of ectopic and orthotopic CT26 tumours. These two techniques can be appropriately used for tumour treatment followup, with a preference for US imaging, due to its short acquisition time and simplicity of use.

Lipidic spherulites: formulation optimisation by paired optical and cryoelectron microscopy.

Objective of this study was to assess the various steps leading to spherulite obtention by means of optical and cryoelectron microscopy. The formulation, resting and hydration steps were optimised. Green-based process and organic-based process were compared. It was found that spherulites could be obtained only when two key steps were followed: a prior resting phase of excipients and the shearing stress of the hydrated excipients. Moreover, the new formulation under study formed spherulites in the 100-200 nm range, which is smaller than previously reported spherulites. Such laboratory scale optimised process led the integration of spherulites in a larger number of prospective studies. Indeed, we finally showed that the encapsulated payload of a hydrophobic compound, such as the anti-angiogenic agent fisetin, was increased to a much higher degree than with a liposomal encapsulation.

Synthesis and biological evaluation of enantiomerically pure cyclopropyl analogues of combretastatin A4.

To evaluate the influence of stereochemistry on biological activities of cis-cyclopropyl combretastatin A4 (CA4) analogues, we have prepared several cyclopropyl compounds in their pure enantiomeric forms. The key reactions in our synthesis are the cyclopropanation of a (Z)-alkenylboron compound bearing a chiral auxiliary, and the cross-coupling of both enantiomeric cyclopropyl trifluoroborate salts with aryl and olefinic halides. Three pairs of cis-cyclopropyl CA4 analogues were evaluated for their potential antivascular activities. The diarylcyclopropyl compounds with SR-configuration (-)-1b, (-)-2b and the cyclopropylvinyl enantiomer (+)-3a with RR-configuration were the most potent tubulin polymerization inhibitors. A correlation was noted between anti-tubulin activity and rounding up activity of endothelial cells. The cytotoxic activity on B16 melanoma cells was in the submicromolar range for most compounds, but unlike the anti-tubulin activity, there was no difference in cytotoxic activity between racemic and enantiomerically pure forms for the three series of compounds. Molecular docking studies within the colchicine binding site of tubulin were in good agreement with the tubulin polymerization inhibitory data and confirmed the importance of the configuration of the synthesized cis-cyclopropyl CA4 analogues for potential antivascular activities.

Liposomal encapsulation of the natural flavonoid fisetin improves bioavailability and antitumor efficacy.

The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has shown antiangiogenic and anticancer properties. Because of fisetin limited water solubility, we designed a liposomal formulation and evaluated its biological properties in vitro and in Lewis lung carcinoma (LLC) bearing mice. A liposomal formulation was developed with DOPC and DODA-PEG2000, possessing a diameter in the nanometer range (173.5±2.4nm), a high homogeneity (polydispersity index 0.181±0.016) and high fisetin encapsulation (58%). Liposomal fisetin incubated with LLC cells were internalized, induced a typical fisetin morphological effect and increased the sub-G1 cell distribution. In vivo, liposomal fisetin allowed a 47-fold increase in relative bioavailability compared to free fisetin. The effect of liposomal fisetin on LLC tumor growth in mice at low dose (21mg/kg) allowed a higher tumor growth delay (3.3 days) compared to free fisetin at the same dose (1.6 day). Optimization of liposomal fisetin therapy was attempted by co-treatment with cyclophosphamide which led to a significant improvement in tumor growth delay (7.2 days) compared to cyclophosphamide with control liposomes (4.2 days). In conclusion, fisetin liposomes markedly improved fisetin bioavailability and anticancer efficacy in mice and this formulation could facilitate the administration of this flavonoid in the clinical setting.

Synthesis and in vitro evaluation of potential anticancer activity of mono- and bis-1,2,3-triazole derivatives of bis-alkynes.

In order to find new molecules with cytotoxic activity against cancer cells, we prepared bis-akyne amides derived from propiolic acid. The bis-alkynes were then transformed in their mono-1,2,3-triazole analogs onto the amide side, due to its greater reactivity, using a catalyst-free Huisgen's reaction. The mono-triazoles were then subjected to the copper (I)-catalyzed version of the previous reaction (CuAAC), using a supported catalyst, to produce bis-triazoles. All products were obtained pure after simple trituration or filtration procedures. All synthetic compounds were tested in vitro for their cytotoxic activity using B16 melanoma cells. Four compounds (7, 23, 25 and 33) showed activities in the micromolar range (<21 µM) whereas three compounds (3, 22 and 38) presented activity at low micromolar concentrations (<10 µM), and two analogs (2 and 13) were active at nanomolar levels (<1 µM).

Vascular density and endothelial cell expression of integrin alpha v beta 3 and E-selectin in murine tumours.

The endothelial cell adhesion molecules, including the integrin alpha v beta 3 (αvß3) and E-selectin, are involved in the process of angiogenesis required for tumour growth, cell migration and metastasis. The purpose of this study was to assess and compare widely used tumour models to select the ones most suitable for angiogenesis research. Fifteen murine tumours were selected including melanoma (B16), colon (C26, C38, C51), mammary (MA13, MA16, MA16/Adr, MA17, MA17/Adr, MA25, MA44), pancreatic (PO2, PO3), Glasgow osteogenic sarcoma (GOS) and Lewis lung carcinoma (LLC). The tumour vascular density, assessed using the platelet endothelial cell adhesion molecule 1 (PECAM-1; CD31) immunostaining, revealed that B16 melanoma was poorly vascularized (<5 %), whereas the colon and mammary tumours were well vascularized (5-15 %). The most vascularized tumours (>15 %) were the pancreatic tumours (PO2 and PO3), the sarcoma (GOS) and the lung tumour (LLC). The integrin αvß3 and E-selectin, evaluated by immunohistology, showed that 7/15 tumours expressed the αvß3 integrin which was homogeneously distributed on all tumour sections (B16, C26, MA17/Adr, MA25, MA44, PO2, LLC). E-selectin was expressed in 4/15 tumours and its expression was restricted to the tumour periphery. Only 2/15 tumours (B16 and C26) were shown to express both integrin αvß3 and E-selectin. In conclusion, these data not only contribute to a better understanding of the tumour biology of murine tumours but can also guide the choice of appropriate models for antiangiogenic therapy, for selective drug delivery to tumours and the validation of tumour imaging modalities targeting these endothelial cell adhesion molecules.

Colon tumor growth and antivascular treatment in mice: complementary assessment with MR elastography and diffusion-weighted MR imaging.

PURPOSE: To investigate the potential value of magnetic resonance (MR) elastography and diffusion-weighted (DW) MR imaging in the detection of microstructural changes of murine colon tumors during growth and antivascular treatment. MATERIALS AND METHODS: The study was approved by the regional ethics committee for animal care. Sixty Balb-C mice, bearing ectopic and orthotopic colon tumors, were monitored for 3 weeks with high-resolution T2-weighted MR imaging, three-dimensional steady-state MR elastography, and DW MR imaging at 7 T. The same imaging protocol was performed 24 hours after injection of combretastatin A4 phosphate (CA4P) in 12 mice. The absolute value of the complex shear modulus (|G*|) and the apparent diffusion coefficient (ADC) were measured in the viable zones of tumors and compared with microvessel density (MVD), cellularity, and micronecrosis by using the Pearson correlation coefficient. RESULTS: During tumor growth, |G*| increase was correlated with MVD (r = 0.70 [P = .08] and r = 0.78 [P = .002], for both the ectopic and orthotopic models, respectively). Moreover, the ectopic tumors displayed decreased ADC, which correlated with increased cellularity (r = 0.77, P = .04), whereas no changes in ADC and cellularity were observed in orthotopic tumors. After CA4P administration, |G*| decreased in the ectopic model (P < .0001), similar to the MVD evolution (P = .03), whereas no significant changes in |G*| (P = .7) and MVD (P = .6) were observed in the orthotopic model. ADC increased in both models (P = .047 and P = .01 for the ectopic and the orthotopic models, respectively) in relation to increased micronecrosis. CONCLUSION: Imaging of mechanical properties and diffusivity provide complementary information during tumor growth and regression that are respectively linked to vascularity and tumor cell alterations, including cellularity and micronecrosis.

In vitro and in vivo biological evaluation of new 4,5-disubstituted 1,2,3-triazoles as cis-constrained analogs of combretastatin A4.

To find new and better antivascular agents for cancer therapy, a series of combretastatin A4 (CA4) analogs were prepared from 1,3-diaryl-2-nitroprop-1-enes (6-12) obtained in a two-step synthesis from appropriate arylaldehydes and 2-aryl-1-nitroethanes (4 or 5). Treatment of these 1,3-diaryl-2-nitroprop-1-enes 6-12 by sodium azide in DMSO yielded the targeted compounds. The synthesized 1,2,3-triazoles disubstituted in 4- and 5-positions by one benzyl group and one aryl nucleus have also been tested for biological activities involved in antivascular action. It was found that several new compounds exhibited interesting biological activities in the nanomolar or low micromolar range, in terms of rounding up of endothelial cells, inhibition of tubulin polymerization, and cytotoxicity on B16 melanoma cancer cells. In silico docking studies of 11 and 19 within the active site of tubulin were also carried out in order to rationalize the inhibitory properties of these compounds and further understand their inhibition mechanism. In vivo evaluation of compounds 11 and 19 in mice bearing colon 26 carcinoma indicated modest anticancer activity.

Nanoemulsion formulation of fisetin improves bioavailability and antitumour activity in mice.

The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has shown antitumour activity but its administration is complicated by its low water solubility. Our aim was to incorporate fisetin into a nanoemulsion to improve its pharmacokinetics and therapeutic efficacy. Solubility and emulsification tests allowed to develop an optimal nanoemulsion composed of Miglyol 812N/Labrasol/Tween 80/Lipoid E80/water (10%/10%/2.5%/1.2%/76.3%). The nanoemulsion had an oil droplet diameter of 153 ± 2 nm, a negative zeta potential (-28.4 ± 0.6 mV) and a polydispersity index of 0.129. The nanoemulsion was stable at 4 °C for 30 days, but phase separation occurred at 20 °C. Pharmacokinetic studies in mice revealed that the fisetin nanoemulsion injected intravenously (13 mg/kg) showed no significant difference in systemic exposure compared to free fisetin. However, when the fisetin nanoemulsion was administered intraperitoneally, a 24-fold increase in fisetin relative bioavailability was noted, compared to free fisetin. Additionally, the antitumour activity of the fisetin nanoemulsion in Lewis lung carcinoma bearing mice occurred at lower doses (36.6 mg/kg) compared to free fisetin (223 mg/kg). In conclusion, we have developed a stable nanoemulsion of fisetin and have shown that it could improve its relative bioavailability and antitumour activity.

Development of a liposomal formulation of the natural flavonoid fisetin.

The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has been shown to possess antiangiogenic and anticancer properties. Because of the limited water solubility of fisetin, our aim was to design and optimize a liposomal formulation that could facilitate its in vivo administration, taking into account the availability and cost of the various components. Several methods were evaluated such as probe sonication, homogeneization, film hydration and lipid cake formation. A selection of lipid and lipid-PEG was also performed via their incorporation in different formulations based on the size of the liposomes, their polydispersity index (PDI) and the fisetin encapsulation yield. An optimal liposomal formulation was developed with P90G and DODA-GLY-PEG2000, possessing a diameter in the nanometer scale (175nm), a high homogeneity (PDI 0.12) and a high fisetin encapsulation (73%). Fisetin liposomes were stable over 59 days for their particle diameter and still retained 80% of their original fisetin content on day 32. Moreover, liposomal fisetin retained the cytotoxicity and typical morphological effect of free fisetin in different tumour and endothelial cell lines. In conclusion, based on its physico-chemical properties and retention of fisetin biological effects, the developed liposomal fisetin preparation is therefore suitable for in vivo administration.

Ferrocenyl chalcone difluoridoborates inhibit HIV-1 integrase and display low activity towards cancer and endothelial cells.

We report here the discovery of a potent series of HIV-1 integrase (IN) inhibitors based on the ferrocenyl chalcone difluoridoborate structure. Ten new compounds have been synthesized and were generally found to have similar inhibitory activities against the IN 3' processing and strand transfer (ST) processes. IC(50) values were found to be in the low micromolar range, and significantly lower than those found for the non-coordinated ferrocenyl chalcones and other ferrocene molecules. The ferrocenyl chalcone difluoridoborates furthermore exhibited low cytotoxicity against cancer cells and low morphological activity against epithelial cells.

Fisetin disposition and metabolism in mice: Identification of geraldol as an active metabolite.

Although the natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has been recently identified as an anticancer agent with antiangiogenic properties in mice, its in vivo pharmacokinetics and metabolism are presently not characterized. Our purpose was to determine the pharmacokinetics and metabolism of fisetin in mice and determine the biological activity of a detected fisetin metabolite. After fisetin administration of an efficacious dose of 223 mg/kg i.p. in mice, the maximum fisetin concentration reached 2.5 µg/ml at 15 min and the plasma concentration declined biphasically with a rapid half-life of 0.09 h and a terminal half-life of 3.1h. Three metabolites were detected, one of which was a glucuronide of fisetin (M1), whereas another glucuronide (M2) was a glucuronide of a previously unknown fisetin metabolite (M3). HPLC-MS/MS analysis indicated that M3 was a methoxylated metabolite of fisetin (MW=300 Da). The UV spectrum of M3 was identical to that of fisetin and standard 3,4',7-trihydroxy-3'-methoxyflavone (geraldol). In addition, because M3 co-eluted with standard geraldol in 4 different chromatographic ternary gradient conditions, M3 was therefore assigned to geraldol. Of interest, this metabolite was shown to achieve higher concentrations than fisetin in Lewis lung tumors. We also compared the cytotoxic and antiangiogenic activities of fisetin and geraldol in vitro and it was found that the latter was more cytotoxic than the parent compound toward tumor cells, and that it could also inhibit endothelial cells migration and proliferation. In conclusion, these results suggest that fisetin metabolism plays an important role in its in vivo anticancer activities.

Synthesis and structure-activity relationships of constrained heterocyclic analogues of combretastatin A4.

A series of combretastatin A4 (CA4) analogues with a lactam or lactone ring fused to the trimethoxyphenyl or the B-phenyl moiety were synthesized in an efficient and stereoselective manner by using a domino Heck-Suzuki-Miyaura coupling reaction. The vascular-disrupting potential of these conformationally restricted CA4 analogues was assessed by various in vitro assays: inhibition of tubulin polymerization, modification of endothelial cell morphology, and disruption of endothelial cell cords. Compounds were also evaluated for their growth inhibitory effects against murine and human tumor cells. B-ring-constrained derivatives that contain an oxindole ring (in contrast to compounds with a benzofuranone ring) as well as analogues bearing a six-membered lactone core fused to the trimethoxyphenyl ring are endowed with significant biological activity. The most potent compound of this series (oxindole 9 b) is of particular interest, as it combines chemical stability and a biological activity profile characteristic of a vascular-disrupting agent.

Identification and induction of cytochrome P450s involved in the metabolism of flavone-8-acetic acid in mice.

The metabolism of flavone-8-acetic acid (FAA) has been hypothesized to be partly responsible for its potent anticancer activity in mice. The purpose of this study was to identify the mouse enzymes involved in FAA Phase I metabolism and evaluate their possible induction in vivo by FAA. Mouse microsomes metabolized FAA into 6 metabolites: 3',4'-dihydrodiol-FAA, 5,6-epoxy-FAA, 4'-OH-FAA, 3'-OH-FAA, 3',4'-epoxy-FAA and 6-OH-FAA. Using Cyp-specific inhibitors (furafylline, Cyp1a2; α-naphthoflavone, Cyp1b1; tranylcypromine, Cyp2b9; quercetin, Cyp2c29; quinidine, 2d9; diethyldithiocarbamate, Cyp2e1; ketoconazole, Cyp3a11), the formation of 5,6-epoxy-FAA was mainly attributed to Cyps 1a2, 1b1, 2b9, 2c29 and 2e1, whereas the 3',4'-epoxy-FAA was formed by Cyps 2b9 and 3a11. The 4'-OH-FAA was generated by Cyps 1a2, 1b1, 2b9 and 2e1, and the 6-OH-FAA was formed by Cyps 1b1 and 2c9. Using the epoxide scavenger N-acetyl cysteine, 4'-OH-FAA, 3'-OH-FAA and 6-OH-FAA were shown to derive partly from non enzymatic isomerisation of their corresponding epoxides. The specific epoxide hydrolase inhibitor elaidamide allowed the confirmation that 3',4'-dihydrodiol-FAA was formed via the epoxide hydrolase. FAA treatment in vivo in mice led to a significant increase in the hepatic expression of Cyp1a2 (1.9-fold), 2e1 (2.1-fold), 2b10 (3.2-fold), 2d9 (2.3-fold) and 3a11 (2.2-fold), as evaluated by qRT-PCR. In conclusion, several Cyps were shown to be involved in FAA metabolism, particularly Cyps 3a11 and 2b9 which were responsible for the formation of the principal metabolites (5,6-epoxy-FAA, 3',4'-epoxy-FAA), and that FAA could induce the expression of several Cyps after in vivo administration. The possible implication of these enzymes in the in vivo anticancer activity of FAA in mice is discussed.

Improved antiangiogenic and antitumour activity of the combination of the natural flavonoid fisetin and cyclophosphamide in Lewis lung carcinoma-bearing mice.

PURPOSE: The natural flavonoid fisetin was recently identified as a lead compound that stabilizes endothelial cell microtubules. In this study, we investigated the antiproliferative and antiangiogenic properties of fisetin in vitro and in vivo. METHODS: Fisetin cytotoxicity was evaluated using Lewis lung carcinoma cells (LLC), endothelial cells and NIH 3T3 cells. Endothelial cell (EC) migration and capillary-like structure formation were evaluated using EAhy 926 cells. In vivo tumour growth inhibition studies were performed using LLC-bearing mice treated with fisetin and/or cyclophosphamide (CPA). RESULTS: The fisetin IC(50) was 59 µM for LLC and 77 µM for EC cells, compared to 210 µM for normal NIH 3T3 cells (24 h). Fisetin inhibited EC migration and capillary-like structure formation at non-cytotoxic concentrations (22-44 µM). In mice, fisetin inhibited angiogenesis assessed using the Matrigel plug assay. In LLC-bearing mice, fisetin produced a 67% tumour growth inhibition (223 mg/kg, intraperitoneal), similar to the 66% produced by low-dose CPA (30 mg/kg, subcutaneous). When fisetin and CPA were combined, however, a marked improvement in antitumour activity was observed (92% tumour growth inhibition), with low systemic toxicity. Tumour histology showed decreased microvessel density with either fisetin or CPA alone, and a dramatic decrease after the fisetin/CPA combination. CONCLUSIONS: We have shown that fisetin not only displays in vitro and in vivo antiangiogenic properties, but also can markedly improve the in vivo antitumour effect of CPA. We propose that this drug combination associating a non-toxic dietary flavonoid with a cytotoxic agent could advantageously be used in the treatment of solid tumours.

Domino approach to 2-aroyltrimethoxyindoles as novel heterocyclic combretastatin A4 analogues.

Two series of 2-aroyltrimethoxyindoles were designed to investigate the effects of the replacement of the trimethoxyphenyl ring of phenstatin with a trimethoxyindole moiety. These compounds were efficiently prepared through a domino palladium-catalyzed sequence from 2-gem-dibromovinylanilines substituted by three methoxy groups and arylboronic acids under carbon monoxide atmosphere. These novel heterocyclic combretastatin A4 analogues were evaluated for their cell growth inhibitory properties and their ability to inhibit the tubulin polymerization.