Tetrazole derivatives as potent immunomodulatory agents in tumor microenvironment.

Twenty-seven compounds bearing a tetrazole ring as a central unit have been designed, synthetized and biologically evaluated. Studies have been performed in order to compare the effect of tetrazole derivatives bearing amine electron-donor or nitro electron-acceptor groups. The antiproliferative activity has been determined in monoculture studies on tumor cell lines HT-29, A-549, MCF-7 and on non-tumor cell line HEK-293 as well as in co-culture studies (HT-29/THP-1). All the compounds have been studied as PD-L1 (Programmed Death Ligand 1), VEGFR-2 (Vascular Endothelial Growth Factor 2), CD-47 (Cluster of Differentiation 47) and c-Myc inhibitors. The effect on TNF-α secretion has also been determined. Bromoderivatives 23, 24 and chloroderivatives 26, 27 have demonstrated an apoptotic effect on HT-29 cancer cells. Compounds bearing an amine group have shown very promising effects as TME immunomodulatory agents.

Aryl azoles based scaffolds for disrupting tumor microenvironment.

Thirty-nine aryl azoles, thirteen triazoles and twenty-seven tetrazoles, have been synthetized and biologically evaluated to determine their activity as tumor microenvironment disruptors. Antiproliferative studies have been performed on tumor cell lines HT-29, A-549 and MCF-7 and on non-tumor cell line HEK-293. It has been studied in HT-29 the expression levels of biological targets which are involved in tumor microenvironment processes, such as PD-L1, CD-47, c-Myc and VEGFR-2. In addition, antiproliferative activity was evaluated when HT-29 were co-cultured with THP-1 monocytes and the secretion levels of IL-6 were also determined in these co-cultures. The angiogenesis effect of some selected compounds on HMEC-1 was also evaluated as well as their action against vasculogenic mimicry on HEK-293. Compounds bearing an amino group in the phenyl ring and a halogen atom in the benzyl ring showed promising results as tumor microenvironment disrupting agents. The most outstanding compound decrease dramatically the population of HT-29 cells when co-cultured with THP-1 monocytes and the levels of IL-6 secreted, as well as it showed moderate effects over PD-L1, CD-47 and c-Myc.

Unveiling the Potential of BenzylethyleneAryl-Urea Scaffolds for the Design of New Onco Immunomodulating Agents.

This work focuses on the development of thirteen benzylethylenearyl ureas and one carbamate. After the synthesis and purification of the compounds, we studied their antiproliferative action on cell lines, such as HEK-293, and cancer ones, such as HT-29, MCF-7 or A-549, on the immune Jurkat T-cells and endothelial cells HMEC-1. Compounds C.1, C.3, C.12 and C.14 were selected for further biological studies to establish their potential as immunomodulating agents. Some of the derivatives exhibited significant inhibitory effects on both targets: PD-L1 and VEGFR-2 in the HT-29 cell line, showing that urea C.12 is active against both targets. Some compounds could inhibit more than 50% of cancer cell proliferation compared to non-treated ones when assessed in co-cultures using HT-29 and THP-1 cells. In addition, they significantly reduced CD11b expression, which is a promising target for immune modulation in anticancer immunotherapies.

Exploring BenzylethoxyAryl Urea Scaffolds for Multitarget Immunomodulation Therapies.

Thirteen benzylethoxyaryl ureas have been synthesized and biologically evaluated as multitarget inhibitors of VEGFR-2 and PD-L1 proteins to overcome resistance phenomena offered by cancer. The antiproliferative activity of these molecules on several tumor cell lines (HT-29 and A549), on the endothelial cell line HMEC-1, on immune cells (Jurkat T) and on the non-tumor cell line HEK-293 has been determined. Selective indexes (SI) have been also determined and compounds bearing p-substituted phenyl urea unit together with a diaryl carbamate exhibited high SI values. Further studies on these selected compounds to determine their potential as small molecule immune potentiators (SMIPs) and as antitumor agents have been performed. From these studies, we have concluded that the designed ureas have good tumor antiangiogenic properties, exhibit good inhibition of CD11b expression, and regulate pathways involved in CD8 T-cell activity. These properties suggest that these compounds could be potentially useful in the development of new cancer immune treatments.

Synthesis and Biological Evaluation of Potential Oncoimmunomodulator Agents.

Fourteen triazole-scaffold derivatives were synthetized and biologically evaluated as potential oncoimmunomodultator agents by targeting both PD-L1 and c-Myc. First, the antiproliferative activity of these molecules on the monocultures of several tumor cell lines (HT-29, A-549, and MCF-7) and on the non-tumor cell line HEK-293 was studied. Then, the effects on the mentioned biological targets were also evaluated. Finally, the effect on cancer cell viability when the molecules were co-cultured with immune cells (Jurkat T cells or THP-1) was also determined. Compounds bearing a bromoophenyl group were selected because of their excellent results, and their effect on IL-6 secretion was also studied. In conclusion, we found compounds that are capable of downregulating c-Myc, as well as influencing and altering the distribution of PD-L1 in tumor cells; the compounds are thus capable of influencing the behavior of defensive cells towards cancer cells. p-Bromophenyltriazol 3 is the most active of these as a PD-L1 and c-Myc downregulator and as a potential immunomodulator agent. Moreover, it exhibits an interesting action on inflammation-related cytokine IL-6.

Synthesis and Biological Evaluation of Small Molecules as Potential Anticancer Multitarget Agents.

Twenty-six triazole-based derivatives were designed for targeting both PD-L1 (programmed death receptor ligand 1) and VEGFR-2 (vascular endothelial growth factor receptor 2). These compounds were synthetized and biologically evaluated as multitarget inhibitors of VEGFR-2, PD-L1 and c-Myc proteins. The antiproliferative activity of these molecules on several tumor cell lines (HT-29, A-549, and MCF-7) and on the non-tumor cell line HEK-293 was determined. The effects on the abovementioned biological targets were evaluated for some selected compounds. Compound 23, bearing a p-chlorophenyl group, showed better results than sorafenib in regard to the downregulation of VEGFR-2 and a similar effect to BMS-8 on both PD-L1 and c-Myc proteins. The antiangiogenic and antivascular activities of chloro derivatives were also established by endothelial microtube formation assay on Matrigel®.

The widespread presence of a family of fish virulence plasmids in Vibrio vulnificus stresses its relevance as a zoonotic pathogen linked to fish farms.

Vibrio vulnificus is a pathogen of public health concern that causes either primary septicemia after ingestion of raw shellfish or secondary septicemia after wound exposure to seawater. In consequence, shellfish and seawater are considered its main reservoirs. However, there is one aspect of its biology that is systematically overlooked: its association with fish in its natural environment. This association led in 1975 to the emergence of a zoonotic clade within phylogenetic lineage 2 following successive outbreaks of vibriosis in farmed eels. Although this clade is now worldwide distributed, no new zoonotic clades were subsequently reported. In this work, we have performed phylogenetic, genomic and functional studies to show that other zoonotic clades are in fact present in 4 of the 5 lineages of the species. Further, we associate these clades, most of them previously but incompletely described, with the acquisition of a family of fish virulence plasmids containing genes essential for resistance to the immune system of certain teleosts of interest in aquaculture. Consequently, our results provide several pieces of evidence about the importance of this species as a zoonotic agent linked to fish farms, as well as on the relevance of these artificial environments acting as drivers that accelerate the evolution of the species.

Aryl Urea Based Scaffolds for Multitarget Drug Discovery in Anticancer Immunotherapies.

Twenty-one styryl and phenethyl aryl ureas have been synthetized and biologically evaluated as multitarget inhibitors of Vascular endothelial growth factor receptor-2 VEGFR-2 and programmed death-ligand-1 (PD-L1) proteins in order to overcome resistance phenomena offered by cancer. The antiproliferative activity of these molecules on several tumor cell lines (HT-29, MCF-7, HeLa and A549), on the endothelial cell line human microvascular endothelial cells (HMEC)-1 and on the non-tumor cell line human embryonic kidney cells (HEK)-293 has been determined. Some derivatives were evaluated for their antiangiogenic properties such as their ability to inhibit microvessel formation using HMEC-1 or their effect on VEGFR-2 in both cancer and endothelial cell lines. In addition, the immunomodulator action of a number of selected compounds was also studied on PD-L1 and c-Myc proteins. Compounds 16 and 23 (Z) and (E)-styryl p-bromophenyl urea, respectively, showed better results than sorafenib in down-regulation of VEGFR-2 and also improved the effect of the anti-PD-L1 compound BMS-8 on both targets, PD-L1 and c-Myc proteins.

Synthesis of N-acyl Derivatives of Aminocombretastatin A-4 and Study of their Interaction with Tubulin and Downregulation of c-Myc.

BACKGROUND: Six N-acyl derivatives of aminocombretastatin A-4 have been synthesized and evaluated according to their interaction with tubulin and as c-Myc downregulators. AIMS: In search of new promising anti-cancer agents. OBJECTIVE: This study is focused on the synthesis and the biological evaluation of N-acyl derivatives of aminocombretastatin A-4 (CA-4). Docking studies were carried out to find out whether the synthetic derivatives could bind to tubulin at the colchicine site in a conformation similar to that of CA- 4. The synthetic derivatives' effect on the proliferation of several cancer cells and non-cancer cells has been measured. Their effect on tubulin polymerization, cell cycle distribution, the microtubule network and c-Myc expression has also been evaluated. METHODS: A set of six N-acyl derivatives was achieved by means of a peptide-type coupling of aminocombretastatin A-4 and the corresponding carboxylic acid. The synthetic compounds' ability to inhibit cell proliferation was measured by MTT assay against three human carcinoma cell lines (colorectal HT-29, lung A549, and breast adenocarcinoma MCF-7) and one non-tumor cell line (HEK- 293). Turbidimetry time-course measurements evaluated the inhibition of tubulin polymerization. The action of the synthetic derivatives on cell cycle distribution was measured by flow cytometry and their effects on the microtubule network were determined by immunofluorescence microscopy. Finally, the downregulation of the synthetic derivatives on c-Myc protein was quantified by ELISA assay, while the effect on c-Myc gene was measured by RT-qPCR analysis. RESULTS: Derivatives bearing pentanoyl (compound 2), hexanoyl (compound 3), and heptanoyl (compound 4) side chains show anti-proliferative activities on the HT-29 line in the low nanomolar range, with values similar to that exhibited by AmCA-4 but far exceeding those of CA-4. Compounds 1 (butanoyl side chain) and 2-3 inhibit tubulin polymerization in vitro in a manner similar to that of CA-4 and AmCA-4 whereas compounds 4, 5 (octanoyl side chain) and 6 (dodecanoyl side chain) may be considered as partial inhibitors of tubulin polymerization. While all derivatives are able to accumulate cells in G2/M phase, compounds with the longest acyl chains (5 and 6) are the least active ones in this particular action. Moreover, compounds 2-3 were the most active ones as c-Myc downregulators. CONCLUSION: Our studies show that the most active compounds in the disruption of the microtubule network are also the most potent ones in the downregulation of c-Myc expression. Other: Compounds 2 and 3 are good candidates for in vivo studies as they combine the best antimitotic and c-Myc downregulation activities at low doses.

N-alpha-Aminoacyl Colchicines as Promising Anticancer Agents.

BACKGROUND: In the last years, many efforts have been made to find colchicine derivatives with reduced toxicity. Additionally, the deregulation of amino acid uptake by cancer cells provides an opportunity to improve anticancer drug effectiveness. OBJECTIVE: To design new colchicine derivatives with reduced cytotoxicity and enhanced selectivity by means of introducing aminoacyl groups. METHODS: 34 colchicine analogues bearing L- and D-amino acid pendants were synthetized and characterized by NMR, IR and MS techniques. Cytotoxicity and antimitotic properties were assessed by spectrophotometry and cell cycle assays. Oncogene downregulation was studied by RTqPCR whereas in vivo studies were performed in SCID mice. RESULTS: Compounds exhibit high antiproliferative activities at the nanomolar level while being, in general, less cytotoxic than colchicine. Most compounds inhibit the polymerization of tubulin in a way similar to colchicine itself, with L-amino acid derivatives being the most active in the inhibition of tubulin polymerization. All selected compounds caused cell cycle arrest at the G2/M phase when tested at 1 µM. More specifically, Boc-L-proline derivative 6 arrested half of the population and showed one of the highest Selectivity Indexes. Derivatives 1 (Boc-glycine), 27 (D-leucine) and 31 (Boc-glycine-glycine) proved fairly active in downregulating the expression of the c-Myc, hTERT and VEGF oncogenes, with compound 6 (Boc-L-proline) having the highest activity. This compound was shown to exert a potent anti-tumor effect when administered intraperitoneally (LD50 > 100 mg/kg for 6, compared with 2.5 mg/kg for colchicine). CONCLUSION: Compound 6 offers an opportunity to be used in cancer therapy with less toxicity problems than colchicine.

Synthesis of Combretastatin A-4 and 3'-Aminocombretastatin A-4 derivatives with Aminoacid Containing Pendants and Study of Their Interaction with Tubulin and as Downregulators of the VEGF, hTERT and c-Myc Gene Expression.

Natural product combretastatin A-4 (CA-4) and its nitrogenated analogue 3'-aminocombretastatin A-4 (AmCA-4) have shown promising antitumor activities. In this study, a range of CA-4 and AmCA-4 derivatives containing amino acid pendants have been synthesized in order to compare their biological actions with those of their parent compounds. Thus, inhibition of cell proliferation on tumor cell lines HT-29, MCF-7 and A-549, as well as on the nontumor cell line HEK-273; in vitro tubulin polymerization; mitotic cell arrest; action on the microtubule cell network and inhibition of VEGF, hTERT, and c-Myc genes have been evaluated. Some AmCA-4 derivatives bearing L-amino acids exhibited inhibition of cell proliferation at low nanomolar levels exceeding the values shown by AmCA-4. Furthermore, while CA-4 and AmCA-4 derivatives do not show significant effects on the in vitro tubulin polymerization and cell cycle arrest, some selected CA-4 and AmCA-4 derivatives are able to cause total depolymerization of the microtubule network on A-549 cells. The best results were obtained in the inhibition of gene expression, particularly on the VEGF gene, in which some AmCA-4 derivatives greatly exceeded the inhibition values achieved by the parent compound.

Novel multitarget inhibitors with antiangiogenic and immunomodulator properties.

By means of docking studies, seventeen compounds T.1-T17 have been designed and evaluated as multitarget inhibitors of VEGFR-2 and PD-L1 proteins in order to overcome resistance phenomena offered by cancer. All these designed molecules display a urea moiety as a common structural feature and eight of them (T.1-T8) further contain a 1,2,3-triazol moiety. The antiproliferative activity of these molecules on several tumor cell lines (HT-29, MCF-7, HeLa, A549, HL-60), on the endothelial cell line HMEC-1 and on the non-tumor cell line HEK-293 has been determined. The urea derivatives were also evaluated for their antiangiogenic properties, whereby their ability to inhibit tubulogenesis and kinase activity employing flow cytometry, ELISA, immunofluorescence and western blot techniques was measured. In addition, these techniques were also employed to investigate the immunomodulator action of the synthetic compounds on the inhibition of PD-L1 and c-Myc proteins. Compound T.2, 1-(3-chlorophenyl)-3-(2-(4-(4-methoxybenzyl)-1H-1,2,3-triazol-1-yl)ethyl)urea, has shown similar results to sorafenib in both down-regulation of VEGFR-2 and inhibition of the kinase activity of this receptor. Furthermore, compound T.14, (E)-1-(4-chlorophenyl)-3-(3-(4-methoxystyryl)phenyl)urea, improves the effect of T.2 as regards tube formation of endothelial cells and inhibition of VEGFR-2 tyrosine kinase activity. In addition, T.14 improves the effect of the experimental drug BMS-8 in the inhibition of PD-L1 and c-Myc proteins.

Arylpyridines, arylpyrimidines and related compounds as potential modulator agents of the VEGF, hTERT and c-Myc oncogenes.

Twenty-four derivatives structurally related to honokiol have been synthesized and biologically evaluated. IC50 values were determined towards the HT-29, MCF-7 and HEK-293 cell lines. Some of these derivatives exhibited comparable or lower IC50 values than honokiol towards the HT-29 and MCF-7 cell lines or else higher selectivity indexes than the natural product. Twelve selected derivatives were evaluated for their ability to inhibit the expression of the VEGFA, hTERT and c-Myc genes and also to inhibit the production of total c-Myc protein and the secretion of the VEGF protein. One of the most promising compounds, 3-(2,4-dimethoxyphenyl)pyridine, may be a good candidate for further studies as an anticancer agent as it is able to improve the effect shown by honokiol in downregulating all gene expression and protein production at a safe concentration for non-tumor cells.

Synthesis and in-vitro Evaluation of S-allyl Cysteine Ester - Caffeic Acid Amide Hybrids as Potential Anticancer Agents.

We have synthesized a series of S-allyl cysteine ester-caffeic acid amide hybrids and evaluated them in order to determine their possible anticancer activity and selectivity in colorectal cancer, which is still one of the leading causes of morbidity and mortality worldwide. All compounds were tested against SW480 human colon adenocarcinoma cells and the non-malignant CHO-K1 cell line. Among the tested compounds, hybrids 6e, 9a, 9b, 9c, and 9e exhibited the highest effect on viability (IC50 SW480-48h= 0.18, 0.12, 0.12, 0.11, and 0.12 mM, respectively) and selectivity (SI = 10.3, 1.5, >83.33, >90.91 and >83.33, respectively) in a time- and concentration-dependent manner. Besides, our results were even better as regards lead compounds (S-allyl cysteine and caffeic acid) and the standard drug (5-FU). Additionally, these five compounds induced mitochondrial depolarization that could be related with an apoptotic process. Moreover, hybrids 6e, 9a, and 9e induced cell cycle arrest in G2/M phase, and compound 9c in S- phase, which suggests that these hybrid compounds could have also a cytostatic effect in SW480 cell line. The SAR analysis showed that hydroxyl groups increased the activity. Besides, there was not a clear relationship between the antitumor properties and the length of the alkyl chain. Since hybrid compounds were much more selective than the conventional drug (5-FU), this makes them promising candidates for further studies against colorectal cancer.

New N,C-Diaryl-1,2,4-triazol-3-ones: Synthesis and Evaluation as Anticancer Agents.

BACKGROUND: A set of 2,5-diaryl-1,2,4-triazol-3-ones was synthesized in two steps and evaluated as regards their activity in some relevant biological targets related to cancer. OBJECTIVE: This study is focused on the synthesis and the biological evaluation of 2,5-diaryl-1,2,4- triazol-3-ones. In this sense, the effect of the synthetic triazolones on the proliferation of HT-29 and A549 cancer cells and on HEK non-cancer cells has been measured. In addition, the effects of triazolones on the expression of hTERT, c-Myc and PD-L1 genes and on the production of c-Myc and PD-L1 proteins have also been evaluated. METHOD: A set of 2,5-diaryl-1,2,4-triazol-3-ones was synthesized in two steps. Firstly, N- (aminocarbonyl)-3-methoxybenzamide was prepared by coupling 3-methoxybenzoic acid and cyanamide followed by aqueous HCl hydrolysis. Then, the 2,5-diaryl-1,2,4-triazol-3-ones were obtained upon reaction of N-(aminocarbonyl)-3-methoxybenzamide with arylhydrazines in decaline at 170ºC. The ability of the triazolones to inhibit cell proliferation was measured against two human carcinoma cell lines (colorectal HT-29 and lung A549), and one non-tumor cell line (HEK- 293) by MTT assay. The downregulation of the synthetic triazolones on the expression of the hTERT, c-Myc and PD-L1 genes was measured by an RT-qPCR analysis. Their ability to regulate the expression of the c-Myc and PD-L1 proteins, as well as their direct interaction with c-Myc protein, was determined by the ELISA method. Finally, the direct interaction of triazolones with PD-L1 protein was assessed by the thermal shift assay. RESULTS: Ten 2,5-diaryl-1,2,4-triazol-3-ones were synthesized and characterized by spectroscopic methods. A thorough study by 1H, 13C, 15N and 19F NMR spectroscopy showed that all the synthetic compounds exist as 4H-triazolones and not as hydroxytriazoles or 1H-triazolones. Some triazolones showed relatively high activities together with very poor toxicity in non-tumor cell line HEK-293. 2-(2-fluorophenyl)-5-(3-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (4) was particularly active in downregulating c-Myc and PD-L1 gene expression although 2-(4- chloro-2-fluorophenyl)-5-(3-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (8) is the one that combines the best downregulatory activities in the three genes studied. Considering protein expression, the most active compounds are 2-(4-fluorophenyl)-5-(3-methoxyphenyl)-2,4-dihydro- 3H-1,2,4-triazol-3-one (5) and 2-(2,4,6-trifluorophenyl)-5-(3-methoxyphenyl)-2,4-dihydro-3H- 1,2,4-triazol-3-one (10) (c-Myc expression) and 2-(2,3,5,6-tetrafluorophenyl)-5-(3-methoxyphenyl)- 2,4-dihydro-3H-1,2,4-triazol-3-one (11) and (8) (PD-L1 expression). CONCLUSION: Some of the triazolones studied have shown relevant activities in the inhibition of the hTERT, c-Myc and PD-L1 genes, and in the inhibition of c-Myc and PD-L1 protein secretion, the 2-(4-chloro-2-fluorophenyl)-5-(3-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazol-3-one (8) was found to be a particularly promising lead compound.

Synthesis and biological evaluation as antiangiogenic agents of ureas derived from 3'-aminocombretastatin A-4.

Twenty-six compounds derived from 3'-aminocombretastatin A-4 (AmCA-4) containing a urea fragment mimicking the structure of Sorafenib, have been synthesized and evaluated as antiangiogenic compounds. Antiproliferative activity of all the synthetic ureas has been measured on tumor cell lines HT-29, MCF-7, HeLa, A-549 and HL-60 as well as on the endothelial cell line HMEC-1 and on the non-tumor cell line HEK-293. Preliminary docking studies were developed in order to predict which ureas show better interactions with the protein VEGFR-2. Then, the selected derivatives were evaluated in terms of their apoptotic effect and antiangiogenic properties. In this regard, VEGFR-2/ligand interactions were determined by flow cytometry and immunofluorescence techniques. Inhibition of VEGFR-2 tyrosine kinase activity in both the A-549 and HMEC-1 cell lines was also carried out. In addition, tube formation inhibition was studied in endothelial cells. Ortho-chloro substituted urea 5 and ortho-bromo substituted urea 8 were the most active ones in both down-regulation of VEGFR-2 and inhibition of the kinase activity of this receptor, with better results than those obtained with sunitinib and sorafenib.

Synthesis and biological evaluation of cyclic derivatives of combretastatin A-4 containing group 14 elements.

Several tricyclic compounds inspired by the structure of combretastatin A-4 and bearing group 14 elements have been synthesized by homocoupling lithiated aryl fragments followed by ring-closing metathesis. These tricyclic compounds and their diolefin precursors were evaluated for their antiproliferative action on the tumor cell lines HT-29, MCF-7, HeLa and A-549 and on the non-tumor cell line HEK-293. In addition, their effects on the cell cycle were also measured. The tricyclic compounds show antiproliferative activity similar to that of combretastatin A-4, even though they are not so active in arresting the cell cycle. However, some diolefin precursors are able to cause accumulation of cells in the G2/M phase in a higher percentage than combretastatin A-4 itself. Inhibition of endothelial tube formation and VEGFR-2 phosphorylation of some selected compounds is comparable to that of combretastatin A-4, particularly those of tin-containing compounds 23c and 26c, whose actions exceed those of sorafenib, a clinically used VEGFR-2 inhibitor.

Arylureas derived from colchicine: Enhancement of colchicine oncogene downregulation activity.

Our efforts to get therapeutically useful colchicine derivatives for the treatment of cancer have led us to synthetize and biologically evaluate twenty-seven N,N'-disubstituted ureas containing a colchicine moiety and an aryl fragment. The cytotoxicity of the compounds, their ability to inhibit the expression of oncogenes related to telomerase activation and to the VEGF/VEGFR-2 autocrine process, such as c-MYC, hTERT and VEGF and their capability to downregulate c-MYC and VEGFR-2 proteins and the secretion of VEGF have been measured. In these biological evaluations, we have found that the change of the acetyl group in colchicines for an N-arylurea unit causes a great improvement in anticancer properties. The most promising derivatives were compounds 6 (o-Cl) and 14 (o,o-di-F) as they were able to downregulate all the tested targets at a concentration below their IC50 values. Thus, the arylurea unit enhances the potential of colchicine as an anticancer agent.

Effects on tubulin polymerization and down-regulation of c-Myc, hTERT and VEGF genes by colchicine haloacetyl and haloaroyl derivatives.

Several colchicine analogues in which the N-acetyl residue has been replaced by haloacetyl, cyclohexylacetyl, phenylacetyl and various aroyl moieties have been synthesized. The cytotoxic activities of the synthesized compounds have been measured on three tumor cell lines (HT-29, MCF-7 and A549) and on one non-tumor cell line (HEK-293). These compounds exhibit high antiproliferative activities at the nanomolar level, in many cases with a higher potency than colchicine itself. Some of the compounds, particularly the haloacetyl derivatives, inhibit the polymerization of tubulin in a similar manner as colchicine. As regards the cell cycle, the most active compounds are the chlorobenzoyl and bromobenzoyl derivatives, which cause cell cycle arrest at the G2/M phase when tested at 20 nM, and the bromoacetyl derivative, which arrests the cell cycle at 15 nM. In addition, these colchicine derivatives have shown fairly active downregulating the expression of the c-Myc, hTERT and VEGF genes, as well as VEGF protein secretion, at very low concentrations.

Synthesis and biological evaluation of carbamates derived from aminocombretastatin A-4 as vascular disrupting agents.

A series of twenty-six carbamates derived from aminocombretastatin A-4 (AmCA-4) were synthesized and evaluated for their capacity to affect cell proliferation, tubulin polymerization, mitotic cell arrest, microtubule network organization, apoptosis and endothelial tubular structures in vitro. The anti-proliferative activity of the synthetic carbamates was measured on several human tumor cell lines (i.e. HT-29, MCF-7, HeLa, A-549, MDA-MB-231, HL-60) as well as on the endothelial cell line HMEC-1 and the non-tumor cell line HEK-293. The compounds showed anti-proliferative activity in the nanomolar range thereby exceeding by far the activity of combretastatin A-4 (CA-4) and, in some cases, the activity of AmCA-4. The most active compounds proved to be the carbamates bearing chloro, bromo or methoxy groups in the meta position of the phenyl ring. Moreover, all carbamates inhibited in vitro tubulin polymerization, in a similar manner to that of CA-4 and AmCA-4 by interacting with the colchicine binding site in tubulin. The synthetic carbamates proved as active as AmCA-4 in causing mitotic arrest, as assessed in A549 human lung cancer cells, and disruption of the microtubule cellular network. Some selected carbamates induced apoptosis at concentrations as low as 10 nM, being more active than AmCA-4. Finally, these selected carbamates displayed a vascular disrupting activity on endothelial cells in a dose-dependent manner. In conclusion, our data indicate that carbamates derived from aminocombretastatin A-4 represent interesting lead compounds for the design of vascular disrupting agents.