Arylamidonaphtalene sulfonate compounds as a novel class of heparanase inhibitors.

The search for antimetastatic agents for cancer therapy may involve the ability of new compounds to maintain the tissue extracellular matrix integrity. Among known factors, heparanase, an endoglucuronidase responsible for heparan sulfate cleavage, is a promising target whose inhibition could represent a strong obstacle for metastatic cancerous mechanisms. The antimetastatic activity of some suramin derivatives reported in literature suggests a possible involvement of the heparanase enzyme. To confirm such hypothesis, we have investigated FCE27266, a molecule known for its antiangiogenic and antimetastatic properties. Other new derivatives were also synthesized and investigated. Our findings revealed that FCE27266 as well as some derivatives have a strong heparanase inhibition activity, together with no cytotoxic power. Moreover, a FCE27266 analogue (SST0546NA1; 17a) resulted also positive to lower gene expression of some proangiogenic factors.

Inhibition of activated STAT5 in Bcr/Abl expressing leukemia cells with new pimozide derivatives.

STATs are transcription factors acting as intracellular signaling after stimulation with cytokines, growth factors and hormones. STAT5 is also constitutively active in many forms of cancers, including chronic myelogenous leukemia, acute lymphoblastic leukemia and Hodgkin's lymphoma. Recently, literature reported that the neuroleptic drug pimozide inhibits STAT5 phosphorylation inducing apoptosis in CML cells. We undertook an investigation from pimozide structure, obtaining simple derivatives with cytotoxic and STAT5-inhibitory activity, two of them markedly more potent than pimozide.

Development of hemiasterlin derivatives as potential anticancer agents that inhibit tubulin polymerization and synergize with a stilbene tubulin inhibitor.

Hemiasterlins are cytotoxic tripeptides with antimicrotubule activity originally isolated from marine sponges. We have developed new hemiasterlin derivatives BF65 and BF78 that are highly potent to induce cancer cell death in the low nanomolar range. Examination of their mechanisms of cell cycle arrest and disruption of microtubules revealed an unusual characteristic in addition to anti-tubulin effect. Immunofluorescence staining revealed that A549 lung carcinoma cells treated with BF65 or BF78 exhibited both monopolar and multipolar mitotic spindles. Centrosomes were separated with short spindle microtubules in cells with multipolar spindles. In vitro tubulin polymerization assay confirmed that both BF65 and BF78 were highly potent to inhibit tubulin polymerization. These two compounds induced the formation of monoastral spindles suggesting that they might be inhibitors of mitotic kinesins such as KSP/Eg5. However, kinetic measurement of microtubule activated kinesin ATPase activity demonstrated that unlike the positive control monastrol, neither BF65 nor BF78 suppressed KSP/Eg5 activity. Hence the effect may be a variant form of tubulin inhibition. Similar to vinca alkaloids, BF compounds synergized with a colchicine site microtubule inhibitor stilbene 5c both in vitro and in vivo, which may provide a potential drug combination in the future clinical application.

Synthesis and biological activity of a novel class nicotinic acetylcholine receptors (nAChRs) ligands structurally related to anatoxin-a.

The introduction of the isoxazole ring as bioisosteric replacement of the acetyl group of anatoxin-a led to a new series of derivatives binding to nicotinic acetylcholine receptors. Bulkier substitutions than methyl at the 3 position of isoxazole were shown to be detrimental for the activity. The binding potency of the most interesting compounds with α1, α7 and α3ß4 receptor subtypes, was, anyway, only at micromolar level. Moreover, differently from known derivatives with pyridine, isoxazole condensed to azabicyclo ring led to no activity.

Versatile synthesis of new cytotoxic agents structurally related to hemiasterlins.

A representative series of structural analogues of the antimitotic tripeptides hemiasterlins have been synthesized. The key-step of this synthetic strategy consists of an Ag(2)O-promoted nucleophilic substitution on a common precursor, a chiral non-racemic 2-bromoacyl derivative. Simple variation of nucleophile substituents allows a rapid and stereocontrolled development of new series of derivatives. Some reported compounds showed potent biological activity as growth inhibitors of cancer cell lines and tubulin polymerization inhibitors.

Pro-apoptotic activity of novel synthetic isoxazole derivatives exhibiting inhibitory activity against tumor cell growth in vitro.

In order to develop potential anticancer agents stimulating apoptosis, novel 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives have been synthetized. The original structures of geldanamycin and radicicol, which are known natural heat shock protein (HSP) inhibitors, were deeply modified because both of them exhibit several drawbacks, such as poor solubility, hepatotoxicity, intrinsic chemical instability or deprivation of the in vivo activity. This novel class of synthetic compounds containing the isoxazole nucleus exhibited potent and selective inhibition of HSP90 in previous studies. Biological assays (focusing on in vitro antiproliferative effects and pro-apoptotic activity) in human erythroleukemic K562 cells (as a model system referring to tumor cells grown in suspension), glioblastoma U251-MG and glioblastoma temozolomide (TMZ)-resistant T98G cell lines (two model systems referring to tumor cells grown attached to the flask), were performed. Almost all isoxazole derivatives demonstrated significant antiproliferative and pro-apoptotic activities, showing induction of both early and late apoptosis of K562 cells. Different effects were observed on the glioma U251-MG and T98G cells, depending on the structure of the analogues. Antiproliferative and pro-apoptotic activities in K562 cells were associated with the activation of the erythroid differentiation program. The present study demonstrated that 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives should be considered for in vivo studies focusing on the development of anticancer drugs acting, at least partially, via activation of apoptosis.

Inhibition of cell proliferation by a resveratrol analog in human pancreatic and breast cancer cells.

Resveratrol has been reported to possess cancer preventive properties. In this study, we analyzed anti-tumor activity of a newly synthesized resveratrol analog, cis-3,4',5-trimethoxy-3'-hydroxystilbene (hereafter called 11b) towards breast and pancreatic cancer cell lines. 11b treatments reduced the proliferation of human pancreatic and breast cancer cells, arrested cells in the G2/M phase, and increased the percentage of cells in the subG1/G0 fraction. The 11b treatments also increased the total levels of mitotic checkpoint proteins such as BubR1, Aurora B, Cyclin B, and phosphorylated histone H3. Mechanistically, 11b blocks microtubule polymerization in vitro and it disturbed microtubule networks in both pancreatic and breast cancer cell lines. Computational modeling of the 11b-tubulin interaction indicates that the dimethoxyphenyl group of 11b can bind to the colchicine binding site of tubulin. Our studies show that the 11b treatment effects occur at lower concentrations than similar effects associated with resveratrol treatments and that microtubules may be the primary target for the observed effects of 11b. These studies suggest that 11b should be further examined as a potentially potent clinical chemotherapeutic agent for treating pancreatic and breast cancer patients.

Design, synthesis and biological evaluation of novel stilbene-based antitumor agents.

A series of novel stilbene derivatives has been synthesized and studied with the main goal to investigate SAR of the amino compound 1a, as well as to improve its water solubility, a potentially negative aspect of the molecule that could be a serious obstacle for a pre-clinical development. We have obtained derivatives with good cytotoxic activity, in particular, the derivatives 5c and 6b could represent two novel leads for further investigation. Compound 8b, a morpholino-carbamate derivative, prodrug of 1a, has a very good solubility in water, and is active in suppressing growth of tumor cells at a concentration of 5000 nM, which is a concentration 100 times higher than the parent stilbene 1a.

Hydropathic analysis and biological evaluation of stilbene derivatives as colchicine site microtubule inhibitors with anti-leukemic activity.

The crucial role of the microtubule in cell division has identified tubulin as a target for the development of therapeutics for cancer; in particular, tubulin is a target for antineoplastic agents that act by interfering with the dynamic stability of microtubules. A molecular modeling study was carried out to accurately represent the complex structure and the binding mode of a new class of stilbene-based tubulin inhibitors that bind at the alphabeta-tubulin colchicine site. Computational docking along with HINT (Hydropathic INTeractions) score analysis fitted these inhibitors into the colchicine site and revealed detailed structure-activity information useful for inhibitor design. Quantitative analysis of the results was in good agreement with the in vitro antiproliferative activity of these derivatives (ranging from 3 nM to 100 muM) such that calculated and measured free energies of binding correlate with an r(2) of 0.89 (standard error +/- 0.85 kcal mol(-1)). This correlation suggests that the activity of unknown compounds may be predicted.

Antitumor effects of curcumin and structurally beta-diketone modified analogs on multidrug resistant cancer cells.

Using concepts of bioisostery a series of curcumin analogs were synthesized: the diketonic system of the compound was elaborated into enaminones, oximes, and the isoxazole heterocycle. The cell growth inhibitory and apoptosis inducing effects of the new analogs were evaluated by in vitro assays in the hepatocellular carcinoma HA22T/VGH cells, as well as in the MCF-7 breast cancer cell line and in its multidrug resistant (MDR) variant MCF-7R. Increased antitumor activity on all cell lines was found with the isoxazole analog and especially with the benzyl oxime derivative; in the HA22T/VGH cell model, the latter compound inhibited constitutive NF-kappaB activation.

Novel combretastatin analogues endowed with antitumor activity.

We studied the anticancer activity of a series of new combretastatin derivatives with B-ring modifications. The structure-activity relationship (SAR) information confirmed the importance of cis-stereochemistry and of a phenolic moiety in B-ring. We selected the benzo[b]thiophene and benzofuran combretastatin analogues 11 (ST2151) and 13 (ST2179) and their phosphate prodrugs (29 and 30) for their high antitumor activity in in vitro and in vivo models. Cell exposure to IC50 of 11, 13, and CA-4 led to the arrest of various cell types in the G2/M phase of the cell cycle and induction of apoptosis. Mainly, 11 and 13 induced the formation of multinucleated cells with abnormal chromatin distribution, with only a minimal effect on the microtubule organization, with respect to CA-4. Interestingly, both the pharmacokinetic profile of 29 and its in vivo antitumor effect and those of 30, active even after oral administration, suggest additional pharmacological differences between these compounds and CA-4P.

A novel hybrid drug between two potent anti-tubulin agents as a potential prolonged anticancer approach.

We report the design, synthesis and biological characterisation of a novel hybrid drug by conjugation of two tubulin inhibitors, a hemiasterlin derivative A (H-Mpa-Tle-Aha-OH), obtained by condensation of three non-natural amino acids, and cis-3,4',5-trimethoxy-3'aminostilbene (B). As we have previously demonstrated synergy between A and B, we used a monocarbonyl derivative of triethylene glycol as linker (L) to synthesise compounds A-L and A-L-B; via HPLC we analysed the release of its potential hydrolysis products A, A-L, B and B-L in physiological fluids: the hybrid A-L-B undergo hydrolysis in rat whole blood of the ester bond between A and L (half-life=118.2±9.5min) but not the carbamate bond between B and L; the hydrolysis product B-L was further hydrolyzed, but with a slower rate (half-life=288±12min). The compound A-L was the faster hydrolyzed conjugate (half-life=25.4±1.1min). The inhibitory activity of the compounds against SKOV3 ovarian cancer cell growth was analysed. The IC50 values were 7.48±1.27nM for A, 40.3±6.28nM for B, 738±38.5nM for A-L and 37.9±2.11nM for A-L-B. The anticancer effect of A-L-B was evidenced to be obtained via microtubule dynamics suppression. Finally, we stated the expression of the active efflux transporters P-gp (ABCB1) and MRP1 (ABCC1) in the human normal colon epithelial NCM460 cell line by reverse-transcription PCR. Via permeation studies across NCM460 monolayers we demonstrate the poor aptitude of A to interact with active efflux transporters (AET): indeed, the ratio between its permeability coefficients for the basolateral (B)→apical (A) and B→A transport was 1.5±0.1, near to the ratio of taltobulin (1.12±0.06), an hemiasterlin derivative able to elude AETs, and significantly different form the ratio of celiprolol (3.4±0.2), an AET substrate.

Hemiasterlin derivative (R)(S)(S)-BF65 and Akt inhibitor MK-2206 synergistically inhibit SKOV3 ovarian cancer cell growth.

We reported previously that a hemiasterlin derivative BF65 is a potent anticancer agent that can inhibit microtubule assembly. Here we show that a more potent stereospecific diastereomer (R)(S)(S)-BF65 can synergize with an allosteric Akt inhibitor MK-2206 to suppress the growth of SKOV3 ovarian cancer cells with constitutively active Akt. (R)(S)(S)-BF65 induced mitotic arrest and MK-2206 caused G0/G1 arrest, while the combination of both induced simultaneous G0/G1 and G2/M cell cycle arrest. (R)(S)(S)-BF65 induced phosphorylation and inactivation of Bcl-2, and downregulated Mcl-1, consequently may lead to apoptosis. (R)(S)(S)-BF65 inhibited mitogen-activated protein kinases (MAPKs), which may stimulate cell proliferation upon activation. (R)(S)(S)-BF65 also induced DNA damage after long-term treatment. MK-2206 is known to inhibit phosphorylation and activation of Akt and suppress cancer cell growth. The combination of (R)(S)(S)-BF65 and MK-2206 also inhibited the Akt pathway. Interestingly, MK-2206 upregulated Bcl-2 and induced activation of MAPKs in SKOV3 cells; however, when combined with (R)(S)(S)-BF65, these prosurvival effects were reversed. The combination also more significantly decreased Mcl-1 protein, increased PARP cleavage, and induced γ-H2AX, a DNA damage marker. Remarkably, MK-2206 enhanced the microtubule depolymerization effect of (R)(S)(S)-BF65. The combination of (R)(S)(S)-BF65 and MK-2206 also markedly inhibited cell migration. Thus, MK-2206 synergizes with (R)(S)(S)-BF65 to inhibit SKOV3 cell growth via downregulating the Akt signaling pathway, and enhancing the microtubule disruption effect of (R)(S)(S)-BF65. (R)(S)(S)-BF65 in turn suppresses Bcl-2 and MAPKs induced by MK-2206. (R)(S)(S)-BF65 and MK-2206 compensate each other leading to increased apoptosis and enhanced cytotoxicity, and may also suppress cancer cell invasion.

Heterocyclic and phenyl double-bond-locked combretastatin analogues possessing potent apoptosis-inducing activity in HL60 and in MDR cell lines.

Two new series of combretastatin (CA-4) analogues have been prepared. The alkenyl motif of CA-4 was replaced either by a five-membered heterocyclic (isoxazoline or isoxazole) or by a six-membered ring (pyridine or benzene). The new compounds have been evaluated for their effects on tubulin assembly and for cytotoxic and apoptotic activities. Five compounds (18b, 20a, 21a, 34b, and 35b) demonstrated an attractive profile of cytotoxicity (IC50 < 1 microM) and apoptosis-inducing activity but poor antitubulin activity. The isoxazoline derivatives 18b, 20a, and 21a, demonstrated potent apoptotic activity different from that of natural CA-4. Their ability to block most cells in the G2 phase suggests that these compounds could act on targets different from the mitotic spindle. This would indicate activation of both the intrinsic and the extrinsic apoptotic pathways. The data suggest unambiguously that structural alteration of the stilbene motif of CA-4 can be extremely effective in producing potent apoptosis-inducing agents.

Studies on the apoptotic activity of natural and synthetic retinoids: discovery of a new class of synthetic terphenyls that potently support cell growth and inhibit apoptosis in neuronal and HL-60 cells.

New terphenyl derivatives have been synthesized and tested for their effect on cell survival in serum-free cultures. These compounds protected HL60 cells from death and supported their growth with an activity higher than that of the natural 14-hydroxy-retro-retinol. Terphenyls 26 and 28 also possess antiapoptotic activity on neuronal cells, proving them as possible candidates for the treatment of neurodegenerative and ischemic diseases.

Synthesis and pharmacology of 6-substituted benztropines: discovery of novel dopamine uptake inhibitors possessing low binding affinity to the dopamine transporter.

A series of 6alpha- and 6beta-substituted benztropines were synthesized. A marked enantioselectivity was observed for the 6beta-methoxylated benztropines, the (1R)-isomers being more potent than the corresponding (1S) compounds. The racemic 6alpha-methoxy-3-(4',4' '-difluorodiphenylmethoxy)tropane (5 g) was the most potent compound. It has been found that modifications at the 6-position of benztropine might reduce the DAT binding affinity, maintaining otherwise a significant dopamine uptake inhibitory activity. A reinvestigation of the absolute configuration of 6beta-methoxytropinone proved the 6R configuration for the (+)-enantiomer.

Synthesis and biological evaluation of resveratrol and analogues as apoptosis-inducing agents.

Resveratrol 1 (3,4',5-trihydroxy-trans-stilbene), a phytoalexin present in grapes and other food products, has recently been suggested as a potential cancer chemopreventive agent based on its striking inhibitory effects on cellular events associated with cancer initiation, promotion, and progression. This triphenolic stilbene has also displayed in vitro growth inhibition in a number of human cancer cell lines. In this context, a series of cis- and trans-stilbene-based resveratrols were prepared with the aim of discovering new lead compounds with clinical potential. All the synthesized compounds were tested in vitro for cell growth inhibition and the ability to induce apoptosis in HL60 promyelocytic leukemia cells. The tested trans-stilbene derivatives were less potent than their corresponding cis isomers, except for trans-resveratrol, whose cis isomer was less active. The best results were obtained with compounds 11b and 7b, the cis-3,5-dimethoxy derivatives of rhapontigenin 10a (3,5,3'-trihydroxy-4'methoxy-trans-stilbene) and its 3'-amino derivative 10b, respectively, which showed apoptotic activity at nanomolar concentrations. The corresponding trans isomers 12b and 8b were less active both as antiproliferative and as apoptosis-inducing agents. Of interest, 11b and 7b were active toward resistant HL60R cells and their activity was higher than that of several classic chemotherapeutic agents. The flow cytometry assay showed that at 50 nM compounds 7b or 11b were able to recruit almost all cells in the apoptotic sub-G(0)-G(1) peek, thus suggesting that the main mechanism of cytotoxicity of these compounds could be the activation of apoptosis. These data indicate unambiguously that structural alteration of the stilbene motif of resveratrol can be extremely effective in producing potent apoptosis-inducing agents.

4,5,6,7-Tetrahydro-isoxazolo-[4,5-c]-pyridines as a new class of cytotoxic Hsp90 inhibitors.

Hsp90 is considered an interesting therapeutic target for anticancer drug development. Here we describe a new class of 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine compounds. A small library of derivatives has been synthesized and investigated. Some reported compounds show interesting properties combining both notable binding to Hsp90 and potent cell growth inhibitory activity. N-5 substitution with a 2,4 resorcinol carboxamide appears crucial for activity. Moreover, a derivative bearing a hydroxamic acid residue bound to C-3 amide portion was found to inhibit both Hsp90 and HDAC6.

Novel 3,4-isoxazolediamides as potent inhibitors of chaperone heat shock protein 90.

A structural investigation on the isoxazole scaffold led to the discovery of 3,4-isoxazolediamide compounds endowed with potent Hsp90 inhibitory properties. We have found that compounds possessing a nitrogen atom directly attached to the C-4 heterocycle ring possess in vitro Hsp90 inhibitory properties at least comparable to those of the structurally related 4,5-diarylisoxazole derivatives. A group of compounds from this series of diamides combine potent binding affinity and cell growth inhibitory activity in both series of alkyl- and aryl- or heteroarylamides, with IC50 in the low nanomolar range. The 3,4-isoxazolediamides were also very effective in causing dramatic depletion of the examined client proteins and, as expected for the Hsp90 inhibitors, always induced a very strong increase in the expression levels of the chaperone Hsp70. In vivo studies against human epidermoid carcinoma A431 showed an antitumor effect of morpholine derivative 73 comparable to that induced by the reference compound 10.

Lack of nucleophilic addition in the isoxazole and pyrazole diketone modified analogs of curcumin; implications for their antitumor and chemosensitizing activities.

Curcumin (CUR) can be considered as a good lead compound for the design of new anticancer drugs. Further, structure-activity relationship studies may clarify the importance of the redox activities in the antitumor effects of the drug. We have elaborated the alpha,beta-unsaturated 1,3-diketone moiety of CUR into the isoxazole (ISO) and pyrazole (PYR) derivatives. These derivatives should be much less prone to nucleophilic addition than CUR and benzyl mercaptan addition analyses showed that indeed they do not form isolable conjugated products. When compared with CUR, ISO and PYR exhibited increased cell growth inhibitory and pro-apoptotic effects in liver cancer HA22T/VGH cells as well as in other tumor cell types; in contrast to CUR, the antitumor effects of ISO or PYR were not influenced by concomitant administration of N-acetylcysteine, as a source of -SH groups, or buthionine sulfoximine, as an inhibitor of glutathione synthesis. Further, treatment with CUR, but not with ISO or PYR, significantly decreased the content of reduced glutathione in the HA22T/VGH cells. Finally, ISO and PYR lacked the ability of the parent compound to sensitize the HA22T/VGH cells to cisplatin (CIS), an effect which appeared to occur through an interaction of CUR and CIS at the level of the -SH groups. Thus, the ability of interacting with cell thiols might not be requested for the more potent antitumor activities of new diketone modified CUR derivatives, which might rely on other mechanisms, though possibly devoid of chemosensitization capabilities.