Giuseppina Grisolia

Novel A-Ring and B-Ring Modified Combretastatin A-4 (CA-4) Analogues Endowed with Interesting Cytotoxic Activity

A novel class of combretastatins, modified at A-ring or both A- and B-rings, mainly by replacement with benzofuran or benzo[b]thiophene, were synthesized. The new heterocombretastatins showed good cytotoxic activity on BMEC and H-460 cell lines. The aminocombretastatin 9f potently inhibits cell growth of BMEC and combretastatin-resistant HT-29 cell lines, with potential interest to treat colon carcinoma. Heterocombretastatins 9a,b inhibit tubulin polymerization similarly to CA-4 by having a binding to colchicine site five times stronger.

Novel Terphenyls and 3,5-Diaryl Isoxazole Derivatives Endowed with Growth Supporting and Antiapoptotic Properties

A new study on terphenyl and diaryl-isoxazole and -isoxazoline derivatives, maintaining a common 3-adamantyl-4-hydroxyphenyl moiety, has been conducted to find compounds with growth supporting and antiapoptotic properties. Unexpectedly, diphenyisoxazole derivatives bearing a nitro group replacing the carboxylic function have been found with the highest cell protective activity within the series, in complete and in serum-free conditions. Inhibition of apoptosis induced by daunorubicin has also been observed for the most active compound.

A convenient synthesis of unsymmetrically substituted terphenyls of biologically active stilbenes via a double Suzuki cross-coupling protocol

A double Suzuki cross-coupling protocol has been devised as a practical route to a variety of terphenyls. Good chemoselectivity was observed. Unsymmetrically substituted triphenylenes were also easily prepared.

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.

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.

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

SummaryHemiasterlins 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.

Development of water soluble derivatives of cis-3, 4′, 5-trimethoxy-3′-aminostilbene for optimization and use in cancer therapy

SummaryColchicine site tubulin inhibitors are currently developed as vascular disrupting agents (VDAs). However, they were found to have cardiotoxicity in clinical trials. To overcome the problem, we developed a stilbene derivative, cis-3, 4′, 5-trimethoxy-3′-aminostilbene (stilbene 5c), which is highly potent and has no bone marrow and cardiac toxicity in mice. Here we attempt to optimize stilbene 5c using computer-based drug design and synthesize derivatives with benzimidazole or indole group. Biological evaluation showed that they are weaker than stilbene 5c without better water solubility. Alternative approach was thus adopted to make prodrugs of stilbene 5c. A water-soluble prodrug PD7 was synthesized by addition of a morpholino group with carbamate linkage to the amino group of stilbene 5c. In vitro studies show that PD7 induces mitotic arrest and disrupts microtubule similar to stilbene 5c. The cell signaling events in Cdc2, p53, Akt, and aurora kinase are similar in cells treated with stilbene 5c, CA4 or PD7, suggesting that they share the same mechanism. Although PD7 is less effective than stilbene 5c in vitro, the biological activity of PD7 as a single agent is similar to that of stilbene 5c. Combination of PD7 with VEGF inhibitor bevacizumab significantly enhances the therapeutic efficacy of PD7 in mouse xenograft model. These data suggest that PD7 could be a good candidate for further pre-clinical and clinical development as a new VDA for cancer therapy.

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 α3β4 receptor subtypes, was, anyway, only at micromolar level. Moreover, differently from known derivatives with pyridine, isoxazole condensed to azabicyclo ring led to no activity.

Abstract 731: 4-Amino-substituted 5-resorcinol-isoxazole derivatives, a novel class of Hsp90 inhibitor: Synthesis and evaluation of in vitro activity

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Hsp90 (heat shock protein 90) is a component of a molecular chaperone complex, involved in the folding, maturation and stabilisation of key signalling molecules which control cell proliferation, survival and transformation. It works by a modulation of a set of cancer-associated proteins, collectively referred to as “clients”. Inhibition of Hsp90 causes simultaneous destabilization and eventual degradation of client proteins that result in suppression of tumor growth. This observation led to the idea, among pharmaceutical chemists, to consider Hsp90 as a potential target for a new strategy in human cancer therapy. After pioneering studies with natural products (geldamycin and radicicol), many selective Hsp90 inhibitors from various institutions have entered in clinical trials. One of these classes of inhibitors is the 4,5-diaryl-isoxazole with a resorcinol in position 5 and a second substituted aryl in position 4. This scaffold was considered a prerequisite for the activity on Hsp90. VER-52296/NVP-AUY922, currently in Phase II clinical trials, belongs to diaryl-isoxazole class. In our project, we systematically investigated - with in silico pre-screening, in concert with structural information from X-ray protein crystallography - all possible modifications to make on the isoxazole scaffold. We have mainly focused our attention on the importance of an amide portion at the 4 position. Now we have identified a new class of synthetic small molecule Hsp90 inhibitors, characterized by a 4-amino-substituted 5-monoaryl-isoxazole (5-resorcinol-isoxazole) scaffold. We turned our attention to the 4 position of isoxazole where unexpectedly, we have found that compounds possessing a nitrogen atom directly attached to the isoxazole ring are endowed of activity against Hsp90 similar or even better than the diaryl analogues. Here we describe design, synthesis and preliminary in vitro biological profile of a small library of these derivatives. Examples from this series of compounds have activities ranging from low to high nM against Hsp90 as measured in a fluorescence polarization (FP) competitive binding assay, and are active in human cancer cell lines where they inhibit cell proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 731.