Massimo Castorina

Synthesis and in vitro antitumor activity of new 4,5-dihydropyrazole derivatives

A series of 3,5-diaryl-4,5-dihydropyrazole regioisomers, and their 1-acetylated derivatives, bearing a 3,4,5-trimethoxyphenyl moiety combined with a variety of substituted phenyl rings, was synthesized and evaluated for antitumor activity. Results of the in vitro assay against a non-small cell lung carcinoma cell line (NCI-H460) showed several compounds to be endowed with cytotoxicity in micromolar to sub-micromolar range, depending on substitution pattern and position of aryl rings on 4,5-dihydropyrazole core. Potent and selective activity was also observed in the NCI 60 human cancer cell line panel. 5-(3,4,5-Trimethoxyphenyl)pyrazolines 31 and 39 were found to possess potent antiproliferative activity against SR and MDA-MB-435, with GI(50) inhibitory values in nanomolar range. Structure-activity relationships revealed that introduction of a (hydroxy)acetyl group at N-1 of inactive 5-(3,4,5-trimethoxyphenyl)pyrazolines, results in a clear in vitro activating effect. Compound 31 (IC(50)=5.16 microM) showed inhibition of tubulin polymerization comparable to that of CA-4 (IC(50)=4.92 microM).

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 tumor-targeted RGD peptide-camptothecin conjugates: synthesis and biological evaluation.

Five RGD peptide-camptothecin (CPT) conjugates were designed and synthesized with the purpose to improve the therapeutic index of this antitumoral drug family. New RGD cyclopeptides were selected on the basis of their high affinity to alpha(v) integrin receptors overexpressed by tumor cells and their metabolic stability. The conjugates can be divided in two groups: in the first the peptide was attached to the drug through an amide bond, in the second through a hydrazone bond. The main difference between the two spacers lies in their acid stability. Affinity to the receptors was maintained for all conjugates and their internalization into tumor cells was demonstrated. The first group conjugates showed lower in vitro and in vivo activity than the parent drug, probably due to the excessive stability of the amide bond, even inside the tumor cells. Conversely, the hydrazone conjugates exhibited in vitro tumor cell inhibition similar to the parent drug, indicating high conversion in the culture medium and/or inside the cells, but their poor solubility hampered in vivo experiments. On the basis of these results, information was acquired for additional development of derivatives with different linkers and better solubility for in vivo evaluation.

Acetyl-L-carnitine affects aged brain receptorial system in rodents

Acetyl-L-carnitine (ALCAR), the acetyl ester of carnitine, is regarded as a compound of considerable interest because of its capacity to counteract several physiological and pathological modifications typical of brain ageing processes. In particular, it has been demonstrated that ALCAR can counteract the age-dependent reduction of several receptors in the central nervous system of rodents, such as the NMDA receptorial system, the Nerve Growth Factor (NGF) receptors, those of glucocorticoids, neurotransmitters and others, thereby enhancing the efficiency of synaptic transmission, which is considerably slowed down by ageing. The present review thus postulates the importance of ALCAR administration in preserving and/or facilitating the functionality of carnitines, the concentrations of which are diminished in the brain of old animals.

Synthesis and Evaluation of New Hsp90 Inhibitors Based on a 1,4,5-Trisubstituted 1,2,3-Triazole Scaffold

Ruthenium catalyzed 1,3-cycloaddition (click chemistry) of an azido moiety installed on dihydroxycumene scaffold with differently substituted aryl propiolates gave a new family of 1,4,5-trisubstituted triazole carboxylic acid derivatives that showed high affinity toward Hsp90 associated with cell proliferation inhibition, both in nanomolar range. The 1,5 arrangement of the resorcinol, the aryl moieties, and the presence of an alkyl (secondary) amide in position 4 of the triazole ring were essential to get high activity. Docking simulations suggested that the triazoles penetrate the Hsp90 ATP binding site. Some 1,4,5-trisubstituted triazole carboxamides induced dramatic depletion of the examined client proteins and a very strong increase in the expression levels of the chaperone Hsp70. In vitro metabolic stability and in vivo preliminary studies on selected compounds have shown promising results comparable to the potent Hsp90 inhibitor NVP-AUY922. One of them, (compound 18, SST0287CL1) was selected for further investigation as the most promising drug candidate.

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.

Conjugates of a Novel 7-Substituted Camptothecin with RGD-Peptides as αvβ3 Integrin Ligands: An Approach to Tumor-Targeted Therapy

Eight conjugates of a novel camptothecin derivative (Namitecan, NMT) with RGD peptides have been synthesized and biologically evaluated. This study focused on factors that optimize the drug linkage to the transport vector. The different linkages investigated consist of heterofunctional glycol fragments and a lysosomally cleavable peptide. The linkage length and conformation were systematically modified with the purpose to understand their effect on receptor affinity, systemic stability, cytotoxicity, and solubility of the corresponding conjugates. Among the new conjugates prepared, C6 and C7 showed high receptor affinity and tumor cell adhesion, acceptable stability in murine blood, and high cytotoxic activity (IC₅₀ = 8 nM). The rationale, synthetic strategy, and preliminary biological results will be presented.

A cluster analysis study of acetyl-l-carnitine effect on NMDA receptors in aging

Aging is associated with a reduction in the maximum density of n-methyl-d-aspartate (NMDA)-sensitive glutamate binding sites in the hippocampus of Fischer 344 rats. This study was designed to investigate the effect of acetyl-l-carnitine (ALCAR) on NMDA receptors in the old rat (24 months) after chronic or single-dose treatments. The number of NMDA receptors was significantly decreased in the old rat hippocampus by 19.5% compared with the young rat. A six-month treatment with ALCAR in the old rat attenuated the loss of NMDA binding sites in the hippocampus. A single-dose treatment with ALCAR in the old rat increased the Bmax value by 35%, while no change was observed in the young group. We conclude that ALCAR can exert two actions: a trophic/neuro-preserving one when chronically administered during aging, and a stimulatory one when given at a single dose in the aged rat.

A Potent Integrin Antagonist from a Small Library of Cyclic RGD Pentapeptide Mimics Including Benzyl-Substituted Azabicycloalkane Amino Acids

A small library of cyclic RGD pentapeptide mimics, including benzyl‐substituted azabicycloalkane amino acids, was synthesized with the aim of developing active and selective integrin antagonists. In vitro binding assays established one particular compound with affinity for both the αvβ3 and the αvβ5 integrins. The synthesis in solution and the in vitro screening of these RGD derivatives, as well as the determination of the conformational properties of the integrin ligands by spectroscopic and computational methods are described.

MPTP and convulsive responses in rodents

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg/kg s.c. for 5 days) to mice resulted in complete abolishment of strychnine seizure and of the tonic phase of the maximal electroshock response. Bicuculline and picrotoxin convulsions were not significantly affected by MPTP treatment. The severity of the pentylenetetrazole seizures was mildly, but significantly affected in the protective way. MPTP depleted neostriatal dopamine and its metabolites, together with hippocampal norepinephrine. No nigral neuronal loss was detected histologically. Strychnine seizures and the tonic phase of the maximal electroshock response are thought to depend mostly on hindbrain (bulbo-spinal) structures. Thus, these experiments suggest that a caudally projecting system originates from the substantia nigra, pars compacta, and/or locus coeruleus, controlling seizures that involve bulbo-spinal centers. While neostriatal dopamine depletion offers a good index of seizure resistance, its role in the protection from seizures remains to be established.