Tiziana Brunetti

Isolation and pharmacological activities of the Tecoma stans alkaloids

Tecoma stans is a plant traditionally used in Mexico for the control of diabetes. Amongst the alkaloids isolated from the plant harvested in Egypt, Tecomine was shown to be one of the compounds responsible for the hypoglycemic action. Given the interest in substances able to treat type II diabetes, we isolated the main alkaloids present in the plant growing in Egypt and Brazil and tested them in vivo on db/db mice. Contrary to previous literature reports on different animal models, Tecomine was unable to modify glycemia; the only effect seen being a decrease in plasma cholesterol levels. On the contrary, when tested in vitro on glucose uptake in white adipocytes, the compound showed a marked effect. The two other alkaloids isolated, namely 5beta-Hydroxyskitanthine, early called Base C, and Boschniakine were inactive both in vivo and in vitro assays.

Exploring bis-(indolyl)methane moiety as an alternative and innovative CAP group in the design of histone deacetylase (HDAC) inhibitors

In order to gather further knowledge about the structural requirements on histone deacetylase inhibitors (HDACi), starting from the schematic model of the common pharmacophore that characterizes this class of molecules (surface recognition CAP group-connection unit-linker region-Zinc Binding Group), we designed and synthesized a series of hydroxamic acids containing a bis-(indolyl)methane moiety. HDAC inhibition profile and antiproliferative activity were evaluated.

N-Hydroxy-(4-oxime)-cinnamide: A versatile scaffold for the synthesis of novel histone deacetilase (HDAC) inhibitors

With the aim to discover novel HDAC inhibitors with high potency and good safety profiles, we have designed a small library based on a N-hydroxy-(4-oxime)-cinnamide scaffold. We describe the synthesis of these novel compounds and some preliminary in vitro cytotoxic activity on three tumor cell lines, NB4, H460 and HCT116, as well as their inhibitory activity against class I, II and IV HDAC. Several 4-oxime derivatives demonstrated a promising inhibitory activity on HDAC6 and HDAC8 coupled to a good selectivity profile.

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.

Characterization and pharmacological actions of tecostanine, an alkaloid of Tecoma stans

Tecostanine (1) was isolated from Tecoma stans leaves. Its sterochemistry was elucidated as well as its antihyperglycemic activity and its affinity to opioid and nicotinic receptors. The oxalate salt of 1 did not significantly affect blood glucose levels in normoglycaemic and hyperglycaemic rats. It did not appear to interact with opioid receptors (mu type) and showed only moderate affinity to the nicotinic receptor.

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.

New retinoid derivatives as back-ups of Adarotene

Adarotene belongs to the so-called class of atypical retinoids. The presence of the phenolic hydroxyl group on Adarotene structure allows a rapid O-glucuronidation as a major mechanism of elimination of the drug, favoring a fast excretion of its glucuronide metabolite in the urines. A series of ether, carbamate and ester derivatives was synthesized. All of them were studied and evaluated for their stability at different pH. The cytotoxic activity in vitro on NCI-H460 non-small cell lung carcinoma and A2780 ovarian tumor cell lines was also tested. A potential back-up of Adarotene has been selected to be evaluated in tumor models.

2-{3-[2-(4-chlorophenyl)ethoxy]-phenylthio}-2-methylpropanoic acid : a fibrate-like compound with hypolipidemic and antidiabetic activity

Diabetes is a complex metabolic disease which has reached epidemic proportions; its continuously increasing incidence is a consequence of the excessive caloric intake and lack of physical activity that characterize the western lifestyle. The progressive impairment of insulin sensitivity and eventual deterioration of b-cell function are the underlying causes of the overt disease. The classical and commonly used treatments for lowering glycemia are the insulin secretagogues, such as the sulfonylureas, and metformin, an insulin sensitizer with a mechanism of action that is still under investigation. In recent years rosiglitazone and pioglitazone have entered the market as insulin sensitizers and anti-hyperglycemic drugs. They are representatives of the new thiazolidinedione (TDZ) class of agents, which elicit their effects through activation of the g isoform of the nuclear peroxisome proliferator-activated receptor (PPARg). 6] The activity of TDZs in humans is accompanied by weight gain and risk of edema, whereas the liver toxicity observed with troglitazone, the first marketed (and soon thereafter withdrawn) representative of this family, seems to be more the result of its particular chemical structure than of its TDZ class-related mechanism of action. Despite an improvement in the ability to control glycemia, the prevention and management of the negative cardiovascular aspects of the disease remain the focus of open primary research, as such aspects rank diabetes the fourth leading cause of mortality in developed countries. In this context, a drug that is able to effectively correct dyslipidemia as well as hyperglycemia and insulin sensitivity is certainly of great interest. Fibrates such as fenofibrate and bezafibrate have been widely used for decades as hypolipidemic, antiatherosclerotic, and cardioprotective agents, and are known to act through the activation of the a isoform of PPAR (PPARa). For these reasons, compounds that activate both PPARa and PPARg receptors (PPARa/g mixed agonists) are considered to be very promising targets (ref. [15] and references therein), but only a few of them bear fibrate-related structures. In the last few years, new hypolipidemic PPARa agonists with improved potency and selectivity have also appeared, such as GW9578 and its closely related analogue GW7647, both of which are characterized by a thioisobutyrate moiety. During our search for new hypolipidemic and anti-hyperglycemic agents, we found quite unexpectedly that compound 3 (Scheme 1), which is structurally related to the fibrates, considerably improves diabetic conditions in C57BL/KsJ db/db diabetic mice (Table 1). In particular, the decrease in glucose level was similar to that effected by rosiglitazone, even if 3 was administered at a higher dose (25 instead of 5 mgkg , twice a day by oral gavage for 25 days), with greatly improved homogeneity of values. This last point should not be underestimated, as in our experience, not all animals in experimental groups respond to rosiglitazone and similar compounds. The same has also been reported in studies with humans. In an oral glucose tolerance test (OGTT) after 19 days of treatment, the glycemic area under curve (AUC) was considerably lower in the case of 3 with respect to control and fibrate results, and [a] Dr. F. Giannessi Department of Endocrinology and Metabolism Sigma-Tau S.p.A Via Pontina km 30400, 00040 Pomezia, Rome (Italy) Fax: (+39)06-9139-3988 E-mail : fabio.giannessi@sigma-tau.it [b] Prof. F. De Angelis Department of Chemistry, Chemical Engineering, and Materials University of L’Aquila, Coppito, 67010 L’Aquila (Italy) Fax: (+39)0862-433753 E-mail : deangeli@univaq.it [c] Dr. N. Dell’Uomo, Dr. E. Tassoni, Dr. T. Brunetti, Dr. M. O. Tinti Department of Chemical Research Sigma-Tau S.p.A (Italy) [d] Dr. P. Pessotto, Dr. A. F. Sciarroni, Dr. F. M. Milazzo, A. Peschechera Department of Endocrinology and Metabolism Sigma-Tau S.p.A (Italy) [e] Dr. P. Carminati Director of Research & Development Department Sigma-Tau S.p.A (Italy) Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author.

Abstract 3896: New heat shock protein 90 inhibitors based on the 1,2,3-triazole structure

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Hsp90 (heat shock protein 90) is a component of a molecular chaperone complex, involved in the folding, maturation and stabilisation of key signalling proteins which control cell proliferation, survival and transformation. It works by modulating a set of cancer-associated proteins, that are often over-expressed and/or mutated in tumor cells, collectively referred as ‘‘clients’’. Inhibition of Hsp90 causes destabilization and eventual degradation of client proteins involved in cell cycle, tumor growth, angiogenesis and apoptosis. Therefore, their modulation results in suppression of tumor growth by multiple parallel mechanisms. Consequently, inhibition of Hsp90 is believed to be a potential target for cancer therapy, also supported by a number of molecules currently under investigation in different phase of clinical trials. We recently synthesized 1,4,5-trisubstituted-1,2,3-triazole derivatives and surprisingly found that these new molecules demonstrated to be extremely active both on the biological target and on human tumor cell lines, even at nanomolar concentration. 3D QSAR analysis was also performed in order to rationalize HSP90 binding data. The overall profile of this new class of 1,2,3-triazoles, including stereoselective synthesis and a comprehensive pharmacological in vitro characterization, will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3896. doi:1538-7445.AM2012-3896

Derivatives of R-Aminocarnitine without Ammonium Moiety as Liver Carnitine Palmitoyltransferase I (L-CPT I) Inhibitors

Type II diabetes is a chronic and progressive metabolic disorder with potentially life-threatening consequences, usually arising due to resistance to insulin action in the setting of an inadequate compensatory insulin secretory response. In type II diabetes, increased hepatic fatty acid oxidation by formation of high levels of acetyl-coenzyme A (CoA), ATP and NADH, over-regulates gluconeogenesis and thus hepatic glucose production. The transport of fatty acids into the mitochondria is regulated by two membrane-bound, carnitine-dependent, long-chain acyltranferases, known as carnitine palmitoyltransferases (CPT I and CPT II). CPT I, the outer mitochondrial membrane enzyme—present in two isoforms: liver (L-CPT I) and muscle (M-CPT I)—catalyzes the formation of long-chain acylcarnitines. CPT II, the inner mitochondrial membrane enzyme present in a single isoform, reconverts long-chain acylcarnitines into longchain acyl CoA thioesters. Oxirane carboxylates, such as etomoxir and methyl 2-tetradecylglycidate, were reported to be irreversible inhibitors of CPT systems, exhibiting good activity as hypoglycemic agents, but at the time they were discontinued probably owing to cardiac hypertrophy due to a lack of selectivity toward the L-CPT I isoform. More recently, this assumption has been questioned since inhibition of the ubiquitously present CPT II isoform has been put forward as a therapeutic target for the treatment of diabetes and associated pathologies. We previously published the identification of selective LCPT I inhibitor ST1326 (teglicar) 8] and the backup analogue ST2425, both with carnitine-related backbones, and their antihyperketotic/antihyperglycemic activity. It was also reported that inhibition of hypothalamic lipid oxidation via intracerebroventricular infusion of ST1326 results in cellular accumulation of long-chain fatty acyl-CoA within the arcuate nucleus of the hypothalamus, leading to marked inhibition of food intake and glucose production, with a significant anorectic response mirrored by an early occurrence of satiety onset. Unfortunately, the physicochemical properties of ST1326 and the other previously synthesized betaine compounds were not very appropriate for crossing the blood–brain barrier (BBB). Here, we report the synthesis and biological characterization of new derivatives of R-aminocarnitine, where the quaternary ammonium moiety of the aminocarnitine backbone of the lead compounds ST1326 and ST2425 is replaced by groups without permanent cationic charges. Such compounds, with tertiary amino or alkyl groups, were designed to increase the ability of the agent to cross the BBB with the intention of achieving CPT inhibition in the central nervous system (CNS), with the aim of overcoming the limitations explained above. Owing to the lack of the quaternary ammonium cation, the new compounds were also expected to be less tensioactive; however, their biological activity, liver/heart selectivity and CPT II inhibition remained to be evaluated. Amino analogues 8–11 were prepared from starting material 1 according to known procedures (Scheme 1). When the appropriate isocyanate used in step d was not commercially available, it was prepared from the corresponding carboxylic acid via a Curtius reaction; 4-[(3-hexyloxy)phenoxy]butylisocyanate is not commercially available and so was prepared as reported in the patent literature. Using the same starting material (1), tert-butyl analogues were also synthesized following the procedure described in Reference [10] , where nucleophilic substitution of 1 was carried out by adding lithium di-tertbutyl cuprate (Scheme 2). Compounds 15 and 16 were then obtained in about 70 % yield from ester 14 after reaction with the appropriate isocyanate and subsequent acid hydrolysis. Compounds 17 and 18, where the ammonium group of the aminocarnitine backbone is replaced by an isopropyl group, were synthesized from the commercially available l-b-homoleucine hydrochloride and the appropriate isocyanate in 80% and 28% yield, respectively (Scheme 3). For in vitro efficacy and selectivity evaluation, IC50 values against L-CPT I were determined in rat liver mitochondria for all new compounds. The most active derivatives were further investigated for their inhibition of M-CPT I using heart mitochondria. Furthermore, these same derivatives, i.e. those that exhibited selectivity for the liver isoform, were assayed for their ED50 values for ketogenesis inhibition in hepatocytes; since b-hydroxybutyrate (b-HBA) is the main product of liver [a] Dr. E. Tassoni, Dr. G. Gallo, Dr. S. Vincenti, Dr. L. Mastrofrancesco, Dr. T. Brunetti, Dr. W. Cabri Chemistry & Analytical Department, Sigma-Tau S.p.A. Via Pontina Km 30.400, 00040 Pomezia (Italy) E-mail : emanuela.tassoni@sigma-tau.it [b] Dr. R. Conti, Dr. F. Giannessi Endocrinology & Metabolism Department, Sigma-Tau S.p.A. Via Pontina Km 30.400, 00040 Pomezia (Italy) E-mail : fabio.giannessi@sigma-tau.it Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201100289.