Fabio Giannessi

Selective Reversible Inhibition of Liver Carnitine Palmitoyl-Transferase 1 by Teglicar Reduces Gluconeogenesis and Improves Glucose Homeostasis

OBJECTIVE We have developed a new antihyperglycemic agent (teglicar) through the selective and reversible inhibition of the liver isoform of carnitine palmitoyl-transferase 1 (L-CPT1). RESEARCH DESIGN AND METHODS Glucose production was investigated in isolated hepatocytes and during pancreatic clamps in healthy rats. Chronic treatments on C57BL/6J, db/db, high-fat fed mice, and rats were performed to understand glucose metabolism and insulin sensitivity. RESULTS In isolated hepatocytes, teglicar concentration dependently reduced ketone bodies and glucose production up to 72 and 50%, respectively. In rats, teglicar reduced the endogenous glucose production (−62%) without affecting peripheral glucose utilization. Heart 2-[3H]deoxyglucose uptake in mice was also not affected, confirming in vivo the drug selectivity toward L-CPT1. Chronic treatment in db/db mice (50 mg/kg/bid; 45 days) reduced postabsorptive glycemia (−38%), water consumption (−31%), and fructosamine (−30%). Such antidiabetic activity was associated with an improved insulin sensitivity assessed by the insulin tolerance test. A significant 50% increase in hepatic triglyceride content (HTGC) was found, although plasma alanineaminotransferase was not altered. In addition, long-term teglicar administration to high-fat fed C57BL/6J mice normalized glycemia (−19%) and insulinemia (−53%). Long-term teglicar administration (30 days, 80 mg/kg) in healthy overnight-fasted rats slightly reduced basal glycemia (−20%, ns), reduced basal insulin levels by 60%, doubled triglycerides, and increased free-fatty acids (+53%). HTGC was markedly increased, but liver and peripheral insulin sensitivity assessed by hyperinsulinemiceuglycemic clamp were not affected. CONCLUSIONS Teglicar, in vitro and in animal models, reduces gluconeogenesis and improves glucose homeostasis, refreshing the interest in selective and reversible L-CPT1 inhibition as a potential antihyperglycemic approach.

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.

Reversible Carnitine Palmitoyltransferase Inhibitors with Broad Chemical Diversity as Potential Antidiabetic Agents

A series of carnitine related compounds of general formula XCH(2)CHZRCH(2)Y were evaluated as CPT I inhibitors in intact rat liver (L-CPT I) and heart mitochondria (M-CPT I). Derivative 27 (ZR = -HNSO(2)R, R = C(12), X = trimethylammonium, Y = carboxylate, (R) form) showed the highest activity (IC(50) = 0.7 microM) along with a good selectivity (M-CPT I/L-CPTI IC(50) ratio = 4.86). Diabetic db/db mice treated orally with 27 showed a significant reduction of serum glucose levels.

In Vivo Imaging Reveals Selective Peroxisome Proliferator Activated Receptor Modulator Activity of the Synthetic Ligand 3-(1-(4-Chlorobenzyl)-3-t-butylthio-5-isopropylindol-2-yl)-2,2-dimethylpropanoic acid (MK-886)

We report here the finding of a new pharmacological activity of a well known antagonist of peroxisome proliferator-activated receptors (PPARs). PPARs belong to the family of nuclear receptors playing a relevant role in mammalian physiology and are currently believed to represent a major target for the development of innovative drugs for metabolic and inflammatory diseases. In the present study, the application of reporter animal technology was instrumental to obtain the global pharmacological profiling indispensable to unraveling 3-(1-(4-chlorobenzyl)-3-t-butylthio-5-isopropylindol-2-yl)-2,2-dimethylpropanoic acid (MK-886)-selective PPAR modulator (SPPARM) activity not underlined by previous traditional, cell-based studies. The results of this study, demonstrating the usefulness of reporter mice, may open new avenues for the development of innovative drugs for cardiovascular, endocrine, neural, and skeletal systems characterized by limited side effects.

Improvement of statin‐associated myotoxicity by L‐carnitine

Although statins represent the mainstay therapy for the treatment of hypercholesterolemia and have a positive tolerability and safety profile, the occurrence of statinassociated myopathy, an accepted class effect of these drugs, may bemore common than originally thought. In this regard, a recent paper by Phillips and collegues on the myotoxic effect of statin has been followed by a number of interesting letters and authors’ responses addressing several issues of statin myotoxicity [1,2]. Among them, Toma reasoned that L-carnitine (LC) treatment might have a favorable impact on the incidence of statin-associated therapy [2]. We have recently completed a study in young rodents treated with myotoxic doses of simvastatin, a useful model for statin myotoxicity studies [3], in combination or not with LC and our results strengthen Toma’s proposal in addressing the preventive or therapeutic effect of LC on statin-associated myopathy in human clinical trials. Indeed, our study shows that, at the highest dose of simvastatin, creatine kinase (CK) plasma levels were significantly elevated in treated animals compared with controls and that carnitine coadministration efficiently counteracted plasma CK levels in the former group (Table 1). Statin treatment at the two highest doses also caused a significant elevation of glutamic oxalacetate transaminase (GOT) and glutamic pyruvate transaminase (GPT), which may reflect both hepatic and muscle insult. LC administration did not lead to a significant correction of these two enzymatic indices in statin-treated groups, although, at the highest dose of simvastatin, LC seems to oppose GOT and GPT elevations (Table 1). The beneficial effect of carnitine administration in the simvastatin-treated animals does not seem to be linked to an alteration of the pharmacokinetic properties of the statin, since the cholesterollowering effect observed at the highest dose of simvastatin was not affected by carnitine (Table 1). Statin-treated animals were also treadmill-tested at a constant 15 angle and a speed of 10 m min. As expected, their walking capacity was severely impaired at the highest statin dose (90% of rats fell more than three times over a period of 5 min walking) compared with controls (no fall over a period of 5 min walking). A marked improvement by adding carnitine to statin treatment was observed (60% of rats fell more than three times over a period of 5 min walking). Themajority of the specificmyotoxic actions described in the literature seem to indicate that muscle membrane represents a common pathogenic target of statins’ mode of action [4]. Since several cellular functions rely on the integrity of biological membranes (i.e. ionic homeostasis, signal transduction, com-

Inversion of Configuration of (S)‐β‐Hydroxy‐γ‐butyrolactone with Total Retention of the Enantiomeric Purity

In this paper we report the inversion of configuration of (S)-β-hydroxy-γ-butyrolactone [(S)-1] to its (R) enantiomer (R)-1, with total retention of the enantiomeric purity, by a four-step procedure. The (R)-β-hydroxy-γ-butyrolactone [(R)-1] was thus synthetized with an overall chemical yield of 47% and > 97% ee. This transformation opens an economic route to the production of (R)-GABOB and (R)-carnitine, among other biologically active compounds, from a D-hexose source, or, alternatively, from the industrial waste compound (S)-carnitine. During the reaction sequence, the intermediate β-lactone 4 is also prepared, which is now under investigation as a chiral synthon for new synthetic applications.

Aminocarnitine ureidic derivatives as inhibitors of carnitine palmitoyltransferase I.

Type II diabetes is a complex metabolic disorder characterized by insulin resistance and impaired b-cell function. It arises as a consequence of obesity, sedentary lifestyle and aging, with resulting hyperglycemia, blood pressure elevation and dyslipidemia. Moreover, in type II diabetes, highly increased hepatic fatty acid oxidation generates high levels of acetyl-coenzyme A (acetyl-CoA), ATP and NADH, which in turn upregulate gluconeogenesis and thus hepatic glucose production. The transport of fatty acids into mitochondria is regulated by membrane-bound carnitine palmitoyltransferases (CPT) I and II. CPT I, the outer mitochondrial membrane enzyme that is present as two isoforms known as liver (L-CPT I) and muscle (MCPT I), catalyzes the formation of long-chain acylcarnitines. CPT II, the inner mitochondrial membrane enzyme present as a single isoform, converts long-chain acylcarnitines back into long-chain acetyl-CoA thioesters. CPT inhibitors, by lowering the level of acetyl-CoA, indirectly reduce liver gluconeogenesis. Oxirane carboxylates, such as etomoxir and methyl 2-tetradecylglycidate, previously identified as irreversible inhibitors of CPT, were found to induce cardiac hypertrophy due to a lack of liver and muscle isoform selectivity. In previously published studies, Novartis (formerly Sandoz) described an alkylphosphate derivative of carnitine as a CPT I inhibitor, and we described the identification of highly selective L-CPT I inhibitors. Teglicar (1, ST1326) was chosen from these inhibitors for preclinical and clinical development as an antiketotic and antidiabetic agent. The ureidic functional group present in teglicar was advantageous in terms of efficacy and selectivity towards the liver isoform of CPT, in comparison with other investigated moieties. Taking these findings into consideration, we decided to continue our studies on L-CPT I inhibitors with a series of new aminocarnitine ureidic derivatives, exploring the effects of aromatic functionalities in the straight-chain alkyl group of 1. The aim was to obtain new inhibitors with improved efficacy, while maintaining the high selectivity for the liver over the muscle isoform of CPT I, and/or with limited tensioactivity (an unwanted characteristic of this class of molecules arising from the long alkyl chain and ionic head). Oxygenated substitutions were introduced for their water coordinating properties, which could favorably limit packing of the molecules in micelle formation. Accordingly, the compounds were synthesized starting from aminocarnitine and isocyanates or the corresponding carboxylic acids. For the most interesting molecule identified, the phosphonium analogue was also prepared in order to investigate the effects of ammonium group substitution with the bioisoster phosphonium on the activity profile. In order to explore the effects of aryloxy substituents in the alkyl chain of 1, ortho and meta oxygen functionalities on an aromatic ring were inserted in derivatives 2, 3 and 5, in an attempt to obtain a lower packing of the molecules. These hexyloxy-phenoxyalkyl derivatives were prepared from carboxylic acids synthesized according to the standard procedures described in Scheme 1, subsequently transformed into isocyanates using diphenyl phosphoryl azide, or alternatively following classical activation as acyl chlorides, substitution with sodium azide and Curtius transposition. The phosphonium analogue 4 was also prepared following the same procedure, using (R)-4-trimethylphosphonio-3-aminobutyrate, prepared from d-aspartic acid as described in the literature, instead of aminocarnitine. Moreover, the effect of the aryl group adjacent to the ureidic functionality was explored in derivatives 6, 10 and 11, having an alkyloxy or a small alkyl chain as the substituent, and with a methylene group spacer between the ureido and aryl group, as in derivatives 7 and 8, having an alkyloxy and/or a benzyloxy substituent. Conversely, a derivative with an aryloxy group at the end of a long chain was also prepared (compound 9). All of these compounds were prepared starting from the isocyanate or the corresponding carboxylic acid, according to the procedure summarized in Scheme 2 (see Supporting Information for more details). A three-dimensional homology model for human liver CPT I (hL-CPT I) was built using the crystallographic structure of murine carnitine acetyltransferase (CAT) co-crystallized with CoA and hexanoylcarnitine (PDB code: 2H3W). The sequence identity between the two enzymes is 33 %. Moreover, the acyl pocket in human liver CPT I is characterized by an insertion of 14 amino acids (between 690 and 707), compared with other [a] Dr. E. Tassoni, Dr. G. Gallo, Dr. S. Vincenti, Dr. N. Dell’Uomo, Dr. L. Mastrofrancesco, Dr. W. Cabri Chemistry & Analytical Department, Sigma-Tau S.p.A. Via Pontina Km 30.400, 00040 Pomezia (Italy) Fax: (+ 39) 069-139-3638 E-mail : emanuela.tassoni@sigma-tau.it [b] Dr. R. Conti, Dr. R. Ricciolini, Dr. F. Giannessi Endocrinology & Metabolism Department, Sigma-Tau S.p.A. Via Pontina Km 30.400, 00040 Pomezia (Italy) Fax: (+ 39) 069-139-3988 E-mail : fabio.giannessi@sigma-tau.it [c] 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://dx.doi.org/10.1002/cmdc.200900535.

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.

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.