Ademaglitazone synthetic ligand of the gamma receptor activated by peroxisomal proliferators with insulin-sensitizing activity and low adipogenic capacity, and its use in the pharmacological treatment of type 2 diabetes mellitus and insulin resistance induced by obesity

Abstract

The compound ADEMAGLITAZONE, with the IUPAC nomenclature (Z)-5-[(5-bromo-1-ethyl-1H-indol-3- yl)methylene]-3-ethylthiazolidine-2,4-dione, possesses insulin-sensiti zing properties and limited capacity to promote adipogenesis. The compound ADEMAGLITAZONE is asynthetic and selective ligand of PPAR-γ, obtained through the condensation of the pharmacophore thiazolidine-2, 4-dione with an Indole moiety. The compound ADEMAGLITAZONE is intended for use in the treatment o f type 2 diabetes mellitus and obesity-induced insulin resistance.

Description

[0001] DESCRIPTION

[0002] "ADEMAGLITAZONE synthetic ligand of the gamma receptor activated by peroxisomal prol i f erators with insulinsensiti zing activity and low adipogenic capacity, and its use in the pharmacological treatment of type 2 diabetes mellitus and insulin resistance induced by obesity"

[0003] The present invention pertains to a speci fic compound and its pharmacological applications , as well as a pharmaceutical formulation containing this compound . Speci fically, the present invention pertains to the compound ADEMAGLITAZONE , or ( Z ) -5- [ ( 5-bromo- l-ethyl- lH-indol-3- yl ) methylene ] -3-ethylthiazolidine-2 , 4-dione ] according to the IUPAC nomenclature , a synthetic ligand of the peroxisome proli f erator-activated receptor gamma ( PPAR-y) , exhibiting insulin-sensiti zing properties and limited capacity to promote adipogenesis .

[0004] The present invention further pertains to the use of the compound ADEMAGLITAZONE in the pharmacological treatment of type 2 diabetes mellitus and obesity-induced insulin resistance . Finally, the present invention pertains to a pharmacological formulation containing the ADEMAGLITAZONE compound for oral administration .

[0005] Obesity-induced type 2 diabetes mellitus ( T2DM) is arguably the largest global epidemic in history . According to estimates , at present , over 468 million people are af fected by T2DM, with an increase that could reach nearly one billion people by 2050 . The main factors contributing to this increase include genetic predisposition, malnutrition ( defined as excessive calorie intake and / or poor diet quality) , ageing, smoking, and alcohol consumption . From a pathophysiological perspective , insulin resistance is the first metabolic alteration that occurs in obese individuals . Without a compensatory increase in insulin secretion, thi s leads to high glucose levels and excessive concentrations of free fatty acids in the blood . In addition to T2DM, insulinresistant obese individuals also have an increased risk of developing malignancies , severe or fatal infections ( including the recent SARS-CoV-2 virus infection-related disease ) , and cardiovascular di seases , resulting in a substantial reduction in quality of li fe and li fe expectancy . Currently, the two main types of orally administered insulinsensiti zing drugs used in the treatment of T2DM, are : - Metformin, burdened by a high risk of gastrointestinal intolerance and a low anti- hyperglycemic ef ficacy;

[0006] - Synthetic ligands of PPAR-y, also known as thiazolidinediones or glitazones , to which the compound according to the present invention belongs .

[0007] Through the modulation of multiple genes involved in glucose and lipid metabolism, partially mediated by the full or partial agonism of PPAR-y ( a nuclear receptor with almost ubiquitous expression involved in tissue insulin sensitivity) thiazolidinediones are able to enhance tissue insulin sensitivity, promote the uptake of free fatty acids by adipocytes , as well as increase adiponectin secretion and reduce the release of pro-inflammatory adipokines and cytokines , such as interleukin ( IL ) - 6 , by these cells .

[0008] Over the years , three drugs belonging to the glitazone class have been marketed : Troglitazone , Rosiglitazone and Pioglitazone , all derived from thiazolidine-2 , 4-dione , and di f fering from each other in the chemical structure of the side chain linked to the thiazolidinedione pharmacophore .

[0009] However, the benefit / risk profiles of these drugs , which were revised following reports of cases of congestive heart failure requiring hospitali zation and / or resulting in death during pharmacovigilance procedures , led to the withdrawal of the marketing authori zation for potent antidiabetic drugs containing Rosiglitazone by the European Medicines Agency (EMA) , while Troglitazone was voluntarily withdrawn by the manufacturing company due to the risk of severe hepatotoxicity .

[0010] Currently, the only available glitazone on the market as a second- or third-line therapy for T2DM, with only modest anti-hyperglycemic ef ficacy, is Pioglitazone .

[0011] The purpose of the present invention is to provide a compound and a pharmaceutical formulation that exhibits insulin-sensiti zing activity and weak adipogenic capacity, as well as reduced risk of toxicity and adverse events , for the pharmacological treatment of T2DM and obesity-induced insulin resistance .

[0012] According to the present invention, a compound is produced as defined in claim 1 .

[0013] For a better understanding of the present invention, reference is now made to the accompanying figures , provided as examples and not intended to be limiting :

[0014] - Figure 1 shows the structural formula of the compound ADEMAGLITAZONE according to the invention;

[0015] - Figures 2A, 2B, 2C and 2D show the superior af finity and speci ficity of the binding of the compound ADEMAGLITAZONE to the PPAR-y receptor compared to Rosiglitazone through computational modeling studies , according to the invention;

[0016] - Figure 3 shows the organic synthesis process of the compound ADEMAGLITAZONE , according to the invention;

[0017] - Figures 4A and 4B show experimental results of the treatment with the compound ADEMAGLITAZONE on adiponectin transcription and circulating adiponectin hormone release , respectively, using control samples treated with Rosiglitazone or the vehicle alone ;

[0018] - Figures 5A and 5B show experimental results of the ef fect of treatment with ADEMAGLITAZONE compared to Rosiglitazone and the vehicle alone (untreated controls ) on systemic insulin tolerance ( lU / kg of Lispro insulin was intraperitoneally administered to diet-induced obese mice after at least 4h of fasting, followed by blood glucose monitoring at regular 15- minute intervals for 60 minutes ) , and on body weight of exposed animals with weekly measurements .

[0019] The compound according to the present invention consists of ADEMAGLITAZONE , with the IUPAC nomenclature ( Z ) - 5- [ ( 5-bromo- l -ethyl- IH-indol- 3-yl ) methylene ] -3- ethylthiazolidine-2 , 4-dione ] , and the structural formula shown in Figure 1 . The compound ADEMAGLITAZONE is a synthetic ligand of the peroxisome proli f erator-activated receptor gamma, which exhibits insulin-sensiti zing activity and limited capacity to promote adipogenesis .

[0020] The compound according to the invention has an innovative chemical structure based on the re-engineering of the pharmacophore of thiazolidinediones ( or glitazones ) , condensed with an N-heterocyclic side chain ( Indole ) .

[0021] Advantageously, the chemical structure of the compound according to the invention of fers improved bio- pharmacological activity and a reduced risk of toxicity compared to conventional synthetic ligands of PPAR-y . The Applicant has substantiated these findings through in sili ca computational modeling analysis and in vi tro and in vi vo preclinical studies .

[0022] The compound according to the invention is a synthetic and selective ligand of PPAR-y, derived from thiazolidine- 2 , 4-dione and containing an N-heterocyclic indole ring in the side chain .

[0023] The Applicant carried out experiments revealing that the oral administration of the compound according to the invention in mouse models of obesity and T2DM, is associated with an anti-diabetic and insulin-sensiti zing response not inferior to that of Rosiglitazone . The advantage over the known compound Rosiglitazone is the lower risk of toxicity and adverse events associated with the use of ADEMAGLITAZONE .

[0024] The compound ADEMAGLITAZONE represents a substantial innovation in the treatment of T2DM and obesity-induced insulin resistance .

[0025] Over the years , several studies have been conducted on the biology of the nuclear receptor PPAR-y, its regulatory ef fects on the transcription of genes involved in glycol ipid metabolism and the biological activity of insulin, and the di f ferent binding af f inities / modes of full or partial agonism of its synthetic ligands , namely thiazolidinediones or glitazones . Recently, in sili ca computational modeling analysis , in vi tro screening tests on validated cell models for the study of glucose metabolism ( i . e . , the human hepatocarcinoma cell line HepG2 ) , together with preclinical in vi vo comparative ef ficacy studies in diet-induced obese ( DIO) mice , have led to the synthesis and identification of the compound according to the invention, a synthetic PPAR-y ligand for oral administration, with high insulinsensiti zing activity and low risk of toxicity, including the ability to stimulate adipogenesis and promote weight gain, hitherto typical of Rosiglitazone and marketed glitazones .

[0026] According to one aspect of the invention, the synthesis process of ADEMAGLITAZONE di f fers from known and marketed glitazones , as it originates from the condensation of the thiazolidine-2 , 4-done pharmacophore with an N-heterocyclic ring ( indole ) .

[0027] In fact , computational modeling studies conducted by the Applicant with speci fic reference to ADEMAGLITAZONE ( Fig . 2A, 2B, 2C and 2D) have shown that the inclusion of an N-heterocyclic system, such as an Indole moiety, within the chemical structure of glitazones , results in increased af finity and speci ficity of binding of these molecules to the PPAR-y receptor, leading to an enhancement of their biological activity without , however, increasing the risk of toxicity but rather reducing it . In sili co computational modeling analyses were performed in the ligand binding domain ( LBD) of PPAR-y, using the Glide Suite Docking package ( Glide , Schrodinger, LLC, New York, NY) and the Schrodinger Induced Fit Docking ( I FD) protocol . The starting point for molecular docking simulations was the crystal structure o f the Rosiglitazone / PPAR-y complex ( PDB ID : 2PRG) . Analyses of static ligand-receptor interaction models obtained via the IDF protocol were subsequently validated by molecular dynamics studies , calculating the Root Mean Square Deviation (RMSD) on the a carbons of the ligand . The receptor grid was centered on the centroid of the complexed ligand, with an energy window of 2 . 5 kcal / mol to perform a full conformational search . To examine conformational flexibility, protein residues within a radius of 5 A from the ligand-engaging poses ( 20 poses analyzed) in the LBD of PPAR-y were analyzed using the Prime molecular dynamics module ( Prime , Schrodinger, LLC, New York, NY) . In computational conformational analyses using the I FD protocol , ADEMAGLITAZONE was found to geometrically reproduce the mode of binding of Rosiglitazone to the LBD of PPAR-y ( Fig . 2A, 2B and 2C ) , by positioning two carbonyl groups of the thiazolidine ring towards protein residues considered crucial for binding . The ligand-receptor interaction analysis was further refined with molecular dynamics simulations ( duration 100ns ) . These simulations revealed direct interactions of ADEMAGLITAZONE , through hydrogen bonds , with H449 , one of the key protein residues of the PPAR-y LBD, for 49% of the simulation time . In addition, intermolecular interactions were identi fied through hydrogen bonds between the carbonyl group of the thiazolidine ring of ADEMAGLITAZONE and residues S289 , Y327 , K367 , H449 and Y473 of the PPAR-y LBD, as well as hydrophobic contacts between the ethyl group of the thiazolidine-2 , 4- done pharmacophore and the residue F282 , and between the indole ring of ADEMAGLITAZONE and residues C285 and L330 , which contribute to the stabili zation of the ligand-receptor complex. Differences in the stability of the ADEMAGLITAZONE / PPAR-y and Rosiglitazone / PPAR-y complexes were analyzed by RMSD analysis, documenting a greater stability of the ADEMAGLITAZONE / PPAR-y complex compared to the Rosiglitazone / PPAR-y complex and the native apo (unbound) form of the PPAR-y receptor (Fig. 2D) .

[0028] The synthesis process of the compound according to the invention is shown in figure 3, and involves a Knoevenagel condensation reaction between an active methylene group of the thiazolidine-2 , 4-dione pharmacophore and indole-3- aldehyde in refluxing acetonitrile, using piperidine acetate as a catalyst.

[0029] Alkylation of the synthesized intermediate is then carried out using diethyl sulfate as the alkylating agent and potassium carbonate as the base in dimethylformamide, resulting in the formation of ( Z ) -5- [ ( 5-bromo-l-ethyl-lH- indol-3-yl) methylene] -3-ethyl thiazolidine-2 , 4 -di one, herein referred to as ADEMAGLITAZONE .

[0030] Glitazones are drugs known for their ability to increase the production of adiponectin (a pro-lipolytic hormone released by adipocytes) , following adipogenesis activation. Circulating adiponectin, in turn, can influence insulin signaling in peripheral tissues, overall exerting an insulin-sensitizing effect. As evidenced in in vitro studies carried out by the Applicant on differentiated 3T3-L1 murine adipocytes, treatment with lOpM of ADEMAGLITAZONE for 24h did not result in an increase in adiponectin mRNA synthesis, unlike Rosiglitazone (Fig. 4A) .

[0031] The findings were also confirmed in preliminary in vivo studies on male DIO mice, treated with oral ADEMAGLITAZONE at a dose of 30mg / kg for 5 weeks, in which no differences in serum adiponectin concentrations were observed compared to untreated control mice. In contrast, treatment with Rosiglitazone at the same dose of ADEMAGLITAZONE led to an increase in circulating adiponectin concentrations. Comparative experimental results are shown in Figure 4B.

[0032] Despite the weak adipogenic activity of ADEMIGLITAZONE compared to Rosiglitazone, as evidenced by its lack of direct effect on adiponectin production and release by murine adipocytes, the synthetic ligand according to the invention acts as a potent systemic insulin-sensitizing agent, with efficacy not inferior to that of Rosiglitazone, as demonstrated by additional experimental results, shown in Figure 5A, obtained through insulin tolerance tests in male DIO mice undergoing 5 weeks of treatment with oral ADEMAGLITAZONE at a dose of 30 mg / kg. However, unlike what was observed with Rosiglitazone , in vivo studies on DIO mice have revealed that the compound according to the invention did not induce weight gain in treated animals compared to untreated controls , as shown in Figure 5B .

[0033] This neutral ef fect on weight represents an advantage of the compound ADEMAGLITAZONE and its use in the pharmacological treatment of obesity-induced insulin resistance and T2DM .

[0034] Overall , the experimental results obtained by the Applicant show that , unlike the known synthetic PPAR-y ligands such as Rosiglitazone , the systemic insulinsensiti zing activity of ADEMAGLITAZONE does not depend on increased adiponectin release from adipose tissue , but rather on the activation of di f ferent signal pathways , that may not be linked to PPAR-y agonism . In fact , there are numerous pro-inflammatory adipokines and cytokines released by a dys functional visceral adipose tissue in obesity . These hormones contribute to the low-grade systemic pro- inflammatory state present in obese patients and, through binding to their own receptors , they activate the JAK-STAT signal pathway, which deeply interferes with the transduction of the insulin signal in target cells , thus contributing to the pathogenesis of insulin resistance . Experimental results have shown that ADMAGLITAZONE is able to restore a normal insulin s ignaling, as indicated by the rapid phosphorylation of protein kinase B / Akt at Thr308 and Ser473 , in several human cell lines rendered insulinresistant by prolonged treatment with palmitic acid, and to signi ficantly improve the circulating pro-inflammatory adipokine and cytokine profile in DIO mice . These ef fects were more pronounced compared to those obtained with Rosiglitazone , without any observed cytotoxicity or lethality at the tested doses .

[0035] The Applicant has experimentally demonstrated that the visceral adipose tissue of obese individuals becomes resistant to the action of insulin due to the down-regulation of the insulin receptor expression . This molecular alteration is at the basis of visceral fat dys function in obesity and leads to an abnormal release of adipokines and cytokines into the systemic circulation, which can interfere , positively or negatively, with insulin signal ing in target organs . As a result , there is a reduction in peripheral glucose uptake and a failure to suppress its endogenous production, which are two crucial factors in the pathogenesis of chronic hyperglycemia in T2DM .

[0036] The present invention also relates to a pharmaceutical composition comprising the compound ADEMAGLITAZONE , having the formula already speci fied herein, and suitable for oral administration .

[0037] The compound ADEMAGLITAZONE according to the invention is advantageously capable of improving the condition o f insulin resistance induced by obesity with potent antidiabetic activity and a low risk of toxicity and adverse events , including the weight gain tendency associated with conventional glitazones .

[0038] Lastly, it is evident that the compound ADEMAGLITAZONE , which binds to the peroxisome proli f erator-activated receptor gamma and exhibits insulin-sensiti zing activity and weak adipogenic capacity, and its use in pharmacological treatment of type 2 diabetes mellitus and obesity-induced insulin resistance , as well as the pharmaceutical formulation containing the compound and its synthesis process , may undergo modi fications and variations without departing from the protective scope of the present invention, as defined in the accompanying claims .

Claims

CLAIMS1. The compound ADEMAGLITAZONE, with the IUPAC nomenclature :(Z) -5- [ ( 5-bromo-l -ethyl- IH-indol- 3-yl ) methylene] -3- ethylthiazolidine-2 , 4-dione ] and structure:Wherein said compound is a synthetic ligand of the peroxisome prolif erator-activated receptor gamma (PPAR-y) , exhibiting insulin-sensitizing properties and limited capacity to promote adipogenesis.

2. Compound according to claim 1, characterized by being produced through re-engineering of the thiazolidinedione pharmacophore, which is condensed with an N-heterocyclic side chain, enabling its binding to PPAR-y with a high level of affinity and specificity.

3. Compound according to claim 1, characterized in being a synthetic and selective ligand of PPAR-y, which derives from thiazolidine-2 , 4-dione and contains an indole N- heterocyclic ring in the side chain.4 . Synthesis process of the compound according to claim 1 , characteri zed in involving the condensation of the pharmacophore thiazolidine-2 , 4-dione with an Indole moiety .5 . Synthesis process of the compound according to claim 1 , characteri zed in comprising the following steps :- a Knoevenagel condensation reaction between an active methylene group of the pharmacophore thiazolidine-2 , 4-dione and indole-3-aldehyde in refluxing acetonitrile , in the presence of piperidine acetate as a catalyst ; - alkylation of the resulting intermediate using diethyl sul fate as the alkylating agent and potassium carbonate as the base in dimethyl formamide . This results in the formation of the compound ( Z ) -5- [ ( 5-bromo- l-ethyl- lH- indol-3-yl ) methylene ] -3-ethyl thiazolidine-2 , 4 -di one . 6 . Compound according to claim 1 , for the use in the treatment of type 2 diabetes mellitus and obesity-induced insulin resistance .7 . A pharmaceutical formulation containing the compound according to claim 1 , intended for oral administration .

Images