INTRODUCTION:

Type 2 diabetes (T2DM) have major health care burden around the world. In 2012, More than 371 million people were diagnosed with diabetes. Still half of people with diabetes are undiagnosed. 4.8 million People died due to diabetes. More than 471 billion USD $ were spent on healthcare for diabetes. Ninety percent of these patients suffer from T2DM, which is characterized by a resistance to insulin. This resistance to insulin is developed years before the diagnosis of T2DM. The preservation of glycemic control relies on the pancreas’s ability to overcome tissue resistance by simply increasing its production of insulin. However, the increased stress on the insulin-secreting pancreatic b-cell from glucotoxicity, lipotoxicity, inflammatory cytokines and genetic sensitivities leads to their failure. The micro and macro-vascular complication associated with T2DM are directly correlated with the magnitude and duration of the hyperglycemia¹.

Major current pharmacotherapies for T2DM include sulfonylureas, metformin, thiazolidinediones (PPARγ agonist), gliptins (DPPIV inhibitors) and liragutide (GLP-1 agonist). These pharmacotherapies have their own limitations with respect to their efficacy or side effects like nausea, diarrhea, hypoglycemia, weight gain, fluid retention and cardiovascular complications ². This makes discovery of new and safe treatments essential for T2DM. Protein tyrosine phosphatase 1B (PTP1B) contribute to diabetes and obesity ^3–5. PTP1B knockout mice exhibit phenotypes of increased insulin sensitivity, improved glucose tolerance and resistance to high fat induced weight gain all without any adverse effects ^6,
7.Designing selective PTP1B inhibitors is a big challenge, T-cell protein tyrosine phosphatase (TCPTP), a major hurdle in the development of safe and effective PTP1B inhibitors ^{8, 9}. PTP1B active site contains phosphotyrosine (pTyr) containing two negative charges at physiological pH. Therefore most of the competitive PTP1B inhibitors have high charge density mimicking pTyr which limits their drug-like properties with limited cell permeability or bioavailability.

Over the past two decades, numerous PTP1B inhibitors have been developed while two compounds, ertiprotafib and trodusquemine, were progressed to clinical trials. However, ertiprotafib had been discontinued in phase II clinical trials due to lack of efficacy and side effect ^{10, 11}. The further development of most PTP1B inhibitors was restricted due to their low cell permeability and poor bioavailability. Therefore, there is a need to develop novel potential drug scaffolds targeting PTP1B with desirable physicochemical properties and in vivo efficacies.

A focused library approach was used to identify highly potent and selective PTP1B inhibitors that are capable of bridging and simultaneously associating with both the active site and an adjacent peripheral site ¹². A ‘linked fragment’ approach was employed to develop potent and selective PTP1B inhibitors that can engage both the active site and the second aryl phosphate-binding site^13-15. Structure-based modeling has been used to target unique PTP1Bconformations for inhibitor development with both high affinity and selectivity¹⁶. A secondary allosteric site has recently been described for PTP1B, and several small-molecule inhibitors that occupy this site stabilize an inactive conformation of PTP1B¹⁷. The most straightforward approach is to reduce the number of negative charges, so that a less-charged derivative might be able to penetrate the cell membrane. Another approach to increase cell permeability is to enhance the hydrophobic character of the compounds. The prodrug approach has been widely used to deliver compounds containing one or more carboxylic acid group(s). The corresponding methyl or ethyl esters are called prodrugs, and they are much easier to pass through the cell membrane. Once inside the cell, the prodrugs are hydrolyzed to regenerate the original inhibitors.

Compounds of the thiazolidinedione (TZD) class have aroused considerable interest as antihyperglycemic compounds and aldose reductase inhibitors ^18-20. Some of these compounds (such as pioglitazone and rosiglitazone) are insulin-sensitizing agents acting as peroxisome proliferatoractivated receptor γ (PPARγ) agonists ¹⁹, and they have been shown to be effective in treating type II diabetes in clinical situation. In addition, some 2,4-TZDs have proved to be PTP1B inhibitors ²¹. TZD moiety and substituted biphenyl scaffold were found to be effective ²². Here we describe our extended efforts on this SAR studies which leading to more potent PTP1B inhibitors with antihyperglycemic activity in vivo. Our goal was to discover novel, potent, cell permeable and orally bio-available PTP1B inhibitors by designing low molecular weight, non-phosphonate and mono carboxylic acid phosphotyrosyl (pTyr) mimetics inhibitors based on known literature compound (Fig. 1). IC50 0.53±0.10 (µmol/L)²³.

Fig. 1 Structure of reference PTP1B inhibitor

MATERIALS AND METHODS:

General procedure for Synthesis of compounds

Synthesis of 4-bromobenzohydrazide:In 100 ml RBF, a solution of hydrazine hydrate (10 ml) and ethyl 4-bromobenzoate (2 gm) in ethanol was stirred at 90 °C for 4 hours. The reaction mixture was cooled and poured into cold water. The crude product was filtered, dried at 100 °C and finally recrystallised from ethanol.

Synthesis of ethyl [2-(4-bromobenzoyl) hydrazino](oxo)acetate:2-(4-bromophenyl)acetohydrazide was dissolved in tetrahydrofuran (10 mL) at room temperature. Ethyl malonyl chloride and tri ethylamine was added and the reaction stirred at room temperature for 20 minutes. Solvents were removed in vacuo to give the title compound.

Synthesis of ethyl 5-(4-bromophenyl)-1,3,4-oxadiazole-2-carboxylate:A suspension of ethyl [2-(4-bromobenzoyl)hydrazino](oxo)acetate in POCl3 (3 mL) was stirred at 90° C for 3 h. The resulting clear solution was quenched with ice-water, solid obtained was filtered washed with water, dried to give title compound.

Synthesis of ethyl 5-(Ar)-1,3,4-oxadiazole-2-carboxylate: To a stirred solution of ethyl ethyl 5-(4-bromophenyl)-1,3,4-oxadiazole-2-carboxylate and Ar- boronic acid in dioxane is added 2M K2CO3 at room temperature, then reaction mixture is purged with argon gas for 30 min, followed by triphenyl phosphine palladium is added at same temperature and reaction mixture is stirred at 110 °C for 16 h. Then the reaction mixture is filtered through diatomaceous earth, diluted with water and extracted with EtOAc (2x30 mL). Combined organic layer is washed with water (2x50 mL) and brine solution (2x50 mL), then dried over anhydrous Na2SO4 and evaporated to give crude compound. Crude compound is purified by column chromatography (100:200 silica mesh) (eluent-5.6% EtOAc/hexane), gave a title compound.

Synthesis of 5-(Ar)-1,3,4-oxadiazole-2-carboxylic acid:To a stirred solution of ethyl 5-(Ar)-1,3,4-oxadiazole-2-carboxylate in ethanol (5 mL) is added 5N NaOH) at room temperature, then reaction mixture is stirred for 2 h at room temperature. The reaction mixture is evaporated under reduced pressure and the residue is triturated with ether/n-pentane (1:1) mixture and decanted. This material is dissolved in water and acidified with citric acid solution to about 5 pH. The solid precipitated is filtered and freeze dried to give the title compound as off white solid.

Animals

Male C57BL/6J mice were purchased from Laxmi Biofarms Pvt. Ltd. Ale Phata, Pune, India. The research work was conducted in accordance with the internationally accepted principles for laboratory animal use and care. The rats were housed under good hygienic conditions in the Animal house under standard conditions of temperature (24±1)0 C, relative humidity (65 ±10) % and 12 hrs light, 12 hrs dark cycle. The rats were fed with standard pellet diet and drinking water ad libitum. The animals were allowed to acclimatize to experimental conditions by housing them for 8-10 days prior to the experiments. All animal use was in compliance Experimental Animal Care issued by the Committee for Purpose of Control and Supervision of Experiments on Animal (CPCSEA).

Oral glucose tolerance test (OGTT)

Day before the study animals were randomized in different groups (Six mice per group) on the basis of body weight. Animals were orally gavaged BID with the compound-9 (0.3. 1, 3, 10, 30 and 100 mg/kg, p.o.) and rosiglitazone- 10 mg/kg, p.o. for 7 days. An oral glucose tolerance test (OGTT) performed on day 7. In OGTT assay, 16 hr fasted mice were treated with vehicle or compounds (10ml/kg, PO) after fasting blood glucose (t=−60 min) measurement. The mice were then gavaged with an oral bolus of glucose (2 g/kg). We measured the basal blood glucose level and then at 0, 10, 30, 60 and 120 min for OGTT from tail blood using glucometer (Bayer –Contour TS). The blood glucose excursion profile from 0 to 120 min was used to integrate an area under the curve (AUC) for each treatment. Percent inhibition values for each treatment were generated. Statistical analyses of the obtained data were performed using One way ANOVA followed by Dunnett’s test and Two way ANOVA followed by Bonferroni test using GraphPad prism ver. 5 software.

Enzyme-based assay for PTP1B

A colorimetric assay to measure inhibition against PTP1B was performed in 96-well plates. In this assay, the tested compounds were solubilized in DMSO and serially diluted for the concentrations ranging from 0.03 µM to 1000 µM. The assays were carried out in a final volume of 100 μL containing 50 mmol/L MOPS, pH 6.5, 2 mmol/L pNPP, 30 nmol/L GSTPTP1B and serially diluted compounds (2% DMSO). The catalysis of pNPP was continuously monitored on a SpectraMax 340 microplate reader at 405 nm for 2 min at 30°C. The IC50 value was calculated from the nonlinear curve fitting of the percent inhibition vs the inhibitor concentration [µM] using graph-pad prism software²³results is summarized in Figure 5.

Structure designing of Compound

Molecular modeling was used to understand the binding mode of proposed compounds. Molecular docking study was carried out in PDB ID: 1Q1M corresponding to PTP1B. Docking was carried out in Molegro Virtual Docker (MVD)²⁴. Figure 2 shows reference compound docked into active site of PTP1B (pdb id 1Q1M). Critical interactions seen for reference compound are; Acid group interacts with Arg 221 and Gln 266 (h-bond, with side chain), while –CO Interacts with backbone –NH of Gly 220. Substituted phenyl rings appears to have Hydrophobic interactions with Tyr 46, Val 49 &cation-pi interactions with Arg 45, Arg 47.

Fig. 5 Effect of NCE-9 on Enzyme-based assay for PTP-1B inhibition

Compound 9 was evaluated in vitro for their inhibitory activity against PTP1B (Fig. 5). As illustrated in Table Fig. 5, NCE-9 shows good inhibitory activity, with IC50 values 0.46 µmol/L.As compounds 9 showed good Inhibitory activities for PTP1B, we further evaluated them in mouse pharmacodynamics study

Fig. 6 Effect of NCE-9 on blood glucose in OGTT in C57BL/6J mice after one week of bid treatment

All values are expressed as Blood glucose in mg/dl (Mean ± SEM), n = 6. Vertical lines represent SEM. All data are subjected to Two Way ANOVA followed by Bonferroni’spost test. ###p<0.001: Rosiglitazone vs Vehicle, ^ p<0.05: NCE-9 (10 mpk) vs Vehicle, θθθθ p<0.0001, θ p<0.05: NCE-9 (30 mpk) vs Vehicle, ππππ p<0.0001, πππ p<0.001: NCE-9 (100 mpk) vs Vehicle

Fig. 7 Effect of NCE-9 on AUC Glucose in OGTT in C57BL/6J mice after one week of bid treatment

All values are expressed as AUC Glucose(0-120 min) in mg/dl*min (Mean ± SEM),n = 6. Vertical lines represent SEM. All data are subjected to One Way ANOVA followed by Dunnett’spost test. ** p<0.01 vs Vehicle

Compound 9 in OGTT significantly improved after prolonged treatment, and the area under the curve(AUC) was decreased(Fig. 7). The blood glucose level declined more rapidly than in Rosiglitazone treated mice (10 mg/kg)(Fig. 6).

CONCLUSION:

In summary, series of compounds containing disubstituted oxadiazole were prioritized and synthesized, based on molecular modeling studies. Compounds were prioritized based on quantitative and qualitative analysis. Qualitatative Analysis: Based on lean Moldock, Rerank and h bond scores. Quantitative Analysis: Based on overlay (on reference compound) and interaction with Arg 221 Lean values are calculated by dividing MolDock score, Rerank score &Hbond score with number of heavy atoms to find the contribution of each when binding to receptor. Molegro Virtual Docker1 was used for docking analysis. Novel oxadiazole series with di-substitution showed the better inhibition toward PTP1B. Compound 9 found to be the best among these disubstituted oxadiazole reported with 0.46 µmol/L in PTP1B enzyme assay. Also compound 9 improved glucose tolerance in OGTT which is comparable with Rosiglitazone. Further investigation of these derivatives with change of pharmacophore and in vitro assays of the disubstituted oxadiazole series are in progress and will be reported in due course.

ACKNOWLEDGEMENT:

The authors would like to thank management and staff of the Department of Pharmacology, Faculty of Pharmacy, KCTS’ R. G. Sapkal College of Pharmacy, Shri Neminath Jain Bramhacharyashram's Shriman Sureshdada Jain College of Pharmacy, Jain Gurukul, Chandwad, Nashik, 423101 Maharashtra, India for providing facility to carry out research work.

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Received on 21.03.2014 Accepted on 28.04.2014

Asian J. Pharm. Tech. 2014; Vol. 4: Issue 2, Pg 43-49