Bioorganic Chemistry 72 (2017) 345–358

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Bioorganic Chemistry

journal homepage: www.elsevier .com/locate /bioorg

Design, synthesis and anti-diabetic activity of triazolotriazine derivativesas dipeptidyl peptidase-4 (DPP-4) inhibitors

http://dx.doi.org/10.1016/j.bioorg.2017.03.0040045-2068/� 2017 Published by Elsevier Inc.

Abbreviations: CMC, carboxy methyl cellulose; DMSO, dimethyl sulfoxide; DPP-4, dipeptidyl peptidase-4; 3D-QSAR, 3-dimensional quantitative structure activityrelationship; HFD, high-fat diet; HPLC, high performance liquid chromatography;STZ, streptozotocin; T2DM, type 2 diabetes mellitus.⇑ Corresponding author at: Department of Pharmaceutical Chemistry, Institute of

Pharmacy, Nirma University, S.G. Highway, Chharodi, Ahmedabad 382481, India.E-mail address: ghate72@gmail.com (M.D. Ghate).

Bhumika D. Patel a, Shraddha V. Bhadada b, Manjunath D. Ghate a,⇑aDepartment of Pharmaceutical Chemistry, Institute of Pharmacy, Nirma University, Ahmedabad 382481, Gujarat, IndiabDepartment of Pharmacology, Institute of Pharmacy, Nirma University, Ahmedabad 382481, Gujarat, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 7 December 2016Revised 2 March 2017Accepted 3 March 2017Available online 6 March 2017

Keywords:Dipeptidyl peptidase-4 inhibitors3D-QSARPharmacophore modelingMolecular dockingTriazolotriazine derivativesType 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is one of the major global metabolic disorders characterized by insulinresistance and chronic hyperglycemia. Inhibition of the enzyme, dipeptidyl peptidase-4 (DPP-4) has beenproved as successful and safe therapy for the treatment of T2DM since last decade. In order to designnovel DPP-4 inhibitors, various in silico studies such as 3D-QSAR, pharmacophore modeling and virtualscreening were performed and on the basis of the combined results of them, total 50 triazolo[5,1-c][1,2,4]triazine derivatives were designed and mapped on the best pharmacophore model. From this, best25 derivatives were docked onto the active site of DPP-4 enzyme and in silico ADMET properties were alsopredicted. Finally, top 17 derivatives were synthesized and characterized using FT-IR, Mass, 1H NMR and13C NMR spectroscopy. Purity of compounds was checked using HPLC. These derivatives were then eval-uated for in vitro DPP-4 inhibition. The most promising compound 15q showed 28.05 lMDPP-4 IC50 with8–10-fold selectivity over DPP-8 and DPP-9 so selected for further in vivo anti-diabetic evaluation. DuringOGTT in normal C57BL/6J mice, compound 15q reduced blood glucose excursion in a dose-dependentmanner. Chronic treatment for 28 days with compound 15q improved the serum glucose levels in type2 diabetic Sprague Dawley rats wherein diabetes was induced by high fat diet and low dose streptozo-tocin. This suggested that compound 15q is a moderately potent and selective hit molecule which canbe further optimized structurally to increase the efficacy and overall pharmacological profile as DPP-4inhibitor.

� 2017 Published by Elsevier Inc.

1. Introduction

Type 2 diabetes mellitus (T2DM) is one of the major globalmetabolic disorders characterized by insulin resistance andchronic hyperglycemia. India ranks second highest for diabeticpatients with around 69.1 million people suffering from diabetesin 2015. By 2040, this number will be increased up to 123.5 millionas predicted by the International Diabetes Federation (IDF) [1].Conventionally sulfonylureas, meglitinides, thiazolidinediones,biguanides and a-glucosidase inhibitors are used for treatment ofT2DM but most of them cause common side effects such as hypo-glycemia and weight gain. Many newer anti-diabetic therapies

such as 11b-hydroxysteroid dehydrogenase 1 inhibitors, sodium–glucose co-transporter 2 inhibitors, glucagon-receptor antagonists,dipeptidyl peptidase-4 (DPP-4) inhibitors, metabolic inhibitors ofhepatic glucose, pancreatic-G-protein-coupled fatty-acid-receptoragonists, and insulin-releasing glucokinase activators haveemerged to overcome such side effects [2]. Out of these, inhibitionof the DPP-4 enzyme has been proved as successful, safe and wellestablished therapy for the treatment of T2DM since last decade[3]. DPP-4 enzyme deactivates the natural hypoglycemic incretin,glucagon like peptide (GLP)-1, so inhibition of this enzyme restoresglucose homeostasis in diabetic patients through various actions ofGLP-1 like increased insulin biosynthesis, increased b-cells prolif-eration and decreased their apoptosis, and decreased glucagonsecretion and gluconeogenesis. Starting from the discovery of firstDPP-4 inhibitor sitagliptin in 2006, till date total ten DPP-4 inhibi-tors (1–10, Fig. 1) are in market with more or less similar efficacybut with different selectivity and pharmacokinetic profile. Theyhave proven themselves as effective and well tolerated treatment

Fig. 1. Marketed DPP-4 inhibitors.

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for T2DM without side effects of weight gain or hypoglycemia.Instead they have many cardiovascular benefits.

Currently, research is going on for discovery of longer actingDPP-4 inhibitors that are amenable for once-weekly dosing toimprove patient compliance in T2DM. Such a long time sustainedDPP-4 inhibition may result in better coverage for glucose-simulated insulin secretion due to the consistently higher levelsof GLP-1 and overall produce greater therapeutic benefit. Two suchdrug molecules, Trelagliptin [4] 9 and Omarigliptin [5] 10 gotrecent market approval in Japan in March 2015 and September2015 respectively. More such new weekly acting DPP-4 inhibitorsare required in market. So, in our initial efforts of developing suchnovel DPP-4 inhibitors, various heterocyclic scaffolds reported forDPP-4 inhibition so far had been reviewed thoroughly and basedon their medicinal chemistry approaches, we summarized keyin silico features to design potent and selective DPP-4 inhibitors[6]. Later, we performed 3D-QSAR, pharmacophore modeling,virtual screening and molecular docking analysis on DPP-4 inhibi-tors which were already published [7,8]. Results of all the methodswere combined to design triazolotriazine derivatives as novelnon-peptidomimetic DPP-4 inhibitors. Till now, triazolotriazinederivatives have been well explored as adenosine A2a receptorantagonists, CYP1A1 inhibitors, c-Met inhibitors, etc. [9–13] butfor the first time, we are reporting triazolotriazine derivatives asDPP-4 inhibitors for the treatment of T2DM. In the present study,we reported design, synthesis and pharmacological evaluation ofa series of [1,2,4]triazolo[5,1-c][1,2,4]triazine derivatives as

Fig. 2. Stepwise graphical representation

DPP-4 inhibitors. These derivatives were evaluated in vitro forDPP-4 inhibition. In vivo anti-diabetic activity was also carriedout in animal models.

2. Results and discussion

2.1. Design of DPP-4 inhibitors

In our earlier published in silico studies, 3D-QSAR study wascarried out on a series of 36 quinoline/isoquinoline based non-peptidomimetic DPP-4 inhibitors [7]. On the basis of the CoMFA/CoMSIA model contour maps, significant regions for steric, electro-static, hydrophobic and H-bond interactions were identified. Inanother study, structure and ligand based pharmacophore modelswere generated, validated and used for virtual screening to findnovel hit structures [8]. Designing of the novel molecules was car-ried out on the basis of pharmacophore features of the best vali-dated model A, structural features of hits obtained throughvirtual screening and contour map analysis of 3D-QSAR model asshown in Fig. 2. Heterocyclic ring, [1,2,4]triazolo[1,5-a]pyrimidine,present in the hit molecule Asinex ASN 09858221 was replacedwith its probable bioisosteric ring [1,2,4]triazolo[5,1-c][1,2,4]tri-azine ring with an aim to explore it for DPP-4 inhibition for the firsttime. Total 50 different [1,2,4]triazolo[5,1-c][1,2,4]triazine deriva-tives were designed initially. Various R, R1 and R2 substitutionswere chosen on the basis of the identified structural features

of in silico designing of molecules.

Table 1Designed and synthesized 7-bromo-3-substituted-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine derivatives with their docking score and in vitro enzyme inhibition data.

Sr. no R1 Docking score %DPP-4 enzyme inhibitionb DPP-4* IC50 lM DPP-8* IC50 lM DPP-9* IC50 lM

-PLP1a 1 lM 10 lM 100 lM

15a 4-OCH3 63.19 8.5 11.1 12.2 ND ND ND15b 4-CH3 65.68 5.9 11.0 10.6 ND ND ND15c 4-CN 68.29 12.7 25.4 48.3 166.4 1176.5 1923.415d 4-Cl 63.96 8.3 15.1 16.6 ND ND ND15e 4-C6H5 72.58 8.1 17.9 27.0 ND ND ND15f 3-F 66.85 7.4 7.6 13.5 ND ND ND15g 4-OH 62.02 9.4 4.4 3.5 ND ND ND15h 4-CF3 72.03 9.6 0.7 19.5 ND ND ND15i 3-NO2 73.15 11.0 10.8 11.9 ND ND ND15J 3-OCH3 69.13 7.3 12.4 18.3 ND ND ND15k 3-CN 70.66 7.1 3.5 18.2 ND ND ND15l 2,4-di-F 67.9 3.3 23.6 30.6 ND ND ND15m 3,4-di-Cl 70.49 �11.2 �3.1 18.2 ND ND ND15n 4-F, 3-Cl 69.69 �5.9 6.2 11.9 ND ND ND15o 2-OCH3 67.06 �4.1 16.0 16.3 ND ND ND15p – 72.72 12.3 9.3 34.6 ND ND ND15q – 58.88 17.7 20.5 53.3 28.05 230 275Sitagliptin 64.88 0.01 lM 0.1 lM 0.3 lM 0.018 48c >100c

13.8 74.2 84.8

Bold values represent most two potent compounds in a series.ND: Not determined.

a Higher –PLP(Piecewise Linear Potential) scores indicate better protein-ligand binding.b DPP-4, DPP-8 and DPP-9 inhibitory activity was determined by fluorescence-based enzyme assay. IC50 was determined using Graph Pad prism software.c Reported literature value (Ref. [20]).

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essential for activity. All the designed molecules were thenscreened against pharmacophore A using Discovery Studio 2.1[14] and from the retrieved hits, top 25 molecules with pharma-cophore Fit values �2.5 were docked into DPP-4 active site usingLigandFit module [15] of Discovery Studio 2.1. The X-ray crystallo-graphic structure of DPP-4 (PDB ID: 3KWF) was retrieved from thePDB databank and used for molecular docking simulations [16].Docking score (–PLP; Piecewise Linear Potential) and various inter-actions between designed molecules and crucial amino acids at thetarget site were analyzed and finally, a series of best 17 com-pounds, 7-bromo-3-substituted-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine derivatives as shown in Table 1 was synthesized.Molecular docking studies of them indicated that P1 and P2 frag-ments of the molecules got fit into the complimentary target pock-ets, S1 and S2 respectively of DPP-4 active site. Proposed bindinginteractions of molecules as presented in Fig. 3 indicated thatANH of the triazolotriazine ring at position 1 makes crucial Hbonding interaction and possibly ionic interactions with Glu205/Glu206 amino acids at S2 site in most of the compounds. The sub-stituted phenyl ring at position 3 occupied the hydrophobic S1pocket lined with the residues Tyr 631, Val 656, Trp 659, Tyr662, Tyr 666, Asn710, Val 711, etc.

2.2. Chemistry

The designed series of 7-bromo-3-substituted-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine derivatives 15a-q was synthesizedin three steps as per Scheme 1.

In first step of the synthesis, bromination of 1,2,4-triazole 11was carried out using two equivalent moles of bromine in an aque-ous solution of equimolar KBr. KHCO3 was added in equimolar con-centration and reaction mixture was heated at 80 �C with stirring.The reaction was also tried with other bases such as K2CO3 andCs2CO3 using different conditions. However, the reaction wasoptimized in stirring condition at 80 �C using KHCO3 and it gotcompleted within 30–40 min with a good yield of 3,5-dibromo-1H-1,2,4-triazole 12 [17]. In second step, simple nucleophilic sub-stitution reaction occurred between 3,5-dibromo-1H-1,2,4-triazole12 and fifteen differently substituted phenacyl bromides 13a-o togive intermediate 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-substituted-ethanone derivatives 14a-o. Phenacyl bromides having sub-stituted phenyl ring with various electron releasing groups such asAOCH3, CH3, and 4-OH and electron withdrawing groups such asACN, ACl, AF, and ANO2 were selected. Apart from these, twoother related chemicals, 2-(bromoacetyl) naphthalene 13p and

Fig. 3. Proposed interactions of designed molecules at DPP-4 active site.

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2-(bromoacetyl) benzofuran 13q were also reacted with 3,5-dibromo-1H-1,2,4-triazole 12 to give intermediate 14p and 14q.Reaction started with nucleophilic attack of N-1 of intermediate12 on a carbon of 13a-qwith removal of HBr. Different polar proticsolvents such as methanol and ethanol and polar aprotic solventsuch as acetone and DMF were tried in the presence of mild base,K2CO3 at different conditions viz. stirring, heating and reflux. Thebest yield and purity of compounds 14a-qwere obtained with stir-ring in acetone using K2CO3 as base at 0 �C initially and then atroom temperature. Reaction completed in around 4–6 h with agood yield of compounds 14a-q [18]. The final third step of ringclosing reaction also proceeded via nucleophilic substitution reac-tion. Reaction between intermediate 14a-q and hydrazine hydrateNH2NH2�H2O ended up into the designed series of 7-bromo-3-substituted-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]traizine deriva-tives 15a-q with removal of HBr and H2O [18]. Maximum yieldwas obtained by refluxing for about 8–9 h in methanol and thenallowed to cool.

Structures of all intermediates and final products were con-firmed by FTIR, NMR and ESI-MS spectroscopy (Supporting infor-mation). In FTIR analysis, all the 14a-q intermediates showedAC@O stretch peak in the range of 1710–1680 cm�1 which got dis-appeared in final compounds 15a-q, indicating ring closure withformation of triazolotriazine ring. The ANH stretch of newly intro-duced secondary cyclic amine was observed in the range of 3400–3200 cm�1 in all the compounds 15a-q. Strong ANH bending wasalso observed around 1588–1570 cm�1. Due to the presence oftwo bromine atoms in all intermediates, their mass spectra (ESI-MS) showed characteristic [M+1]+, [M+3]+ and [M+5]+ peak pat-

tern whereas in final compounds, due to removal of one bromineas HBr, only [M+1]+ and [M+3]+ peaks were observed which wereof almost equal or near to equal in intensity. The significant fea-ture of 1H NMR spectrum of intermediate 12 was singlet ANH pro-ton peak at d (ppm) 7.26 which disappeared when it wasconverted into compounds 14a-q. The aromatic protons of com-pounds 14a-q appeared in the range of d (ppm) 7–8 with charac-teristic splitting pattern. The ACH2A protons of substitutedphenacyl bromide chain appeared at around d (ppm) 5.5 as singlet.1H NMR spectra of final compounds 15a-q additionally showedthe appearance of the cyclic ANH proton at around d (ppm) 11which came from hydrazine hydrate. In proton decoupled 13CNMR analysis of compounds 15a-q, all the carbon of differentenvironments appeared at different d (ppm) value as singlet. C-7of the triazolotriazine scaffold which is attached with bromineappeared at around 147 ppm, C-9 appeared at around 137 ppm,C-4 present as cyclic ACH2A appeared at around 44 ppm and C-3 which is substituted with aromatic ring, appeared at around133 ppm. All the aromatic carbons of phenyl ring appeared inthe range of 125–150 ppm depending upon their substitutions.Structure was also confirmed by single crystal X-ray crystallogra-phy of compound 15p (crystallized from THF and hexane, support-ing information) as anticipated in an ORTEP image shown in Fig. 4(CCDC 1474029). Various XRD parameters of compound 15p areshown in Table 2 while detailed description of results is providedin supporting information. Purity of all final compounds wasdetermined by HPLC analysis and was found to be more or equalto 95%. The retention time and purity of each compound are pre-sented in supporting information.

Scheme 1. Synthetic scheme for 7-bromo-3-substituted [1,2,4]triazolo[5,1-c][1,2,4]triazine derivatives. Reagents and Conditions: (a) H2O, KHCO3, Br2 in aqueous solution ofKBr, Stirring, 30–40 min, 80 �C (b) K2CO3, Stirring, 4–6 h, RT. (c) NH2NH2�H2O 80%, MeOH, Reflux, 8–9 h.

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2.3. Pharmacological evaluation

2.3.1. In vitro DPP-4, DPP-8 and DPP-9 assay and structure activityRelationship (SAR) studies

Data on the DPP-4 inhibitory activity during in vitro assay forthe synthesized compounds 15a-q and standard drug, sitagliptinare shown in Table 1. Sitagliptin produced 74.2% DPP-4 inhibition

at 0.1 lM concentration while most of the synthesized derivativesproduced less than 40% inhibition except compounds 15c and 15qwhich showed 48.3 and 53.3% inhibition respectively at 100 lMconcentration. These data suggested that although compounds15c and 15qwere found to be less efficacious compared to sitaglip-tin, but compared to other molecules with substituted phenyl ringas P1 fragment, the fused heterocyclic ring, benzofuran (compound

Fig. 4. ORTEP image of compound 15p.

Table 2Crystal data, data collection and refinement parameters for compound 15p.

Sr. no. Parameter Compound 15p

1 Empirical formula C14H10BrN5

2 MW 327.013 Color, habit Colorless, chip4 Crystal system Triclinic5 Crystal dimensions 0.660 � 0.400 � 0.300 mm6 Space group P-1 (#2)7 Z 48 a (�A) 6.7005(8) Å9 b (�A) 9.742(2) Å10 c (�A) 20.978(3) Å11 a (deg) 90.0000�12 b (deg) 90.0000�13 c (deg) 102.348(3)�14 V (�A3) 1337.7(3) Å3

15 Density (g/cm3) 1.629 g/cm3

16 T 20.0 �C17 X-ray wavelength 0.71075 Å18 l (MoKa) 30.785 cm�1

19 No. of reflns collected Total: 12,991Unique: 6058 (Rint = 0.0392)

20 R1 (obsd) 0.050021 wR2 (all) 0.122922 2hmax 54.9�

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15q) as P1 fragment produced more inhibition of enzyme. Thecompound 15q exhibited the highest 53.3% in vitro DPP-4 inhibi-tion. The probable reason could be the better fit of benzofuran ringwith more surface area into the S1 pocket compared to phenyl ringas per docking study. Compound 15p having naphthalene ringinstead of phenyl ring at 3rd position of triazolotriazine ring alsoproduced good inhibition (34.6%) but it was less than compound15q. So, another reason for good inhibitory activity of compound15q could be the improved aqueous solubility. The predicted clogP[19] value of compound 15q was 2.12 while that of compound 15pwas 3.45. Compound 15c having cyano group at para position ofphenyl ring produced 48% in vitro DPP-4 inhibition which mightbe due to the presence of cyano group. It can be proposed thatcyano group might give interaction with Ser630 of catalytic triadin the S1 pocket and thereby giving better affinity compared toothers. This cyano substituted phenyl ring represents the sameP1 fragment as found in marketed drug, alogliptin. Fig. 5a repre-sents the surface map of DPP-4 active site with docked poses ofcompounds 15q, 15p and 15c while Fig. 5b shows the proposedbinding interactions of compound 15c at the DPP-4 active site.

Except compounds 15q, 15c and 15p, remaining compoundswere found to be inactive with low inhibition of DPP-4 (10–30%)at 100 lM concentration. Out of these, compounds 15a, 15b, 15g,

15j, and 15o possess electron releasing groups (i.e. AOH, ACH3

and AOCH3) at 4th (para), 3rd (meta) or 2nd (ortho) positions of phe-nyl ring while compounds 15d, 15h, 15i, 15l, 15m, and 15n pos-sess electron withdrawing groups (i.e. ACl, ACN, AF, ACF3,ANO2) at the same positions. Thus, it might be concluded that dif-ference in electronegativity of molecules does not have majoreffect on DPP-4 inhibition. Rather, it was observed that bulk ofthe aromatic ring (as P1 fragment) has a good correlation withactivity. Due to lesser bulk of substituted phenyl ring present inabove inactive compounds, hydrophobic S1 pocket of DPP-4 activesite was not fully occupied as compared to best compound 15qwith benzofuran ring as P1 fragment which might be the probablereason for their lesser activity.

The best two active compounds 15q and 15c were selected forfurther DPP-4 IC50 determination and it was found to be28.05 lM and 166.4 lM respectively as shown in Table 1. Theywere also tested against DPP-4 homologues, DPP-8 and DPP-9 inthe DPP-4 gene family. The IC50 value of compound 15q againstDPP-8 was found to be 230 lM while against DPP-9, it was foundto be 275 lM. The IC50 value of compound 15c against DPP-8was found to be 1176.5 lM while against DPP-9, it was1923.4 lM. Thus, compared to sitagliptin (IC50 = 18 nM; DPP-4,48,000 nM; DPP-8, >100,000 nM; DPP-9) [20], most active com-pound 15q was found to be less potent and moderately selectivefor DPP-4 inhibition over DPP-8/9.

2.3.2. In vivo oral glucose tolerance test in normal C57BL/6J miceAlthough in vitro potency and selectivity of most potent com-

pound 15q for DPP-4 inhibition were found to be moderate, con-sidering it as novel heterocyclic derivative and early hit structurefor DPP-4 inhibition, further in vivo biological evaluation was car-ried out to know anti-hyperglycemic activity. Initial in vivo phar-macodynamic study was carried out in normal male C57BL/6Jmice using OGTT (oral glucose tolerance test). Here, compound15q was administered orally as suspension in 0.5% CMC to mice30 min prior to an oral glucose (2 g/kg) load. Serum glucose levelswere estimated at different time intervals from 0 to 180 min. Fol-lowing oral administration of 5, 10, and 20 mg/kg dose of com-pound 15q, serum glucose levels of mice started decreasing from30 min post glucose load (Fig. 6a). The major difference in OGTTresults has been observed in the value of Tmax between the glucosecontrol and compound 15q. The value of Tmax of Compound 15q atall the dose levels was 30 min while the same in case of glucosecontrol and sitagliptin was 15 min which indicated that compound15q is having different pharmacodynamic behavior compared tositagliptin. This increased value of Tmax suggested that compound15q might decrease the glucose absorption from the gut wall into

Fig. 5. (a) The surface map of DPP-4 active site with docked poses of compounds 15q, 15p and 15c. (b) Proposed interactions of compound 15c at the DPP-4 active site. Pinkcolored molecule is compound 15c, Green colored molecule is compound 15p and red colored molecule is compound 15q.

Fig. 6. (a) Effects of compound 15q on the change of serum glucose levels in C57BL/6 mice after oral glucose load. Data are presented as means ± SEM of 6 animals.* P < 0.05 versus glucose control, two-way ANOVA followed by Bonferroni’s posttest.(b) AUC glucose plot of compound 15q from 0 to 180 min in C57BL/6 mice after oralglucose load. Data are presented as means ± SEM of 6 animals. * P < 0.05 versusglucose control, one-way ANOVA followed by Dunnett’s posttest.

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the blood stream in the mice. Overall, glucose-induced blood glu-cose excursion was reasonably inhibited by compound 15q. Thetreatment resulted in reduced glucose AUC by 19.6%, 33.6% and47.1%, respectively in a dose-dependent manner while 27.8%reduction was observed in the AUC by sitagliptin 3 mg/kg as shownin Fig. 6b. Individually each dose produced significant reduction inserum AUC glucose levels compared to glucose control animals.However, 20 mg/kg dose was found to be significantly differentagainst standard drug, sitagliptin dose of 3 mg/kg at all-time inter-vals and produced almost 47% reduction in serum glucose AUC.These results suggested that compound 15q may improve theblood glucose tolerance in vivo in acute single dose study whereinnormal mice were under elevated serum glucose condition due tooral glucose load. Thus, the dose of compound 15q selected for thefurther chronic in vivo study was 20 mg/kg.

2.3.3. In vivo anti hyperglycemic evaluation in HFD/STZ induced T2DMmodel

The in vivo antihyperglycemic activity of compound 15q wasevaluated in a chronic model of high fat diet fed/streptozotocin(HFD/STZ) induced type II diabetes. As illustrated in Fig. 7, signifi-cant increase in serum glucose levels was observed in the diabeticcontrol animals at the end of the study as compared to normal con-trol animals which was significantly reduced in diabetic treated i.e.compound 15q 14 mg/kg treated and diabetic standard i.e. sitaglip-tin 2 mg/kg treated groups. There was no significant differencebetween the diabetic treated and diabetic standard animals sug-gesting that compound 15q at a dose of 14 mg/kg produced similaranti-hyperglycemic effect to sitagliptin, 2 mg/kg. It was observedthat only sitagliptin produced significant reduction in the serumglucose levels on the 14th day while compound 15q did not showany significant difference, when compared to their respective glu-cose levels at day 0. Again, significant difference was observed indiabetic treated group between glucose levels of day 14 and day28 while it was non-significant in diabetic standard group. Thisindicates that, the onset of action of compound 15q is almost after14 days of daily treatment while sitagliptin produced its anti-hyperglycemic effect within 14 days and maintained the glucoselevels throughout the study period of 28 days. There is no statisti-cal difference in glucose levels between normal control and controltreated group on 14th as well as 28th day of treatment suggesting

Fig. 7. Effects of compound 15q at 14 mg/kg dose on the change of serum glucose levels in HFD/STZ rats after multiple once daily oral dosing for 28 days. Data are presentedas means ± SEM of 6 animals. * P < 0.05 versus normal control from 28 day, # P < 0.05 versus diabetic control from 28 day, two way ANOVA followed by Bonferroni’s posttest.

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that, compound 15q is not having any hypoglycemic side effectduring chronic treatment.

In conclusion, although compound 15q was found to be veryless potent for in vitro DPP-4 inhibition, it produces good in vivoanti-hyperglycemic effect which might be possibly due to someoff-target binding other than DPP-4. Thus, further studies arerequired to explore other possible anti-diabetic mechanism ofaction of molecule.

3. Experimental

3.1. Chemistry

All chemicals and solvents were purchased from commercialsources like Sigma Aldrich, TCI Chemicals, Merck, Spectrochem,SD-Fine and Himedia. They were used without further purification.When needed, the solvents were dried over 3 Å or 4 Å molecularsieves and then distilled. Reaction progress was monitored usingsilica gel pre-coated analytical thin-layer chromatography (TLC)plates (without fluorescent indicator) from Merck. Melting pointswere determined on a SMP 203 digital melting point apparatusfrom Lab Intelligence Appliances. Infrared spectra of the com-pounds were recorded on a JASCO FTIR 6100 spectrometer byKBr dispersion method. Mass spectra were recorded using aWater’s micro mass Q-Tof micro spectrometer with an ESI source.1H NMR spectra of all the intermediates and final compounds wererecorded using Bruker AVANCE 400 MHz NMR spectrometers. 13CNMR spectra of the compounds were recorded using a BrukerAVANCE 100 MHz NMR spectrometer. Chemical shifts were mea-sured in parts per million (ppm) downfield from an internaltetramethylsilane (TMS) standard. Single crystal X-ray intensitydata for compound 15p were made on a Rigaku SCX mini diffrac-tometer using graphite monochromated Mo-Ka radiation. Purityof the compounds was checked using high performance liquidchromatography (HPLC) analysis which was carried out at kmax

220 nm using column ODS C-18, 150 mm � 4.6 mm � 4l on AGI-LENT 1100 series.

3.1.1. Procedure for synthesis of 3,5-dibromo-1H-1,2,4-triazole (12)[17]

In the solution of 1.0 g (14 mmol) 1,2,4-triazole 11 in 25 mL ofwater, 3.4 g (34 mmol) of KHCO3 was added and resulting solutionwas heated to 80 �C for 30–40 min. To this solution, 1.6 mL

(31 mmol) of bromine in an aqueous solution of KBr (1.2 g KBr in2.4 mL water) was added dropwise with constant stirring. Thereaction mixture was stirred for further 15–20 min to eliminatethe excess of bromine. The reaction mixture was cooled to roomtemperature and acidified with conc. HCl. The resulting precipi-tates were filtered off and washed with ice cold water. Compound12 was recrystallized from hot water as white solids. Yield 71.06%;m.p. 190–195 �C; FT-IR (KBr, cm�1): 3099, 2903, 2242.8, 2116.5,1516.7, 1427, 1342.2., 1269.9, 1130, 1014.3, 986.4, 832, 718.3.701.9. 1H NMR (400 MHz, CDCl3): d 1.25 (s, 1H). ESI-MS calculatedfor (C2HBr2N3) [M+1]+ 225.85, found 223.8 [M�1]+, 225.8 [M+1]+,227.9 [M+3]+.

3.1.2. General procedure for synthesis of 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-substituted-ethanone (14a-q) [18]

The mixture of 1 g (4.4 mmol) 3,5-dibromo -1H-1,2,4-triazole 2and 0.6 g (4.4 mmol) K2CO3 in 10 mL acetone was cooled to 0 �Cwith constant stirring. Various substituted phenacyl bromides(13a-o, 4.4 mmol) or equivalent reactants (13p, 13q, 4.4 mmol)were added slowly in portions at 0 �C with stirring. After completeaddition, reaction mixture was stirred at room temperature for 4–6 h. The progress of reaction was monitored by TLC. After comple-tion of chemical reaction, the reaction mixture was filtered. Theresidue was washed with acetone and combined filtrate was con-centrated under reduced pressure to get the solids which werethen recrystallized by co-solvent recrystallization method usingethyl acetate-petroleum ether to obtain the pure solids (14a-q).

3.1.2.1. 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-(4-methoxyphenyl)ethanone (14a). Yield: 80%; white solid; m.p. 147–151 �C; FT-IR(KBr, cm�1): 1067.41, 1125.26, (ACAOAC stretch), 1683.55 (AC@Ostretch); 1H NMR (400 MHz, CDCl3): d 3.86 (s, 3H), 5.99 (s, 2H), 7.13(d, J = 8.9 Hz, 2H), 8.04 (d, J = 8.9 Hz, 2H). ESI-MS calculated for(C11H9Br2N3O2) [M+1]+ 373.91, found 374.1 [M+1]+, 376 [M+3]+,378 [M+5]+.

3.1.2.2. 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-p-tolylethanone(14b). Yield: 78%; white solid; m.p. 152–156 �C; FT-IR (KBr,cm�1): 1687.41 (AC@O stretch); 1H NMR (400 MHz, CDCl3): d2.46 (s, 3H), 5.59 (s, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.88 (d,J = 8.3 Hz, 2H). ESI-MS calculated for (C11H9Br2N3O) [M+1]+

357.91, found 358 [M+1]+, 360 [M+3]+, 362 [M+5]+.

354 B.D. Patel et al. / Bioorganic Chemistry 72 (2017) 345–358

3.1.2.3. 4-(2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)acetyl)benzonitrile(14c). Yield: 75%; buff yellow solid; m.p. 213–217 �C; FT-IR (KBr,cm�1): 1708.62 (AC@O stretch), 2232.2 (ACN stretch); 1H NMR(400 MHz, CDCl3): d 5.56 (s, 2H), 7.82 (d, J = 8.32 Hz, 2H), 8.03 (d,J = 8.3 Hz, 2H). ESI-MS calculated for (C11H6Br2N4O) [M+1]+

368.89, found 367 [M�H]+, 368.9 [M+1]+, 370.8 [M+3]+.

3.1.2.4. 1-(4-chlorophenyl)-2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)ethanone (14d). Yield: 83%; pale yellow solid; m.p. 158–163 �C;FT-IR (KBr, cm�1): 1702.84 (AC@O stretch); 1H NMR (400 MHz,CDCl3): d 5.62 (s, 2H), 7.94 (d, J = 9.0 Hz, 2H), 7.56 (d, J = 9.1 Hz,2H). ESI-MS calculated for (C10H6Br2ClN3O) [M+1]+ 377.86, found378 [M+1]+, 380 [M+3]+, 381.9 [M+5]+.

3.1.2.5. 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-(4-phenylphenyl)ethanone (14e). Yield: 84%; pale yellow solid; m.p. 155–160 �C;FT-IR (KBr, cm�1): 1692.23 (AC@O stretch); 1H NMR (400 MHz,CDCl3): d 5.58 (s, 2H), 7.45–7.35 (m, 3H), 7.58 (d, J = 7.2 Hz, 2H),7.71 (d, J = 8.3 Hz, 2H), 7.98 (d, J = 8.3 Hz, 2H). ESI-MS calculatedfor (C16H11Br2N3O) [M+1]+ 419.93, found 420.3 [M+1]+, 422.1 [M+3]+, 424.1 [M+5]+.

3.1.2.6. 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-(3-fluorophenyl)ethanone (14f). Yield: 75%; pale yellow solid; m.p. 94–98 �C; FT-IR (KBr, cm�1): 1697.05 (AC@O stretch); 1H NMR (400 MHz,CDCl3): d 5.52 (s, 2H), 7.36–7.31 (m, 1H), 7.52–7.47 (m, 1H), 7.68(d, 1H), 7.62–7.58 (m, 1H). ESI-MS calculated for (C10H6Br2FN3O)[M+1]+ 361.89, found 362.1 [M+1]+, 364.1 [M+3]+, 366.1 [M+5]+.

3.1.2.7. 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-(4-hydroxyphenyl)ethanone (14g). Yield: 68%; white solid; m.p. 199–204 �C; FT-IR(KBr, cm�1): 1671.98 (AC@O stretch), 3399.89 (AOH stretch); 1HNMR (400 MHz, CDCl3): d 1.97 (s, 1H), 5.49 (s, 2H), 6.90 (d,J = 7.9 Hz, 2H), 7.86 (d, J = 7.9 Hz, 2H). ESI-MS calculated for (C10H7-Br2N3O2) [M+1]+ 359.89, found 360.1 [M+1]+, 362 [M+3]+, 364.1 [M+5]+.

3.1.2.8. 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-(3-nitrophenyl)etha-none (14h). Yield: 65%; buff yellow solid; m.p. 139–143 �C; FT-IR(KBr, cm�1): 1530.24 and 1350.89 (ANAO stretch), 1697.05 (AC@Ostretch); 1H NMR (400 MHz, CDCl3): d 5.77 (s, 2H), 7.78–7.74 (t,1H), 8.32 (d, J = 7.7 Hz, 1H), 8.49 (d, J = 8.2 Hz, 1H), 8.79 (s, 1H).ESI-MS calculated for (C10H6Br2N4O3) [M+1]+ 388.88, found 388.4[M+1]+, 390.5 [M+3]+, 392.5 [M+5]+.

3.1.2.9. 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-(3-methoxyphenyl)ethanone (14i). Yield: 84%; white solid; m.p. 116–120 �C; FT-IR(KBr, cm�1): 1127.19 (CAOAC Stretch), 1692.23 (AC@O stretch);1H NMR (400 MHz, CDCl3): d 3.80 (s, 3H), 5.54 (s, 2H), 7.47–7.14(m, 4H). ESI-MS calculated for (C11H9Br2N3O2) [M+1]+ 373.91,found 373.5 [M+1]+, 375.4 [M+3]+, 377.5 [M+5]+.

3.1.2.10. 3-(2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)acetyl)benzonitrile(14J). Yield: 73%; yellow solid; m.p. 128–132 �C; FT-IR (KBr,cm�1): 1698.98 (AC@O stretch), 2236.06 (ACN stretch); 1H NMR(400 MHz, CDCl3): d 5.56 (s, 2H), 7.69–7.65 (t, 1H), 7.92–7.89 (m,1H), 8.15–8.13 (m, 1H), 8.19 (s, 1H). ESI-MS calculated for (C11H6-Br2N4O) [M+1]+ 368.89, found 368.9 [M+1]+, 370.9 [M+3]+, 372.9[M+5]+.

3.1.2.11. 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-(2,4-difluo-rophenyl)ethanone (14k). Yield: 85%; red solid; m.p. 130–135 �C;FT-IR (KBr, cm�1): 1697.05 (AC@O stretch), 1H NMR (400 MHz,CDCl3): d 5.45 (s, 2H), 6.95–6.89 (m, 1H), 7.03–6.98 (m, 1H),8.02–7.97 (m, 1H). ESI-MS calculated for (C10H5Br2F2N3O) [M+1]+

379.88, found 379.6 [M+1]+, 381.7 [M+3]+, 383.7 [M+5]+.

3.1.2.12. 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-(3,4-dichlorophe-nyl)ethanone (14l). Yield: 81%; pale yellow solids; m.p. 191–196 �C; FT-IR (KBr, cm�1): 1707.66 (AC@O stretch), 1H NMR(400 MHz, CDCl3): d 5.50 (s, 2H), 7.60 (d, J = 8.4 Hz, 1H), 7.74 (d,J = 8.4 Hz, 1H), 7.99 (s, 1H). ESI-MS calculated for (C10H5Br2Cl2N3O)[M+1]+ 411.82, found 411.6 [M+1]+, 413.5 [M+3]+, 415.5 [M+5]+.

3.1.2.13. 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-(4-(trifluo-romethyl)phenyl)ethanone (14m). Yield: 78%; yellow solids; m.p.120–123 �C; FT-IR (KBr, cm�1): 1673.66 (AC@O stretch), 1H NMR(400 MHz, CDCl3): d 5.65 (s, 2H), 7.85 (d, J = 8.3 Hz, 2H), 8.11 (d,J = 8.2 Hz, 2H). ESI-MS calculated for (C11H6Br2F3N3O) [M+1]+

411.88, found 411.9 [M+1]+, 414.0 [M+3]+, 415.8 [M+5]+.

3.1.2.14. 1-(3-chloro-4-fluorophenyl)-2-(3,5-dibromo-1H-1,2,4-tria-zol-1-yl)ethanone (14n). Yield: 76%; pale yellow solids; m.p. 190–194 �C; FT-IR (KBr, cm�1): 1690.06 (AC@O stretch), 1H NMR(400 MHz, CDCl3): d 5.60 (s, 2H), 7.36 (t, J = 8.4 Hz, 1H), 7.92 (m,J = 2.2 Hz, 1H), 8.09 (d, J = 6.9 Hz, 1H). ESI-MS calculated for (C10H5-Br2ClFN3O) [M+1]+ 395.85, found 395.8 [M+1]+, 397.9 [M+3]+,399.8 [M+5]+.

3.1.2.15. 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-(2-methoxyphenyl)ethanone (14o). Yield: 81%; white solids; m.p. 130–134 �C; FT-IR(KBr, cm�1): 1135.22 (CAOAC Stretch), 1687.34 (AC@O stretch);1H NMR (400 MHz, CDCl3): d 4.03 (s, 3H), 5.60 (s, 2H), 7.11–7.05(m, 2H), 7.63 (t, J = 8.7 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H). ESI-MS cal-culated for (C11H9Br2N3O2) [M+1]+ 373.91, found 373.9 [M+1]+,375.8 [M+3]+, 377.5 [M+5]+.

3.1.2.16. 2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)-1-(naphthalene-2-yl)ethanone (14p). Yield: 84%; pale yellow solid; m.p. 133–137 �C; FT-IR (KBr, cm�1): 1698.98 (AC@O stretch); 1H NMR (400 MHz,CDCl3): d 5.69 (s, 2H), 7.64–7.54 (m, 2H), 7.95–7.85 (m, 4H), 8.42(s, 1H). ESI-MS calculated for (C14H9Br2N3O) [M+1]+ 393.91, found394 [M+1]+, 396 [M+3]+, 398 [M+5]+.

3.1.2.17. 1-(benzofuran-2-yl)-2-(3,5-dibromo-1H-1,2,4-triazol-1-yl)ethanone (14q). Yield: 76%; pale yellow solid; m.p. 151–155 �C;FT-IR (KBr, cm�1): 1100 (CAOAC Stretch), 1675.23 (AC@O stretch);1H NMR (400 MHz, CDCl3): d 5.63 (s, 2H), 7.39 (d, J = 7.9 Hz, 1H),7.63–7.55 (m, 2H), 7.71 (s, 1H), 7.79 (d, J = 7.9 Hz, 1H). ESI-MS cal-culated for (C12H7Br2N3O2) [M+1]+ 383.89, found 383.9 [M+1]+,385.9 [M+3]+, 388 [M+5]+.

3.1.3. General procedure for synthesis of 7-bromo-3-substituted-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine (15a-q) [18]

To a stirred solution of 1 g of 14a-q in methanol (15 mL), NH2-NH2�H2O (80%, 3.6 equivalent) was added. If required, mixture washeated to facilitate the solubility of 14a-q into methanol. Afteraddition of NH2NH2�H2O, reaction mixture was refluxed for 8–10 h. Solids precipitated out in the reaction mixture after coolingwere collected by filtration, washed with methanol and dried toobtain 15a-q as crystalline solids.

3.1.3.1. 7-bromo-3-(4-methoxyphenyl)-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine (15a). Yield: 71%; white solid; m.p. 273–277 �C; FT-IR (KBr, cm�1): 1588.09 (ANH bending), 3231 (ANHstretch); 1H NMR (400 MHz, DMSO-d6): d ppm 3.81 (s, 3H), 5.21(s, 2H), 7.02 (d, J = 8.9 Hz, 2H), 7.71 (d, J = 8.9 Hz, 2H), 11.55 (s,1H, NH). 13C NMR (100 MHz, DMSO-d6): d ppm 160.40, 147.44,137.87, 131.15, 128. 21, 126.54, 113.92, 55.26, 44.69. ESI-MS calcu-lated for (C11H10BrN5O) [M+1]+ 308.0, found 308.0 [M+1]+, 310.0[M+3]+. Elemental anal. calcd for C11H10BrN5O: C, 42.88; H, 3.27;N, 22.73; O, 5.19, found C, 42.84; H, 3.31; N, 22.75; O, 5.16.

B.D. Patel et al. / Bioorganic Chemistry 72 (2017) 345–358 355

3.1.3.2. 7-bromo-3-p-tolyl-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]tri-azine (15b). Yield: 73%; white solid; m.p. 282–286 �C; FT-IR (KBr,cm�1): 1581.34 (ANH, bending), 3120.26 (ANH stretch); 1H NMR(400 MHz, DMSO-d6): d ppm 2.36 (s, 3H), 5.21 (s, 2H), 7.28 (d,J = 8.1 Hz, 2H), 7.65 (d, J = 8.2 Hz, 2H), 11.62 (s, 1H, NH). 13C NMR(100 MHz, DMSO-d6): d ppm 147.40, 139.36, 137.88, 131.25,129.11, 128.33, 124. 92, 44.75, 20.87. ESI-MS calculated for(C11H10BrN5) [M+1]+ 292.01, found 292.1 [M+1]+, 294.1 [M+3]+.Elemental anal. calcd for C11H10BrN5: C, 45.23; H, 3.45; N, 23.97,found C, 45.20; H, 3.48; N, 23.94.

3.1.3.3. 4-(7-bromo-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazin-3-yl)benzonitrile (15c). Yield: 65%; pale yellow solid; m.p. 290–293 �C; FT-IR (KBr, cm�1): 1575.56 (ANH bending), 2229.31(ACN stretch), 2956.34 (ANH stretch); 1H NMR (400 MHz,DMSO-d6): d ppm 5.28 (s, 2H), 8.02–7.86 (m, 4H), 11.97 (s, 1H,NH). 13C NMR (100 MHz, DMSO-d6): d ppm 146.92, 138.21,136.30, 132.45, 128.55, 125.64, 118.59, 111.58, 44.89. ESI-MS cal-culated for (C11H7BrN6) [M�H]+ 300.99, found 300.6 [M�H]+,302.8 [M+1]+. Elemental anal. calcd for C11H7BrN6: C, 43.59; H,2.33; N, 27.73, found C, 43.57; H, 2.36; N, 27.71.

3.1.3.4. 7-bromo-3-(4-chlorophenyl)-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine (15d). Yield: 73%; white solid; m.p. 297–300 �C;FT-IR (KBr, cm�1): 1588.09 (ANH bending), 3211.86 (ANH stretch);1H NMR (400 MHz, DMSO-d6): d ppm 5.25 (s, 2H), 7.77 (d,J = 8.7 Hz, 2H), 7.54 (d, J = 8.7 Hz, 2H), 11.77 (s, 1H, NH). 13C NMR(100 MHz, DMSO-d6): d ppm 147.16, 137.95, 136.78, 134.21,132.84, 128.56, 126.73, 44.79. ESI-MS calculated for (C10H7BrClN5)[M+1]+ 311.96, found 312.1 [M+1]+, 314.1 [M+3]+, 316.0 [M+5]+.Elemental anal. calcd for C10H7BrClN5: C, 38.43; H, 2.26; N, 22.41,found C, 38.46; H, 2.24; N, 22.38.

3.1.3.5. 7-bromo-3-(4-phenylphenyl)-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine (15e). Yield: 76%; white solid; m.p. 279–283 �C;FT-IR (KBr, cm�1): 1575.56 (ANH bending), 3113.51 (ANH stretch);1H NMR (400 MHz, DMSO-d6): d ppm 5.29 (s, 2H), 7.42 (t, 1H), 7.51(t, 2H), 7.85–7.72 (m, 6H), 11.74 (s, 1H, NH). 13C NMR (100 MHz,DMSO-d6): d ppm 147.32, 141.07, 140.10, 139.32, 137.95, 137.44,133.00, 129.01, 127.85, 126.84, 125.58, 44.84. ESI-MS calculatedfor (C16H12BrN5) [M+1]+ 354.03, found 354.3 [M+1]+, 356.2 [M+3]+. Elemental anal. calcd for C16H12BrN5: C, 54.25; H, 3.41; N,19.77, found C, 54.27; H, 3.42; N, 19.79.

3.1.3.6. 7-bromo-3-(3-fluorophenyl)-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine (15f). Yield: 72%; white solid; m.p. 270–273 �C;FT-IR (KBr, cm�1): 1574.59 (ANH bending), 3214.75 (ANH stretch);1H NMR (400 MHz, DMSO-d6): d ppm 5.30 (s, 2H), 7.32–7.27 (t,1H), 7.59–7.48 (m, 3H), 11.75 (s, 1H, NH). 13C NMR (100 MHz,DMSO-d6): d ppm 163.47, 161.05, 147.16, 137.96, 136.73, 130.63,121.16, 116.47, 111.74, 111.51, 44.93. ESI-MS calculated for(C10H7BrFN5) [M+1]+ 295.99, found 296.0 [M+1]+, 298.2 [M+3]+.Elemental anal. calcd for C10H7BrFN5: C, 40.56; H, 2.38; N, 23.65,found C, 40.53; H, 2.41; N, 23.63.

3.1.3.7. 4-(7-bromo-1,4-dihydro-[1,2,4]-triazolo[5,1-c][1,2,4]triazin-3-yl)phenol (15g). Yield: 67%; white solid; m.p. ˃300 �C; FT-IR(KBr, cm�1): 1598.7 (ANH bending), 3159.79 (AOH stretch); 1HNMR (400 MHz, DMSO-d6): d ppm 5.17 (s, 2H), 6.85 (d, J = 8.7 Hz,2H), 7.60 (d, J = 8.6 Hz, 2H), 9.90 (s, 1H, OH), 11.48 (s, 1H, NH).13C NMR (100 MHz, DMSO-d6): d ppm 158.93, 147.52, 137.84,133.54, 130.06, 126.63, 115.30, 44.62. ESI-MS calculated for(C10H8BrN5O) [M+1]+ 293.99, found 294.3 [M+1]+, 296.1 [M+3]+.Elemental anal. calcd for C10H8BrN5O: C, 40.84; H, 2.74; N, 23.81;O, 5.44, found C, 40.81; H, 2.72; N, 23.84; O, 5.48.

3.1.3.8. 7-bromo-3-(3-nitrophenyl)-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine (15h). Yield: 62%; white solid; m.p. 275–278 �C; FT-IR (KBr, cm�1): 1581.34 (ANH bending), 3099.05 (ANH stretch); 1HNMR (400 MHz, DMSO-d6): d ppm 5.34 (s, 2H), 7.79–7.74 (t, 1H),8.14 (d, J = 8.3 Hz, 1H), 8.29 (d, J = 8.2 Hz, 1H), 8.53 (s, 1H), 11.94(s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): d ppm 148.01,147.02, 138.04, 136.06, 135.61, 131.20, 130.27, 123.86, 119.28,45.00. ESI-MS calculated for (C10H7BrN6O2) [M+1]+ 322.98, found323.0 [M+1]+, 325.2 [M+3]+. Elemental anal. calcd for C10H7BrN6O2:C, 37.17; H, 2.18; N, 26.01; O, 9.90, found C, 37.14; H, 2.21; N,26.04; O, 9.87.

3.1.3.9. 7-bromo-3-(3-methoxyphenyl)-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine (15i). Yield: 75%; white solid; m.p. 250–253 �C; FT-IR (KBr, cm�1): 1559.17 (ANH bending), 3094.23(ANH stretch); 1H NMR (400 MHz, DMSO-d6): d ppm 3.80 (s, 3H),5.24 (s, 2H), 7.04 (s, 1H), 7.31 (d, J = 8.1 Hz, 2H), 7.40–7.36 (t,1H), 11.69 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6): d ppm159.30, 147.30, 137.92, 135.37, 131.20, 129.65, 117.47, 115.50,110.01, 55.14, 44.88. ESI-MS calculated for (C11H10BrN5O) [M+1]+

308.01, found 307.6 [M+1]+, 309.6 [M+3]+. Elemental anal. calcdfor C11H10BrN5O: C, 42.88; H, 3.27; N, 22.73; O, 5.19, found C,42.85; H, 3.30; N, 22.77; O, 5.21.

3.1.3.10. 3-(7-bromo-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazin-3-yl)benzonitrile (15J). Yield: 69%; white solid; m.p. 287–291 �C;FT-IR (KBr, cm�1): 1564.95 (ANH bending), 2228.34 (ACN stretch),3119.3 (ANH stretch); 1H NMR (400 MHz, DMSO-d6): d ppm 5.28(s, 2H), 7.69–7.65 (t, 1H), 7.92 (d, J = 7.7 Hz, 1H), 8.10 (d,J = 8.1 Hz, 1H), 8.12 (s, 1H), 11.88 (s, 1H, NH). 13C NMR (100 MHz,DMSO-d6): d ppm 147.04, 138.01, 136.16, 135.15, 132.93, 129.79,129.44, 128.50, 118.44, 111.81, 44.90. ESI-MS calculated for (C11-H7BrN6) [M+1]+ 302.99, found 302.5 [M+1]+, 304.6 [M+3]+. Elemen-tal anal. calcd for C11H7BrN6: C, 43.59; H, 2.33; N, 27.73, found C,43.56; H, 2.35; N, 27.74.

3.1.3.11. 7-bromo-3-(2,4-difluorophenyl)-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine (15k). Yield: 72%; pale yellow solid; m.p.214–218 �C; FT-IR (KBr, cm�1): 1564.95 (ANH bending), 3209.3(ANH stretch); 1H NMR (400 MHz, DMSO-d6): d ppm 5.21 (s, 2H),7.22–7.18 (t, 1H), 7.41–7.35 (t, 1H), 7.83–7.77 (m, 1H), 11.76 (s,1H, NH). 13C NMR (100 MHz, DMSO-d6): d ppm 164.08, 163.96,161.60, 161.48, 159.08, 158.96, 147.32, 137.92, 135.15, 130.34,130.29, 130.24, 130.19, 112.19, 112.01, 111.97, 105.15, 104.89,104.62, 46.38. ESI-MS calculated for (C10H6BrF2N5) [M+1]+

313.98, found 314.8 [M+1]+. Elemental anal. calcd for C10H6BrF2N5:C, 38.24; H, 1.93; N, 22.30, found C, 38.21; H, 1.91; N, 22.32.

3.1.3.12. 7-bromo-3-(3,4-dichlorophenyl)-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine (15l). Yield: 68%; white solid; m.p. 272–276 �C; FT-IR (KBr, cm�1): 1588.09 (ANH bending), 3206.08(ANH stretch); 1H NMR (400 MHz, DMSO-d6): d ppm 5.23 (s, 2H),7.75 (s, 2H), 7.90 (s, 1H), 11.86 (s, 1H, NH). 13C NMR (100 MHz,DMSO-d6): d ppm 146.98, 138.01, 135.77, 134.58, 130.68, 129.64,128.42, 126.67, 125.05, 44.84. ESI-MS calculated for (C10H6BrCl2N5)[M+1]+ 345.92, found 345.5 [M+1]+ 347.5 [M+3]+ 349.5 [M+5]+. Ele-mental anal. calcd for C10H6BrCl2N5: C, 34.61; H, 1.74; N, 20.18,found C, 34.59; H, 1.76; N, 20.15.

3.1.3.13. 7-bromo-3-(4-(trifluoromethyl)phenyl)-1,4-dihydro-[1,2.4]-triazolo[5,1-c][1,2,4]traizine (15m). Yield: 73%; white solid; m.p.275–278 �C; FT-IR (KBr, cm�1): 1583.45 (ANH bending), 3146.08(ANH stretch); 1H NMR (400 MHz, DMSO-d6): d ppm 5.29 (s, 2H),7.95 (d, J = 8.2 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 11.92 (s, 1H, NH).13C NMR (100 MHz, DMSO-d6): d ppm 147.01, 140.66, 138.01,136.42, 128.80, 125.64, 122.69, 119.99, 44.92. ESI-MS calculated

356 B.D. Patel et al. / Bioorganic Chemistry 72 (2017) 345–358

for (C11H7BrF3N5) [M+1]+ 345.98, found 346.0 [M+1]+ 347.8 [M+3]+.Elemental anal. calcd for C11H7BrF3N5: C, 38.17; H, 2.04; N, 20.23,found C, 38.15; H, 2.07; N, 20.20.

3.1.3.14. 7-bromo-3-(3-chloro-4-fluorophenyl)-1,4-dihydro-[1,2,4]tri-azolo[5,1-c][1,2,4]traizine (15n). Yield: 70%; faint yellow solid; m.p.242–246 �C; FT-IR (KBr, cm�1): 1579.02 (ANH bending), 3196.08(ANH stretch); 1H NMR (400 MHz, DMSO-d6): d ppm 5.24 (s, 2H),7.53–7.49 (t, 1H), 7.76 (s, 1H), 7.91 (d, J = 7.1 Hz, 1H), 11.81 (s,1H, NH). 13C NMR (100 MHz, DMSO-d6): d ppm 159.00, 156.52,147.05, 137.99, 135.91, 131.89, 127.36, 125.86, 120.07, 117.20,44.88. ESI-MS calculated for (C10H6BrClFN5) [M+1]+ 329.95, found330.0 [M+1]+ 332.2 [M+3]+. Elemental anal. calcd for C10H6BrClFN5:C, 36.34; H, 1.83; N, 21.19, found C, 36.36; H, 1.81; N, 21.21.

3.1.3.15. 7-bromo-3-(2-methoxyphenyl)-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine (15o). Yield: 78%; white solid; m.p. 257–260 �C; FT-IR (KBr, cm�1): 1564.34 (ANH bending), 3102.20(ANH stretch); 1H NMR (400 MHz, DMSO-d6): d ppm 3.85 (s, 3H),5.13 (s, 2H), 7.04–6.99 (t, 1H), 7.13 (d, J = 8.4 Hz, 1H), 7.45–7.42(t, 1H), 7.51 (d, J = 7.2 Hz, 1H), 11.49 (s, 1H, NH). 13C NMR(100 MHz, DMSO-d6): d ppm 157.58, 148.06, 139.92, 137.84,131.08, 129.36, 123.92, 120.59, 111.87, 55.69, 46.72. ESI-MS calcu-lated for (C11H10BrN5O) [M+1]+ 308.01, found 308.0 [M+1]+, 309.9[M+3]+. Elemental anal. calcd for C11H10BrN5O: C, 42.88; H, 3.27;N, 22.73; O, 5.19, found C, 42.90; H, 3.24; N, 22.76; O, 5.22.

3.1.3.16. 7-bromo-3-(naphthalene-2-yl)-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine (15p). Yield: 75%; white solid; m.p. 254–257 �C; FT-IR (KBr, cm�1): 1576.52 (ANH bending), 3112.55(ANH stretch); 1H NMR (400 MHz, DMSO-d6): d ppm 5.39 (s, 2H),7.58 (s, 1H), 8.43–7.96 (m, 6H), 11.81 (s, 1H, NH). 13C NMR(100 MHz, DMSO-d6): d ppm 147.31, 137.59, 133.24, 131.40,128.49, 128.00, 127.58, 127.26, 127.05, 126.67, 125.97, 124.99,122.13, 44.84. ESI-MS calculated for (C14H10BrN5) [M+1]+ 328.01,found 328.1 [M+1]+, 330.1 [M+3]+. Elemental anal. calcd for C14H10-BrN5: C, 51.24; H, 3.07; N, 21.34, found C, 51.27; H, 3.04; N, 21.36.

3.1.3.17. 3-(benzofuran-2-yl)-7-bromo-1,4-dihydro-[1,2,4]triazolo[5,1-c][1,2,4]triazine (15q). Yield: 76%; pale yellow solid; m.p.280–284 �C; FT-IR (KBr, cm�1): 1572.78 (ANH bending), 3132.42(ANH stretch); 1H NMR (400 MHz, DMSO-d6): d ppm 5.21 (s, 2H),7.32–7.29 (t, 1H) 7.35 (s, 1H), 7.42–7.38 (t, 1H), 7.66 (d,J = 8.2 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 13C NMR (100 MHz, DMSO-d6): d ppm 154.33, 150.09, 147.17, 137.95, 130.57, 127.62,125.98, 123.52, 121.76, 111.31, 106.80, 44.84. ESI-MS calculatedfor (C12H8BrN5O) [M+1]+ 317.99, found 317.7 [M+1]+, 320.2 [M+3]+. Elemental anal. calcd for C12H8BrN5O: C, 45.31; H, 2.53; N,22.01; O, 5.03, found C, 45.34; H, 2.50; N, 22.03; O, 5.00.

3.1.4. X-ray crystallographic analysis of compound 15pThe X-ray crystallographic analysis of compound 15p was

determined on a colorless crystal, with approximate dimensionsof 0.66 mm � 0.40 mm � 0.30 mm, grown from the vapor diffusionmethod using THF and hexane. The crystal structure was solvedand refined using CrystalClear (Rigaku, Orem, UT) [21]. Thedetailed methodology of single crystal XRD is given in supportinginformation.

3.1.5. HPLC analysis of final compounds 15a-qPurity of final compounds 15a-q was checked by HPLC analysis.

A solution of compounds prepared in water: acetonitrile (1:1, 5 ll)was injected into a C18 column (J’SPHERE 4 l; 150 � 4.6 mm) andeluted with a linear gradient of acetonitrile (CAN) in water, bothbuffered with 0.05% trifluoro acetic acid (TFA), using a flow rate

of 1 mL/min, with effluent monitoring by PDA detector at220 nm. A typical gradient of 20–70% of water-ACN mixture, buf-fered with 0.05% TFA was used, over a period of 100 min, with1% gradient change per minute. The retention time and peak purityof chromatographic peak of various compounds were noted down.Table ST-1 in supporting information shows the retention time and% area of each compound. The % area almost corresponds to the%purity of compounds.

3.2. Pharmacological evaluation

3.2.1. In vitro DPP-4, DPP-8 and DPP-9 assay [22–25]In vitro enzyme inhibitory activity was measured by

fluorescence-based assay which employs fluorogenic substrate,Gly-Pro-Aminomethylcoumarin (AMC), to measure DPP (DPP-4,DPP-8 and DPP-9) activity. Cleavage of the peptide bond by DPPreleases the free AMC group resulting in fluorescence that can beanalyzed using an excitation wavelength of 350–360 nm and emis-sion wavelength of 450–465 nm. The assay was performed by mix-ing 30 ll assay buffer (100 mM/L HEPES with 0.1 mg/mL BSA, pH7.5 for DPP-4 assay; Tris HCl 25 mM and 0.1% BSA in deionizedwater, pH 7.5 for DPP-8 and DPP-9 assay), 10 ll of human recom-binant DPP enzyme (BPS Bioscience, San Diego) and 10 ll of testcompounds 15a-q (of various concentrations) with 50 ll of thesubstrate, Gly-Pro-Aminomethylcoumarin (Sigma-Aldrich). Thefinal concentration of the DPP-4 enzyme was 1 ng/well (10 ng/wellfor DPP-8 and DPP-9) and of the substrate was 5 lM per well.Plates were incubated at 37 �C for 30 min kinetic run, and fluores-cence was measured at excitation/emission wavelengths of 360/40,460/40 nm at a sensitivity of 60 using a Synergy HT multi detectionmicroplate reader (BioTek instruments). Enzyme inhibition wasdetermined and expressed as % inhibition. The IC50 values weredetermined for potent compounds using Graph Pad prismsoftware.

3.2.2. In vivo oral glucose tolerance test in normal C57BL/6J mice[22,26]

All experiments were performed in compliance with the rele-vant laws and institutional guidelines, and experimental protocolscarried out in present study were permitted by the InstitutionalAnimal Ethics Committee (IAEC) of Institute of Pharmacy, NirmaUniversity, Ahmedabad, India (protocol number: IP/PCHEM/FAC/15-1/021, August 14, 2014 and IP/PCHEM/FAC/17/026, August4, 2015). All experimental methods are in harmony with CPCSEAguidelines, Ministry of Environment and Forests, Government ofIndia.

Male C57BL/6 J mice (30–35 g) were kept on fasting overnightand divided into five groups (n = 6). Initially blood was collectedfrom all animals from retro-orbital plexus and the treatment wasgiven. Compound 15q was administered orally in three differentdoses i.e. 5, 10 and 20 mg/kg body weight against standard sita-gliptin dose of 3 mg/kg. After 30 min. of treatment, all mice weredosed with oral glucose load (2 g/kg). Blood samples were col-lected at the time points of 0, 15, 30, 60, 120 and 180 min whereinglucose administration was done at time point 0. Serum was sep-arated by centrifugation and immediately subjected for the glucoseestimation by God-Pod method using diagnostic kit (Lab Care Diag-nostics, India Limited.). Data analysis was performed using twoway ANOVA followed by Bonferroni test.

3.2.3. In vivo anti hyperglycemic evaluation in HFD/STZ induced T2DMmodel

Male Sprague-Dawley (SD) rats of 160–180 g body weight wereselected for the study. Type-2 diabetes was induced as perpublished protocol of the high-fat diet-fed and low-dose streptozo-

B.D. Patel et al. / Bioorganic Chemistry 72 (2017) 345–358 357

tocin (STZ)-treated rat model of T2DM [27]. High fat diet was givenfor 2 weeks after which STZ (35 mg/kg) was administeredintraperitoneally. Diabetes was induced in the animals after2 weeks which was confirmed by fasting glucose estimation. Ani-mals showing serum glucose levels more than 140 mg/dl wereconsidered diabetic. Animals were then divided into five groups(n = 6) such that their mean basal plasma glucose levels were sim-ilar to each other. Treatment was started on the animals for28 days after confirmation of diabetes. The highest active dose of20 mg/kg for compound 15q and sitagliptin dose of 3 mg/kg inmice were converted into the rat doses [28] i.e. 14 mg/kg and2 mg/kg respectively, for once a day oral dosing. The normal con-trol and diabetic control groups were administered with equalamount of vehicle 0.5% Na-CMC. Serum glucose levels were mea-sured after overnight fasting on day 0, 14 and 28. The results areexpressed as mean ± SEM and data analysis was performed usingtwo-way ANOVA followed by Bonferroni test. A value of p < 0.05was considered statistically significant.

4. Conclusion

A series of 3,7-disubstituted-1,4-dihydro[1,2,4]triazolo[5,1-c][1,2,4]triazines derivatives as DPP-4 inhibitors was designed usingthe combined results of 3D-QSAR, pharmacophore modeling, vir-tual screening and docking studies. All 17 compounds were syn-thesized in good yield and characterized by FTIR, 1H and 13C MRand mass (ESI-MS) spectra. The purity of compounds was deter-mined using HPLC analysis and found to be >95%. During in vitroscreening against DPP-4 enzyme, compound 15q (benzofuranderivative) and 15c (4-cyano phenyl derivative) gave acceptable53.3% and 48.3% DPP-4 inhibition, respectively. The SAR of synthe-sized compounds suggested that bulky and polar heterocyclic ringas P1 fragment would give more activity compared to phenyl ring.The IC50 of best two compounds 15q and 15c was determinedin vitro against DPP-4, DPP-8 and DPP-9. For compound 15q, theywere found as 28.05 lM, 230 lM, and 275 lM respectively whilefor compound 15c, they were found as 166.4 lM, 1176.5 lM and1923.4 lM respectively which suggested that both compoundswere more selective toward DPP-4 compared to DPP-8 and DPP-9. During in vivo OGTT in C57BL/6 mice, compound 15q producedsignificant reduction in serum glucose levels and glucose AUC0–

180min level was significantly inhibited by 19.6%, 33.6% and 47.1%respectively, in a dose-dependent manner from 5 mg/kg, 10 mg/kg and 20 mg/kg dose. Further, at the end of a chronic study(4 weeks) carried out in HFD/STZ induced T2DM model ofSprague-Dawley rats, compound 15q produced significant anti-hyperglycemic effect at 14 mg/kg once a day oral dosing and nohypoglycemic effect was observed in the treated animals. Collec-tively, these results suggest that compound 15q is a moderatelypotent hit for DPP-4 inhibition which can be further modifiedstructurally to enhance its efficacy and selectivity. The hit to leadoptimization efforts are under process in our laboratory. Also, thedifference between in vitro and in vivo results suggests that com-pound 15q might be possibly acting on other diabetic targets thanDPP-4 to produce anti-hyperglycemic effect for which we are plan-ning for detailed mechanistic study in T2DM model.

Acknowledgments

Authors, BP, SB and MG are thankful to Nirma University,Ahmedabad, India, for providing necessary facilities and supportwhile authors MG and BP are thankful to GUJCOST, Gandhinagar,India, to provide financial assistance to carry out the researchwork.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bioorg.2017.03.004.

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