key: cord-1030237-eqqbszqk
authors: Syam, Yasmin M.; Anwar, Manal M.; Abd El-Karim, Somaia S.; Elseginy, Samia A.; Essa, Basma M.; Sakr, Tamer M.
title: New quinoxaline compounds as DPP-4 inhibitors and hypoglycemics: design, synthesis, computational and bio-distribution studies
date: 2021-11-17
journal: RSC advances
DOI: 10.1039/d1ra06799k
sha: 62e8f500ae56ca9ad42fd39bcb6bcdb6759a18eb
doc_id: 1030237
cord_uid: eqqbszqk

The current work represents the design and synthetic approaches of a new set of compounds 6–10 bearing the 1,4-dimethyl-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonamide scaffold. The biological evaluation revealed that most of the new compounds were promising selective dipeptidyl peptidase-IV (DPP-4) inhibitors and in vivo hypoglycemic agents utilizing linagliptin as a standard drug. The acute toxicity examination confirmed the safety profile of all compounds. Molecular docking studies related the significant DPP-4 suppression activity of compounds 9a, 10a, 10f, 10g to their nice fitting in the active pocket of DPP-4. In addition, the molecular dynamic study exhibited the stability of both 10a and 10g within the active site of DPP-4. The QSAR study showed that the difference between the predicted activities is very close to the experimental suppression effect. Moreover, both compounds 10a and 10g obeyed Lipinski's rule, indicating their efficient oral bioavailability. Compound 10a was radiolabeled, forming the (131)I-SQ compound 10a to study the pharmacokinetic profile of this set of compounds. The biodistribution pattern hit the target protein since the tracer accumulated mainly in the visceral organs where DPP-4 is secreted in a high-level, thus with consequent stimulation of insulin secretion, leading to the target hypoglycemic effect.

Diabetes mellitus (DM) is an endocrine and metabolic disorder. It is considered one of the world's biggest health problems with several specic factors related to its pathogenesis. The International Diabetes Federation (IDF) reported that there were 425 million adults suffering from DM worldwide in 2017, with an alarming growth estimate at 700 million in 2045 and 90-95% are expected to be patients with type -2 diabetes mellitus (T2DM). [1] [2] [3] [4] The main causes of T2DM are either deciency in insulin production by the pancreas or inactivation of some proteins participating in the insulin signaling pathway (insulin resistance). [5] [6] [7] [8] Diabetes is usually involved with multiple complications, such as cardiovascular diseases, 9 retinopathy, 10 neuropathy, 11,12 dental and kidney diseases. 13, 14 There are other complications of diabetes, including wound interference cure, 15 ketoacidosis, hyperosmolar coma, 16, 17 as well as reduced immunity to pneumonia and inuenza. 18, 19 Recently, it has been reported that type 2 diabetes is one of the most important diseases that results in a signicant increase of the risk for hospitalization and death in COVID-19 patients. Hypoglycemia and hyperglycemia are both considered as predictors for adverse outcomes in hospitalized patients. In patients with diabetes and SARS-CoV-2 infection, optimal glycemic control should be followed to reduce the likelihood of a serious COVID-19 course. [20] [21] [22] [23] The main goal behind T2DM treatment is to decrease and maintain the level of the glycosylated hemoglobin at less than 7% to prevent the risk of the micro-and macrovascular complications related to the disease. 24 Drugs currently used for T2DM treatment include biguanides, insulin sensitizers (thiazolidinediones), insulin secretagogues (sulphonylureas; meglitinides), and external insulin delivery (insulin analogs). The European Medicines Agency currently approved a-glucosidase inhibitors, glucagon-like peptide-1 (GLP-1) agonists, sodium-dependent glucose transporter 2 inhibitors, and the recent class of dipeptidyl peptidase-4 (DPP-4) inhibitors. 25 DPP-4 is a multifunctional serine peptidase that cleaves Nterminal dipeptides from substrates containing Pro or Ala at the penultimate position. It is expressed in many tissues, such as the kidney, gastrointestinal tract, biliary tract, liver, placenta, uterus, prostate, skin and lymphocytes. 26 DPP4 is mainly released by fully differentiated adipocytes, and its serum levels signicantly correlate with the adipocyte size. Therefore, it risk of heart failure, severe and acute pain of joints, pancreatitis and pancreatic cancer were reported. 43, 44 Accordingly, it is essential to discover new and safer DPP-4 inhibitors that will ultimately encourage their uses in clinics.

Based on its wide spectrum of biological activities, the sulphonamide moiety (-SO 2 NH 2 ) is an efficacious pharmacophore in the medicinal chemistry and drug discovery eld. 45, 46 The sulfonyl functionality is not only able to bind with the amino acid residues of the active pocket of the biological targets with hydrogen bonds, but it also incorporates with the structural core ring and constrains the side chains, allowing their special conformations that perfectly t in the active pockets. 47 Different laboratory research studies have reported that the sulfonamide compounds I and II represented the potent DPP-IV inhibitory activity of the IC 50 values, 6.7 and 39 nM, respectively. 48 (Fig. 2) . Furthermore, Omarigliptin (MK-3102) is a sulfonamide-based DPP-4 inhibitor that is currently under development by Merck & Co. It is a long-acting, oral and once-weekly dosing antidiabetic drug, which makes it unique among the other members of this class. 49 In addition, our previous efforts to develop a new series of tetralin-sulphonamide derivatives introduced potent candidates of selective DPP-4 suppression activity III with IC 50 values in the nanomolar range 50 (Fig. 2) .

The quinoxaline nucleus constitutes a rising biomedical class of low-molecular weight heterocyclic compounds producing potential antidiabetic activity. [51] [52] [53] A chemical library of nearly 32 000 compounds (Korea Chemical Bank) was screened as DPP-4 inhibitors using high throughput screening (HTS) techniques, which resulted in discovering various quinoxalinedione derivatives as potent DPP-IV inhibitors IV (ref. 54) (Fig. 2 ). In addition, the quinoxaline scaffold is bioisosteric to the quinazoline ring, which is a major fragment of the Linagliptin structure, and it constituted the major core of different antidiabetic agents of potent DPP-4 inhibition activity. 55, 56 In view of the synergistic activity that is usually accomplished by molecular hybridization of different bioactive pharmacophores, this work represents the design and synthesis of new derivatives bearing a 1,4-dimethyl-2,3-dioxo-1,2,3,4tetrahydroquinoxaline ring system that is conjugated at position-6 with different alkyl, heterocyclic or substituted aromatic rings via either sulfonamide, sulfonohydrazide and/or sulfonyl hydrazine-carboxy(thio)amide linkers aiming to gain new candidates having potential DPP-4 inhibitory activity, resulting in potent antidiabetic effect (Fig. 3) . The new compounds were evaluated as DPP-4 suppressors, as well as hypoglycemic agents. As representative examples, the inhibitory activity of two promising compounds was assessed against DPP-8/9 to examine the selectivity of the newly synthesized compounds over other DPP family members, such as DPP-8 and DPP-9. A docking study has been performed for the promising active derivatives to nd out the key structural parameters that are vital for the binding modes with the active site of DPP-4 in comparison to linagliptin as a reference drug. In addition, the quantitative structure activity relationship (QSAR) of the active set of new derivatives was performed to detect the descriptors, which might be correlated to the hypoglycemic activity. Furthermore, obeying Lipinski's rule criteria was assessed for the new analogues to nd out their oral bioavailability efficiency. Additionally, a biodistribution study was carried out for one of the promising hypoglycemic candidates as a representative example to investigate its in vivo pharmacokinetic behavior and body organ uptakes, besides its elimination pathway via its 131 I compound complex radiolabeling.

All melting points are uncorrected and were taken in open capillary tubes using the electrothermal apparatus 9100. Elemental microanalyses were carried out at the Microanalytical Unit, Central Services Laboratory, National Research Centre, Dokki, Cairo, Egypt, using a VarioElementar, and were found within AE0.5% of the theoretical values. Infrared spectra were recorded on a Jasco FT/IR-6100, Fourier transform infrared spectrometer at the cm À1 scale using the KBr disc technique at the Central Services Laboratory, National Research Centre, Dokki, Cairo, Egypt. 1 H NMR and 13 C NMR spectra were obtained by using a JEOL AS-500 NMR spectrometer (with operating frequencies of 500 MHz for 1 H using TMS as an internal standard and 125 MHz for 13 C) at the Central Services Laboratory, National Research Centre, Dokki, Cairo, Egypt. Chemical shis (d) are reported in parts per million (ppm) and coupling constants (J) are reported in Hertz (Hz). The mass spectra were measured with a GC MS-Qp1000EX Shimadzu, Cairo University, Cairo, Egypt. The follow up of the reactions and checking the purity of the compounds were made by TLC on silica gelprecoated aluminium sheets (Type 60, F 254, Merck, Darmstadt, Germany) using chloroform/petroleum ether 40-60 (5 : 1, v/v), and the spots were detected by exposure to UV lamp at l 254 nanometer for a few seconds and by iodine vapor. The chemical names given for the prepared compounds are according to the IUPAC system. The starting and the intermediate compounds; quinoxaline-2,3(1H,4H)-dione (1) 1,4-dimethylquinoxaline-2,3(1H,4H)-dione (2) and 1,4-dimethyl-2,3-dioxo-1,2,3,4tetrahydroquinoxaline-6-sulfonyl chloride (3) 1,4-dimethyl-1,4dihydroquinoxaline-2,3-dione (4) and 1,4-dimethyl-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl chloride (5) were prepared according to the reported methods. 57, 58 2.1.1. General method for the preparation of 1,4-dimethyl-2,3-dioxo-N-(heterocyclic/substituted aromatic)-1,2,3,4tetrahydro quinoxaline-6-sulfonamide 6a-f. A mixture of dimethyl-2,3-dioxo-1,2,3,4-tetrahydroquinoxaline-6-sulfonyl chloride (3) (2.88 g, 10 mmol) and the appropriate primary amines, namely, 2-aminothiazole, 4-acetylaniline, 4-aminobenzoic acid, 4-uoroaniline, 4-bromoaniline and/or 2-aminothiophenol (10 mmol) in absolute ethanol (10 mL), was reuxed for 3 h. The formed precipitate was ltered, washed several times with pet. ether, dried and crystallized from ethanol to afford the titled compounds 6a-f, respectively. 2.1.4. General method for the preparation of N 0substituted-1,4-dimethyl-2,3-dioxo-1,2,3,4tetrahydroquinoxaline-6-sulfonohydrazide 9a-c. A solution of the sulfonohydrazide derivative 8 (10 mmol, 2.84 g) and various acid chloride derivatives, namely, acetyl chloride, 2-chloroacetyl chloride and benzoyl chloride (10 mmol) in pyridine (20 mL), was reuxed for 6 h. Aer completion of the reaction, the solution was poured onto ice/H 2 O and neutralized with HCl. The formed precipitate was collected by ltration, washed several times with water, and crystallized from isopropanol to give the corresponding derivatives 9a-c. 

To a solution of the sulfonohydrazide derivative 8 (10 mmol, 2.84 g) in DMF (20 mL) containing a few drops of triethylamine (10 mmol), the appropriate iso/isothiocyanate was added and the reaction mixture was reuxed for 6 h. Aer completion of the reaction, it was poured onto ice/H 2 O and neutralized with HCl. The formed precipitate was collected by ltration, washed several times with water, and crystallized from the proper solvent.

2 

All of the biological data were cited in the details in the ESI † le.

All of the computational studies were cited in the details in the ESI † le.

The details of the radiopharmaceutical evaluation approach were cited in the ESI † le.

The synthetic approaches of the target compounds are depicted in Schemes 1 and 2. The key starting compound, quinoxaline-2,3(1H,4H)-dione (3), was obtained by condensation of the commercially available oxalic acid (1) with o-phenylenediamine (2) in alkaline medium. N-Alkylation of 3 with methyl iodide in the presence of anhydrous K 2 CO 3 led to the formation of the corresponding 1,4-dimethyl analogue 4. 57 Chlorosulfonation of the alkylated derivative 3 with chlorosulfonic acid led to the formation of the corresponding key 6-sulfonyl chloride intermediate 5, 58 which was allowed to react with different heterocyclic/aromatic primary amines, namely, 2-aminothiazole, 4-aminoacetophenone, 4-aminobenzoic acid, 4-uoroaniline, 4-bromoaniline and 2-aminothiophenol, to obtain the corresponding sulfonamide compounds 6a-f, respectively. Furthermore, compound 5 was treated with various sulfa drugs, namely, sulfanilamide, sulfathiazole, sulfapyridine and sulfadiazine in ethanol containing a catalytic amount of triethylamine, to afford the corresponding sulfa derivatives 7a-d, respectively (Scheme 1). Furthermore, hydrazinolysis of the 6-sulfonyl chloride intermediate 5 was carried out by its treatment with hydrazine hydrate in ethanol to get the corresponding 6-sulfonohydrazide derivative 8, which in turn underwent nucleophilic substitution by its reaction with various acid chloride derivatives, namely, acetyl chloride, 2-chloroacetyl chloride and benzoyl chloride, as well as with different iso(thio)cyanates to accomplish the corresponding N-substituted-6-sulfonohydrazide and hydrazinecarbox(thio)amide derivatives 9a-c, 10a-h, respectively (Scheme 2). The structural identication of the new target compounds was performed by microanalytical and IR, 1 H NMR, 13 C NMR and mass spectral data, which were in conformity with their assigned molecular structures. IR spectra exhibited absorption bands in the range of 3344-3174 cm À1 related to the NH groups, 1710-1653 cm À1 assigned to the 2C]O quinoxaline groups, in addition to two bands in the range 1388-1134 cm À1 representing the SO 2 groups. The compounds 6b, 6c, 9a-c and 10ad represented the additional absorption bands at 1710-1653 cm À1 , conrming the presence of the acetyl, carboxylic and carboxamide C]O groups, respectively, while compounds 10e-h revealed absorption bands at the region of 1458-1451 cm À1 related to the C]S functionality. Furthermore, the 1 H NMR data displayed a singlet signal at d 3.40-3.50 ppm integrating the six protons of the 1,4-di-CH 3 groups and a singlet D 2 O exchangeable signal at the range of d 5.92-6.70 ppm due to the sulfonamide NH groups, while the aromatic protons appeared at their expected regions. The compounds 6b, 6c, 6f exhibited additional singlets at d 3.08, 10 .08, 5.41 ppm attributed to CH 3 , OH and SH, respectively. On the other hand, the aromatic zone of compounds 7a-d integrated higher proton numbers attributed to the conjugated phenylsulfonamide moieties. Moreover, the acetyl and benzyl sulfonamide compounds 9a, 9b displayed two singlets at d 2.05 and 4.21 ppm integrated for -COCH 3 and ph-CH 2 protons. In contrast, the hydrazinecarboxamide derivative 10a exhibited an up-eld doublet signal resonating at d 1.09 ppm due to the 2CH 3 -isopropyl. Meanwhile, the methine CH-isopropyl appeared as a multiplet signal at d 4.32 ppm, in addition to three D 2 O exchangeable singlets at d 5.53-5.60 and 7.91 ppm related to the 3NH groups. Furthermore, besides the parent protons at the expected zones, compounds 10b, 10e, 10h exhibited a singlet signal at d 4.20 ppm, characteristic tribletquartet signals at d 1.15, 3.63 ppm and a singlet signal at d 3.70 ppm attributed to the ph-CH 2 , -CH 2 -CH 3 and -OCH 3 groups, respectively. Furthermore, the cyclohexyl protons of compound 10f were represented as three upeld multiplets at d 1.10-1.14, 1.65-1.78, 3.08-3.11 ppm, attributed to 3CH 2 -a, 2CH 2 -b and CH, respectively. 13 the previously reported method. 50 The obtained results are expressed as IC 50 (nM) and summarized in Table 1 .

It could be noted that most of the tested derivatives produced a promising DPP-4 suppression effect, indicating that the quinoxaline-6-sulfonamide scaffold is pivotal for the DPP-4 inhibitory potency. Structure activity relationship analyses revealed the following points:

Conjugation of the 2,3-dioxo-1,2,3,4-tetrahydroquinoxaline motif with various substituted aromatic moieties via the 6sulfonamide linker as compounds 6b-f exhibited DPP-4 inhibitory activity that was approximately equipotent or slightly less than that obtained by the reference drug Linagliptin of IC 50 values ranging from 0.67-1.28 nM, IC 50Linagliptin ; 0.77 nM, while the suppression activity was completely lost by the thiazolyl derivative 6a.

On the other hand, conjugation of the parent 2,3-dioxo-1,2,3,4-tetrahydro quinoxaline-6-sulfonamide with different 4-(heterocyclic) sulfamoylphenyl moieties as compounds 7 led to a signicant inuence on inhibitory activity. The 4-sulfamoylphenyl, the pyridinyl and the pyrimidinyl analogues 7a, c, d represented 1.2-1.6 folds more potent DPP-4 inhibitory effect than that obtained by Linagliptin of IC 50 s; 0.60, 0.48 and 0.48 nM, respectively. The presence of an additional sulfamoyl functionality might be the reason for enhancement of the suppression effect of the latter ligands by forming extra hydrogen bonds with the active pocket of DPP-4. A slight decrease in the potency was detected by the thiazolyl derivative 7b of IC 50 ; 0.93 nM.

Furthermore, the resultant data showed that the elongation of the 6-sulfonamide linker to 6-sulfonohydrazide leading to the formation of the ligands 9a-c produced a robust inuence on the inhibitory activity. The compounds 9a-c exhibited a DPP-4 suppression effect that was about 8-9 folds more potent than Linagliptin (IC 50 ; 0.085, 0.095, 0.095 nM, respectively). The detected pronounced activity of 9a-c might be explained due to the presence of additional N and O atoms acting for extra Hinteraction with the active site of the target enzyme.

Furthermore, an outstanding increase in the DPP-4 suppression potency by 11-18 folds compared to linagliptin was investigated upon prolongation of the 6-sulfonohydrazide linker to sulfonyl-N-substituted hydrazine-1-carbox(thio)amide, creating the corresponding analogues 10 of IC 50 range 0.039-0.068 nM, a result which could be explained due to further bindings with the amino acid residues in the active pocket of the enzyme. The p-chlorophenyl and p-methoxyphenyl derivatives 10d, 10h, respectively, completely lost the activity. The conformation restriction of the latter ligands could explain the dramatic loss of the target activity (Fig. 4) .

Subsequently, compounds 7d, 9a, 10a, 10g showing promising DPP-4 suppression activity were selected as representative examples to perform the selectivity assay over DPP-4 homologues; DPP-8 and DPP-9 in the DPP-4 gene family ( Table  2 ). The results showed that the tested compounds exhibited excellent selectivity over the DPP-4 homologues proteins. All compounds 7d, 9a, 10a, 10g exhibited IC 50 values >100 mM against both DPP-8 and DPP-9. This result revealed that all tested compounds possessed signicant selectivity towards DPP-4 inhibition of about >1000 folds over that of DPP-8/9 (Table 2) .

The in vivo anti-hyperglycemia effects of the new compounds showing promising DPP-4 inhibition activity were evaluated by performing the intraperitoneal glucose tolerance test (IPGTT) in high-fat diet-fed/streptozotocin (HFD/STZ) induced T2DM male albino mice following the previously reported method. The blood glucose levels in (mg dl À1 ) were measured at 1, 2, 3, and 6 hours intervals. 59, 60 Table 3 summarizes the changes in blood glucose levels in the tested diabetic animals following a single dose administration of the investigated compounds. The obtained ndings were in line with DPP-4 inhibition activities. The blood glucose levels were restored to more or less normal levels, with no major differences relative to the control group. The compounds 9 and 10 exhibited the most potent activity, representing glucose levels very close to those obtained by linagliptin throughout the six recorded values post doses administration (Table 3) .

3.2.3. Cytotoxicity bioassay. One of the parameters that distinguishes various drugs from each other is the frequency and the severity of the side effects to the normal cells at the therapeutic doses. Accordingly, it was of interest to study the safety prole of compounds 7d, 9a, 10a, and 10g as representative examples against the normal hTERT-RPE1 cell line. The suppression percentage of the normal cells by each tested compound was determined via MTT 61 (Table 4 ). It was investigated that the tested derivatives exhibited minor cytotoxic effects of 10%, 20%, 10%, and 6.9% on the examined normal cells, representing the promising safety prole of the tested compounds.

The compounds 9a, 10a, 10f, and 10g were selected as representative examples for the most promising compounds to carry a docking score of À4.8 kcal mol À1 and it formed two H-bonds between NH and Glu_205 and Glu_206 at distances 2.9Å and 3.1Å, respectively, and the N of the CN group formed a H-bond with Tyr547 at a distance of 3.2Å, in addition to the p-p interactions between the phenyl group and Phen357, Tyr 585 and Tyr665. Compound 9a formed ve H-bonds with the key residues Arg125, Ser209, Tyr547, Ser630 and His740 with a binding score of À4.9 kcal mol À1 . Compound 10a showed a binding score of À5.6 kcal mol À1 . It formed 6 H-bonds with Glu206, Arg358, Ser630, Tyr547, Tyr662, and Asn710, while compound 10f showed a binding score of À5.1 kcal mol À1 and formed four Hbonds with Glu205, Glu206 and Tyr662. The quinoxaline group of both compounds showed p-p stacking with Phe357. Furthermore, compound 10g showed a binding score of (À7.7 kcal mol À1 ) and illustrated three H-bonds with Glu205 and Tyr662 at a distance of (2.4, 2.9, and 3.1Å), respectively, besides the p-p interactions between the quinoxaline group and Phe357 (Fig. 5 , Table 5 ). Compound 10a showed a good binding mode within the active site of DPP-IV in comparison to the co-crystallized ligand. The isopropyl amide moiety of 10a occupied the position of acetyl pyrrolidine carbonitrile moiety of the native ligand. Meanwhile, the di-methyl quinoxalinedione moiety occupied the position of the open chain of 10a, which links the 2-methylpyrazolo[1,5-a]pyrimidine with the pyrrolidine group. Replacement of the isopropyl amide group of 10a with the cyclohexyl thioamide group 10f showed a different binding mode. The quinoxaline moiety moved away from the position of the native ligand and protruded out of the binding pocket, and this may explain the slight decrease in the activity of 10f (Fig. 6) . Replacement of the cyclohexyl moiety in 10f with a phenyl group as compound 10g showed the good binding mode of 10g (Fig. 6) . The phenyl group occupied the position of the pyrrolidine of the native ligand, and the quinoxaline group occupied the position of pyrazolopyrimidine. The promising activity of 10g may be attributed to its good binding mode within the binding site.

In conclusion, 9a, 10a, 10f and 10g showed good H-bonds prole with the key residues as Glu205, Glu206 and Tyr662, while compound 10g with a phenyl substituent showed the best binding mode with the active site of the target protein, and this explains its good binding score and good inhibitory activity. Fig. 7 RMSD of (A) compound 10a, (B) compound 10g over 100 ns MD simulation.

Molecular dynamics studies (MD) were carried out for two of the most promising compounds 10a and 10g as representative examples in order to study their stability within the active site of DPP-IV. 64 The ligand-receptor complexes of 10a and 10g that came out from the docking simulation were used in the molecular dynamic simulation for 100 ns. Compound 10a showed a smooth root mean square deviation (RMSD), which indicated its stability within the binding pocket. In addition, it could be seen that 10a had two conformations over the MD simulation. The rst conformation showed RMSD around 2.5Å, and the second conformation appeared at 50 ns and showed an RMSD around 1.5Å (Fig. 7A) . Compound 10g showed a smooth RMSD curve and two conformations (Fig. 7B ). The rst one showed a root mean square deviation (RMSD) of 2.5Å and extended from 1 ns to 50 ns, while another conformation extended from 50 ns to 100 ns, illustrating an RMSD around 1.5 A. The MD simulation results for compounds 10a, 10g showed nearly the same pattern, and this might be attributed to the similarity in the structures.

In conclusion, MD of compounds 10a and 10g showed that both compounds are stable over the MD simulation. These results explained the activity of 10a and 10g, which might be attributed to their stability within the active site.

A molecular modelling experiment was performed to develop a hypothetical pharmacophore model for the anti-DPP-IV activity. In order to study the tting of the designed compounds to the developed pharmacophore, a ligand-based pharmacophore model was developed using a training set of nine of diverse chemical structures that showed promising IC 50 values. The hypothetical pharmacophore features used are the hydrogen bond donor (HBD), hydrogen bond acceptor (HBA), ring aromatic (RA) and hydrophobic aromatic feature (HAR).

Sixteen pharmacophore models were generated via exible aligning of the represented training set, and all of the pharmacophore models included at least three chemical features. The top pharmacophore model generated showed 6 features: two HBD, two HBA, one HYP, and one RA feature, as represented in Fig. 8 , with constraint distances and angles between its features listed in Table 5 . The top pharmacophore was selected upon the total mutual similarity score of the molecules (S) and the alignment score of the molecules (F). The top pharmacophore model was generated upon alignment molecules that showed the lowest F and S, as the lower values indicate greater similarity and better alignment. Fitting of the designed active compounds, for example, 10g to the pharmacophore, revealed the presence of the appropriate chemical groups superimposed on the pharmacophore features (Fig. 9) . Compound 10g superimposed with 4 features; two C]O group represented 2HBA, phenyl group represented HYP and phenyl group represented RA.

The QSAR study was performed to investigate the correlation between the biological activity and physicochemical properties of the training set using MOE soware. The 18 compounds that showed activity against DPP-IV were selected as the training set, while inactive compounds (6a, 10d and 10h) were omitted from the analysis. 64,65 2D Descriptors (including the molecular properties, surface area and volume, and topological descriptors) and 3D descriptors, as well as the energies of the highest occupied and the lowest unoccupied molecular orbitals (HOMO and LUMO) were calculated using the QSAR descriptors tool. The pIC 50 was calculated as pIC 50 ¼ Àlog IC 50 . The partial linear regression (PLS) method was applied to search for the optimal QSAR models. The QSAR model was validated employing the leave one-out crossvalidation (LOO) method, in which one compound is removed from the data set and the activity is correlated using the rest of the data set, 66 r 2 (squared correlation coefficient value), r 2 prediction (predictive squared correlation coefficient value), root mean square error (RMSE), and residuals between the predicted and the experimental activity of the test set, and the training set and Zoutlier score were applied to validate the QSAR model.

Eqn (1) represents the best performing QSAR model. PIC 50 ¼ 0.01378 vsurf_Wp4 À 0.00817 vsurf_W5 + 0.68457 MNDO_HOMO + 0.00697 PEOE_VSA_4 + 15.53954. PIC 50 is the negative logarithmic value of the concentration required to produce 50% inhibition of DPP-IV activity. In this model, two vsurf_ descriptors (such as vsurf_W5 and vsurf_Wp4) were contributed for the activity prediction. The vsurf_descriptors are the volume and surface properties, depending on the structural connectivity of the molecules. 67 The vsurf_Wp explains the polar volume of the molecules. In general, Wp1-4 explain the polarizability and dispersion forces. 67 Here, the vsurf_Wp4 has been calculated at the _0.2 kcal mol À1 energy level and its positive contribution explains that the polarizable property on the van der Waals (vdW) surface of the molecules is crucial for the interactions. The vsurf_W descriptor describes the hydrophilic region of the molecules, and W5-8 accounts for the polar and hydrogen bond donor acceptor regions. vsurf_W5 was calculated at À0.3 kcal mol À1 energy level and the negative sign indicates that the molecules should not have many hydrogen-bond donor or acceptor groups. PEOE_VSA_4 is a partial charge descriptor. This descriptor depends on calculating the partial charge of each atom. PEOE is the Partial Equalization of Orbital Electronegativity, which is a method of calculating the atomic partial charges. Meanwhile, PEOE-VSA-4 describes the partial negative charge of the vdW surface area, and its positive charge reveals the presence of partial negative charge on the molecules, which is important. MNDO_HOMO is the energy (eV) of the Highest Occupied Molecular Orbital and its positive sign is important for the biological activity. According to eqn (1), the QSAR model is illustrated graphically by scattering plots of the experimental versus the predicted bioactivity, as shown in Fig. 10 . The method used to build the models was partial least-squares, r 2 ¼ 0.7, r 2 (pred) 0.2. r 2 (pred) is equivalent to q 2 from a leave-1-out crossvalidation root mean square error (RMSE) ¼ 0.09, cross validated RMSE ¼ 0.17. From eqn (1), it could be concluded that the anti DPP-4 inhibition activity of the training set increased by increasing vsurf_Wp4, PEOE_VAS_4 and MANDO_H, while it increased by decreasing vsurf_W5.

QSAR models were evaluated internally by applying the leaveone-out cross-validation, where r 2 (squared correlation coefficient value) is 0.7, and r 2 (pred) is 0.2. The RMSE value was so small ¼ 009. The high value of r 2 and low value of RMSE indicate the reliability of the model. The resulting model was also validated by measuring the residuals between the experimental activity and the predicted activity of the training set (Table 7 ). It is notable that the difference between the predicted activity by the QSAR model is so close to the experimental activity, which is evidence of the efficacy of the model to be applied for the prediction of more effective hits. In addition, the Z-score was used in the model evaluation (Table 6) , and there is no compound that is an outlier.

In order to investigate the oral bioavailability of the best anti-DPP-IV compounds, the Lipinski's criteria of the newly synthesized compounds were calculated. Lipinski's rule is based on oral bioavailable drugs having a molecular weight (MW) of 500 or less, ten or fewer hydrogen bond acceptor sites, ve or fewer hydrogen bond donor sites, and log P should not be greater than 5. In addition, the total polar surface area (PSA) was calculated since it is another key property that has been linked to drug bioavailability. Thus, passively absorbed molecules with Fig. 10 The scatter plots of the experimental versus the predicted bioactivity values of PIC 50 for the training set compounds. Table 8 shows that the best promising compounds 10a and 10g fullled Lipinski's rule and PSA, indicating that these promising compounds have good oral bioavailability.

5. Radiopharmaceutical evaluation of the sulfonylquinoxaline derivative 10a (SQ) compound

The derivative 2-(1,4-dimethyl-2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-6-ylsulfonyl)-N-isopropyl hydrazine-1carboxamide (10a) (SQ) was selected as a representative example for the quinoxaline derivatives to evaluate its biodistribution through in vivo radiolabeling the 131 I-SQ compound, [70] [71] [72] [73] [74] aiming to investigate its pharmacokinetic behavioral prole, its uptake by different body organs, alongside its elimination pathway. The radiosynthesis of the 131 I-SQ compound was optimized with a radiosynthesis yield aer a decay correction of 94% at pH 6, 200 mg chloramine-T amount, 300 mg SQ amount, and 45 min reaction time (Fig. 11 ).

The results tabulated in Table 9 revealed the accumulation of the 131 I-SQ compound in various body organs. Interestingly, the 131 I-SQ compound showed high accumulation in the visceral tissues, especially the stomach and intestine, where DPP-4 is mainly released by adipose tissues and is present in a high level. 27 Thus, the examined derivative SQ (10a) achieved the target strategy of this study. Post-administration, it accumulates mainly in the visceral organs where DPP-4 is secreted in a highlevel post prandial. This leads to the DPP-4 suppression effect, blocking the inactivation of the incretin hormones with the consequent stimulation of insulin secretion and inhibition of glucagon secretion, leading to the control of the glucose level in the body. [75] [76] [77] [78] [79] [80] [81] [82] [83] In addition, it was clear that the excretion pathway of the 131 I-SQ compound is through the renal route. Moreover, it did not show any high accumulation in other body organs.

As an effort to achieve new more potent and safer antidiabetic agents, this work deals with the design and synthesis of new different derivatives bearing a quinoxaline ring system tagged with different substituted sulfonamide linkers conjugating various alkyl, heterocyclic and aromatic functionalities. All of the new ligands were evaluated as in vitro DPP-4 enzyme inhibitors. Most of the compounds exhibited promising suppression activity compared with Linagliptin. Compounds 7d and 9a, 10a, 10g revealed potent selectivity towards DPP-4 inhibition about >1000 folds over than DPP-8/9. Furthermore, the promising compounds were assessed as in vivo hypoglycemic candidates, and they exhibited promising glucose controlling effect, in comparison with linagliptin as a reference drug. Furthermore, the latter derivatives represented a promising safety prole against the healthy cells. Docking studies represented nice binding of the promising compounds 9a, 10a, 10g, 10f with the amino acid residues of the target protein DPP-4. In addition, a molecular dynamic study exhibited the stability of both 10a and 10g within the active site of DPP-4, which might explain their signicant DPP-4 inhibiting effect. Moreover, the computational study included the creation of a hypothetical pharmacophore model for the anti-DPP-4 activity, utilizing a training set of various different chemical structures that showed promising IC 50 values. Additionally, a QSAR study was carried out to explore the correlation between the target suppression activity and physicochemical properties of the training set using MOE soware. It has been found that the difference between the predicted activity by QSAR model is very close to the experimental one. In addition, the pharmacokinetic pattern of compound 10a (SQ) (selected as a representative example) was studied by radiosynthesis of the 131 I-SQ compound 10a, and optimized with a radiosynthesis yield of 94% at pH 6, 200 mg chloramine-T amount, 300 mg SQ amount and 45 min reaction time. The biodistribution pattern of the tracer represented its accumulation in the visceral organs mainly in the stomach where DPP-4 is secreted in a high-level, thus inhibiting the inactivation of the incretin hormones, leading to stimulation of insulin secretion and hypoglycemic effect.

The resultant data highlighted that the quinoxaline -sulfonyl motif could provide a basic nucleus for the development of newer potent antidiabetic active hits of selective DPP-4 suppression activity of lower undesirable side effects. 

The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to inuence the work reported in this paper.

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This work was supported nancially by Joint ASRT-BA Research Grants; project No. 1356 (The joint grant between the Academy of Scientic Research and Technology and Bibliotheca Alexandrina).