Synthesis, anti-diabetic and in silico QSAR analysis of flavone hydrazide Schiff base derivatives

Abstract This study reports synthesis of flavone hydrazide Schiff base derivatives with diverse functionalities for the cure of diabetic mellitus and their a-glucosidase inhibitor and in silico studies. In this regard, Flavone derivatives 1–20 has synthesized and characterized by various spectroscopic techniques. These compounds showed significant potential towards a-glucosidase enzyme inhibition activity and found to be many fold better active than the standard Acarbose (IC50 = 39.45 ± 0.11 µM). The IC50values ranges 1.02–38.1 µM. Among these, compounds 1(IC50 = 4.6 ± 0.23 µM), 2(IC50 = 1.02 ± 0.2 µM), 3(IC50 = 7.1 ± 0.11 µM), 4(IC50 = 8.3 ± 0.34 µM), 5(IC50 = 7.4 ± 0.15 µM), 6(IC50 = 8.5 ± 0.27 µM) and 18 (IC50 = 1.09 ± 0.26 µM) showed highest activity. It was revealed that the analogues having –OH substitution have higher activity than their look likes. The molecular docking analysis revealed that these molecules have high potential to interact with the protein molecule and have high ability to bind with the enzyme. Furthermore, in silico pharmacokinetics, physicochemical studies were also performed for these derivatives. The bioavailability radar analysis explored that of all these compounds have excellent bioavailability for five (5) descriptors, however, the sixth descriptor of instauration is slightly increased in all compounds. Communicated by Ramaswamy H. Sarma


Introduction
International Diabetic Federation (IDF) has reported that in twenty-first century, Diabetic mellitus (DM) is one of the considerable health risk globally. There data revealed that diabetic mellitus is spreading very rapidly and approximately 415 million peoples have suffered by diabetic mellitus and this number is increasing very fast (Bo-Wei et al., 2017). The irregular insulin production of insulin may cause abnormal metabolism of biomolecules (carbohydrates, proteins and lipids) and this phenomenon is responsible for rise up of blood sugar level and this elevated blood sugar level is a marker of diabetic mellitus. This high level of blood sugar is called postprandial hyperglycemia (Mary et al., 2003;Natasha et al., 2012). Prolong elevated sugar level in blood is symptom of diabetes mellitus and related with various other diseases, including retinopathy, atherosclerosis, neuropathy, nephropathy cataracts and impaired wound healing (Atta et al., 2007;Gerich, 1996). Research studies revealed that postprandial hyperglycemia may also responsible for cardiovascular disease (Yao et al., 2010). Different therapeutic drugs are used to maintain the blood sugar level. These drugs act as insulin release stimulator, glucose transporters, gluconeogenesis inhibitor and delaying agents for glucose absorption in the intestine (Stephen, 2008;Tetsuo et al., 1998).
Carbohydrates rich food assimilated by glycoside hydrolases enzymes including a-glucosidase, a-amylase and b-glucuronidase, are responsible for rapid increase the blood sugar level. Complex carbohydrates are break down into absorbable monosaccharides by the action of a-glucosidase enzyme. This enzyme presents in the epithelial mucosa of the small intestine and breakup the glycosidic linkage in complex carbohydrates. The inhibiting of this enzyme prevents the sudden increase of sugar level in blood (Hyperglycemia) (Chiasson & Rabasa, 2004;Qurrat-Ul et al., 2017;Seiya, 1997). Therefore, inhibiting the a-glucosidase action on complex carbohydrates is one of the most attractive choices to cure from diabetes mellitus (Braun et al., 1995;Dwek et al., 2002;Ernst & Werner, 1981;Franco et al., 2002;Karpas et al., 1988;Lajolo et al., 1984;Madariaga et al., 1988;Mc David et al., 1983;Mehta et al., 1998;Sou et al., 2001;Zitzmann et al., 1999).
Miglitol, Acarbose, voglibose and nojirimycin are well known compounds for managing the blood glucose level in diabetic patients. Although these compounds may have ability to inhibit thea-glucosidase enzyme, but have some side effects such as diarrhea, meteorism and flatulence. Furthermore, the absorption of these inhibitors also a disadvantage of these compounds. Therefore, the development of new a-glucosidase inhibitors is need of time (Park et al., 2008;Rawlings et al., 2009;.
Flavone are naturally occurring compounds and their use in our daily diet is highly recommended for health improvement. The structural modifications of naturally occurring flavones may enhance their biological activities . Flavone motif is a part of various pharmaceutically active compounds. Researcher has reported that flavone analogues have high potential to diminish hyperglycemia, and augmentation of insulin secretion (Rahim, Malik, et al., 2015). Kaempferol, apigenin, luteolin and chrysin, flavones have reported for their enhanced a-glucosidase activity (Khan et al., 2014;.The activity is due to their diverse structural feature with multiring system 2. Result and discussion

Rationale of the studies
Earlier our group has reported various compounds such as flavones ethers (A), triazole (B), Phenoxyacetohydrazide (C) and benzimidazole hydrazones (D) and for their a-glucosidase, b-glucoronidase inhibition and antiglycation activity for the cure of diabetics and its complications. Our studies focus on the effect of various substituents having different position on enzyme inhibition involved in diabetics.
In this research, flavone derivatives 1-20 having hydrazone linkage were synthesized and evaluated for their yeast a-glucosidase inhibitory activity. Moreover, this study also revealed interacting pattern of the compounds with a-glucosidase protein. The outcome of these studies, enable us to identify new inhibitors and structural features contributing towards a-glucosidase activity inhibition. Acarbose the standard a-glucosidase inhibitor having multiring structure with hydroxyl (-OH) and -amine (-NH 2 ) and ether (C-O-C) linkages. These groups efficiently bind with protein. In the same manner, our synthesized compounds also have features, i.e., -NH, -OH and COC linkages, whereas, the structural modification can also be seen in term of imine (-C ¼ N-), and a-b unsaturated ring moiety as well as multiaromatic ring system which may further capable to bind with protein via p-p interaction than the standard.

Synthesis of flavone hydrazide
The key intermediate 4-(6-hydroxy-4-oxo-4H-benzopyran-2yl)-benzoic acid hydrazide (Flavone hydrazide) 1 was synthesized in four (4)-steps. The first step is synthesis of chalcone. Chalcone was synthesized by treating 2,5-dihydroxy acetophenone with 4-formylbenzoic acid in the presence of ethanolic potassium hydroxide. In second step, the cyclization of chalcone via I 2 /DMSO methodology was done. In this step, oxidative cyclization of chalcone was done in order to obtain flavone carboxylic acid derivative. In third step, the flavone carboxylic acid derivative was further refluxed with methanol/sulphuric acid mixture to convert the flavone carboxylic acid into flavone ester. In fourth step, this flavone methyl ester was reacted with hydrazine hydrate in order to obtain the target flavone hydrazide (Scheme 1). The structure of target molecule was confirmed by H 1 NMR, C 13 NMR and Elemental (CHN/S) analysis.
For better understanding of structure-activity relationship these compounds were subjected to computational studies.

Homology modeling of a-glucosidase enzyme
The 3D structure of enzyme a-glucosidase from Saccharomyces cerevisiae (Baker's yeast) was developed with the help of homology modeling technique by using target enzyme crystal structure of a-D-glucose bound isomaltase (PDB ID: 3A4A; Supporting Information) (Yousuf et al., 2018).

Molecular docking studies
To observe the binding pattern of potent a-glucosidase inhibitors, molecular docking studies of biologically active compounds against the targeted receptor was performed by using software Patch dock server, which is a rigid docking algorithm works on geometry and shape-based complementary search. The develop homology model of receptor was used to identify the binding pocket through molecular docking studies of drug (acarbose) showing interactions with HIS 280, LYS 156 and ARG 315 (Figure 1), later the active ligands and receptor in (Pdb) file format was submitted as input files, by setting the clustering RMSD (1.5) and selected default protein-drug molecule complex parameters for conducting molecular docking studies. Software Patchdock detects the shape-base molecular surface area of ligand-receptor complex as a patch of (concave, convex and flat surface pieces), which have the high probability to belong from the binding site region, this reduces the number of potential dock solutions as a result ultimately highlight the patches with 'hot spot' amino acid residues (Schneidman-Duhovny et al., 2005). The software generated 100 dock poses for each inhibitor molecule. Top most favorable dock poses were selected to visualize and analyze the ligand-receptor interactions by using software UCSF CHIMERA (Pettersen et al., 2004). Each dock pose of protein-ligand binding was keenly observed and analyze in order to conclude the insightful knowledge of pharmacophore functional groups with existing noncovalent forces of interactions specifically hydrogen bonding within the provided binding pocket of 5Å region.
Compound 2 exhibited the most potent almost (38 folds) higher a-glucosidase inhibitory potential, with (IC 50 1.02 ± 0.2 mg/ mL), comparative to standard drug acarbose (IC 50 39.45 ± 0.11), which is furthermore validated through its various top rank dock pose analysis, in which hydrazide 'N' substituent's, of flavanoids moiety, is making H-bonding with active site residue LYS 156, while the meta-hydroxy substituent is involved to develop another H-bonding with GLN 279, in another dock pose the meta-hydroxy substituent is making two H-bonds with residue TYR 158, however, face to face p-p stacking interactions are also possibly existing and responsible for its most potent biological activity among the all tested compounds Figure 2(a,b).
Compound 18 also exhibited the almost similar more potent a-glucosidase inhibitory potential, with (IC 50 ¼ 1.09 ± 0.2 mg/mL),which is furthermore validated through its various top rank dock pose analysis, hydrazide 'N' substituent's, of flavonoid moiety, is making H-bonding with active site LYS 156, while 5-hydroxymethyl pyridine is making Hbonding with TYR 158, and 3-hydroxy substituent on pyridine is making H-bonding with SER 240, however, parallel,    p-p stacking interactions are also possibly existing, the very slight change in activity was observed due to slightly weak hydrophobic interactions because of methyl group substituent comparative to compound 2 Figure 3.
Compound 1 is the third more potent a-glucosidase inhibitor, with (IC 50 ¼ 4.6 ± 0.23 lg/mL),the binding pattern analysis depicting the presence of two H-bonding b/w the hydroxy substituent and SER 240, while another H-bonding is observed b/w the hydroxy substituent with ASP 242, however, T-shaped and parallel, p-p stacking interactions are also possibly existing between TYR 158 and the hydroxy substituent phenyl ring there is no H-bonding was observed with LYS 156 residue, and we observed slightly lower inhibitory potential comparative to compound 2, Figure 4.
Compound 3 is the fourth more potent inhibitor, with (IC 50 7.1 ± 0.11 lg/mL). The binding pattern analysis showing the presence of H-bonding b/w 'N' of hydrazide with active site amino acid residue LYS 156, while in another dock pose, residue SER 240 is making H-bonding with ortho-hydroxy substituent on phenyl ring, however, T-shape and parallel, p-p stacking interactions are also possibly existing between TYR 158 and substituted phenyl rings conjugated p-electron system Figure 5(a,b).
Compound 5 is the fifth more potent a-glucosidase inhibitor, with (IC 50 ¼7.4 ± 0.15 lg/mL), the binding pattern analysis depicting the presence of H-bonding b/w active site residue LYS 156 and flavanoid (chromone), however, T-shape, p-p stacking interactions are also possibly existing between TYR 158 and para hydroxy-substituted phenyl ring Figure 6.
Compound 4 (IC 50 ¼ 8.3 ± 0.34 lg/mL),is the sixth more effective a-glucosidase inhibitor, the binding pattern analysis showing the presence of H-bonding meta hydroxy substituted phenyl ring with TYR 158, however, p-p stacking interactions are also possibly existing between TYR 158 and meta-hydroxy-substituted phenyl ring, Figure 7.
Compound 6 (IC 50 ¼ 8.5 ± 0.27 lg/mL), is the seventh more potent inhibitor. The binding pattern analysis showing the presence of H-bonding of flavonoid carbonyl oxygen with GLY 160, just adjacent to the active site residue, LYS 156, another H-bonding is also observed b/w hydroxy substituent with residue THR 310 Figure 8.
Compound 7 is the eighth more potent a-glucosidase inhibitor, with (IC 50 ¼ 9.7 ± 0.15 lg/mL), the binding pattern analysis showing the presence of H-bonding of flavonoid carbonyl oxygen with active site residue SER 240, while SER 236, is making two H-bonds with hydrazide 'NH' and     'C ¼ O' within the provided binding pocket of 5Å region Figure 9.
Compound 20 is the ninth more potent a-glucosidase inhibitor, with (IC 50 ¼10.2 ± 0.57 lg/mL),the binding pattern analysis showing the presence of H-bonding of carbonyl oxygen with residue SER 240 A while the active site residues LYS 156 and HIS 280 are present within the provided binding pocket of 5Å region Figure 10.
Compound 8 is the tenth most potent a-glucosidase inhibitor, with (IC 50 ¼ 10.9 ± 0.44 lg/mL),the binding pattern analysis showing the presence of H-bonding of flavanoid carbonyl oxygen with residue GLN 279 while the active site residues LYS 156 and HIS 280 are present within the provided binding pocket of 5Å region Figure 11.

Profiling of pharmacokinetics properties of potent
a-glucosidase inhibitors a-Glucosidase inhibitors were also profiled against p-gp and isozymes of CYP450 through Swiss ADME tools (Daina et al., 2017), to predicts which compound can be inhibitor of p-gp, and against different CytochromeP450 (CYP450) isozymes. P-Glycoprotein is an important protein in pharmaceutical research due to its substantial effect on ADME (Absorption, Distribution, Metabolism and Excretion) properties. In our screen compounds (1, 3-

Physicochemical properties profiling of potent a-glucosidase inhibitors
According to the Lipinski rule there are various physicochemical descriptors to describe the properties of molecules (Lipinski et al., 2001), including molecular weight, numbers of hydrogen bond donor (HBD), number of hydrogen bond accepter (HBA), the octanol/water partition coefficient (log P) should be 500, 5, 10 and 5, respectively, while the Total Polar Surface Area (TPSA) due to nitrogen (N) and sulfur (S) atoms should be in the range 20-130 A 2 . In our case of study all the screened compounds successfully filtered and showed excellent drug ability properties according to Rule of Five (ROF), however, compounds 7 & 8 exceeds WLOGP very slightly greater than 5, while compounds 1, 2 & 15-18 also slightly exceeds the TPSA greater than 130 A 2 Table 4.   All the compounds are showing excellent drug ability properties according to ROF, compounds 7 & 8 very slightly exceeds WLOGP, however, compounds 1, 2 & 15-18 also slightly exceeds the TPSA greater than 130 A 2 .

Brain or Intestinal EstimateD permeation (BOILED-Egg)
The estimation of two pharmacokinetics behavior is pivotal important, i.e., the absorbtion of drug via human gastrointestinal tract and diffusion across the blood brain barriers. In this regards, Brain Or Intestinal EstimateD permeation (BOILED-Egg) method was used as an accurate predictive model for computing, to analyze the lipophilicity and polarity behavior of small organic molecules. It is a plot of Total Polar Surface Area (TPSA) v/s WLOGP (Daina & Zoete, 2016). The derivatives occurred in the yellow part (egg yolk region) have higher probability of blood brain barrier permeation, although the derivatives in the white part have high chances of absorption through gastrointestinal tract (GI).In our screened compounds, all the inhibitors are lying within Egg white area and anticipated to be absorbed by GI, whereas none of the compound is lying within Egg yolk yellow region, therefore, not to be permeate by BBB of the Boiled egg region and also predicted not to be effluated from CNS by p-glycoprotein Figure 12. , was predicted by Drugs mapping bioavailability radar descriptors. The highlighted pink area epitomizes the optimal range of each descriptor, the molecules within the pink region are considered to have significant bioavailability in the body. All the screened compounds are displaying the acceptable five properties of descriptors, except the descriptor of INSATU due to the presence of increase instauration Table 5. All compounds are showing good bioavailability radar of 5 descriptors; however, the sixth descriptor of instauration is slightly increased in all compounds.

Conclusion
Flavone hydrazide Schiff Base derivatives 1-20 has synthesized and evaluated for their a-glucosidase inhibition activity. These compounds showed remarkable inhibition activity. The computational studies reveled that these molecules may interact with the protein molecule via H-bonding and p-p interactions. The pharmacokinetic studies explored that they have high GI absorption (Gastrointestinal Absorption). A number of physicochemical parameters were also studied. Brain or Intestinal EstimateD permeation (BOILED-Egg) study predicted that all the compounds are lying within Egg white  Figure 12. Showing all the compounds are lying within egg white region and predicted to be absorbed by gastrointestinal tract, while none of the compound is lying within egg yolk region, therefore, not to be permeate by BBB of the boiled egg region and predicted not to be effluated from CNS by pglycoproteins.   region and predicted to be absorbed by Gastrointestinal tract, while none of the compound is lying within Egg Yolk region, therefore, not to be permeate by BBB of the Boiled egg region and predicted not to be effluated from Central Nervous System (CNS) by p-glycoproteins. Furthermore, the bioavailability radar analysis explained that all compounds showed good bioavailability radar of 5 descriptors; however, the sixth descriptor of instauration is slightly increased in all compounds. The all studies showed these compound may have potential towards a-glucosidase enzyme inhibition. This work will enable us to identify new inhibitors and structural pharmacophore features contributing towards a-glucosidase inhibitory activity. Therefore, these compounds may use for anti-diabatic drug development process.

Materials and methods
All nuclear magnetic resonance experiments had been carried out using on Advance Bruker 150 & 300 MHz. Elemental analysis and Electron impact mass spectra (EI-MS) was performed on Perkin Elmer 2400-II CHNS/O Elemental Analyzer, United States and Finnigan MAT-311A, Germany, respectively.

Synthesis of flavone-hydrazide
Flavones hydrazide was synthesized by the reaction in four (4) steps Scheme 1.

Synthesis of chalcone
In first step a chalcone derivative was synthesized by refluxing equamolar amount of 2,5-dihydroxy acetophenone (0.1 mole) and 4-formylbenzoic acid (0.1 mole) for 20 h in the presence of ethanolic potassium hydroxide. The precipitates were formed and the solution was filtered off and washed with dist. water.

Oxidation cyclization of chalcone
Four grams of chalcone was mixed with iodine-DMSO solution and refluxed it for 8 h. After evaporation of solvent the obtained precipitates were and washed with Na 2 S 2 O 3 solution. The product was recrystallized with ethanol.

Synthesis of flavone methyl ester
Four grams flavone carboxylic acid was refluxed for 16 h with sulphuric acid and methanol. The solvent was evaporated and washed with dist. water and precipitates were used for next step without any purification.

Synthesis of flavone hydrazide
The flavone methyl ester was refluxed with hydrazine hydrate for 6 h in methanol. After accomplishment of reaction (TLC analysis) and solvent evaporation the obtained solid product was recrystallized via ethanol in order to obtained pure Flavone hydrazide.