Designing of some Novel Methyl 2-((4-(Benzamido)Phenyl)Sulfanyl)-1,2,3,4-tetrahydro-6- Methylpyrimidine-5-carboxylate Derivatives as Potential Glucokinase Activators through Molecular Docking

Aims: Glucokinase (GK) is a cytoplasmic enzyme that metabolizes the glucose to glucose- 6-phosphate docking studies to determine the binding interactions for the best-fit conformations in the binding site of the GK enzyme. Autodockvina 1.1.2 in PyRx 0.8 was used to perform the docking studies of all the designed novel derivatives and native ligand against the crystal structure of GK. Based on the results of docking studies, the selected molecules will be tested for their antidiabetic activity in the animal models. Results: Amongst the designed derivatives, compounds A2, A3, A8, A10, A11, A13, A14, A16, A17, and A18 have shown better binding free energy (between -8.7 to -10.3 kcal/mol) than the native ligand present in the enzyme structure. In present investigation, many molecules had formed strong hydrogen bond with Arg-63 which indicate the potential to activate GK. Conclusion: From above results it has been observed that these designed benzamide derivatives have potential to activate the human GK which enables us to proceed for the syntheses of these derivatives.


INTRODUCTION
Diabetes is a metabolic condition categorized by malfunction of glucose metabolism [1]. It leads to other complications like cardiovascular, peripheral, vascular, ocular, neurologic and renal abnormalities etc [1,2]. The growing problem of diabetes has led to integrated research activities globally for the development of defensive and therapeutic strategies [1,3]. The World Health Organization (WHO) has estimated that ~1.6 and 2.5 million people may die from diabetes in 2015 and 2030 respectively [4,5]. It will be the 5 th foremost reason of death worldwide by 2030 [6][7][8].
The glucose phosphorylating enzyme glucokinase (GK) is a monomeric protein having 465 amino acids (molecular weight =50kD) [9,10]. It maintains glucose homeostasis inside cells, acts as a glucose sensor in pancreatic βcells and as a rate regulatory enzyme for hepatic glucose clearance and glycogen synthesis [11,12]. It has two binding sites, one for binding D-glucose and the other for a putative allosteric activator named glucokinase activator (GKA) [9]. The GKAs intermingle with the identical region of the GK enzyme that is normally affected by the naturally occurring mutations in humans. Newly, it has been reported that GKAs are extremely effective in patients with type 2 diabetes mellitus (T2DM) [13][14][15][16][17].
As a glucokinase activator and in the treatment of T2DM, benzamide nucleus has been described in many publications. We chose the benzamide nucleus for the development of several new GK activators based on this literature. We had designed and developed some novel GK activators constructed on benzamide nucleus. The substitutions on benzamide nucleus were carried out in such a way that strong Hbond and hydrophobic interactions with residues in the allosteric site of GK protein can be targeted. Additionally, the molecules were designed so as to be orally bioavailable by introducing groups like aryl and/or alkyl in the benzamide nucleus.

Designing of Novel Methyl 2-((4-(benzamido) phenyl)sulfanyl)-1,2,3,4tetrahydro-6-Methylpyrimidine-5-Carboxylate Derivatives
The novel derivatives have been designed as per the reaction scheme depicted in Fig. 1. In the first step, N-(4-chlorophenyl)benzamide has been designed by condensing with benzoic acid and 4-chloroaniline in the presence of N,N'-Dicyclohexylcarbodiimide (DCC). In the second step, 1,2,3,4-tetrahydropyrimidine-2-thiol derivatives have been designed using modified Biginelli reaction by using different aromatic/aliphatic aldehydes. In the third step, product of first and second step were condensed to get final novel benzamide derivatives. The structures of the derivatives are shown in Table 1 with the IUPAC names. Step-I: Step      [36]. The complete molecular docking procedure, identification of cavity and active amino acid residues was performed as per the procedure described by S. L. Khan et al., [37][38][39][40]. The identified cavity of the enzyme with cocrystallize ligand molecule is represented in Fig.  2.

RESULTS AND DISCUSSION
The ligand energy (kcal/mol) and binding free energy (kcal/mol) of the derivatives are illustrated in Table 2. The molecular interactions of the derivatives are tabulated in Table 3. The 3D-and 2D-docking poses of the best 10 molecules with GK enzymes are depicted in Table 4.
All the designed novel derivatives were docked on human glucokinase enzyme and the docking results were compared with native ligand present in enzyme (PDB ID 1V4S). The formation of hydrogen bonds with the target can cause more effective conformational changes. Many derivatives showed better binding interactions at allosteric site than the native ligand with the formation of more hydrogen bonds. The native ligand has formed 3 conventional hydrogen bonds with THR-228 (2.21A 0 ), LYS-169 (2.60A 0 ), and ASP-78 (2.04A 0 ); one carbon hydrogen bond withGLY-81 (3.75A 0 ); Pi-Anion bond with ARG-85 (3.57A 0 ), ASP-409 (3.71A 0 ), Pi-Cation bond with ASP-205 (3.95A 0 ) and binding free energy of -7.2 kcal/mol.

CONCLUSION
In the present work, we have designed and developed some novel benzamide derivatives as GK activators for treating T2DM. Neha Charaya et al. have designed, synthesized and evaluated some novel thiazol-2-yl benzamide derivatives as antidiabetic agents [18]. They have reported that this benzamide scaffold can be treated as the primary hits for the expansion of novel, safe, active, and orally bioavailable GK activators to treat T2DM. Saurabh C. Khadse et al. have designed, synthesized and evaluated the series of hetero-substituted sulphonamidobenzamide hybrids as GK activators and concluded that these are safe and could be explored further for better therapeutic efficacy in the treatment of T2DM. They have reported that the hydrogen bonding with Arg-63 amino acid residue is an essential interaction necessary for ideal binding [41]. Kaapjoo Park et al. have reported some novel heteroaryl-containing benzamide derivatives as GK activators. The strong hydrogen bonds with Arg-63, the hydrophobic pocket surrounded by Tyr-214, Tyr-215, Gly-97 and the solvent exposed region with hydrogen bonding to Arg-250 are important for GK activation [42]. In present investigation, many molecules had formed strong hydrogen bond with Arg-63 which indicate the potential to activate GK. From above results it has been observed that these designed benzamide derivatives have potential to activate the human GK which enables us to proceed for the syntheses of these derivatives.
All the designed novel derivatives were docked on the human glucokinase enzyme, and the docking results were compared with the native ligand present in the enzyme (PDB ID 1V4S). The formation of hydrogen bonds with the target can cause more effective conformational changes. Many derivatives showed better binding interactions at allosteric sites than the native ligand with more hydrogen bonds. Amongst the designed derivatives, compounds A2, A3, A8, A10, A11, A13, A14, A16, A17, and A18 have shown better binding free energy (between -8.7 to -10.3 kcal/mol) than the native ligand present in the enzyme structure. From current results, we have concluded that designed derivatives can effectively activate the human GK enzyme, which can be useful in treating T2DM. We will proceed with the syntheses, characterization and screening of these derivatives by oral glucose tolerance test (OGTT) as antidiabetic agents in animal models. In part two of this research work, we will report the synthesis of these derivatives and their pharmacological screening as antidiabetic agents in the animal model.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.