SYNTHESIS AND PRELIMINARY MOLECULAR DOCKING STUDIES OF NOVEL ETHYL-GLYCINATE AMIDE DERIVATIVES

Ethyl glycinate was synthesized by the Fischer esterification protocol, and its amide derivatives; 2-amino-N-(nitrophenyl)acetamide 31, 2-amino-N-(6-methylpyridin-2-yl) acetamide 33, N,N'-(1,4-phenylene)bis-(2-aminoacetamide) 35, N,N'-(6-chloropyrimidine-2,4-diyl)bis-(2-aminoacetamide) 37, and 2,4-(diamino-N’N-6-hydroxypyrimidyl)acetamide 39 respectively were obtained  by coupling reactions of 4-nitroaniline, 2-amino-6-methylpyridine, 1,4-diamino-N,N’-benzene, 2,6-diamino-4-chloropyrimidine and 2,4-diamino-6-hydroxypyrimidine respectively with ethyl glycinate.  These compounds were characterized on the basis of their melting points, UV-Visible, IR, 1HNMR and 13CNMR spectroscopic analyses. The results obtained from the spectra were consistence with the assigned structures of the compounds. The synthesized compounds were subjected to molecular docking with a target protein, 1CVU to compare their binding energies with celecoxib and rofecoxib which are standard drugs that inhibit COX2 enzyme. From the docking results, the binding energies values of the above synthesized compounds are -5.8 kJmol-1, -6.2 kJmol-1, -7.2 kJmol-1, -7.4 kJmol-1 and -7.6 kJmol-1 respectively. Compound 39 showed the highest binding energy of -7.6 kJmol-1, close to celecoxib and rofecoxib with binding energy values of -8.0 kJmol-1 and -8.2 kJmol-1 respectively. This result indicates that compound 39 possess some level of inhibitory activity against COX2.


INTRODUCTION
Amino acids are molecules containing both amino and carboxylic acid groups. There are basically twenty in number namely; glycine 1, alanine 2, serine3, threonine 4, cysteine 5, valine6, leucine7, isoleucine 8, methionine 9, proline10, phenylalanine 11, tyrosine 12, tryptophan 13, aspartic acid 14, glutamic acid 15, asparagine 16, glutamine 17, histidine18, lysine 19, arginine 20, and of all these, glycine1 is the simplest, Young (1994). Out of these, nine of them are classified as essential amino acids, because they cannot be synthesized by the body and are therefore required to be taken in diets namely; histidine, leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine according to Dietary Reference Intakes (2014 Figure 1: Structures and names of the 20 amino acids Glycine 1 is the simplest and a conditionally essential amino acid; its chemical formula is C2H5NO2. It is a white solid with density of 1.607g/mol. It is soluble in pyridine, sparingly soluble in ethanol and insoluble in ether. It was first produced by a French chemist, H. Braconnot from acid hydrolysis of protein in 1820, according to . It has a sweet taste like glucose and can also be produced by alkaline hydrolysis of meat and gelatin with potassium hydroxide. Because of its simplicity, it has only one form, unlike other amino acids that possess the L and D isomers. Wu (2009) reported that glycine supports healthy kidney and liver function as well as the nervous system and serves as a major constituent in extracellular structural proteins (collagen and elastin) in animals. Although glycine has been traditionally classified as a ''nutritionally conditionally essential amino acid'' for mammals (including humans, pigs and rodents) due to the presence of its endogenous synthesis in the body according to Wu, (2010), and Darling et al.(1999), it has been reported that the amount of glycine synthesized in vivo is insufficient to meet metabolic demands in these species according to Jackson (1991), Melendez-Heviaet al. (2009), and Rezaeiet al., (2013).
Other functions of glycine include: protection of the body against hyper toxicity by effectively and successively fighting against ethanol induced toxicity according to Senthilkumar et al (2004), Zeb and Rahman (2017), an effective therapy for shocks, Abello et al (1994), treatment of gastric ulcer by decreasing the acid secretions caused by pylorus ligation, prevention of organ transplanting failure (kidneys) when treated with a solution containing glycine and Carolina, Zeb and Rahman (2017). This mixture helps to protect the kidneys against storage injury as well as long survival after kidney transplantation Yin et al (2002). Glycine is a very successful immunomodulatory that suppresses inflammation. It also prevents aging in human system. Glycine could also help in the correction of erectile dysfunction, enables proper circulation of blood, helps in cholesterol reduction, prevention of diabetes, hair loss, insomnia and menopause, boosting of the immune system, quickens surgery recovery, improves fertility, it also helps in weight loss and well-being. Shortage of glycine in small quantities is not harmful for health but severe shortage may lead to failure of immune response, low growth, abnormal nutrient metabolism as well as other undesirable effects on health, Lewis et al (2005). A typical example of a glycine derivative that can bring about reduction of cholesterol level in the body is dimethylglycine 21.       Cyclooxygenase (COX) officially known as prostaglandin-endoperoxide synthase (PTGS) is an enzymethat is responsiblefor formation of prostanoids, including thromboxane and prostaglandins such as prostacyclin, from arachidonic acid, Kristina et al (2006). Various prostaglandin synthases then convert PGH2 into several different prostaglandins and thromboxanes, Liu et al (2006). These prostaglandins and thromboxanes target specific G protein-coupled receptors and play major roles in regulation of renal function, platelet aggregation, protection of the stomach lining, and other numerous biological tasks, as well as mediation of the cellular inflammatory response, Kristina et al (2006) and Liu et al (2006). These functions are attributed mainly to the first of the two established COX isoforms, the COX-1, while the inflammatory response is largely associated with the inducible isoform, COX-2. Pharmaceutical inhibition of COX can provide relief from the symptoms of inflammation and pain. Those that are specific to the COX-2 isozyme are called COX-2 inhibitors. For example the active metabolite (AM404) of paracetamol believed to provide most or all of its analgesic effects is a COX inhibitor, and this is believed to provide part of its effect, Liu et al (2006) and Högestätt et al (2005). Inhibition of COX-2 produces the analgesic, antipyretic, and antiinflammatory effects typical of non-steroidal anti-inflammatory drugs (NSAIDs), while inhibition of COX-1 is responsible for the antithrombotic effects of aspirin and other nonselective NSAIDs, as well as many of their side effects, such as gastric ulcer formation. The many therapeutically useful effects of COX inhibition have made the NSAIDs among the most widely used drugs of the past century according to Högestätt et al (2005). Since selective COX-2 inhibition can provide analgesic and anti-inflammatory effects with reduced undesirable gastric side effects, COX-2 selective inhibitors such as celecoxib and rofecoxib have become some of the most widely used prescription medications in the developed world. However, recent reports that COX-2 selective inhibitors may increase the risk of heart attack in some patients has caused great concern, and stimulated increased interest in these enzymes, Masferrer et al (1994) and Solomon et al (2002).

Figure 5: Structures of Celecoxib and Rofecoxib
In this paper we have reported the successful synthesis as well the determination of the binding energies of novel ethyl-glycinate amide derivatives with the COX-2 construct which was used to obtain the 1CVU crystal structure for molecular docking in order compare their binding energy with the standard drugs, celecoxib and rofecoxib respectively used as COX-2 inhibitors. This is to determine if these derivatives could also serve as good drug molecules that can inhibit COX-2 enzyme or not.

EXPERIMENTAL
All the reagents were purchased from commercial supplier, Aldrich, and were used without further purification. Melting points were determined with electro thermal melting points apparatus in open capillaries and are uncorrected. UV and Visible spectra were recorded in DMF on a Jenway 6405 UV/Vis spectrophotometer, using matched 1cm quartz cell. IR spectra in (KBr) on a FTIR (NARICT, Zaria), 1 H-NMR and 13 C-NMR on a JEOL Associate E-400 instrument (chemical shift are reported on the δ scale relative to tetramelthylsilane (TMS) as an internal standard) and mass spectra on a Shimadzu QP2010 spectrophotometer. Analytical samples were obtained by column chromatography on aluminum oxide 90 (Merck, 70-230 Mesh ASTM) employing ethano-chloroform (9:1) as eluting solvent before recrystallization. The 3.0 Å resolution X-ray crystal structure of the ovine COX-1/AA complex, pdb entry 1CVU, was used to generate the initial model.

ETHYL GLYCINATE 29
The compound glycine ethyl ester 98 was prepared by using the esterification reaction of glycine 1 with ethanol in the presence of hydrochloric acid as a catalyst, Jiabo and Yaowu (2008) and Fischer and Speier (1895). A mixture of glycine (15.0g, 0.15mol), ethanol (200ml) and conc. hydrochloric acid (7ml) was refluxed for 15h, at 78 o C. At the end of the reaction, the mixture was placed in a water bath and evaporated. The evaporated product was kept in an airtight desiccator for a week and a crystalline whitish product was obtained. This was later recrystallized from ethanol mixed with a little quantity of diethyl ether to precipitate the final product (14.50g, 96.5%). This was further dried for some days and brilliant white crystals were obtained, melting at 188.

2-AMINO-N-(4-NITROPHENYL) ACETAMIDE (31)
The compound, 2-amino-N-(4-nitrophenyl) acetamide 31 was prepared by the reaction of the synthesized glycine ethyl ester 29 (2.0g, 0.02mol) and 4-nitroaniline 30 (2.0g, 0.01mol) with the stoichiometric ratio of 2:2. Both compounds were dissolved in (50ml) ethanol and boiled under reflux for 4h. The crude product was obtained with the help of a rotary evaporator under reduced pressure. This was dried and subjected to column chromatography using a mixture of ethanol and chloroform (9:1) as eluting solvent followed by recrystallization, to give a brownyellowish compound 31,

2-AMINO-N-(6-METHYLPYRIDIN-2-YL) ACETAMIDE 33
The compound 2-Amino-N-(6-methylpyridyl) acetamide 33 was prepared by the reaction of ethyl glycinate 29 (2.0g, 0.02mol) and 2-amino-6-methylpyridine 32 (2.0g, 0.02mol) in the ratio of 2:2 respectively. These compounds were dissolved in 50mlethanol and boiled under reflux for 4h. The crude product was obtained with the help of a rotary evaporator under reduced pressure. This was dried and subjected to column chromatography using a mixture of ethanol and chloroform (9:1) as eluting solvent followed by recrystallization, to give a whitish crystalline compound 33,

PREPARATION OF PROTEIN TARGETS
The 3D structure of the cyclooxygenase active site of COX-2(PDB: 1CVU) was retrieved from the RCSB Protein Data Bank (PDB) (www.rcsb.org/pdb/home/home.do), Picot et al (1994). All bound ligands, cofactors, andwater molecules were removed from the proteins using Discovery Studio Visualizer v16. 1.0. 15,350. All file conversions required for the docking study were performed using the open source chemical toolbox. Open Babel version 2.3.2 (www.openbabel.org). Finally Auto Dock was used to calculate the binding free energy of a given inhibitor conformation in the macromolecular structure.

CONCLUSION
Five new derivatives of ethyl glycinate bearing carboxamide pharmacophores have been synthesized and characterized in this work. All the compounds showed appreciable binding energies ranging from − 5.8 to -7.6 kcal/mol with target protein, 1CVU. Compound showed the highest binding energy of 7.6 kcal/mol. Although the binding energies values were not as high as that of the standard drugs used, these novel compounds could be used as starting materials for the synthesis of drugs that can inhibit COX2 enzyme responsible for causing inflammation in the body.

SOURCES OF FUNDING
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.