Thiazole Based Carbohydrazide Derivatives as α-Amylase Inhibitor and Their Molecular Docking Study

1Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia 2Department of Chemistry, University of Wah, Quaid Avenue, Wah Cantt 47000, Pakistan 3Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia 4Faculty of Applied Science, UiTM Shah Alam, 40450 Shah Alam, Selangor D.E., Malaysia 5Department of Chemistry, Hazara University, Mansehra-21300, Khyber Pakhtunkhwa, Pakistan 6School of Engineering, Monash University (Malaysia Campus), Bandar Sunway 47500, Malaysia 7Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Qassim University, Buraidah 51452, Saudi Arabia


Introduction
-Amylase is a protein enzyme EC.3.2.1.1 which helps in the breakdown of starch to glucose and maltose.Living organism used carbohydrates and sugars as the major energy storage molecules [1,2].The -amylase enzyme is calcium metalloenzyme found in human saliva, serum, and urine.According to clinical chemistry, the presence of parotitis and pancreatitis can be determined by the activity of -amylase in serum and urine, respectively [3].
-Amylase is one of the most critical endoamylases which has ability to hydrolyze the inner -1,4 glycosidic linkages to glucose, dextrin, and maltose while holding the -anomeric setup in the products [4].As an omnipresent enzyme, amylase is delivered by numerous species, including plants, animals, and microorganisms.A huge number of microbial amylases of diverse origins have been broadly studied and widely applied in various areas, e.g., textile, detergent, food, paper, biofuel, etc. Besides, very important clinical applications have been carried out in medicine and for the reduction of toxicity by the environmental pollutants [5,6].
Recently, -amylase has been isolated and characterized from natural sources [7].Researchers have also studied its effects on enzyme stability and analyzed the binding properties to develop new inhibitors [8,9].The thiazole and its derivatives are very much important in nature because they keep versatile biological properties.Thiazole containing molecules are used as an antimicrobial drug, abafungin as antifungal drug, ritonavir as antiretroviral drug, thiazofurin and bleomycine as antineoplastic [10], antihistaminic, niridazole as schistosomicidal, and nitazoxanide as antiprotozoal [11].Our group has already reported thiazole analogs as acetylcholinesterase and butyrylcholinesterase inhibitor [12], with hopes to further explore their potential as alpha-amylase inhibitor.In this regard here in the present study we are going to report thiazole analogs as potent alpha-amylase inhibiter.the reaction mixture was acidified by 3-4 drops glacial acetic to achieve the target compounds (Scheme 1) (1-25) (Table 1).

Results and Discussion
Here in this study each step was monitored by TLC through ethyl acetate/hexane system (ratio 3:7).

𝛼-Amylase
Inhibitory Potential.To carry on our research work on enzyme inhibition [13,14] we have synthesized thiazole based carbohydrazide analogs (1-25) (Table 1) and they were evaluated for -amylase inhibitory activity.All analogs except 11 have -amylase inhibition with IC 50 values from 1.709 ± 0.12 to 3.049 ± 0.25 M against the standard acarbose (IC 50 = 1.637 ± 0.153 M) (Table 2).The structure activity relationship has been also studied and it is mainly based upon the difference of substituents on phenyl ring attached to the carbohydrazide part.The analog 14, a para nitro-substituted analog, was found to be the most active analog among all.The more noteworthy potential of the compound might be due to the presence of nitro group which is a strong electron withdrawing group.When comparing compound 14 (IC 50 value 1.709 ± 0.12 M) with analog 12, an ortho nitro-substituted analog (IC 50 value 2.416 ± 0.04 M), and 13, a meta nitro-substituted analog (IC 50 value 2.544 ± 0.06 M), analog 14 showed greater potential than analogs 12 and 13, which seems to be due to different positioning of nitro group on phenyl ring.
Compound 10, a para hydroxy analog, was found to be the second most dynamic analog within this series with IC 50 value 1.747 ± 0.20 M.The greater potential shown by this compound seems to be due to the hydroxyl group which might be involved in hydrogen bonding with the active site of enzyme.By comparing analog 10 with other hydroxyl analogs like 9, a meta hydroxyl analog (IC 50 = 2.594 ± 0.05 M), 5, a 2,5-dihydroxyl analog (IC 50 = 2.752 ± 0.08 M), 6, a 2,3-dihydroxyl analog (IC 50 = 2.479 ± 0.104 M), 7, a 2,4dihydroxyl analog (IC 50 = 2.481 ± 0.04 M), and 8, a 3,4dihydroxyl analog (IC 50 = 2.613 ± 0.05 M), the compound is superior.The slight activity difference seems to be mainly governed by the position of substituent.Analog 17, a 2,4,6trihydroxyl analog (IC 50 = 2.649 ± 0.08 M), showed potential but analog 11, a 2,3,4-trihydroxyl analog, was found to be completely inactive.This might be due to position difference of hydroxyl group.
Compound 1, a para methyl analog, was the third most active analog among the series with IC 50 value 1.763 ± 0.03 M when compared to 2, an ortho methyl analog (IC 50 = 2.608 ± 0.23 M).The reason for the greater potential is again might be due to position difference.Similar pattern was observed for other substituted analogs like chloro analogs, fluoro analogs, and pyridine analogs.
It was concluded from this study that either EWG or EDG on phenyl portion appeared to have potential while the slight changes in potential was basically due to the position of the substituent as well as the number of substituents which is also a vital factor.In addition, molecular docking analysis was done to get an idea about the binding interaction of the active analogs.

Molecular Docking.
Docking simulation was performed to determine the binding modes of synthesized thiazole based analogs (1-25) targeting the crystal structure of amylase (PDB ID: 4W93) [15].Earlier to docking, the crystal structure of the amylase was arranged utilizing protein preparation technique described in Schrödinger Maestro [15].Crystal structure was recovered from the protein data bank (PDB) and was additionally optimized through eliminating the cofactors; hetero atoms, water molecules, missing atoms, hydrogen bonds, and charges were computed [16].Docking calculations were fulfilled utilizing additional Precision (XP) mode.The docking results came out by advanced analyze method with the help of the XP GS score and each derivative binding mode was visually assessed utilizing PyMOL system [17].
All the 25 compounds of the Schiff bases were docked using glide to show the binding mode of the active amylase inhibitors.Analysis of the docking results based on the Glide XP G Score and binding mode in the active site were done.Among them, only the top four compounds binding modes on the amylase active site are reported.
Figure 1(a) shows the binding mode of compound 14, the side chain imidazole ring of His201 establishes - stacking with the nitrophenyl ring of the compound, and in addition the same ring forms hydrophobic interaction with Tyr151, Leu162, Leu165, Ala198, and Ile235.The dimethyl group attached to the thiazole ring forms hydrophobic interaction with Trp59 and Tyr62.On the other hand, the binding mode of compound 10 (Figure 1(a)) shows that the hydroxyl group in the phenol forms hydrogen bond with side chain oxygen of Glu233.Furthermore, - stacking between thiazole ring and Trp59 was established despite the fact that there is also hydrophobic interaction between the benzothiophenyl ring and Y159, L229, M273, and L319.In addition, the phenyl ring of the compound and aliphatic chain of residues Tyr62, Ala198, and Ile235 form hydrophobic contact.Similarly, the dimethyl group attached to the thiazole ring forms hydrophobic interaction with Trp59, Tyr62, Leu162, and Leu165 (Figure 1(b)).
The preferred binding poses of compound 1 (Figure 1(c)) showed that the compound was totally stabilized by hydrophobic moieties such as toluene and dimethyl groups establishing contact with residues such as Leu162, Leu165, Trp59, Tyr62 and Tyr151, Ala198, Ile235, respectively.Similarly, the binding mode of compound 20 shows the presence of hydrogen bond between the pyridine with the main chain oxygen, and the ring also forms - stacking with His201.Next, the dimethyl group attached to the thiazole ring forms hydrophobic interaction with Trp59, Tyr62, Leu162, and Leu165.Finally, pyridine was also found to be establishing hydrophobic contact with Tyr151, Ala198, and Ile235 as in Figure 1(d).

Conclusion
Thiazole based carbohydrazide derivatives (1-25) have been synthesized and evaluated for -amylase inhibitory potential; 24 out of 25 derivatives displayed significant -amylase inhibitory activity from 1.709 to 3.049 M.The binding interaction of analogs with active site of protein ligands was confirmed through molecular docking study, where the active molecules form stable hydrogen bond network with the key residues in the active site.Consistently, the activity profile of the compounds directly depends on magnitude of hydrogen bonding and hydrophobic contact enhancing the complex stabilization.SAR study was established to know the effect of substitution on aromatic residues of aldehyde toward inhibitions.

Experimental
Avance Bruker 600 MHz was used for nuclear magnetic resonance experiments.Carlo Erba Strumentazion-Mod-1106, Italy was used for Elemental analysis.Precoated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Germany) were utilized for thin layer chromatography.Chromatograms were visualized by UV at 254 and 365 nm.Finnigan MAT-311A, Germany, was utilized for electron impact mass spectra (EI-MS).

General Procedure for the Synthesis of (E)-2-(2,5-
Dimethylthiazol-4-yl)-N  -(arylidene) Acetohydrazide.Synthesis of the series of acetohyrazide was done on basis of previously described reaction procedure in published paper by our group [13].All the derivatives were subjected to 1 H NMR for structural elucidation and confirmation.

Figure 1 :
Figure 1: Binding modes on the amylase active site of top four compounds (14, 10, 1, and 20) by molecular docking study.
SEM a = standard mean error.