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

Abstract

In this study we are going to present thiazole based carbohydrazide in search of potent antidiabetic agent as α-amylase inhibitors. Thiazole based carbohydrazide derivatives 1-25 have been synthesized, characterized by ¹HNMR, ¹³CNMR, and EI-MS, and evaluated for α-amylase inhibition. Except compound 11 all analogs showed α-amylase inhibitory activity with IC₅₀ values from 1.709 ± 0.12 to 3.049 ± 0.25 μM against the standard acarbose (IC₅₀ = 1.637 ± 0.153 μM). Compounds 1, 10, 14, and 20 exhibited outstanding inhibitory potential with IC₅₀ value 1.763 ± 0.03, 1.747 ± 0.20, 1.709 ± 0.12, and 1.948 ± 0.23 μM, respectively, compared with the standard acarbose. Structure activity relationships have been established for the active compounds. To get an idea about the binding interaction of the compounds, molecular docking studies were done.

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1. Introduction

α-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 α-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 α-amylase in serum and urine, respectively [3].

α-Amylase is one of the most critical endoamylases which has ability to hydrolyze the inner α-1,4 glycosidic linkages to glucose, dextrin, and maltose while holding the α-anomeric setup in the products [4]. As an omnipresent enzyme, α-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, α-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.

2. Results and Discussion

2.1. Chemistry

Methyl 2-(2,5-dimethylthiazol-4-yl) acetate and excess amount of hydrazine hydrate were refluxed in ethanol for six hrs to obtaine pure methyl 2-(2,5-dimethylthiazol-4-yl) hydrazide. Intermediate 2-(2,5-dimethylthiazol-4-yl) hydrazide was further treated with different substituted aromatic aldehydes in ethanol as solvent; the reaction mixture was acidified by 3-4 drops glacial acetic to achieve the target compounds (Scheme 1) (1-25) (Table 1). Here in this study each step was monitored by TLC through ethyl acetate/hexane system (ratio 3:7).

Table 1

Thiazole based carbohydrazide analogs (1-25).

NO.	R	NO.	R
1		14
2		15
3		16
4		17
5		18
6		19
7		20
8		21
9		22
10		23
11		24
12		25
13

[figure omitted; refer to PDF]

2.2. 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 α-amylase inhibitory activity. All analogs except 11 have α-amylase inhibition with IC₅₀ values from 1.709 ± 0.12 to 3.049 ± 0.25 μM against the standard acarbose (IC₅₀ = 1.637 ± 0.153 μM) (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.

Table 2

α-Amylase inhibitory potential of thiazole based carbohydrazides.

No.	IC 50 ± SEM a	No.	IC 50 ± SEM a	No.	IC 50 ± SEM a
1	1.763 ± 0.03	10	1.747 ± 0.20	19	2.133 ± 0.12
2	2.608 ± 0.23	11	NA	20	1.948 ± 0.23
3	2.713 ± 0.03	12	2.416 ± 0.04	21	2.40 ± 0.2
4	3.049 ± 0.25	13	2.544 ± 0.06	22	2.385 ± 0.09
5	2.752 ± 0.08	14	1.709 ± 0.12	23	2.797 ± 0.14
6	2.479 ± 0.104	15	2.338 ± 0.07	24	2.909 ± 0.08
7	2.481 ± 0.04	16	2.581 ± 0.03	25	2.703 ± 0.06
8	2.613 ± 0.05	17	2.649 ± 0.08	Acarbose	1.637 ± 0.153
9	2.594 ± 0.05	18	2.525 ± 0.05

$\mathrm{S}\mathrm{E}{\mathrm{M}}^{\mathrm{a}}$ = standard mean error.

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 (IC₅₀ value 1.709 ± 0.12 μM) with analog 12, an ortho nitro-substituted analog (IC₅₀ value 2.416 ± 0.04 μM), and 13, a meta nitro-substituted analog (IC₅₀ value 2.544 ± 0.06 μM), 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 IC₅₀ value 1.747 ± 0.20 μM. 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 (IC₅₀= 2.594 ± 0.05 μM), 5, a 2,5-dihydroxyl analog (IC₅₀= 2.752 ± 0.08 μM), 6, a 2,3-dihydroxyl analog (IC₅₀= 2.479 ± 0.104 μM), 7, a 2,4-dihydroxyl analog (IC₅₀= 2.481 ± 0.04 μM), and 8, a 3,4-dihydroxyl analog (IC₅₀= 2.613 ± 0.05 μM), the compound is superior. The slight activity difference seems to be mainly governed by the position of substituent. Analog 17, a 2,4,6-trihydroxyl analog (IC₅₀= 2.649 ± 0.08 μM), 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 IC₅₀ value 1.763 ± 0.03 μM when compared to 2, an ortho methyl analog (IC₅₀= 2.608 ± 0.23 μM). 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.

2.3. 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 π-π 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.

[figures omitted; refer to PDF]

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, π-π 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 π-π 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).

3. Conclusion

Thiazole based carbohydrazide derivatives (1-25) have been synthesized and evaluated for α-amylase inhibitory potential; 24 out of 25 derivatives displayed significant α-amylase inhibitory activity from 1.709 to 3.049 μM. 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.

4. 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).

4.1. Synthesis of 2-(2,5-Dimethylthiazol-4-yl) Acetohydrazide

Equimolar amount of methyl 2-(2,5-dimethylthiazol-4-yl) acetate (1 mmol) and hydrazine hydrate (1 mmol) were refluxed for 6 hours in ethanol (25 ml) as a solvent. After 6 hours, TLC was done to observe reaction completion. After first step completion, the intermediate and different benzaldehydes were refluxed and methanol was used as a solvent.

Yield: 83%. m. p. 86°C; yellow crystals. ¹H NMR (600 MHz, DMSO): δ 9.13 (s, 1H), 3.69 (s, 2H, NH₂), 3.50 (s, 2H, CH₂), 2.81 (s, 3H), 2.34 (s, 3H); HR-EI-MS: m/z calcd for C₇H₁₁N₃OS, [M]+ 185.2450; Found 185.2462; ¹³C NMR (150 MHz, DMSO): δ 168.1, 159.4, 150.4, 122.5, 36.1, 19.6, 10; Anal. Calcd for C₇H₁₁N₃OS, C, 45.39; H, 5.99; N, 22.68; Found C, 45.37; H, 6.01; N, 22.67.

4.2. 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 ¹H NMR for structural elucidation and confirmation.

4.2.1. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(4-methylbenzylidene) Acetohydrazide (1)

Yield: 170.6%. m. p. 86°C; white powder; ¹H NMR (600 MHz, DMSO): δ 10.10 (s, 1H), 8.37 (s, 1H), 7.94–7.88 (m, 2H), 7.43–7.37 (m, 2H), 3.83 (s, 2H), 2.83 (s, 3H), 2.56 (s, 3H), 2.32 (s, 3H); HR-ESI-MS: m/z calcd for C₁₅H₁₇N₃OS, [M]+ 360.1508; Found 360.1517; ¹³C NMR (150 MHz, DMSO): δ 159.4, 150.4, 144.1, 140.7, 130.7, 129.1, 129.1, 126.1, 126.1, 122.5, 171.0, 36.4, 21.3, 19.6, 10.6; Anal. Calcd for C₁₅H₁₇N₃OS, C, 62.69; H, 5.96; N, 14.62; Found C, 62.60; H, 5.88; N, 14.54.

4.2.2. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(2-methylbenzylidene) Acetohydrazide (2)

Yield: 83%. m. p. 206.2°C; white powder; ¹H NMR (600 MHz, DMSO): δ 10.20 (s, 1H), 8.35 (s, 1H), 7.75–7.68 (m, 2H), 7.23 (d, J = 4.0 Hz, 3H), 3.83 (s, 2H), 2.83 (s, 3H), 2.43 (s, 3H), 2.32 (s, 3H); HR-EI-MS: m/z calcd for C₁₅H₁₇N₃OS, [M]+ 287.3810; Found 287.3801; ¹³C NMR (150 MHz, DMSO): δ 171.0, 159.4, 150.4, 143.3, 135.3, 131.1, 130.9, 129.0, 126.5, 125.8, 122.5, 36.4, 19.6, 18.9, 10.6; Anal. Calcd for C₁₅H₁₇N₃OS, C, 62.69; H, 5.96; N, 14.62; Found C, 62.61; H, 5.87; N, 14.52.

4.2.3. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(3-hydroxy-4-methoxybenzylidene) Acetohydrazide (3)

Yield: 82%. m. p. 187.5°C; white powder; ¹H NMR (600 MHz, DMSO): δ 11.30 (s, 1H), 9.31 (s, 1H), 8.35 (s, 1H), 7.40 (dd, J = 2.1, 1.1 Hz, 1H), 7.11 (ddd, J = 7.5, 2.1, 1.1 Hz, 1H), 7.07-7.02 (m, 2H), 3.74 (s, 2H), 2.84 (s, 3H), 2.32 (s, 3H); HR-EI-MS: m/z calcd for C₁₅H₁₇N₃O₃S, [M]+ 319.3790; Found 319.3779; ¹³C NMR (150 MHz, DMSO): δ 171.0, 159.4, 152.4, 150.4, 147.3, 146.8, 131.1, 122.8, 122.5, 115.9, 112.3, 56.1, 36.4, 19.6, 10.6; Anal. Calcd for C₁₅H₁₇N₃O₃S, C, 56.41; H, 5.37; N, 13.16; Found C, 56.32; H, 5.31; N, 13.09.

4.2.4. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(2-hydroxy-4-methoxybenzylidene) Acetohydrazide (4)

Yield: 84%. m. p. 201°C; yellow; ¹H NMR (600 MHz, DMSO): δ 10.30 (s, 1H), 9.40 (s, 1H), 8.78 (d, J = 6.0 Hz, 1H), 7.75 (dd, J = 7.5, 2.0 Hz, 1H), 6.53 (dd, J = 7.5, 2.0 Hz, 1H), 6.47 (d, J = 2.1 Hz, 1H), 3.86 (m, 3H), 3.50 (s, 2H), 2.83 (s, 3H), 2.33 (s, 3H); HR-EI-MS: m/z calcd for C₁₅H₁₇N₃O₃S, [M]+ 319.3790; Found 319.3782; ¹³C NMR (150 MHz, DMSO): δ 171.0, 164.3, 162.1, 159.4, 150.4, 146.0, 133.4, 122.5, 110.8, 107.0, 103.4, 55.8, 36.4, 19.6, 10.6; Anal. Calcd for C₁₅H₁₇N₃O₃S, C, 56.41; H, 5.37; N, 13.16; Found C, 56.31; H, 5.32; N, 13.11.

4.2.5. (E)-N′-(2,5-Dihydroxybenzylidene)-2-(2,5-dimethylthiazol-4-yl) Acetohydrazide (5)

Yield: 87%. m. p. 180°C; brown; ¹H NMR (600 MHz, DMSO): δ 11.20 (s, 1H), 10.30 (s, 1H), 9.10 (s, 1H), 8.39 (s, 1H), 8.78 (d, J = 6.0 Hz, 1H), 6.78 (d, J = 6.0, 2.0 Hz, 1H), 6.77-6.69 (m, 1H), 3.85 (s, 2H), 2.86 (s, 3H), 2.33 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₅N₃O₃S, [M]+ 305.3520; Found 305.3512; ¹³C NMR (150 MHz, DMSO): δ 171.0, 159.4, 153.7, 151.2, 150.4, 146.0, 122.5, 120.4, 119.9,119.6,116.3, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₅N₃O₃S, C, 55.07; H, 4.95; N, 13.76; Found C, 55.01; H, 4.90; N, 13.68;

4.2.6. (E)-N′-(2,3-Dihydroxybenzylidene)-2-(2,5-dimethylthiazol-4-yl) Acetohydrazide (6)

Yield: 83%. m. p. 163°C; brown; ¹H NMR (600 MHz, DMSO): δ 10.40 (s, 1H), 10.10 (s, 1H), 9.40 (s, 1H), 8.40 (s, 1H), 8.78 (d, J = 6.0 Hz, 1H), 7.01 (d, J = 7.0 Hz, 1H), 6.83-6.76 (m, 1H), 3.83 (s, 2H), 2.84 (s, 3H), 2.32 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₅N₃O₃S, [M]+ 305.3520; Found 305.3508; ¹³C NMR (150 MHz, DMSO): δ 171.0, 159.4, 151.7, 150.4, 146.1, 146.0, 124.7, 122.5, 122.8, 119.9, 119.6, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₅N₃O₃S, C, 55.07; H, 4.95; N, 13.76; Found C, 55.01; H, 4.90; N, 13.71.

4.2.7. (E)-N′-(2,4-Dihydroxybenzylidene)-2-(2,5-dimethylthiazol-4-yl) Acetohydrazide (7)

Yield: 80%. m. p. 207°C; pale yellow; ¹H NMR (600 MHz, DMSO): δ 10.60 (s, 2H), 10.20 (s,1H), 8.80 (s, 1H), 8.60 (d, J = 2.0 Hz, 1H), 7.70 (dd, J = 8.0, 2.0 Hz, 1H), 6.40 (dd, J = 8.0, 2.0 Hz, 1H), 3.40 (s, 2H), 2.80 (s, 3H), 2.35 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₅N₃O₃S, [M]+ 305.3520; Found 305.3505; ¹³C NMR (150 MHz, DMSO): δ 170.0, 158.4, 153.4, 151.0, 150.2, 146.2, 122.1, 120.2, 119.6, 119.3,116.0, 36.6, 19.8, 10.9; Anal. Calcd for C₁₄H₁₅N₃O₃S, C, 55.07; H, 4.95; N, 13.76; Found C, 55.00; H, 4.85; N, 13.65.

4.2.8. (E)-N′-(3,4-Dihydroxybenzylidene)-2-(2,5-dimethylthiazol-4-yl) Acetohydrazide (8)

Yield: 79%. m. p. 216°C; brown cystals; ¹H NMR (600 MHz, DMSO): δ 11.10 (s, 1H), 9.62 (s, 1H), 9.17 (s, 1H), 8.37 (s, 1H), 6.93 (d, J = 7.5 Hz, 1H), 6.88 (dd, J = 2.0 Hz, 1H), 6.82 (d, J = 7.4 Hz, 1H), 3.61 (s, 2H), 2.64 (s, 3H), 2.30 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₅N₃O₃S, [M]+ 305.3520; Found 305.3531; ¹³C NMR (150 MHz, DMSO): δ 171.0, 159.4, 150.4, 149.6, 146.8, 146.1, 131.3, 123.2, 122.5, 117.4, 116.3, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₅N₃O₃S, C, 55.07; H, 4.95; N, 13.76; Found C, 55.00; H, 4.87; N, 13.69.

4.2.9. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(3-hydroxybenzylidene) Acetohydrazide (9)

Yield: 81%. m. p. 157°C; white powder; ¹H NMR (600 MHz, DMSO): δ 10.40 (s, 1H), 10.10 (s, 1H), 8.37 (s, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.09-7.02 (m, 1H), 6.92 (dt, J = 7.5, 1.8 Hz, 1H), 6.87 (d, J = 2.0 Hz, 1H), 3.84 (s, 2H), 2.86 (s, 3H), 2.32 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₅N₃O₂S, [M]+ 289.3530; Found 289.3516; ¹³C NMR (150 MHz, DMSO): δ171.0, 159.4, 158.6, 150.4, 146.8, 138.7, 130.2, 122.5, 121.8, 118.2, 114.9, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₅N₃O₂S, C, 58.11; H, 5.23; N, 14.52; Found C, 58.04; H, 5.11; N, 14.43.

4.2.10. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(4-hydroxybenzylidene) Acetohydrazide (10)

Yield: 85%. m. p. 213°C; white powder; ¹H NMR (600 MHz, DMSO): δ 11.10 (s, 1H), 9.89 (s, 1H), 8.37 (s, 1H), 7.37-7.31 (s, J = 7.5 Hz, 2H), 6.85-6.84 (s, J = 7.5 Hz, 2H), 3.83 (s, 2H), 2.83 (s, 3H), 2.32 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₅N₃O₂S, [M]+ 289.3530; Found 289.3520; ¹³C NMR (150 MHz, DMSO): δ171.0, 160.8, 159.4, 150.4, 144.1, 130.6, 130.6, 126.3, 122.5, 116.0, 116.0, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₅N₃O₂S, C, 58.11; H, 5.23; N, 14.52; Found C, 58.06; H, 5.18; N, 14.44.

4.2.11. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(2,3,4-trihydroxybenzylidene) Acetohydrazide (11)

Yield: 95%. m. p. 213°C; yellow crystals; ¹H NMR (600 MHz, DMSO): δ 11.40 (s, 1H), 11.20 (s, 1H), 10.40 (s, 1H), 9.50 (s, 1H), 11.10 (s, 1H), 6.42 (s, 2H), 3.83 (s, 2H), 2.82 (s, 3H), 2.33 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₅N₃O₄S, [M]+ 321.3510; Found 321.3502; ¹³C NMR (150 MHz, DMSO): δ171.0, 159.4, 153.1, 152.4, 150.4, 146.8, 136.1, 126.4, 122.5, 112.5, 110.0, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₅N₃O₄S, C, 52.33; H, 4.71; N, 13.08; Found C, 52.24; H, 4.60; N, 13.01.

4.2.12. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(2-nitrobenzylidene) Acetohydrazide (12)

Yield: 76%. m. p. 167°C; yellow crystals; ¹H NMR (600 MHz, DMSO): δ 11.30 (s, 1H), 10.50 (s, 1H), 8.35 (d, J = 1.0 Hz, 1H), 8.10 (ddd, J = 7.5 Hz, 1H), 7.93 (d, J = 7.5 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H), 3.51 (s, 2H), 2.84 (s, 3H), 2.33 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₄N₄O₃S, [M]+ 318.3510; Found 318.3502; ¹³C NMR (150 MHz, DMSO):δ171.0, 159.4, 150.4, 147.8, 143.3, 134.9, 131.9, 130.1, 128.4, 124.0, 122.5, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₄N₄O₃S, C, 52.82; H, 4.43; N, 17.60; Found C, 52.74; H, 4.36; N, 17.51.

4.2.13. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(3-nitrobenzylidene) Acetohydrazide (13)

Yield: 86%. m. p. 221°C; brown; ¹H NMR (600 MHz, DMSO): δ 10.40 (s, 1H), 9.40 (s, 1H), 8.48 (d, J = 2.0 Hz, 1H), 8.37 (d, J = 2.0 Hz, 1H), 8.09 (dd, J = 7.5 Hz, 1H), 7.72 (t, J = 7.5 Hz, 1H), 3.84 (s, 2H), 2.83 (s, 3H), 2.33 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₄N₄O₃S, [M]+ 318.3510; Found 318.3502; ¹³C NMR (150 MHz, DMSO): δ 171.0, 159.4, 150.4, 148.0, 142.8, 134.6, 132.5, 129.7, 126.2, 121.6, 122.5, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₄N₄O₃S, C, 52.82; H, 4.43; N, 17.60; Found C, 52.74; H, 4.36; N, 17.53.

4.2.14. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(4-nitrobenzylidene) Acetohydrazide (14)

Yield: 99%. m. p. 167°C; yellow powder; ¹H NMR (600 MHz, DMSO): δ 10.40 (s, 1H), 8.37 (s, 1H), 7.90 (d, J = 8.0 Hz, 2H), 6.96 (d, J = 8.0 Hz, 2H), 3.53 (s, 2H), 2.86 (s, 3H), 2.33 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₄N₄O₃S, [M]+ 318.3510; Found 318.3501; ¹³C NMR (150 MHz, DMS): δ 172.0, 159.5, 150.5, 150.2, 144.1, 139.8, 124.2, 124.2, 124.0, 124.0, 122.5, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₄N₄O₃S, C, 52.82; H, 4.43; N, 17.60; Found C, 52.76; H, 4.36; N, 17.53.

4.2.15. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(2-hydroxy-5-methoxybenzylidene) Acetohydrazide (15)

Yield: 83%. m. p. 89°C; white powder; ¹H NMR (600 MHz, DMSO): δ 10.20 (s, 1H), 9.30 (s, 1H), 8.40 (s, 1H), 7.80 (d, J = 6.0 Hz, 1H), 7.24-7.20 (m, 1H), 6.83 (d, J = 1.0 Hz, 1H), 3.85 (s, 3H), 3.53 (s, 2H), 2.43 (s, 3H), 2.32 (s, 3H); HR-EI-MS: m/z calcd for C₁₅H₁₇N₃O₃S, [M]+ 319.3790; Found 319.3782; ¹³C NMR (150 MHz, DMSO): δ 171.1, 159.3, 153.4, 153.3, 150.4, 146.0, 122.5, 119.5, 118.0, 117.2, 113.5, 55.8, 36.4, 19.6, 10.6; Anal. Calcd for C₁₅H₁₇N₃O₃S, C, 56.41; H, 5.37; N, 13.16; Found C, 56.32; H, 5.30; N, 13.10.

4.2.16. (E)-N′-(3-Bromo-4-fluorobenzylidene)-2-(2,5-dimethylthiazol-4-yl) Acetohydrazide (16)

Yield: 97%. m. p. 255°C; yellow powder; ¹H NMR (600 MHz, DMSO): δ 10.60 (s, 1H), 8.37 (s, 1H), 7.58 (s, 1H), 7.27 (dd, J = 6.0, 7.5 Hz, 1H), 6.97 (dd, J = 6.0, 7.5 Hz, 1H), 3.51 (s, 2H), 2.84 (s, 3H), 2.33 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₃BrFN₃OS, [M]+ 370.2404; Found 370.2396; ¹³C NMR (150 MHz, DMSO): δ 171.0, 167.7, 159.4, 150.4, 146.8, 134.3, 131.5, 129.8, 122.5,117.8, 110.2, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₃BrFN₃OS, C, 45.42; H, 3.54; N, 11.35; Found C, 45.33; H, 3.49; N, 11.31.

4.2.17. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(2,4,6-trihydroxybenzylidene) Acetohydrazide (17)

Yield: 96%. m. p. 141°C; yellow powder; ¹H NMR (600 MHz, DMSO): δ 11.70 (s, 1H), 10.85 (s, 1H), 10.40 (s, 1H), 9.650 (s, 1H), 8.35 (s, 1H), 6.54 (s, 2H), 3.83 (s, 2H), 2.84 (s, 3H), 2.33 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₅N₃O₄S, [M]+ 321.3510; Found 321.3523; ¹³C NMR (150 MHz, DMSO): δ 171.0, 163.9, 163.9, 163.6, 159.4, 150.4, 143.3, 122.5, 106.2, 96.3, 96.3, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₅N₃O₄S, C, 52.33; H, 4.71; N, 13.08; Found C, 52.25; H, 4.66; N, 13.02.

4.2.18. (E)-N′-(3-Chlorobenzylidene)-2-(2,5-dimethylthiazol-4-yl) Acetohydrazide (18)

Yield: 77%. m. p. 141°C; white powder; ¹H NMR (600 MHz, DMSO): δ 10.80 (s, 1H), 8.35 (s, 1H), 7.92 (d, J = 2.0 Hz, 1H), 7.65 (d, J = 2.0 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 7.72 (d, J = 7.5 Hz, 1H), 3.53 (s, 2H), 2.86 (s, 3H), 2.33 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₄ClN₃OS, [M]+ 307.7960; Found 307.7948; ¹³C NMR (150 MHz, DMSO): δ 171.0, 159.4, 150.4, 146.8, 135.1, 134.4, 131.1, 130.2, 127.3, 127.1, 122.5, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₄ClN₃OS, C, 54.63; H, 4.58; N, 13.65; Found C, 54.56; H, 4.49; N, 13.58.

4.2.19. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(pyridin-3-ylmethylene) Acetohydrazide (19)

Yield: 87%. m. p. 88°C; yellow powder; ¹H NMR (600 MHz, DMSO): δ 10.40 (s, 1H), 9.08 (d, J = 2.0 Hz, 1H), 8.71 (dd, J = 5.0, 2.0 Hz, 1H), 8.42 (dt, J = 8.0, 2.0 Hz, 1H), 8.34 (s, 1H), 7.42 (dd, J = 8.0, 5.0 Hz, 1H), 3.82 (s, 2H), 2.83 (s, 3H), 2.32 (s, 3H); HR-EI-MS: m/z calcd for C₁₃H₁₄N₄OS, [M]+ 274.3420; Found 274.3411; ¹³C NMR (150 MHz, DMSO): δ 171.0, 159.4, 151.9, 150.4, 149.0, 143.3, 133.7, 130.4, 123.9, 122.5, 36.4, 19.6, 10.6; Anal. Calcd for C₁₃H₁₄N₄OS, C, 56.92; H, 5.14; N, 20.42; Found C, 56.84; H, 5.19; N, 20.34.

4.2.20. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(pyridin-4-ylmethylene) Acetohydrazide (20)

Yield: 87%. m. p. 98°C; white powder; ¹H NMR (600 MHz, DMSO): δ 10.30 (s, 1H), 8.62 (s, 1H), 8.22 (d, J = 6.0 Hz, 2H), 7.57 (d, J = 6.0 Hz, 2H), 3.53 (s, 2H), 2.84 (s, 3H), 2.32 (s, 3H); HR-EI-MS: m/z calcd for C₁₃H₁₄N₄OS, [M]+ 274.3420; Found 274.3412; ¹³C NMR (150 MHz, DMSO): δ 171.0, 159.4, 150.4, 149.4, 149.4, 146.8, 144.3, 120.4, 120.4, 122.5, 36.4, 19.6, 10.6; Anal. Calcd for C₁₃H₁₄N₄OS, C, 56.92; H, 5.14; N, 20.42; Found C, 56.84; H, 5.10; N, 20.36.

4.2.21. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(2-fluorobenzylidene) Acetohydrazide (21)

Yield: 83%. m. p. 105°C; white milky; ¹H NMR (600 MHz, DMSO): δ 10.40 (s, 1H), 8.40 (s, 1H), 8.20 (d, J = 7.0 Hz, 1H), 7.53-7.46 (m, 1H), 7.28 (d, J = 7.0 Hz, 1H), 7.22 (td, J = 7.5, 2. Hz, 1H), 3.81 (s, 2H), 2.86 (s, 3H), 2.33 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₄FN₃OS, [M]+ 291.3444; Found 291.3436; ¹³C NMR (150 MHz, DMSO): δ 171.0, 159.6, 159.4, 150.4, 143.3, 132.6, 130.8, 124.4, 118.2, 115.6, 122.5, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₄FN₃OS, C, 57.72; H, 4.84; N, 14.42; Found C, 57.65; H, 4.80; N, 14.36.

4.2.22. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(4-fluorobenzylidene) Acetohydrazide (22)

Yield: 85%. m. p. 97°C; white crystals; ¹H NMR (600 MHz, DMSO): δ 10.36 (s, 1H), 8.35 (s, 1H), 7.90 (t, J = 7.0 Hz, 2H), 7.08 (d, J = 7.0 Hz, 2H), 3.52 (s, 2H), 2.45 (s, 3H), 2.33 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₄FN₃OS, [M]+ 291.3444; Found 291.3431; ¹³C NMR (150 MHz, DMSO): δ 171.0, 165.2, 159.4, 150.4, 144.1, 130.8, 130.8, 129.3, 115.6, 115.6, 122.5, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₄FN₃OS, C, 57.72; H, 4.84; N, 14.42; Found C, 57.66; H, 4.80; N, 14.38.

4.2.23. (E)-2-(2,5-Dimethylthiazol-4-yl)-N′-(3-fluorobenzylidene) Acetohydrazide (23)

Yield: 84%. m. p. 97°C; white crystals; ¹H NMR (600 MHz, DMSO): δ 10.20 (s, 1H), 8.38 (s, 1H), 7.59 (td, J = 7.6, 5.7 Hz, 1H), 7.48-7.39 (m, 2H), 7.19-7.13 (m, 1H), 3.54 (s, 2H), 2.52 (s, 3H), 2.30 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₄FN₃OS, [M]+ 291.3444; Found 291.3436; ¹³C NMR (150 MHz, DMSO): δ 171.0, 165.2, 159.4, 150.4, 144.1, 130.8, 130.8, 129.3, 115.6, 115.6, 122.5, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₄FN₃OS, C, 57.72; H, 4.84; N, 14.42; Found C, 57.67; H, 4.81; N, 14.46.

4.2.24. (E)-N′-(2-Chlorobenzylidene)-2-(2,5-dimethylthiazol-4-yl) Acetohydrazide (24)

Yield: 75%. m. p. 127°C; white crystals; ¹H NMR (600 MHz, DMSO): δ 10.20 (s, 1H), 8.35 (s, 1H), 8.20 (d, J = 2.0 Hz, 1H), 7.52-7.44 (m, 2H), 7.35 (td, J = 7.5, 2.0 Hz, 1H), 3.55 (s, 2H), 2.84 (s, 3H), 2.33 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₄ClN₃OS, [M]+ 307.7960; Found 307.7952; ¹³C NMR (150 MHz, DMSO): δ 171.0, 159.4, 150.4, 138.7, 134.7, 133.9, 132.4, 130.1, 127.2, 126.9, 122.5, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₄ClN₃OS, C, 54.63; H, 4.58; N, 13.65; Found C, 54.54; H, 4.54; N, 13.60.

4.2.25. (E)-N′-(4-Chlorobenzylidene)-2-(2,5-dimethylthiazol-4-yl) Acetohydrazide (25)

Yield: 95%. m. p. 120°C; yellow crystals; ¹H NMR (600 MHz, DMSO): δ 11.75 (s, 1H), 8.37 (s, 1H), 7.85 (d, J = 7.5 Hz, 2H),), 7.50 (d, J = 7.5 Hz, 2H), 3.46 (s, 2H), 2.86 (s, 3H), 2.33 (s, 3H); HR-EI-MS: m/z calcd for C₁₄H₁₄ClN₃OS, [M]+ 307.7960; Found 307.7972; ¹³C NMR (150 MHz, DMSO): δ 171.0, 159.4, 150.4, 144.1, 136.6, 131.8, 130.6, 130.6, 128.9, 128.9, 122.5, 36.4, 19.6, 10.6; Anal. Calcd for C₁₄H₁₄ClN₃OS, C, 54.63; H, 4.58; N, 13.65; Found C, 54.58; H, 4.54; N, 13.60.

4.3. α-Amlyase Inhibition Assay

The α-amylase inhibition was estimated by an assay modified from Kwon, Apostolidis & Shetty [18, 19].

Conflicts of Interest

The authors report that there are no conflicts of interest.

Acknowledgments

The authors are thankful to AIMST University, Malaysia, for providing the facilities for this project and the Ministry of Higher Education (MOHE), Malaysia, for funding the computational part (Software and Workstation) through the “TRGS” (Grant No. 600-RMI/TRGS 5/3 (1/2014)-3).

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