New thiosemicarbazide-1,2,3-triazole hybrids as potent α-glucosidase inhibitors: Design, synthesis, and biological evaluation

Article abstract of DOI:10.1016/j.molstruc.2019.04.082

A new series of thiosemicarbazide-1,2,3-triazole hybrids 10a-o has been synthesized, characterized by ¹H NMR, ¹³C NMR, and screened for their in vitro α-glucosidase inhibitory activity. All of the synthesized compounds displayed excellent α-glucosidase inhibitory activity with IC₅₀ values in the range of 75.0 ± 0.5 to 253.0 ± 0.5 μM, as compared to the standard drug acarbose (IC₅₀ = 750.0 ± 1.5 μM). Among the synthesized compounds, compound 10h (IC₅₀ = 75.0 ± 0.5)with 4-methoxy group at phenyl part of thiosemicarbazide moiety and 2,6-dichloro substituents at benzyl moiety was found to be the most potent compound. Kinetic analysis revealed that compound 10h is a competitive inhibitor for α-glucosidase. Docking study of compound 10h in the active site of α-glucosidase showed that this compound interacted with residues His239, His279, Glu304, Gly306, and Arg312.

Full text of DOI:10.1016/j.molstruc.2019.04.082

Accepted Manuscript
New thiosemicarbazide-1,2,3-triazole hybrids as potent α-glucosidase inhibitors:
Design, synthesis, and biological evaluation
Zohreh Bakherad, Maryam Mohammadi-Khanaposhtani, Hojjat Sadeghi-Aliabadi,
Sepideh Rezaei, Afshin Fassihi, Mohammad Bakherad, Hossein Rastegar, Mahmood
Biglar, Lotfollah Saghaie, Bagher Larijani, Mohammad Mahdavi
PII:
S0022-2860(19)30485-5
DOI:
https://doi.org/10.1016/j.molstruc.2019.04.082
MOLSTR 26454
Reference:
To appear in: Journal of Molecular Structure
Received Date: 12 February 2019
Revised Date: 18 April 2019
Accepted Date: 19 April 2019
Please cite this article as: Z. Bakherad, M. Mohammadi-Khanaposhtani, H. Sadeghi-Aliabadi, S.
Rezaei, A. Fassihi, M. Bakherad, H. Rastegar, M. Biglar, L. Saghaie, B. Larijani, M. Mahdavi, New
thiosemicarbazide-1,2,3-triazole hybrids as potent α-glucosidase inhibitors: Design, synthesis,
and biological evaluation, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.04.082.
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ACCEPTED MANUSCRIPT
Graphical Abstract
New thiosemicarbazide-1,2,3-triazole hybrids as potent α-glucosidase inhibitors: design,
synthesis, and biological evaluation
Zohreh Bakherad, Maryam Mohammadi-Khanaposhtani, Hojjat Sadeghi-Aliabadi, Afshin
Fassihi, Mohammad Bakherad, Hossein Rastegar, Mahmood Biglar, Somaye Imanparast,
Mohammad Ali Faramarzi, Lotfollah Saghaie*, Bagher Larijani and Mohammad Mahdavi
A new series of thiosemicarbazide-1,2,3-triazole hybrids 10a-o have been synthesized,
1
characterized by H NMR, and 13C NMR and screened for their in vitro α-glucosidase
inhibitory activity. All of the synthesized compounds displayed excellent α-glucosidase
inhibitory activity. Kinetic analysis and docking study of the most potent compound 10h
were also performed.

ACCEPTED MANUSCRIPT
New Thiosemicarbazide-1,2,3-triazole Hybrids as Potent α-Glucosidase Inhibitors: Design,
Synthesis, and Biological Evaluation
a
b
a
Zohreh Bakherad , Maryam Mohammadi-Khanaposhtani , Hojjat Sadeghi-Aliabadi , Sepideh
c
a
d
e
f
Rezaei , Afshin Fassihi , Mohammad Bakherad , Hossein Rastegar , Mahmood Biglar ,
Lotfollah Saghaie a, *, Bagher Larijani f, Mohammad Mahdavi f, *
a
Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences,
Isfahan University of Medical Sciences, 81746-73461, Isfahan, Iran.
b Cellular and Molecular Biology Research Center, Health Research Institute, Babol University
of Medical Sciences, Babol, Iran
c School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
d School of Chemistry, Shahrood University of Technology, Shahrood, Iran
e Food and Drug Research Institute, Food and Drug Administration, MOHE, Tehran, Iran
f
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
* Corresponding author.
E-mail addresses: saghaie@pharm.mui.ac.ir (L. Saghaie
Mahdavi)
) and momahdavi@tums.ac.ir (M.
1

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Abstract
A new series of thiosemicarbazide-1,2,3-triazole hybrids 10a-o has been synthesized,
1
13
characterized by H NMR, C NMR, and screened for their in vitro α-glucosidase inhibitory
activity. All of the synthesized compounds displayed excellent α-glucosidase inhibitory activity
with IC50 values in the range of 75.0 ± 0.5 to 253.0 ± 0.5 µM, as compared to the standard drug
acarbose (IC50 = 750.0 ± 1.5 µM). Among the synthesized compounds, compound 10h (IC50 =
75.0 ± 0.5) with 4-methoxy group at phenyl part of thiosemicarbazide moiety and 2,6-dichloro
substituents at benzyl moiety was found to be the most potent compound. Kinetic analysis
revealed that compound 10h is a competitive inhibitor for α-glucosidase. Docking study of
compound 10h in the active site of α-glucosidase showed that this compound interacted with
residues His239, His279, Glu304, Gly306, and Arg312.
Keywords: α-Glucosidase inhibitor, Molecular docking, Thiosemicarbazide, 1,2,3-Triazole
1. Introduction
α-Glucosidase is a digestive enzyme, which plays a vital role in the breakdown of disaccharide
and polysaccharides in the intestine [1]. α-Glucosidase inhibitors could diminish postprandial
plasma glucose levels and, as a result, they decreased postprandial hyperglycemia. Therefore, α-
glucosidase is an essential target for the treatment of type 2 diabetes [2]. Furthermore, α-
glucosidase also has been introduced as an attractive therapeutic target for other carbohydrate-
mediated diseases including HIV, cancer, and hepatitis [3-5]. Acarbose, miglitol, and voglibose
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are α-glucosidase inhibitors that have been approved for the clinical use. These drugs have side
effects such as flatulence, pain, bloating, diarrhea, and abdominal discomfort [6]. Hence, the
search for new agents with high α-glucosidase inhibitory activity and low side effects is still in
progress [7-9].
Thiosemicarbazide and thiourea are two sulfur-containing scaffolds, which are found in the
many biologically active compounds with antitumor, antibacterial, antifungal, anticonvulsant,
and antioxidant activities [10-19]. Furthermore, derivatives of these scaffolds such as
thiosemicarbazide derivatives A and thiourea derivatives B have been reported to show high
inhibitory activity against α-glucosidase (Fig.1) [20, 21]. On the other hand, recently, several
groups of α-glucosidase inhibitors containing Schiff-base such as compounds C have been
synthesized (Fig. 1) [22].
1,2,3-Triazole is a five-membered ring with three nitrogen atoms and applied as an important
building block in many biologically active compounds with various pharmacological activities
[23-25]. One of the reported pharmacological effects of 1,2,3-triazole derivatives is α-
glucosidase inhibition [26-29]. In this respect, newly, our research group by using molecular
hybridization reported a new series of 1,2,3-triazole-quinazolinone hybrids D as potent α-
glucosidase inhibitors [30]. Therefore, herein, with focusing on structures A, B, C, and D as
potent α-glucosidase inhibitors, a new series of thiosemicarbazide-1,2,3-triazole hybrids 10a-o
are designed as the follow up efforts in the development of new α-glucosidase inhibitors by
molecular hybridization [31-33]. Designed compounds were synthesized and evaluated against
α-glucosidase. Kinetic and docking studies were also performed to assay the interaction of these
compounds with α-glucosidase.
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2. Method and material
Melting points of the target compounds 10a-o was measured on a Kofler hot stage apparatus. 1H
and 13C NMR spectra of title compounds were determined on a Bruker FT-500 via TMS (internal
standard). IR spectra re-coded using KBr disks on a Nicolet Magna FTIR 550
spectrophotometer. Mass spectra for selected compounds 10a, 10d, and 10l were obtained with
an Agilent Technology (HP) mass spectrometer operating at an ionization potential of 70 eV.
Elemental analysis was performed by an Elementar Analysen system GmbH VarioEL CHN
mode.
2.1. General procedure for the synthesis of 4-phenylthiosemicarbazides 3
A solution of isothiocyanate derivatives 1 (1 mmol) and hydrazine 2 (1 mmol) in Et2O (15 mL)
was stirred at room temperature for 6 h. Later, the reaction mixture was filtered off and
precipitated product was washed with Et2O to obtain pure 4-phenylthiosemicarbazides 3.
2.2. General procedure for the synthesis of 3-methoxy-4-(prop-2-ynyloxy)benzaldehyde 6
A suspension of 4-hydroxy-3-methoxybenzaldehyde 4 (1 mmol) and K2CO3 (1 mmol) in DMF
(5 ml) was stirred at room temperature for 1 h. At that point, it was added to a solution of
propargyl bromide 5 (1.2 mmol) in DMF (10 ml) in a dropwise manner, and the reaction mixture
was stirred at room temperature for 2 h. Once the reaction was completed (checked by TLC), it
was poured into crushed ice, and the obtained mixture was filtered off and residue was
recrystallized in ethanol to obtain 3-methoxy-4-(prop-2-ynyloxy) benzaldehyde 6.
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2.3. General procedure for the synthesis of 1-(3-methoxy-4-(prop-2-ynyloxy)benzylidene)-4-
phenylthiosemicarbazides 7
A mixture of 4-phenylthiosemicarbazides 3 (1 mmol) and 3-methoxy-4-(prop-2-ynyloxy)
benzaldehyde 6 (1 mmol) at ethanol in the presence of PTSA (para-toluene sulfonic acid) was
refluxed for 6 h. Then, the obtained mixture was filtered off and residue was recrystallized in the
ethyl acetate/n-hexane to gain pure 1-(3-methoxy-4-(prop-2-ynyloxy) benzylidene) -4-
phenylthiosemicarbazides 7.
2.4. General procedure for the synthesis of thiosemicarbazide-1,2,3-triazole derivatives 10a-o
At first, azide derivatives 9 were prepared in situ. For this purpose, a solution of benzyl halides 8
(1.1 mmol), sodium azide (0.9 mmol), and Et3N (1.3 mmol) in the mixture of water plus t-BuOH
(10mL, 1:1) was stirred at room temperature for 1 h. Then and there, a mixture of 1-(3-methoxy-
4-(prop-2-ynyloxy)benzylidene)-4-phenylthiosemicarbazides 7 (1 mmol), sodium ascorbate, and
CuSO4 .5H2O (7 mol %) was added to the prepared benzyl azide derivative 9, and obtained
mixture was stirred at room temperature for 24-48 h. Upon completion of the reaction
(monitored by TLC), reaction mixture was diluted with cold water and poured into crushed ice.
Then, precipitated products 10a-o were filtered off, washed with water, and purified by
recrystallization in ethanol.
2.4.1. (E)-1-(4-((3-(2,4-dichlorobenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzylidene)-
4-phenylthiosemicarbazide (10a)
White Powder; Mp: 157-159°C; Yield: 72% ; 1H NMR (500MHz, CHCl3-d6, 25 °C, TMS) (δ,
ppm): 3.91 (s, 3H; OCH3), 5.34 (s, 2H; CH2N), 5.64 (s, 2H; CH2O), 7.11 (d, J = 8.3 Hz, 1H; H5),
7.15-7.18 (m, 2H; H4'',6), 7.25 (s, 1H; H2), 7.27-7.29 (m, 2H; H5',6'), 7.43 (d, J = 7.9 Hz, 2H;
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H2'',6''), 7.71 (s, 1H; CHtriazole), 7.91 (s, 1H; H-C=N), 9.16 (s, 1H; NH-Ph), 10.15 (s, 1H; NH-
C=S); 13C NMR (CHCl3-d6, 25 °C, TMS) (δ, ppm): , 51.4, 56.0, 62.9, 109.4, 113.8, 122.3, 123.3,
124.6, 126.2, 126.7, 128.0,128.9, 129.9,130.9, 131.3, 134.3, 135.7, 137.9, 143.1, 144.2, 150.0,
150.1, 175.8; IR (KBr, cm-1): 3429, 3302 (N-H stretching), 3144 (Aromatic C-H stretching),
1546 (C=N), 1506 (C=C), 1265 (C-N), 1201 (C=S), 1137 (C-O); MS (70 eV): m/z = 540 [M+];
Anal. Calcd. For C25H22Cl2N6O2S: C, 55.46; H, 4.10; N, 15.52; Found: C, 55.21; H, 4.32; N,
15.71.
2.4.2. (E)-1-(4-((3-(2,6-dichlorobenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzylidene)-
4-phenylthiosemicarbazide(10b)
1
Cream to white Powder; Mp: 220-222 °C; Yield: 88% ; H NMR (500MHz, CHCl3-d6, 25 °C,
TMS) (δ, ppm): 3.91 (s, 3H; OCH3), 5.32 (s, 2H; CH2N), 5.88 (s, 2H; CH2O), 7.13-7.18 (m, 2H;
H5,6), 7.24-7.28 (m, 2H; H2,4'),7.32 (t, J = 7.7 Hz, 1H; H4), 7.41-7.45 (m, 4H; H3'',5'', 2'',6''), 7.67-
7.68 (m, 3H; H3',5', triazole), 7.86 (s, 1H; H-C=N), 9.15 (s, 1H; NH-Ph), 9.82 (s, 1H; NH-C=S); 13C
NMR (CHCl3-d6, 25 °C, TMS) (δ, ppm): 49.0, 56.5, 62.9, 109.1, 114.1, 122.2, 123.0, 124.6,
126.2, 126.7, 128.8, 128.9, 130.0, 131.1, 133.8, 136.8, 137.9, 139.9, 143.0, 143.5, 149.9, 150.2,
175.6; IR (KBr, cm-1): 3438, 3294 (N-H stretching), 3150 (Aromatic C-H stretching), 1595
(C=N), 1551 (C=C), 1267 (C-N), 1199 (C=S), 1138 (C-O); Anal. Calcd. for C25H22Cl2N6O2S: C,
55.46; H, 4.10; N, 15.52; Found C, 55.61; H, 4.27; N, 15.39.
2.4.3. (E)-1-(4-((3-(3,4-dichlorobenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzylidene)-
4-phenylthiosemicarbazide (10c)
1
White Powder; Mp: 178-180 °C; Yield: 88% ; H NMR (500MHz, CHCl3-d6, 25 °C, TMS) (δ,
ppm): 3.92 (s, 3H; OCH3), 5.34 (s, 2H; CH2N), 5.50 (s, 2H; CH2O), 7.11 (t, J = 8.2 Hz, 2H;
6

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H3'',5''), 7.18 (d, J = 8.3, 1H; H5), 7.25- 7.28 (t, J = 7.7 Hz, 2H; H4'',2), 7.37 (s, 1H, H2'), 7.42-7.47
(m, 3H; H6,5',6'), 7.63 (s, 1H, Htriazole), 7.67 (d, J = 7.5 Hz, 2H, H2'', 6''), 7.87 (s, 1H; H-C=N), 9.15
13
(s, 1H; NH-Ph), 9.85 (s, 1H; NH-C=S); C NMR (CHCl3-d6, 25 °C, TMS) (δ, ppm): 53.4, 56.1,
62.9, 109.1, 109.2, 113.6, 121, 122.2, 123, 124.6, 126.7, 127.3, 128.8, 130.0, 134.4, 137.9, 143.0,
144.4, 146.0, 150.0, 175.8 ; IR (KBr, cm-1): 3387, 3302 (N-H stretching), 3139 (Aromatic C-H
stretching), 1595 (C=N), 1544 (C=C), 1266 (C-N), 1198 (C=S), 1135 (C-O); Anal. Calcd. for
C25H22Cl2N6O2S: C, 55.46; H, 4.10; N, 15.52; Found C, 55.24; H, 4.02; N, 15.72.
2.4.4. (E)-1-(4-((3-(2-methylbenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzylidene)-4-
(4-methoxyphenyl)thiosemicarbazide (10d)
1
Cream Powder; Mp: 94-96 °C; Yield: 79%; H NMR (500MHz, CHCl3-d6, 25 °C, TMS) (δ,
ppm): 3.84 (s, 3H, OCH3), 3.88 (s, 3H; OCH3), 5.31 (s, 2H; CH2N), 5.55 (s, 2H; CH2O), 6.94 (d,
J = 8.1 Hz, 2H; H3'',5''), 7.10 (d, J = 8.4, 1H; H5), 7.16 (d, J = 7.2, 2H; H2'',6''), 7.21-7.24 (m, 3H,
H2, 4', 5'), 7.29 (d, J = 9.4 Hz, 1H; H3'), 7.48-7.50 (m, 3H; H6,6',triazole), 7.90 (s, 1H; H-C=N), 9.00 (s,
13
1H; NH-Ph), 10.16 (s, 1H; NH-C=S); C NMR (CHCl3-d6, 25 °C, TMS) (δ, ppm): 19.2, 52.3,
55.5, 56.3, 63.1, 109.4, 113.8, 114.1, 122.2, 122.8, 126.6, 126.7, 127.0, 129.2, 129.5, 131.1,
132.3, 134.6, 136.9, 143.1, 143.8, 149.0, 150.0, 158.0, 176.4; IR (KBr, cm-1): 3434, 3302 (N-H
stretching), 3147 (Aromatic C-H stretching), 1596 (C=N), 1510 (C=C), 1262 (C-N), 1199 (C=S),
1136 (C-O); MS (70 eV): m/z = 516 [M+]; Anal. Calcd. for C27H28N6O3S: C, 62.77; H, 5.46; N,
16.27 Found C, 63.10; H, 5.33; N, 15.97.
2.4.5. (E)-1-(4-((3-(3-methoxybenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzylidene)-4-
(4-methoxyphenyl)thiosemicarbazide (10e)
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1
White to cream Powder; Mp: 125-127 °C; Yield: 90% ; H NMR (500MHz, CHCl3-d6, 25 °C,
TMS) (δ, ppm): 3.78 (s, 3H, OCH3), 3.84 (s, 3H; OCH3), 3.89 (s, 3H; OCH3), 5.32 (s, 2H;
CH2N), 5.51 (s, 2H; CH2O), 6.80 (m, 1H; H2'), 6.86 (d, J =7.9 Hz, 1H; H4'), 6.90 (d, J = 7.9 Hz,
1H; H6'), 6.95 (d, J = 8.1 Hz, 2H, H3'',5''), 7.09 (d, J = 8.3 Hz, 1H, H5), 7.16 (d, J = 8.3 Hz, 1H,
H6), 7.23 (s, 1H, H2), 7.28-7.29 (m, 1H, H5'), 7.49 (d, J = 8.1 Hz, 2H; H2'',6''), 7.60 (s, 1H;
Htriazole), 7.89 (s, 1H; H-C=N), 9.00 (s, 1H; NH-Ph), 10.17 (s, 1H; NH-C=S); 13C NMR (CHCl3-
d6, 25 °C, TMS) (δ, ppm): 54.2, 55.3, 55.5, 56.1, 63.1, 109.2, 113.7, 113.8, 114.1, 114.3, 120.3,
122.1, 123.0, 126.8, 127.1, 130.2, 130.8, 135.8, 143.1, 144.0, 149.9, 150.1, 158.1, 160.2, 175.8;
IR (KBr, cm-1): 3441, 3261 (N-H stretching), 3133 (Aromatic C-H stretching), 1595 (C=N),
1511 (C=C), 1265 (C-N), 1201 (C=S), 1139 (C-O); Anal. Calcd. for C27H28N6O4S: C, 60.89; H,
5.30; N, 15.78 Found C, 60.53; H, 5.21; N, 16.31.
2.4.6. (E)-1-(4-((3-(2-chlorobenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzylidene)-4-(4-
methoxyphenyl)thiosemicarbazide (10f)
1
White to cream Powder; Mp: 133-135 °C; Yield: 82% ; H NMR (500MHz, CHCl3-d6, 25 °C,
TMS) (δ, ppm): 3.84 (s, 3H, OCH3), 3.90 (s, 3H; OCH3), 5.33 (s, 2H; CH2N), 5.68 (s, 2H;
CH2O), 6.90 (d, J = 8.7 Hz, 2H; H3'',5''), 7.10 (d, J =8.4 Hz, 1H; H5), 7.16 (d, J = 8.4 Hz, 1H; H6),
7.21 (d, J = 7.5 Hz, 1H, H6'), 7.24 (s, 1H, H2), 7.26-7.28 (m, 1H, H4'), 7.33 (t, J = 7.7 Hz, 1H,
H5'), 7.45 (d, J = 7.7 Hz, 1H, H3'), 7.50 (d, J = 8.7 Hz, 2H; H2'',6''), 7.69 (s, 1H; Htriazole), 7.88 (s,
13
1H; H-C=N), 9.00 (s, 1H; NH-Ph), 10.03 (s, 1H; NH-C=S); C NMR (CHCl3-d6, 25 °C, TMS)
(δ, ppm): 51.6, 51.6, 55.3, 56.3, 63.3, 109.2, 113.8, 114.1, 122.1, 123.3, 126.8, 127.0, 127.6,
129.9, 130.3, 130.5, 130.8, 132.2, 133.6, 143.1, 144.0, 149.9, 150.1, 158.1, 176.4; IR (KBr, cm-
1): 3436, 3294 (N-H stretching), 3149 (Aromatic C-H stretching), 1595 (C=N), 1504 (C=C),
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1267 (C-N), 1199 (C=S), 1138 (C-O); Anal. Calcd. for C26H25ClN6O3S: C, 58.15; H, 4.69; N,
15.65 Found C, 57.95; H, 4.83; N, 15.48
2.4.7. (E)-1-(4-((3-(2,4-dichlorobenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzylidene)-
4-(4-methoxyphenyl)thiosemicarbazide (10g)
1
White Powder; Mp: 164-166 °C; Yield: 73% ; H NMR (500MHz, CHCl3-d6, 25 °C, TMS) (δ,
ppm): 3.84 (s, 3H, OCH3), 3.90 (s, 3H; OCH3), 5.33 (s, 2H; CH2N), 5.64 (s, 2H; CH2O), 6.95 (d,
J = 8.9 Hz, 2H; H3'',5''), 7.09 (d, J =8.3 Hz, 1H; H5), 7.14-7.17 (m, 2H; H2,6), 7.24-7.27 (m, 1H,
H5', 6'), 7.46 (s, 1H, H3'), 7.49 (d, J = 8.9 Hz, 2H, H2'',6''), 7.70 (s, 1H; Htriazole), 7.90 (s, 1H; H-
C=N), 9.00 (s, 1H; NH-Ph), 10.18 (s, 1H; NH-C=S); 13C NMR (CHCl3-d6, 25 °C, TMS) (δ,
ppm): 50.6, 55.6, 56.3, 63.1, 109.2, 113.3, 114.1, 121.7, 123.3, 126.9, 127.0, 127.8, 129.8, 130.8,
131.4, 134.4, 136.0, 137.9, 142.7, 144.3, 150.1, 158.3, 176.6; IR (KBr, cm-1): 3437, 3292 (N-H
stretching), 3142 (Aromatic C-H stretching), 1595 (C=N), 1509 (C=C), 1261 (C-N), 1194 (C=S),
1137 (C-O); Anal. Calcd. for C26H24Cl2N6O3S: C, 54.64; H, 4.23; N, 14.71 Found C, 54.89; H,
4.37; N, 14.43.
2.4.8. (E)-1-(4-((3-(2,6-dichlorobenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzylidene)-
4-(4-methoxyphenyl)thiosemicarbazid (10h)
1
White Powder; Mp: 184-186 °C; Yield: 74% ; H NMR (500MHz, CHCl3-d6, 25 °C, TMS) (δ,
ppm): 3.85 (s, 3H, OCH3), 3.91 (s, 3H; OCH3), 5.31 (s, 2H; CH2N), 5.87 (s, 2H; CH2O), 6.96 (d,
J = 8.4 Hz, 2H; H3'',5''), 7.12-7.17 (m, 2H; H5,6), 7.24 (s, 1H; H2), 7.32 (t, J = 8.4 Hz, 1H, H4'),
7.42 (d, J = 8.4 Hz, 2H, H2'',6''), 7.50 (d, J = 8.4 Hz, 2H, H3',5'), 7.68 (s, 1H; Htriazole), 7.85 (s, 1H;
13
H-C=N), 9.00 (s, 1H; NH-Ph), 9.83 (s, 1H; NH-C=S); C NMR (CHCl3-d6, 25 °C, TMS) (δ,
ppm): 49.2, 55.5, 56.6, 62.9, 109.0, 113.8, 114.1, 122.2, 123, 124.6, 126.7, 126.9, 128.9, 130.7,
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131.1, 134.2, 136.9, 139.4, 143.0, 143.5, 149.6, 150.1, 158.1, 173.5; IR (KBr, cm-1): 3425, 3304
(N-H stretching), 3147 (Aromatic C-H stretching), 1594 (C=N), 1511 (C=C), 1262 (C-N), 1198
(C=S), 1138 (C-O); Anal. Calcd. for C26H24Cl2N6O3S: C, 54.64; H, 4.23; N, 14.71 Found C,
54.43; H, 4.37; N, 14.93
2.4.9. (E)-1-(4-((3-(3,4-dichlorobenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzylidene)-
4-(4-methoxyphenyl)thiosemicarbazide (10i)
1
White to cream Powder; Mp: 107-108 °C; Yield: 72% ; H NMR (500MHz, CHCl3-d6, 25 °C,
TMS) (δ, ppm): 3.84 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 5.33 (s, 2H; CH2O), 5.50 (s, 2H;
CH2O), 6.95 (d, J = 8.8 Hz, 2H; H3'',5''), 7.08 (d, J = 8.3 Hz, 1H, H5), 7.12 (d, J = 9.0 , 1H; H6'),
7.16 (d, J = 9.0 Hz, 1H; H5'), 7.24 (s, 1H, H2), 7.37 (s, 1H, H2'), 7.44-7.49 (m, 3H, H6,2'',6'',), 7.64
13
(s, 1H; Htriazole), 7.90 (s, 1H; H-C=N), 9.00 (s, 1H; NH-Ph), 10.21 (s, 1H; NH-C=S); C NMR
(CHCl3-d6, 25 °C, TMS) (δ, ppm): 55.9, 55.5, 56.1, 63.0, 109.2, 113.7, 114.1, 122.1, 123.0,
126.9, 127.0, 127.1, 127.3, 130.0, 130.8, 131.1, 133.2, 134.5, 143.1, 144.4, 149.9, 158.1, 176.4;
IR (KBr, cm-1): 3309 (N-H stretching), 3142 (Aromatic C-H stretching), 1594 (C=N), 1511
(C=C), 1261 (C-N), 1200 (C=S), 1133 (C-O); Anal. Calcd. for C26H24Cl2N6O3S: C, 54.64; H,
12.41; N, 14.71 Found C, 54.81; H, 12.57; N, 14.63
2.4.10. (E)-1-(4-((3-(4-bromobenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzylidene)-4-
(4-methoxyphenyl)thiosemicarbazide (10j)
1
Cream Powder; Mp: 116-118 °C; Yield: 79% ; H NMR (500MHz, CHCl3-d6, 25 °C, TMS) (δ,
ppm): 3.84 (s, 3H, OCH3), 3.90 (s, 3H; OCH3), 5.32 (s, 2H; CH2N), 5.49 (s, 2H; CH2O), 6.94 (d,
J = 7.0 Hz, 2H; H3'',5''), 7.15 (d, J = 7.0 Hz, 2H, H2'',6''), 7.24 (s, 1H; H2), 7.35 (d, H =8.3 Hz, 1H;
H5), 7.46-7.52 (m, 5H, H6, 2',3',5',6'), 7.14 (d, J = 8.3, 1H, H6), 7.23 (s, 1H, H2), 7.28-7.31 (m, 1H,
10

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H5'), 7.39 (d, J = 8.7 Hz, 2H; H2'',6''), 7.60 (s, 1H; Htriazole), 7.90 (s, 1H; H-C=N), 9.00 (s, 1H; NH-
Ph), 10.20 (s, 1H; NH-C=S); 13C NMR (CHCl3-d6, 25 °C, TMS) (δ, ppm):53.4, 55.6, 56.3, 62.9,
109.1, 113.7, 113.9, 114.1, 121.7, 122.3, 123.0, 127.0, 129.6, 130.7, 132.2, 132.4, 132.6, 133.4,
143.0, 145.4, 149.4, 150.1, 158.0, 176.4; IR (KBr, cm-1): 3435, 3325 (N-H stretching), 3144
(Aromatic C-H stretching), 1597 (C=N), 1513 (C=C), 1260 (C-N), 1196 (C=S), 1137 (C-O);
Anal. Calcd. for C26H25BrN6O3S: C, 53.70; H, 4.33; N, 14.45 Found C, 53.93; H, 4.49; N, 14.29.
2.4.11. (E)-1-(4-((3-(3-methoxybenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzylidene)-4-
(4-(dimethylamino)phenyl)thiosemicarbazide (10k)
1
Cream to white Powder; Mp: 143-145 °C; Yield: 82% ; H NMR (500MHz, CHCl3-d6, 25 °C,
TMS) (δ, ppm): 2.98 (s, 6H, NCH3), 3.78 (s, 3H; OCH3), 3.89 (s, 3H; OCH3), 5.31 (s, 2H;
CH2N), 5.50 (s, 2H; CH2O), 6.76 (d, J = 8.8 Hz, 2H; H3'',5''), 6.79 (s, 1H, H2'), 6.86 (d, J = 7.0 Hz,
1H; H4'), 6.90 (d, J =7.0 Hz, 1H; H6'), 7.08 (d, J = 8.3 Hz, 1H, H5), 7.14 (d, J = 8.3, 1H, H6), 7.23
(s, 1H, H2), 7.28-7.31 (m, 1H, H5'), 7.39 (d, J = 8.7 Hz, 2H; H2'',6''), 7.59 (s, 1H; Htriazole), 7.89 (s,
13
1H; H-C=N), 8.95 (s, 1H; NH-Ph), 10.23 (s, 1H; NH-C=S); C NMR (CHCl3-d6, 25 °C, TMS)
(δ, ppm): 40.5, 54.2, 55.3, 56.0, 63.1, 109.2, 112.4, 112.8, 113.7, 113.7, 114.3, 120.3, 122.0,
123.0, 126.8, 127.0, 130.2, 135.8, 137.7, 142.8, 144.1, 149.3, 150.0, 160.1, 176.6; IR (KBr, cm-
1): 3440, 3259 (N-H stretching), 3127 (Aromatic C-H stretching), 1591 (C=N), 1511 (C=C),
1263 (C-N), 1201 (C=S), 1140 (C-O); Anal. Calcd. for C28H31N7O3S: C, 61.63; H, 5.73; N, 17.97
Found C, 61.51; H, 5.84; N, 17.73.
2.4.12. (E)-1-(4-((3-(2-chlorobenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzylidene)-4-
(4-(dimethylamino)phenyl)thiosemicarbazide(10l)
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Light green Powder; Mp: 175-177 °C; Yield: 85% ; 1H NMR (500MHz, CHCl3-d6, 25 °C, TMS)
(δ, ppm): 2.98 (s, 6H, NCH3), 3.88 (s, 3H; OCH3), 5.33 (s, 2H; CH2N), 5.67 (s, 2H; CH2O), 6.76
(d, J = 8.8 Hz, 2H; H3'',5''), 7.08 (d, J = 8.3 Hz, 1H, H5), 7.15 (d, J = 8.3 Hz, 1H; H6), 7.20 (d, J
=7.5 Hz, 1H; H6'), 7.24-7.27 (m, 2H, H2,4'), 7.32 (t, J = 7.5, 1H, H5'), 7.39 (d, J = 8.7 Hz, 2H;
H2'',6''), 7.69 (d, J = 7.5 Hz, 1H, H3'), 7.69 (s, 1H; Htriazole), 7.93 (s, 1H; H-C=N), 8.96 (s, 1H; NH-
Ph), 10.48 (s, 1H; NH-C=S); 13C NMR (CHCl3-d6, 25 °C, TMS) (δ, ppm): 40.8, 51.6, 56.3, 62.9,
107.8, 109.2, 110.2, 112.4, 113.8, 122.0, 123.3, 126.9, 127.6, 127.7, 129.9, 130.3, 130.4, 132.2,
136.0, 142.9, 144.1, 149.4, 149.8, 176.4; IR (KBr, cm-1): 3440, 3259 (N-H stretching), 3127
(Aromatic C-H stretching), 1591 (C=N), 1511 (C=C), 1263 (C-N), 1201 (C=S), 1140 (C-O); MS
(70 eV): m/z = 550 [M+]; Anal. Calcd. for C28H31N7O3S: C, 61.63; H, 5.73; N, 17.97 Found C, 61.51;
H, 5.84; N, 17.73.
2.4.13.
(E)-1-(4-((3-(2,4-dichlorobenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-
methoxybenzylidene)-4-(4-(dimethylamino)phenyl)thiosemicarbazide (10m)
Light green Powder; Mp: 189-191 °C; Yield: 81% ; 1H NMR (500MHz, CHCl3-d6, 25 °C, TMS)
(δ, ppm): 2.99 (s, 6H, NCH3), 3.91 (s, 3H; OCH3), 5.33 (s, 2H; CH2N), 5.64 (s, 2H; CH2O), 6.76
(d, J = 8.8 Hz, 2H; H3'',5''), 7.09 (d, J = 8.3 Hz, 1H; H5), 7.11-7.16 (m, 2H; H5',6'), 7.25-7.28 (m,
2H, H2,6), 7.39 (d, J = 8.8 Hz, 2H, H2'',6''), 7.46 (s, 1H, H3'), 7.70 (s, 1H; Htriazole), 7.89 (s, 1H; H-
C=N), 8.95 (s, 1H; NH-Ph), 10.19 (s, 1H; NH-C=S); 13C NMR (CHCl3-d6, 25 °C, TMS) (δ,
ppm): 40.6, 50.7, 56.4, 62.9, 109.1, 112.4, 113.7, 122.1, 123.3, 126.8, 126.9, 127.0, 128.0, 129.8,
130.9, 131.2, 134.2, 135.7, 142.8, 144.2, 149.4, 149.8, 149.9, 176.5; IR (KBr, cm-1): 3306 (N-H
stretching), 3144 (Aromatic C-H stretching), 1591 (C=N), 1514 (C=C), 1267 (C-N), 1200 (C=S),
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1134 (C-O); Anal. Calcd. for C27H27Cl2N7O2S: C, 55.48; H, 4.66; N, 16.77 Found C, 55.79; H, 4.49;
N, 16.61.
2.4.14.
(E)-1-(4-((3-(3,4-dichlorobenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-
methoxybenzylidene)-4-(4-(dimethylamino)phenyl)thiosemicarbazide (10n)
Light green Powder; Mp: 135-137 °C; Yield: 85% ; 1H NMR (500MHz, CHCl3-d6, 25 °C, TMS)
(δ, ppm): 2.99 (s, 6H, NCH3), 3.90 (s, 3H; OCH3), 5.33 (s, 2H; CH2N), 5.49 (s, 2H; CH2O), 6.76
(d, J = 8.7 Hz, 2H; H3'',5''), 7.07 (d, J = 8.2 Hz, 1H; H5), 7.11-7.15 (m, 2H; H5',6'), 7.24 (s, 1H, H2),
7.38-7.40 (m, 3H, H 2',2'',6''), 7.45 (d, J = 8.2 Hz, 1H, H6), 7.63 (s, 1H; Htriazole), 7.88 (s, 1H; H-
C=N), 8.95 (s, 1H; NH-Ph), 10.13 (s, 1H; NH-C=S); 13C NMR (CHCl3-d6, 25 °C, TMS) (δ,
ppm): 40.8, 52.9, 56.1, 62.9, 109.0, 109.1, 112.4, 113.7, 122.0, 123.0, 126.8, 126.9, 127.1, 127.3,
130.0, 131.1, 133.2, 134.6, 142.8, 144.5, 149.4, 149.7, 150.0, 176.4.; IR (KBr, cm-1): 3434, 3316
(N-H stretching), 3141 (Aromatic C-H stretching), 1595 (C=N), 1520 (C=C), 1267 (C-N), 1198
(C=S), 1134 (C-O); Anal. Calcd. for C27H27Cl2N7O2S: C, 55.48; H, 12.13; N, 16.77 Found C, 55.69;
H, 12.21; N, 16.61
2.4.15. (E)-1-(4-((3-(4-bromobenzyl)-3H-1,2,3-triazol-4-yl)methoxy)-3-methoxybenzylidene)-4-
(4-(dimethylamino)phenyl)thiosemicarbazide (10o)
1
White Powder; Mp: 120-122 °C; Yield: 80% ; H NMR (500MHz, CHCl3-d6, 25 °C, TMS) (δ,
ppm): 3.00 (s, 6H, NCH3), 4.00 (s, 3H; OCH3), 5.33 (s, 2H; CH2N), 5.50 (s, 2H; CH2O), 6.77 (d,
J = 8.8 Hz, 2H; H3'',5''), 7.09 (d, J = 8.3, 1H; H5), 7.14- 7.17 (m, 3H; H2'',6'',6), 7.25 (s, 1H, H2),
7.40 (d, J = 8.6 Hz, 2H; H2',6'), 7.52 (d, J = 8.3 Hz, 2H; H3',5'), 7.59 (s, 1H, Htriazole), 7.84 (s, 1H;
13
H-C=N), 8.93 (s, 1H; NH-Ph), 9.85 (s, 1H; NH-C=S); C NMR (CHCl3-d6, 25 °C, TMS) (δ,
ppm): 40.5, 53.7, 56.2, 63.0, 106.8, 109.1, 109.3, 112.5, 113.7, 122.1, 122.9, 123.0, 126.7, 126.9,
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129.7, 132.1, 132.4, 142.5, 143.8, 144.3, 144.9, 149.3, 149.9, 176.6; IR (KBr, cm-1): 3415, 3293
(N-H stretching), 3142 (Aromatic C-H stretching), 1594 (C=N), 1543 (C=C), 1266 (C-N), 1196
(C=S), 1135 (C-O); Anal. Calcd. for C27H28BrN7O2S: C, 54.55; H, 4.75; N, 16.49 Found C,
54.13; H, 4.91; N, 16.82.
2.5. α-Glucosidase inhibition assay
The α-glucosidase inhibitory activities of thiosemicarbazide-1,2,3-triazole derivatives 10a-o
were determined according to the literature protocol [31]. The Saccharomyces cerevisiae form of
the α-glucosidase enzyme (EC 3.2.1.20) and p-nitrophenyl glucopyranoside (pNPG) as substrate
were prepared in potassium phosphate buffer (pH 6.8, 50 mM). The enzymatic reaction mixture
composed of enzyme buffer (20 ꢀL, 1 U/mL), the substrate (25 ꢀL, 0.5 mM), the test compound
(20 ꢀL), and potassium phosphate buffer (135 ꢀL) was incubated at 37 °C for 30 min. The
enzymatic activity of α-glucosidase was evaluated by spectrophotometer (Gen5, Power wave
xs2, BioTek, America) at a wavelength of 405 nm.
2.6. Kinetic study
Compound 10h (0, 35, 55 and 75 ꢀM, 20µL) was added to the enzyme solution (1 U/Ml, 20µL)
at 30 °C and incubated for 15 min. The substrate (pNGP) at different concentrations (1-4 mM)
was then added to initiate the enzyme reaction and change in absorbance was measured for 20
min at 405 nm by using spectrophotometer (Gen5, Power wave xs2, BioTek, America) [31].
2.7. Molecular docking study
Docking study of the selected compounds 10b, 10h, and 10m was carried out according to the
literature protocol [33].
3. Results and discussion
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3.1. Chemistry
The synthetic route for the synthesis of thiosemicarbazide-1,2,3-triazole hybrids 10a-o has been
depicted in Scheme 1. Firstly, a mixture of isothiocyanat derivatives 1 and hydrazine 2 in
diethylether (Et2O) was stirred at room temperature for 6 h, to afford 4-phenylthiosemicarbazides
3. On the other hand, 3-methoxy-4-(prop-2-ynyloxy)benzaldehyde 6 was produced by a reaction
between 4-hydroxy-3-methoxybenzaldehyde 4 and propargyl bromide 5 in presence of K2CO3 in
DMF at room temperature. In the next step, the 4-phenylthiosemicarbazides 3 reacted with 3-
methoxy-4-(prop-2-ynyloxy)benzaldehyde 6 in ethanol at room temperature, to give 1-(3-
methoxy-4-(prop-2-ynyloxy)benzylidene)-4-phenylthiosemicarbazides 7. The latter compounds
were prone to be participated in click reaction. Accordingly, various benzyl halide derivatives 8
and sodium azide reacted in the mixture of H2O and t-BuOH (1:1) in the presence of Et3N at
room temperature. Then, a mixture of 1-(3-methoxy-4-(prop-2-ynyloxy)benzylidene)-4-
phenylthiosemicarbazides 7, sodium ascorbate, and CuSO4 .5H2O was added to the freshly
prepared benzyl azide derivatives 9 and the reaction was continued at room temperature to afford
the desired compounds 10a-o.
1
The structures of the synthesized compounds 10a-o were determined by H and 13C NMR,
DEPT, IR, and elemental analysis. The IR spectra showed the expected absorption of stretching
bands for NH at 3261-3441 cm-1. Furthermore, these compounds displayed characteristic
stretches of C=N and bonds around 1546-1597 cm-1 and 1194-1201 cm-1, respectively. 1H NMR
spectra of the synthesized compounds showed signals at 6.75-7.68 ppm corresponding to protons
related to phenyl groups, besides a singlet in the region of 7.49-7.71 ppm attributed to triazole
proton. In addition, the two singlets appeared at 5.31-5.34 ppm and 5.49-5.88 ppm, related to
benzyl protons and -OCH2- group connected to triazole ring, respectively. The signals of protons
15

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belonging to the NH connected to the phenyl ring and -C=S group appeared around 8.95-9.16
ppm and 9.84-10.48 ppm, respectively. Moreover, the singlet in the region of 7.85-7.92 ppm
related to imine proton (H-C=N) confirms the success of the reaction between thiosemicarbazide
and vaniline carbaldehyde. Similarly in 13CNMR spectra, the azomethane group (H-C=N)
occurred at 142.5-143.8 ppm as well as the C=S group appeared at 173.5-176.6 ppm.
All title compounds 10a-o could exist in either the E or Z isomeric form of the imono bond (-
CH=N-). In this regard, compound 10f was selected for elucidation of isomeric structure of the
synthesized compounds by 2D NMR. In 2D NMR NOESY spectrum, the special correlation
between hydrogen of –NH-C=S at 10.03 ppm and azomethine (H-C=N-) at 7.88, as well as no
spatial interaction between the hydrogen in position 2 of the aromatic ring (-CH=C-OCH3) at
7.24 ppm and the hydrogen azomethine (H-C=N-) at 7.88, indicated the presence of a E isomeric
form, which corroborates the NOESY spectrum of E-thiosemicarbazone presented in literature
[34].
3.2. α-Glucosidase inhibitory activity
Synthesized compounds 10a-o were evaluated against α-glucosidase to check their in vitro
inhibitory activity. As can be seen in Table 1, thiosemicarbazide-1,2,3-triazole derivatives 10a-o
can be divided into three groups; (i) phenylthiosemicarbazides 10a-c, (ii) 4-
methoxyphenylthiosemicarbazides 10d-j, and (iii) 4-dimethylaminophenylthiosemicarbazides
10l-o. In each group, the substituents on the phenyl ring of benzyl moiety were altered to
optimize activity against α-glucosidase.
Achieved results showed that all the synthesized compounds have excellent inhibitory activity in
the range of IC50 = 75.0 ± 0.5-253.0 ± 0.5 µM, when compared to the standard drug acarbose
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(IC50 = 750.0 ± 1.5 µM) (Table 1). 4-Methoxyphenylthiosemicarbazides 10h, 10f and 10j
displayed the highest activity (75.0 ± 0.5, 90.0 ± 0.4, and 96.3 ± 0.9 ꢀM, respectively).
The inhibitory activity of phenylthiosemicarbazide derivatives 10a-c against α-glucosidase
demonstrated that 2,6-dichloro derivative 10b (IC50 = 99.5 ± 0.6 µM) have the most potent
activity, while 3,4-dichloro derivative 10c (IC50 = 253.0 ± 0.5 µM) were less active compounds
amongst all the synthesized compounds. Changing the second chloro group position of
compound 10b from C-6 to C-4, producing compound 10a (IC50 = 208.3 ± 0.9 µM) diminished
the activity drastically.
In the 4-methoxyphenylthiosemicarbazide series (compounds 10d-j), 2,6-dichloro, 2-Cl, and 4-
Br substituted compounds 10h, 10f, and 10j showed the highest activity in this series and among
all the synthesized compounds. 3-Methoxy derivative 10e showed good inhibitory activity in the
second series. Moreover, the inhibitory activity of remaining substituted compounds of this
series based on the substituent type is in the order of 2,4-dichloro (10g) > 3,4-dichloro (10i) > 2-
CH3 (10d).
In the third group, 3-methoxy derivative 10k showed good inhibitory activity against α-
glucosidase. Compound 10l (IC50 = 217.4 ± 0.8 µM) with chloro substituent on 2-position of
benzyl moiety showed moderate activity in comparison to other synthesized compounds. Adding
the second chlorine atom to benzyl moiety of compound 10l led to an inhibitory activity
improvement as observed in the compounds 10m and 10n. For that matter, the most potent
compound in 4-dimethylaminophenylthiosemicarbazide series is 2,4-dichloro derivative 10m
with IC50 value 124.5 ± 1.0 µM. The less active compound also in this series was 4-bromo
derivative 10o (IC50 = 231.4 ± 1.0± 0.8 µM).
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The results revealed phenylthiosemicarbazides were less active than their analogs of the second
and third groups. Moreover, 4-methoxyphenylthiosemicarbazide derivatives except for
compound 10g were more potent than their 4-dimethylaminophenylthiosemicarbazide analogs
(compounds 10f, 10i, and 10j vs. compounds 10l, 10n, and 10o, respectively). Inhibitory
activities in 2, 4-dichloro derivatives 10g of the second series and 10m of the third series were
approximately the same.
3.3. Enzyme kinetic study
A kinetic study was carried out on the most potent compound 10h to evaluate the inhibition
mode of synthesized compounds on α-glucosidase. For this purpose, the reaction rates were
measured in the absence and presence of different inhibitor concentrations (compound 10h) with
various concentrations of substrate (p-nitrophenyl glucopyranoside), and then graphs were drawn
by the Lineweaver–Burk plot (Fig. 1a). As the concentrations of compound 10h increased, Vmax
values did not change; however, Km value increased, thereby indicating that compound 10h was
a competitive inhibitor for α-glucosidase (Fig. 2a). The Ki value was calculated directly by
secondary re-plotting of Lineweaver–Burk plots against the different concentrations of
compound 10h (Fig. 2b). The results proved that compound 10h was a competitive inhibitor
against α-glucosidase with a Ki of 74.5 µM. Furthermore, according to plots 2a and 2b, the
concentrations of substrate and inhibitor are variable and the rate of reaction is dependent on the
reactant concentrations, therefore the reaction order is considered as first order kinetics.
3.4. Docking study
Docking simulation was employed to investigate the interaction pose of the synthesized
compounds in the active site of α-glucosidase. For this propose, the most potent compound
among all of the synthesized compound (10h of the second series) and the most potent
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compounds of the first and third series (compounds 10b and 10m, respectively) were selected.
Due to the lack of a crystallographic structure for Saccharomyces cerevisiae α-glucosidase (used
in the in vitro evaluation), a homology model of this enzyme was constructed using SWISS-
MODEL Repository [33]. The superposed structure of the standard inhibitor acarbose and the
selected compounds 10b, 10h, and 10m in the active site of the modeled α-glucosidase is shown
in Fig. 3a. The detailed interaction mode of acarbose revealed that this drug established
interactions with residues Asn241, Thr307, His279, Glu304, Pro309, Ser308, Arg312, Thr301,
and Gln322 in the active site of α-glucosidase (Fig. 3b).
As depicted in Fig. 4a, in the most potent compound 10h, the 4-methoxy substituent on phenyl
group formed a hydrogen bond with residue Gly306. In this compound, an NH unit of
thiosemicarbazide moiety interacted with residue Glu304 via electrostatic interaction. 3-
Methoxybenzylidene moiety of the compound 10h created two π-cation interactions with His279
and His239 as well as a π-π interaction with His279. The latter residue also formed a hydrogen
bond with the nitrogen atom of 1,2,3-triazole ring. Furthermore, an interaction between the 2-
chloro substituent of 2, 6-dichlorobenzyl group and Arg312 was also observed.
NH units of thiosemicarbazide moiety of the compound 10b formed three hydrogen bonds with
Thr307 and Glu304. The latter residue also showed an electrostatic interaction with one of the
NH units of thiosemicarbazide moiety. Phenyl ring of 3-methoxybenzylidene moiety of
compound 10b interacted with His279 via a π-cation interaction while this moiety in the
compound 10h formed three interactions with His279 and His239. 1,2,3-triazole ring of
compound 10b formed π-π and π-cation interactions with His 239 while this ring in the
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compound 10h created a hydrogen bond with His279. The 2-chloro substituent of the 2,6-
dichlorobenzyl group in compound 10b, similar to compound 10h, interacted with Arg312.
As can be seen in Fig. 4c, thiosemicarbazide moiety of the compound 10m established a
hydrogen bonds as well as electrostatic interaction with residues Thr307 and Glu304,
respectively. 3-methoxybenzylidene moiety of this compound, unlike compounds 10h and 10b,
cannot interact with active site. 1,2,3-Triazole ring of compound 10m interacted with residue
Arg312 through a hydrogen bond. The 2-chloro substituent of the 2,4-dichlorobenzyl group in
compound 10m interacted with Asp408.
4. Conclusion
In conclusion, we designed and synthesized a novel series of thiosemicarbazide-1,2,3-triazole
hybrids 10a-o. Further, α-glucosidase inhibitory activity of the synthesized compounds 10a-o
was evaluated. The obtained results showed that all the synthesized thiosemicarbazide-1,2,3-
triazole hybrids are more potent than standard drug acarbose. Kinetic study of the most potent
compound 10h indicated that it competitively inhibited α-glucosidase. Furthermore, docking
study showed that selected compounds 10b, 10h, and 10m interacted with important residues in
the active site of α-glucosidase. In summary, the results have shown that these
thiosemicarbazide-1,2,3-triazole hybrids are a new class of α-glucosidase inhibitors.
Acknowledgements
This research has been supported by a grant from the Research Council of Tehran University of
Medical Sciences (Grant No. 98-01-192-41780).
References
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Table 1
In vitro α-glucosidase inhibitory activity of thiosemicarbazide-1,2,3-triazole hybrids 10a-o.
H
H
N
N
OCH3
O
N
R2
S
R1
N
N
N
Compound
10a
R1
R2
IC50 (µM)a
208.3 ± 0.9
99.5 ± 0.6
253.0 ± 0.5
143.7 ± 0.6
104.4 ± 1.2
90.0 ± 0.4
125.2 ± 1.3
75.0 ± 0.5
135.6 ± 0.8
96.3 ± 0.9
155.0 ± 0.5
217.4 ± 0.8
124.5 ± 1.0
194.0 ± 0.7
231.4 ± 1.0
750.0 ± 1.5
H
2,4-Dichloro
2,6-Dichloro
3,4-Dichloro
2-CH3
H
10b
H
10c
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
N(CH3)2
-
10d
3-OCH3
10e
2-Cl
10f
2,4-Dichloro
2,6-Dichloro
3,4-Dichloro
4-Br
10g
10h
10i
10j
3-OCH3
10k
2-Cl
10l
2,4-Dichloro
3,4-Dichloro
4-Br
10m
10n
10o
Acarbose
-
a Values are the mean ± SD. All experiments were performed at least three times.
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S
N
R
N
H
HN
N
Br
B
N
R1
R2
R
NH
NH
Cl
N
S
R
O
HN
O
N
O
O
N
N
N
N
NH
S
HN
N
H
N
O
NH
A
C
Designed Compounds
R1
O
N
N
S
N
N
N
R2
D
Fig. 1. Rational design of new thiosemicarbazide-1,2,3-triazole hybrids as potent α-glucosidase
inhibitors based on molecular hybridization.
28