A new series of Schiff base derivatives bearing 1,2,3‐triazole

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1 of 10 https://doi.org/10.1002/ardp.201900034

F U L L P A P E R

A new series of Schiff base derivatives bearing

1,2,3 ‐ triazole: Design, synthesis, molecular docking, and α‐ glucosidase inhibition

Ensieh Nasli ‐ Esfahani

¹

| Maryam Mohammadi ‐ Khanaposhtani

²

| Sepideh Rezaei

³

|

Yaghoub Sarrafi

⁴

| Zeinab Sharafi

⁵

| Nasser Samadi

⁶

| Mohammad Ali Faramarzi

⁶

|

Fatemeh Bandarian

¹

| Haleh Hamedifar

⁷

| Bagher Larijani

⁸

| Mirhamed Hajimiri

⁹

|

Mohammad Mahdavi

⁸

1Diabetes Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran

2Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran

3School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

4Faculty of Chemistry, University of Mazandaran, Babolsar, Iran

5Razi Herbal Medicines Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran

6Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, Tehran, Iran

7CinnaGen Medical Biotechnology Research Center, Alborz University of Medical Sciences, Karaj, Iran

8Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran

9Nano Alvand Company, Avicenna Tech Park, Tehran University of Medical Sciences, Tehran, Iran

Correspondence

Dr. Mirhamed Hajimiri, Nano Alvand Company, Avicenna Tech Park, Tehran University of Medical Sciences, Tehran 1439955991, Iran.

Email: [email protected]

Dr. Mohammad Mahdavi, Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran 71877, Iran.

Email: [email protected]

Abstract

A series of new Schiff bases bearing 1,2,3

‐

triazole

12a‒o

was designed, synthesized, and evaluated as

α‐

glucosidase inhibitors. All the synthesized compounds showed promising inhibition against

α‐

glucosidase and were more potent than the standard drug acarbose. The kinetic study on the most potent compound

12n

showed that this compound acted as a competitive

α‐

glucosidase inhibitor. The docking study revealed that the synthesized compounds interacted with the important residues in the active site of

α‐

glucosidase.

K E Y W O R D S

1,2,3‐triazole, antidiabetic activity, molecular docking, Schiff base,α‐glucosidase

1 | I N T R O D U C T I O N

Theα‐glucosidase is one of the most important hydrolase enzymes, which exist in the human intestinal cells. This enzyme is essential for the hydrolysis of poly‐and disaccharides into glucose monomers, which are absorbed into the blood.^[1]In type 2 diabetes, in which the body becomes resistant to insulin, the use ofα‐glucosidase inhibitors can be useful to reduce postprandial hyperglycemia.^[2]Availableα‐glucosidase inhibitors such as acarbose and miglitol cause diarrhea and other intestinal disturbances.^[3] In contrast, there are several reports showing that α‐glucosidase inhibitors can be useful for the treatment of carbohydrate‐

mediated diseases such as cancer, Alzheimer’s, hepatitis, and viral infections.^[4–6] Therefore, the design and development of new α‐glucosidase inhibitors with high efficacy and low side effect are attractive for medicinal chemists.^[7^–^9]

A Schiff base unit is a valuable pharmacophore that has been found in numerous biologically active compounds such as analgesic, antioxidant, antileishmanial, and antidiabetic agents.^[10–13]Recently, several series of α‐glucosidase inhibitors containing Schiff bases such as compoundsA,B, andChave been reported (Figure 1).^[14–16]In contrast, 1,2,3‐triazoles are an important class of heterocycles, which have a wide spectrum of pharmaceutical applications. 1,2,3‐Triazole derivatives exhibit various

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biological activities such as antibacterial, antifungal, anticancer, antidiabetic potentials.^[17–20]Some of these derivatives have also shown significant inhibitory activity againstα‐glucosidase.^[21–23]In this regard, we have recently reported a series of 1,2,3‐triazole derivativesDwith highα‐glucosidase inhibitory activity (Figure 1).^[24]

Herein, in view of the α‐glucosidase inhibitory activities of compounds containing Schiff base or 1,2,3‐triazole and in continuation to our interest in the discovery of potent α‐glucosidase inhibitors, we reported the design and synthesis of a series of new Schiff bases bearing 1,2,3‐triazole12a–o.^[25–27]All the synthesized derivatives12a–owere screened for their in vitro α‐glucosidase inhibitory activities. Moreover, kinetic and docking studies were also performed to further evaluate of interactions of these compounds in the active site ofα‐glucosidase.

2 | R E S U L T S A N D D I S C U S S I O N 2.1 | Chemistry

The designed compounds 12a–owere synthesized according to Scheme 1. First, a solution of 2‐aminobenzoic acid 1, benzoyl

chlorides 2, and triethylamine (NEt3) in ethanol was stirred at room temperature for 12 hr. After that, acetic anhydride was added to the solution, and stirring was continued for another 12 hr at room temperature to afford 2‐phenyl‐4H‐benzo[d][1,3]‐ oxazin‐4‐ones3. Compounds3easily reacted with hydrazine4at room temperature and afforded 2‐benzamidobenzohydrazides5.

In contrast, 3‐methoxy‐4‐(prop‐2‐ynyloxy)benzaldehyde 8 was prepared by the reaction between 4‐hydroxy‐3‐methoxybenzaldehyde 6and propargyl bromide7in the presence of K2CO3in dimethylformamide (DMF) at room temperature. In the next step, 2‐benzamidobenzohydrazides5reacted with 3‐methoxy‐4‐(prop‐ 2‐ynyloxy)benzaldehyde 8 in ethanol at room temperature to produce (E)‐N′(4‐(prop‐2‐ynyloxy)benzylidene)‐2‐benzamidobenzohydrazides 9. The latter compounds participated in a click reaction. For this purpose, different benzyl halide derivatives10 and sodium azide reacted in the presence of Et3N in the mixture of H2O andtert‐butyl alcohol (t‐BuOH; 1:1) at room temperature.

Then, the mixture of compounds 9, CuSO4·5H2O, and sodium ascorbate was added to the freshly prepared azide derivatives 11 and the reaction was continued at room temperature for 24–48 hr to produce the target compounds12a–o.

F I G U R E 1 Design strategy of novel Schiff base derivatives bearing 1,2,3‐triazole12a–oas newα‐glucosidase inhibitors based on molecular hybridization of pharmacophoric units of reportedα‐glucosidase inhibitorsA–D

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2.2 | Biology

2.2.1 | In vitro α‐ glucosidase inhibitory activity

The synthesized compounds12a–owere evaluated for their in vitro α‐glucosidase inhibition, in comparison to the standard drug acarbose. The obtained results are summarized in Table 1. All the synthesized compounds displayed excellentα‐glucosidase inhibitory activity with IC50values in the range of 107.1 ± 1.4 to 374.7 ± 1.3 µM, as compared with acarbose (IC50= 750.0 ± 1.5 µM).

Structurally, the synthesized compounds 12a–owere divided into two series based on the structure of 2‐benzamide moiety: unsubstituted benzamide derivatives 12a–h and 4‐methoxybenzamide derivatives 12i–o. In each series, the substituents on the pendant benzyl group were altered to optimize theα‐glucosidase inhibitory activity.

In the first series, the compound12awith a pendant unsubstituent phenyl group showed good inhibitory activity. Introduction of the

methoxy substituent as an electron‐donating group or chlorine atom as an electron‐withdrawing substituent onto the 2‐position of the pendant phenyl group led to a significant decrease in the inhibitory activity, as observed in the compounds12band12c, respectively. By comparing the IC50values of the 2‐chloro derivative 12c with the 2,3‐dichloro derivative 12d or 2,6‐dichloro derivative 12e, it could be concluded that the introduction of second chlorine atom onto the 3‐position of the pendant phenyl group significantly increased inhibitory activity. Interestingly, the 3‐chloro derivative12fshowed the most potent activity in unsubstituted benzamide series while the 2‐chloro derivative 12c was the less active halo‐substituted compound. Replacement of the 3‐chloro substituent of the compound 12fwith the 3‐bromo substituent, as in the compound12g, decreased inhibitory activity. Moreover, the translocation of bromine atom of 3‐to 4‐position of the pendant phenyl ring had no significant effect on inhibitory activity (compound12gvs.12h).

X = Cl, Br

S C H E M E 1 Reagents and conditions for the synthesis of compounds12a–o. (a) NEt3, ethanol, room temperature, 12 hr. (b) Acetic anhydride, room temperature, 12 hr. (c) Ethanol, room temperature, 12 hr. (d) K2CO3, DMF, room temperature, 3 hr. (e) Ethanol, room temperature, 24 hr. (f) NaN3, NEt3, H2O/t‐BuOH, 1 hr; (g) CuSO4·5H2O, sodium ascorbate, room temperature, 24‐48 hr. DMF, dimethylformamide;t‐BuOH,tert‐butyl alcohol

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The observed IC50 values of the second series 12i–o against α‐glucosidase revealed that the better result was obtained with the 3‐chloro substituent (compound12n). Similar to the unsubstituted benzamide series, the 2,3‐dichloro derivative 12lwas more active than the 2‐chloro derivative 12kwhile the 2,6‐dichloro derivative 12m, unlike the first series, was less active than the 2‐chloro derivative 12k. The presence of electron‐donating methyl or an electron‐withdrawing bromo substituent instead of hydrogen on the 4‐position of the pendant benzyl group led to the increase of inhibitory activity, as demonstrated in the compounds12i,12j, and 12oin the order of Br > CH3> H.

The comparison of IC50values of the first series derivatives with their analogs of the second series revealed that the introduction of a methoxy group onto the 4‐position of 2‐benzamide led to a significant increase in inhibitory activity againstα‐glucosidase (Table 1).

2.2.2 | Enzyme kinetic study

To gain further insight into the mechanism of action of the Schiff bases bearing 1,2,3‐triazole12a–o, a kinetic study of the compound12nas the most potent compound against α‐glucosidase was performed (Figure 2). This study revealed that no change was observed inVmax

12b H 2‐OCH3 374.7 ± 1.3 12j OCH3 4‐CH3 154.7 ± 1.8

12c H 2‐Cl 275.7 ± 1.5 12k OCH₃ 2‐Cl 189.2 ± 0.7

12d H 2,3‐Cl₂ 160.0 ± 1.7 12l OCH₃ 2,3‐Cl₂ 147.5 ± 0.8

12e H 2,6‐Cl2 243.0 ± 1.2 12m OCH3 2,6‐Cl2 204.5 ± 1.0

12f H 3‐Cl 122.0 ± 1.5 12n OCH₃ 3‐Cl 107.1 ± 1.4

12g H 3‐Br 217.0 ± 1.2 12o OCH₃ 4‐Br 131.6 ± 1.3

12h H 4‐Br 209.0 ± 1.2 Acarbose – – 750.0 ± 1.5

aValues are the mean ± SD. All experiments were performed at least three times

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F I G U R E 2 Kinetics ofα‐glucosidase inhibition by compound12n. (a) The Lineweaver–Burk plot in the absence and presence of different concentrations of compound12n. (b) The secondary plot betweenKmand various concentrations of compound12n

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values by increasing the concentration of the compound12nwhileKm

values increased (Figure 2a). Therefore, this compound was a competitive inhibitor againstα‐glucosidase. On the basis of the obtained results, theKivalue of the compound12nwas 113μM (Figure 2b).

2.3 | Docking study

To expose the interaction modes of the synthesized compounds and Saccharomyces cerevisiaeα‐glucosidase enzyme at the molecular level, a docking study was performed by AutoDock Tools (version 1.5.6). Since, the crystallographic structure of this form of enzyme has not been so far published, a homology model of S. cerevisiae α‐glucosidase was built according to previous research.^[27]The superposed structures of acarbose (the standard inhibitor) and the most potent compounds12nand12fin the active site of α‐glucosidase are shown in Figure 3. Acarbose as a standard drug with binding energy =−4.04 kcal/mol interacted with

residues Asn241, His279, Thr301, Pro309, Ser308, Glu304, Thr307, Arg312, and Gln322 in this modeledα‐glucosidase.^[27]

The theoretical binding mode between the most potent compound 12n and α‐glucosidase is shown in Figure 4a. The NH unit of the compound12nformed three hydrogen bonds with Glu304 (2.634 Å), Thr307 (2.272 Å), and Ser308 (2.733 Å) and nitrogen atom of the 1,2,3‐triazole ring established a hydrogen bond with Arg312 (2.141 Å). A π–anion interaction can be observed between the 3‐chlorophenyl group and Asp408. Furthermore, the compound12n showed several weak hydrophobic interactions with residues Thr301, Val305, Pro309, and Arg312.

Elimination of the 4‐methoxy substituent of the 2‐benzamide moiety from the compound12n, as in the compound12f, changed the interaction mode (Figure 4a vs. 4b). The NH unit of the compound12f, unlike the compound12n, could not interact with the active site of enzyme. In the case of the compound12f, a hydrogen bond can be observed between nitrogen atom of the 1,2,3‐triazole ring and Arg312 (2.071 Å). It is worthy F I G U R E 3 Acarbose (cyan) and the most potent compounds12n(pink) and12f(yellow) superimposed in the active site pocket

F I G U R E 4 The interaction modes of compounds (a)12nand (b)12fin the active site pocket

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3 | C O N C L U S I O N

In summary, a series of new Schiff base derivatives bearing 1,2,3‐triazole12a–owas synthesized and evaluated asα‐glucosidase inhibitors. In vitro assay revealed that all the synthesized compounds were more potent than the standard drug acarbose. These compounds showed a two‐to sevenfold higher activity than acarbose. The kinetic study of the most potent compound 12n demonstrated that this compound could inhibitα‐glucosidase in a competitive manner. Docking simulations of the synthesized compounds confirmed that they well interacted with the active site residues ofα‐glucosidase.

4 | E X P E R I M E N T A L 4.1 | Chemistry 4.1.1 | General

Melting points of the target compounds12a–owere measured on a Kofler hot stage apparatus and were uncorrected.¹H and¹³C nuclear magnetic resonance (NMR) spectra of the synthesized compounds were determined on a Bruker FT‐500, using TMS as an internal standard. IR spectra were recoded using KBr disks on a Nicolet Magna FTIR 550 spectrophotometer. Elemental analysis was carried out with an Elementar Analysen System GmbH VarioEL CHN mode.

The NMR spectra of the investigated compounds are provided as Supporting Information. The InChI codes of the investigated compounds together with some biological activity data are also provided as Supporting Information.

4.1.2 | General procedure for the synthesis of 2 ‐ phenyl ‐ 4H ‐ benzo[d][1,3]oxazin ‐ 4 ‐ ones 3

A solution of 2‐aminobenzoic acid 1 (1 mmol), benzoyl chlorides 2 (1 mmol), and NEt3 (1 mmol) in ethanol (15 ml) was stirred at room temperature for 12 hr. Then, acetic anhydride (3 ml) was added to the solution, and stirring was continued at room temperature for a further

‐

4.1.4 | General procedure for the synthesis of 3 ‐ methoxy ‐ 4 ‐ (prop ‐ 2 ‐ ynyloxy)benzaldehyde 8

A suspension of 4‐hydroxy‐3‐methoxybenzaldehyde6(1 mmol) and K2CO3(1 mmol) in DMF (5 ml) was stirred at room temperature for 1 hr. Then, it was added to a solution of propargyl bromide 7 (1.2 mmol) in DMF (10 ml) in a dropwise manner and the reaction mixture was stirred at room temperature for 2 hr. Subsequently, the reaction mixture was poured into crushed ice and the obtained precipitate was filtered off. The precipitate was then recrystallized in ethanol to obtain 3‐methoxy‐4‐(prop‐2‐ynyloxy)benzaldehyde8.^[30]

4.1.5 | General procedure for the synthesis of (E) ‐ N ′‐ (4 ‐ (prop ‐ 2 ‐ ynyloxy)benzylidene) ‐ 2 ‐ benzamidobenzohydrazides 9

A mixture of 2‐benzamidobenzohydrazides5(1 mmol) and 3‐methoxy‐4‐ (prop‐2‐ynyloxy)benzaldehyde8(1 mmol) was stirred at room temperature for 24 hr. Then, it was poured into crushed ice and the participated product was filtered off and recrystallized in ethanol to obtain pure (E)‐ N′‐(4‐(prop‐2‐ynyloxy)benzylidene)‐2‐benzamidobenzohydrazides9.

4.1.6 | General procedure for the synthesis of compounds 12a – o

At first, benzyl azide derivatives 11 were prepared in situ.

For this purpose, a solution of benzyl halides 10 (1.1 mmol), sodium azide (0.9 mmol), and Et3N (1.3 mmol) in the mixture of water/t‐BuOH (8 ml, 1:1) was stirred at room temperature for 1 hr.

Subsequently, the mixture of (E)‐N′‐(4‐(prop‐2‐ynyloxy)benzylidene)‐ 2‐benzamidobenzohydrazides 9 (1 mmol), CuSO4·5H2O (7 mol %), and sodium ascorbate was added to the freshly prepared benzyl azide derivatives11and stirred at room temperature for 24–48 hr.

Upon completion of the reaction (monitored by thin‐layer chromato- graphy), the obtained reaction mixture was diluted with cold water and poured into crushed ice. Then, the precipitated products12a–o were filtered off, washed with cold water, and purified by recrystallization in ethanol.

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(E)‐N′‐(4‐((1‐Benzyl‐1H‐1,2,3‐triazol‐4‐yl)methoxy)‐3‐methoxybenzylidene)‐2‐benzamidobenzohydrazide (12a)

Pale yellow crystals; yield, 72%; mp, 196–198°C. IR (KBr, cm⁻¹):

3312, 3302, 3103, 1653, 1644. ¹H NMR (400 MHz, deuterated dimethyl sulfoxide [DMSO‐d6]):δ3.80 (s, 3H, OMe), 5.19 (s, 2H, CH2), 5.62 (s, 2H, CH2), 7.23 (s, 2H), 7.27–7.29 (m, 1H), 7.31–7.40 (m, 6H), 7.58–7.65 (m, 4H), 7.89 (d, J= 7.2 Hz, 1H), 7.96 (d,J= 6.8 Hz, 2H), 8.30 (s, 1H), 8.39 (s, 1H), 8.56 (d,J= 8 Hz, 1H), 11.92 (s, 1H, NH), 12.02 (s, 1H, NH). ¹³C NMR (100 MHz, DMSO‐d6):δ53.30, 55.94, 62.07, 108.93, 113.59, 121.17, 151.53, 122.53, 123.63, 125.43, 127.52, 127.61, 128.46, 128.65, 129.07, 129.25, 129.41, 132.58, 132.96, 134,89, 136.45, 139.69, 143.10, 149.73, 149.76, 150.07, 165.02, 165.20. Analytically calculated (Anal. calcd.) for C32H28N6O4: C, 68.56; H, 5.03; N, 14.99. Found: C, 65.39; H, 5.16; N, 15.08.

(E)‐N′‐(4‐((1‐(2‐Methoxybenzyl)‐1H‐1,2,3‐triazol‐4‐yl)methoxy)‐3‐ methoxybenzylidene)‐2‐benzamidobenzohydrazide (12b)

3311, 3308, 3098, 1651, 1646.¹H NMR (400 MHz, DMSO‐d6):δ3.73 (s, 3H, OMe), 3.80 (s, 3H, OMe), 5.19 (s, 2H, CH2), 5.58 (s, 2H, CH2), 6.87–6.90 (m, 2H), 6.91 (s, 1H), 7.23 (s, 2H), 7.27–7.31 (m, 2H), 7.38 (s, 1H), 7.58–7.65 (m, 4H), 7.89 (d,J= 7.2 Hz, 1H), 7.96 (d,J= 6.8 Hz, 2H), 8.30 (s, 1H), 8.38 (s, 1H), 8.55 (d,J= 8 Hz, 1H), 11.91 (s, 1H, NH), 12.01 (s, 1H, NH). ¹³C NMR (100 MHz, DMSO‐d6):δ53.21, 55.53, 55.58, 62.06, 108.94, 113.57, 113.98, 114.21, 120.52, 121.18, 121.53, 122.53, 123.63, 125.46, 127.51, 129.06, 129.41, 130.25, 130.40, 132.58, 132.96, 134.89, 137.88, 139.69, 143.09, 149.76, 150.06, 159.90, 165.02, 165.19. Anal. calcd. for C33H30N6O5: C, 67.11; H, 5.12; N, 14.23. Found: C, 67.21; H, 5.03; N, 14.19.

(E)‐N′‐(4‐((1‐(2‐Chlorobenzyl)‐1H‐1,2,3‐triazol‐4‐yl)methoxy)‐3‐ methoxybenzylidene)‐2‐benzamidobenzohydrazide (12c)

3311, 3309, 3109, 1649, 1641. ¹H NMR (400 MHz, DMSO‐d6):δ 3.80 (s, 3H, OMe), 5.19 (s, 2H, CH2), 5.73 (s, 2H, CH2), 7.23 (s, 2H), 7.24–7.28 (m, 2H), 7.36–7.41 (m, 3H), 7.53 (d, 1H), 7.58–7.65 (m, 4H), 7.89 (d,J= 6.8 Hz, 1H), 7.95 (d,J= 8.4 Hz, 2H), 8.27 (s, 1H), 8.38 (s, 1H), 8.54 (d,J= 8 Hz, 1H), 11.90 (s, 1H, NH), 12.03 (s, 1H, NH).¹³C NMR (100 MHz, DMSO‐d6):δ51.09, 55.96, 62.00, 108.96, 113.66, 121.21, 121.57, 122.54, 123.67, 125.86, 127.51, 127.63, 128.22, 129.05, 129.41, 130.12, 130.79, 131.05, 132.60, 132.97, 133.12, 133.65, 134.86, 139.64, 142.94, 149.75, 149.77, 150.03, 165.05, 165.23. Anal. calcd. for C32H27ClN6O4: C, 64.59; H, 4.57; N, 14.12.

Found: C, 64.75; H, 4.62; N, 14.25.

(E)‐N′‐(4‐((1‐(2,3‐Dichlorobenzyl)‐1H‐1,2,3‐triazol‐4‐yl)methoxy)‐3‐ methoxybenzylidene)‐2‐benzamidobenzohydrazide (12d)

3308, 3307, 3105, 1645, 1643. ¹H NMR (400 MHz, DMSO‐d6):δ 3.81 (s, 3H, OMe), 5.21 (s, 2H, CH2), 5.79 (s, 2H, CH2), 7.18 (d, J= 7.6 Hz, 1H), 7.23 (s, 2H), 7.27–7.30 (m, 1H), 7.36 (s, 1H), 7.38–7.42 (m, 1H), 7.58–7.65 (m, 3H), 7.67 (d,J= 7.2 Hz, 2H), 7.90 (d,J= 8 Hz, 1H), 7.96 (d, J= 7.2 Hz, 2H), 8.31 (s, 1H), 8.39 (s, 1H), 8.56 (d,

J= 8.4 Hz, 1H), 11.93 (s, 1H, NH), 12.03 (s, 1H, NH). C NMR (100 MHz, DMSO‐d6):δ51.63, 55.91, 62.04, 108.98, 113.71, 121.16, 121.53, 122.52, 123.62, 126.01, 127.51, 127.68, 129.07, 129.12, 129.40, 129.48, 131.06, 131.22, 132.58, 132.70, 132.96, 134.89, 136.31, 139.70, 143.03, 149.73, 149.80, 150.03, 165.02, 165.22.

Anal. calcd. for C32H26Cl2N6O4: C, 61.06; H, 4.16; N, 13.35. Found: C, 61.16; H, 4.29; N, 13.43.

(E)‐N′‐(4‐((1‐(2,6‐Dichlorobenzyl)‐1H‐1,2,3‐triazol‐4‐yl)methoxy)‐3‐ methoxybenzylidene)‐2‐benzamidobenzohydrazide (12e)

3316, 3306, 3107, 1649, 1640. ¹H NMR (400 MHz, DMSO‐d6): δ 3.80 (s, 3H, OMe), 5.17 (s, 2H, CH2), 5.82 (s, 2H, CH2), 7.23 (s, 2H), 7.25–7.29 (m, 1H), 7.36 (s, 1H), 7.46–7.50 (m, 1H), 7.58–7.65 (m, 6H), 7.90 (d,J= 8 Hz, 1H), 7.95 (d,J= 8 Hz, 2H), 8.27 (s, 1H), 8.39 (s, 1H), 8.56 (d,J= 8 Hz, 1H), 11.92 (s, 1H, NH), 12.02 (s, 1H, NH).¹³C NMR (100 MHz, DMSO‐d6):δ49.09, 55.94, 61.89, 108.93, 113.55, 121.16, 121.52, 122.51, 123.62, 125.73, 127.51, 127.61, 129.07, 129.40, 129.47, 130.80, 132.15, 132.58, 132.95, 134.89, 136.40, 139.70, 142.61, 149.73, 150.07, 165.02, 165.21. Anal. calcd. for C32H26Cl2N6O4: C, 61.06; H, 4.16; N, 13.35. Found: C, 60.94; H, 4.05; N, 13.27.

(E)‐N′‐(4‐((1‐(3‐Chlorobenzyl)‐1H‐1,2,3‐triazol‐4‐yl)methoxy)‐3‐ methoxybenzylidene)‐2‐benzamidobenzohydrazide (12f)

3313, 3308, 3109, 1653, 1643.¹H NMR (400 MHz, DMSO‐d6):δ3.81 (s, 3H, OMe), 5.20 (s, 2H, CH2), 5.65 (s, 2H, CH2), 7.23 (s, 2H), 7.36 (s, 1H), 7.40–7.42 (m, 3H), 7.58–7.65 (m, 4H), 7.89 (d,J= 7.2 Hz, 1H), 7.96 (d,J= 8 Hz, 2H), 8.35 (s, 1H), 8.39 (s, 1H), 8.56 (d,J= 8.4 Hz, 1H), 11.93 (s, 1H, NH), 12.02 (s, 1H, NH).¹³C NMR (100 MHz, DMSO‐d6):

δ 52.51, 55.96, 62.07, 108.96, 113.62, 121.20, 121.52, 122.52, 123.62, 125.61, 127.21, 128.36, 128.65, 129.07, 129.41, 131.20, 132.58, 132.95, 133.75, 134.90, 138.83, 139.70, 143.21, 149.72, 149.78, 150.03, 165.03, 165.21. Anal. calcd. for C32H27ClN6O4: C, 64.59; H, 4.57; N, 14.12. Found: C, 64.41; H, 4.66; N, 14.20.

(E)‐N′‐(4‐((1‐(3‐Bromobenzyl)‐1H‐1,2,3‐triazol‐4‐yl)methoxy)‐3‐ methoxybenzylidene)‐2‐benzamidobenzohydrazide (12g)

3311, 3307, 3106, 1659, 1646.¹H NMR (400 MHz, DMSO‐d6):δ3.79 (s, 3H, OMe), 5.19 (s, 2H, CH2), 5.64 (s, 2H, CH2), 7.22 (s, 2H), 7.25–7.28 (m, 1H), 7.30–7.37 (m, 3H), 7.53–7.63 (m, 6H), 7.90 (d, J= 7.6 Hz, 1H), 7.96 (d,J= 7.2 Hz, 2H), 8.35 (s, 1H), 8.39 (s, 1H), 8.55 (d,J= 8.4 Hz, 1H), 11.92 (s, 1H, NH), 12.04 (s, 1H, NH). ¹³C NMR (100 MHz, DMSO‐d6):δ52.47, 55.87, 62.03, 108.95, 113.58, 121.15, 121.55, 122.33, 122.59, 123.65, 125.60, 127.51, 127.59, 127.63, 129.04, 129.40, 131.21, 131.46, 131.55, 132.59, 132.98, 134.85, 139.03, 139.67, 143.21, 149.76, 150.02, 165.04, 165.25. Anal. calcd.

for C32H27BrN6O4: C, 60.10; H, 4.26; N, 13.14. Found: C, 60.16; H, 4.35; N, 13.05.

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(E)‐N′‐(4‐((1‐Benzyl‐1H‐1,2,3‐triazol‐4‐yl)methoxy)‐3‐methoxybenzylidene)‐2‐(4‐methoxybenzamido)benzohydrazide (12i)

3311, 3309, 3114, 1657, 1645. ¹H NMR (400 MHz, DMSO‐d6):δ 2.40 (s, 3H, OMe), 3.80 (s, 3H, OMe), 5.19 (s, 2H, CH2), 5.62 (s, 2H, CH2), 7.23 (s, 2H), 7.25–7.29 (m, 1H), 7.32–7.41 (m, 8H), 7.36 (s, 1H), 7.60–7.64 (m, 1H), 7.85 (d,J= 8 Hz, 2H), 7.90 (d,J= 8 Hz, 1H), 8.30 (s, 1H), 8.39 (s, 1H), 8.57 (d,J= 8 Hz, 1H), 11.91 (s, 1H, NH), 12.01 (s, 1H, NH). ¹³C NMR (100 MHz, DMSO‐d6):δ21.49, 53.30, 55.93, 62.08, 108.92, 113.58, 120.90, 121.39, 122.55, 123.44, 125.43, 127.53, 127.63, 128.46, 128.64, 129.05, 129.24, 129.92, 132.09, 132.96, 136.45, 139.86, 142.69, 143.11, 149.76, 150.07, 164.90, 165.26.

Anal. calcd. for C33H30N6O5: C, 67.11; H, 5.12; N, 14.23. Found: C, 67.22; H, 5.21; N, 14.16.

(E)‐N′‐(4‐((1‐(4‐Methylbenzyl)‐1H‐1,2,3‐triazol‐4‐yl)methoxy)‐3‐ methoxybenzylidene)‐2‐(4‐methoxybenzamido)benzohydrazide (12j) Pale yellow crystals; yield, 81%; mp, 207–209°C. IR (KBr, cm⁻¹):

3313, 3310, 3108, 1660, 1641.¹H NMR (400 MHz, DMSO‐d6):δ2.28 (s, 3H, CH3), 2.40 (s, 3H, OMe), 3.80 (s, 3H, OMe), 5.18 (s, 2H, CH2), 5.56 (s, 2H, CH2), 7.18 (d,J= 8 Hz, 2H), 7.19–7.29 (m, 4H), 7.36 (s, 1H), 7.40 (d,J= 8 Hz, 2H), 7.60–7.64 (m, 1H), 7.85 (d,J= 8 Hz, 2H), 7.89 (d,J= 8 Hz, 1H), 8.26 (s, 1H), 8.39 (s, 1H), 8.57 (d,J= 8 Hz, 1H), 11.90 (s, 1H, NH), 12.02 (s, 1H, NH).¹³C NMR (100 MHz, DMSO‐d6):

δ21.18, 21.52, 53.10, 55.88, 62.06, 108.92, 113.58, 116.82, 120.91, 121.40, 122.53, 123.45, 125.28, 127.53, 127.61, 128.51, 129.77, 129.93, 132.09, 132.97, 133.44, 138.00, 139.85, 142.70, 143.06, 149.76, 150.07, 164.90, 165.25. Anal. calcd. for C34H32N6O5: C, 67.54; H, 5.33; N, 13.90. Found: C, 67.39; H, 5.41; N, 14.01.

(E)‐N′‐(4‐((1‐(2‐Chlorobenzyl)‐1H‐1,2,3‐triazol‐4‐yl)methoxy)‐3‐ methoxybenzylidene)‐2‐(4‐methoxybenzamido)benzohydrazide (12k) Pale yellow crystals; yield, 76%; mp, 211–213°C. IR (KBr, cm⁻¹):

3317, 3306, 3102, 1656, 1649.¹H NMR (400 MHz, DMSO‐d6):δ2.39 (s, 3H, OMe), 3.80 (s, 3H, OMe), 5.20 (s, 2H, CH2), 5.73 (s, 2H, CH2), 7.23 (s, 2H), 7.24–7.28 (m, 2H), 7.35–7.42 (m, 5H), 7.46–7.50 (m, 1H), 7.52 (d,J= 8.8 Hz, 1H), 7.60–7.64 (m, 1H), 7.85 (d,J= 8 Hz, 2H), 7.89 (d,J= 8 Hz, 1H), 8.27 (s, 1H), 8.38 (s, 1H), 8.56 (d,J= 8 Hz, 1H), 11.90 (s, 1H, NH), 12.03 (s, 1H, NH). ¹³C NMR (100 MHz, DMSO‐d6):δ

– –

7.58–7.65 (m, 3H), 7.67 (d,J= 7.2 Hz, 2H), 7.86 (d,J= 8 Hz, 2H), 7.95 (d,J= 7.2 Hz, 1H), 8.30 (s, 1H), 8.38 (s, 1H), 8.55 (d,J= 8 Hz, 1H), 11.90 (s, 1H, NH), 12.03 (s, 1H, NH).¹³C NMR (100 MHz, DMSO‐d6):

δ 51.64, 55.96, 62.00, 108.96, 113.69, 121.20, 121.43, 122.56, 123.66, 126.02, 127.53, 127.66, 129.03, 129.13, 129.41, 129.52, 131.08, 131.24, 132.60, 132.70, 132.97, 134.85, 136.27, 139.79, 143.01, 149.75, 149.79, 150.02, 165.05, 165.29. Anal. calcd. for C33H28Cl2N6O5: C, 60.10; H, 4.28; N, 12.74. Found: C, 60.18;

H, 4.31; N, 12.83.

(E)‐N′‐(4‐((1‐(2,6‐Dichlorobenzyl)‐1H‐1,2,3‐triazol‐4‐yl)methoxy)‐3‐ methoxybenzylidene)‐2‐(4‐methoxybenzamido)benzo-

hydrazide (12m)

3312, 3303, 3105, 1659, 1643.¹H NMR (400 MHz, DMSO‐d6):δ2.39 (s, 3H, OMe), 3.80 (s, 3H, OMe), 5.16 (s, 2H, CH2), 5.82 (s, 2H, CH2), 7.23 (s, 2H), 7.25–7.28 (m, 1H), 7.37 (s, 1H), 7.39 (d,J= 8 Hz, 2H), 7.46–7.50 (m, 1H), 7.58 (d,J= 7.6 Hz, 2H), 7.59–7.64 (m, 1H), 7.85 (d, J= 8 Hz, 2H), 7.88 (d,J= 6.8 Hz, 1H), 8.27 (s, 1H), 8.38 (s, 1H), 8.55 (d, J= 8 Hz, 1H), 11.89 (s, 1H, NH), 12.03 (s, 1H, NH). ¹³C NMR (100 MHz, DMSO‐d6):δ21.51, 49.08, 55.87, 61.85, 108.91, 113.52, 120.92, 121.42, 122.56, 123.49, 125.75, 127.53, 127.60, 129.03, 129.47, 129.93, 130.77, 132.05, 132.16, 132.98, 136.40, 139.80, 142.60, 142.73, 149.72, 149.75, 150.06, 164.93, 165.28. Anal. calcd.

for C33H28Cl2N6O5: C, 60.10; H, 4.28; N, 12.74. Found: C, 60.01; H, 4.19; N, 12.86.

(E)‐N′‐(4‐((1‐(3‐Chlorobenzyl)‐1H‐1,2,3‐triazol‐4‐yl)-

methoxy)‐3‐methoxybenzylidene)‐2‐(4‐methoxybenzamido)benzohydrazide (12n)

3307, 3306, 3115, 1664, 1645.¹H NMR (400 MHz, DMSO‐d6):δ2.39 (s, 3H, OMe), 3.81 (s, 3H, OMe), 5.20 (s, 2H, CH2), 5.65 (s, 2H, CH2), 7.23 (d, 2H), 7.27–7.30 (m, 2H), 7.37 (s, 1H), 7.39–7.42 (m, 5H), 7.60–7.64 (m, 1H), 7.86 (d,J= 8 Hz, 2H), 7.90 (d,J= 8 Hz, 1H), 8.35 (s, 1H), 8.39 (s, 1H), 8.57 (d,J= 8 Hz, 1H), 11.92 (s, 1H, NH), 12.02 (s, 1H, NH).¹³C NMR (100 MHz, DMSO‐d6):δ21.48, 52.51, 55.94, 62.07, 108.92, 113.61, 120.91, 121.39, 122.55, 123.44, 125.59, 127.20, 127.53, 127.66, 128.35, 128.64, 129.05, 129.92, 131.19, 132.10,

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132.96, 133.76, 138.82, 139.86, 142.69, 143.21, 149.73, 149.78, 150.03, 164.90, 165.27. Anal. calcd. for C33H29ClN6O5: C, 63.41;

H, 4.68; N, 13.44. Found: C, 63.52; H, 4.56; N, 13.62.

(E)‐N′‐(4‐((1‐(4‐Bromobenzyl)‐1H‐1,2,3‐triazol‐4‐yl)methoxy)‐ 3‐methoxybenzylidene)‐2‐(4‐methoxybenzamido)benzohydrazide (12o)

Pale yellow crystals; yield, 78%; mp, 222–226°C; IR (KBr, cm⁻¹):

3319, 3305, 3109, 1668, 1646.¹H NMR (400 MHz, DMSO‐d6):δ2.39 (s, 3H, OMe), 3.81 (s, 3H, OMe), 5.19 (s, 2H, CH2), 5.61 (s, 2H, CH2), 7.23 (s, 2H), 7.25–7.30 (m, 3H), 7.37 (s, 1H), 7.40 (d,J= 8 Hz, 2H), 7.59 (d,J= 8 Hz, 2H), 7.61–7.64 (m, 1H), 7.86 (d,J= 8 Hz, 1H), 7.90 (d, J= 8 Hz, 2H), 8.31 (s, 1H), 8.39 (s, 1H), 8.57 (d,J= 8 Hz, 1H), 11.91 (s, 1H, NH), 12.02 (s, 1H, NH).¹³C NMR (100 MHz, DMSO‐d6):δ21.52, 52.54, 55.94, 62.07, 108.94, 113.60, 120.90, 121.39, 121.94, 122.53, 123.44, 125.49, 127.53, 127.65, 129.04, 129.92, 130.72, 132.09, 132.18, 132.96, 135.85, 139.86, 142.69, 143.16, 179.77, 150.05, 164.90, 165.26. Anal. calcd. for C33H29BrN6O5: C, 59.20;

H, 4.37; N, 12.55. Found: C, 59.16; H, 4.42; N, 12.63.

4.2 | Biological assays

4.2.1 | α‐ Glucosidase inhibition assay

α‐Glucosidase (EC3.2.1.20, S. cerevisiae, 20 U/mg) and p‐nitro- phenyl‐α‐^D‐glucoside (PNP‐α‐^D‐Glc) as substrate were purchased from Sigma‐Aldrich. An enzyme solution was prepared in potassium phosphate buffer (pH 6.8 and 50 mM), and the synthesized compounds 12a–o were dissolved in DMSO (10%

final concentration). Various concentrations of these compounds (20 ml), prepared enzyme (20 ml), and buffer (135 ml) were added to a 96‐well plate and incubated at 37°C for 10 min. Then, PNP‐α‐^D‐Glc (25 ml, 4 mM) was added to the plate and allowed to incubate at 37°C for 20 min. The change in absorbance was measured at 405 nm by using a spectrophotometer (Gen5, PowerWave XS2, BioTek). DMSO and acarbose were used respectively as a control and standard inhibitor. The percentage of enzyme inhibition was determined, and IC50 values were obtained by using the Logit method.^[24]

4.2.2 | Kinetic study

The inhibition mode of the synthesized compounds against α‐glucosidase was investigated. In this regard, the enzyme solution (1 U/ml) was incubated with the most potent compound 12nat 0, 70, 90, and 110 µM for 15 min at 30°C. The reaction was then initiated by adding different concentrations of PNP‐α‐^D‐Glc (1–10 mM) as substrate, and change in absorbance was measured for 20 min at 405 nm by using spectrophotometer (Gen5, PowerWave XS2, BioTek). A Lineweaver–Burk plot was generated to identify the type of inhibition and the Michaelis–Menten constant (Km) value was determined from the plot between reciprocal of the substrate concentration (1/[S]) and reciprocal of the enzyme rate (1/V) over various inhibitor

concentrations. The experimental inhibitor constant (Ki) value was constructed by secondary plots of the inhibitor concentration [I] versusKm.

4.3 | Docking study

Building the homology model ofα‐glucosidase and docking study of the most potent compounds 12nand12fin the active site of the modeledα‐glucosidase was performed by AutoDock Tools, according to the previously described method.^[27]

O R C I D

Mohammad Mahdavi http://orcid.org/0000-0002-4171-7310

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