Next Article in Journal
Emerging Applications of Nanotechnology in Healthcare and Medicine
Previous Article in Journal
Hexamethyldisiloxane Removal from Biogas Using a Fe3O4–Urea-Modified Three-Dimensional Graphene Aerogel
Previous Article in Special Issue
Green Synthesis, Characterization, Antioxidant, Antibacterial and Enzyme Inhibition Effects of Chestnut (Castanea sativa) Honey-Mediated Silver Nanoparticles
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 28
Issue 18
10.3390/molecules28186623
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

A Combined Experimental and Computational Study of Novel Benzotriazinone Carboxamides as Alpha-Glucosidase Inhibitors

by
Zunera Khalid
1,2,
Syed Salman Shafqat
3,*,
Hafiz Adnan Ahmad
2,
Munawar Ali Munawar
2,4,*,
Sadaf Mutahir
5,
Safaa M. Elkholi
6,
Syed Rizwan Shafqat
7,
Rahila Huma
1 and
Abdullah Mohammed Asiri
8
1
Department of Chemistry, Kinnaird College for Women, Lahore 54000, Pakistan
2
School of Chemistry, University of the Punjab, Lahore 54590, Pakistan
3
Department of Chemistry, Division of Science and Technology, University of Education, Lahore 54770, Pakistan
4
Department of Basic and Applied Chemistry, Faculty of Science and Technology, University of Central Punjab, Lahore 54000, Pakistan
5
School of Chemistry and Chemical Engineering, Linyi University, Linyi 276000, China
6
Department of Rehabilitation Sciences, College of Health and Rehabilitation Sciences, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia
7
Department of Chemistry, University of Sialkot, Sialkot 51310, Pakistan
8
Chemistry Department, Faculty of Science, King Abdulaziz University, Jeddah 64274, Saudi Arabia
*
Authors to whom correspondence should be addressed.
Molecules 2023, 28(18), 6623; https://doi.org/10.3390/molecules28186623
Submission received: 29 May 2023 / Revised: 26 June 2023 / Accepted: 3 July 2023 / Published: 14 September 2023
(This article belongs to the Special Issue Design, Synthesis and Structure-Activity Relationship Studies of Enzyme Inhibitors)
Browse Figures
Versions Notes

Abstract

:
Diabetes is a chronic metabolic disorder of the endocrine system characterized by persistent hyperglycemia appears due to the deficiency or ineffective use of insulin. The glucose level of diabetic patients increases after every meal and medically recommended drugs are used to control hyperglycemia. Alpha-glucosidase inhibitors are used as antidiabetic medicine to delay the hydrolysis of complex carbohydrates. Acarbose, miglitol, and voglibose are commercial drugs but patients suffer side effects of flatulence, bloating, diarrhea, and loss of hunger. To explore a new antidiabetic drug, a series of benzotriazinone carboxamides was synthesized and their alpha-glucosidase inhibition potentials were measured using in vitro experiments. The compounds 14k and 14l were found to be strong inhibitors compared to the standard drug acarbose with IC50 values of 27.13 ± 0.12 and 32.14 ± 0.11 μM, respectively. In silico study of 14k and 14l was carried out using molecular docking to identify the type of interactions developed between these compounds and enzyme sites. Both potent compounds 14k and 14l exhibited effective docking scores by making their interactions with selected amino acid residues. Chemical hardness and orbital energy gap values were investigated using DFT studies and results depicted affinity of 14k and 14l towards biological molecules. All computational findings were found to be in good agreement with in vitro results.

1. Introduction

Diabetes is a multifactorial metabolic chronic disease characterized by hyperglycemia. It is prevalent in all continents and affects an individual’s life with no distinction of age and gender [1,2]. Previously, it was known as an adult-onset disease, but in the last two decades, many children and teenagers have been reported as victims of this ailment [3]. According to a World Health Organization report, nearly 250 million people suffer from this disease and this population will increase to 366 million by 2030 [4]. This continuous increase in diabetic patients is an alarming situation for the world [5,6]. Early indications of diabetes are frequent urination, excessive thirst, shortness of breath, and nausea but continued uncontrolled glucose levels cause complications such as vascular syndrome, retinopathy, cardiomyopathy, neuropathy, and nephropathy [3,7,8]. Alpha-glucosidase inhibitor drugs are used to suppress the digestion and absorption of carbohydrates [9]. Acarbose (Glucobay, Precose, Prandase), voglibose (Basen), and miglitol (Glyset) are famous alpha-glucosidase inhibitor drugs, but they induce side effects such as bloating, flatulence, diarrhea, and loss of appetite. The synthetic route of marketed drugs is a long process and chemists are working to discover new class of alpha-glucosidase inhibitors [10].
Heterocyclic compounds are widely used as pharmaceutical drugs and 1,2,3-benzotriazin-4(3H)-one is one of the emerging nuclei for use as a bioactive scaffold. Previously, several analogues of benzotriazinone 1 have been reported as antibacterial 2 [11], anticonvulsant 3 [12], anti-inflammatory 4 [13], anticancer 5 [14], and anti-HIV 6 [15] (Figure 1). Moreover, different benzotriazinone derivatives have been studied for their inhibition potency against hydrolase aminopeptidase [16], acetylcholinesterase [17], chorismate mutase [18], HepG2 [14], 4-hydroxyphenylpyruvate dioxygenase [19], and matrix metalloprotease [5]. Carboxamide is a popular pharmaceutical due to its diverse spectrum of activities. Carbamazepine [20], oxcarbazepine [21], rufinamide [22], and meloxicam [23] are recognized carboxamide drugs. Nateglinide and anagliptin are commercial antidiabetic pills [24,25]. In the recent literature, compounds like acridine-9-carboxamide and 6-amino-pyrazolo[1,5-a]pyrimidine-3-carboxamide have been explored as effective alpha-glucosidase inhibitors [9,26].
Recent trends in medicinal research include molecular hybridization of two or more enzyme inhibitors to synthesize new, effective drugs [27,28]. This methodology has been used to prepare influential hybrids of benzotriazinone with other bioactive moieties. Our group is working on the discovery of new alpha-glucosidase inhibitors with better therapeutic activity. In this regard, two series of sulfonamides were synthesized in previous work and their inhibition potential was measured [5,29]. Among previously studied analogues, compounds 7 and 8 (Figure 2) were found to be most potent inhibitors compared to the commercial drug acarbose. In further continuation of work to develop better antidiabetic drugs, a series of benzotriazinone carboxamide derivatives (14a–14n) were prepared and evaluated for their enzyme inhibition potential. Molecular docking studies were also utilized to explain in vitro studies through ligand binding inside enzyme cavity. Electronic parameters of all newly synthesized hybrids were analyzed using DFT studies to explore structure competence in interaction with biomolecules.

2. Result and Discussion

2.1. Chemistry

A series of N-alkyl/phenyl-4-(4-oxobenzo[1,2,3]triazin-3(4H)-yl)butanamides (14a–14n) were synthesized at room temperature by utilizing isatin 9 as an inexpensive starting material. In the first step, hydrogen peroxide and formic acid were applied for the oxidation of 9 to isatoic anhydride 10. Afterwards, 4-aminobutyric acid was taken in a water: triethylamine mixture and treated with anhydride 10 to afford N-(carboxybutyl)anthranilamide 11 that was subsequently diazotized with NaNO2/HCl solution to formulate 4-(4-oxobenzo[1,2,3]triazin-3(4H)-yl)butanoic acid 12 (Scheme 1). Further, it was reacted with benzotriazole and thionyl chloride to prepare the main precursor 13 and was used with different amines or anilines to afford different carboxamide derivatives 14a–14n (Table 1).

2.2. Spectroscopic Analysis

Chemical structures of all compounds were characterized using FT-IR, 1H-NMR, 13C-NMR, and EIMS spectroscopic techniques. In FT-IR spectrum, isatoic anhydride 10 was confirmed by two carbonyl bands at 1765, 1722 cm−1 and these values were found to be consistent with the literature [29]. Further, this compound was used with 4-amino butyric acid to afford 11, which was subsequently diazotized without purification, and product 12 was obtained. 1H-NMR showed one multiplet at 2.25 ppm and two triplets were seen at 2.48 and 4.54 ppm. Four proton signals were observed in the aryl region as two doublets and two triplets. Absence of NH and NH2 peaks in 1H-NMR spectrum confirmed cyclization of 11 to benzotriazinone 12. Furthermore, the MS result depicted the highest fragmentation peak at m/z 187 due to the removal of the carboxylic group. A hydroxyl moiety of acid 12 was replaced by benzotriazole and structure 13 was obtained as the product. In the 1HNMR spectrum, three proton signals were observed in the region of 2.55–4.69 ppm and eight protons appeared in the aryl region of 7.48–8.31 ppm. 13C-NMR also verified this structure 13 by twelve carbon peaks between 114.37–146.61 ppm and two carbonyl carbon peaks at 155.74 and 171.37 ppm. EIMS spectra confirmed this structure by the molecular ion peak at m/z 333. Further, this compound was used to prepare a novel series of alkyl and aryl carboxamides 14a–14n. 1H-NMR spectra of all carboxamides showed an NH peak in the range of 5.95–8.26 ppm.
Table 1. Reaction conditions for the preparation of benzotriazinone carboxamides.
Table 1. Reaction conditions for the preparation of benzotriazinone carboxamides.
Sr. No.CodesMolecular StructuresTimeTemperature (°C)Yield
(%)
1. 14aMolecules 28 06623 i00110 min2591
2. 14bMolecules 28 06623 i00210 min2585
3. 14cMolecules 28 06623 i00315 min2586
4. 14dMolecules 28 06623 i00420 min2585
5. 14eMolecules 28 06623 i00525 min2583
6. 14fMolecules 28 06623 i00630 min2584
7. 14gMolecules 28 06623 i00724 h2575
8. 14hMolecules 28 06623 i00824 h2571
9. 14iMolecules 28 06623 i00924 h2569
10. 14jMolecules 28 06623 i01024 h2571
11. 14kMolecules 28 06623 i01124 h2573
12. 14lMolecules 28 06623 i01224 h2572
13. 14mMolecules 28 06623 i01324 h2576
14. 14nMolecules 28 06623 i01424 h2580

2.3. Enzyme Inhibition Assay

The alpha-glucosidase inhibitory potential of all synthesized compounds, 14a–14n, were evaluated through their in vitro assays and results revealed moderate to excellent activity. The experimental results of all compounds are shown in Table 2. Acarbose was taken as a positive control and the inhibition results of all tested compounds were expressed as percentage inhibition and IC50 values. Among all compounds, 14k and 14l were found to be potent inhibitors with IC50 values of 27.13 ± 0.12 μM and 32.14 ± 0.11 μM in comparison to the standard drug acarbose with an IC50 value of 37.38 ± 0.12 μM. The nature of the group and its relative position on the phenyl ring of carboxamides affect inhibition potential. Compound 14k was observed as more effective than 14l due to methyl substituent at the para position, which enables it to be a better fit in the enzyme cavity. Moreover, compound 14g, with two methyl substituents, was noticed to be less efficient than 14k. Compounds 14g–14n showed good inhibition compared to 14a–14f, which demonstrates the importance of aryl groups in carboxamide analogues. Compound 14j possessed a chloro group at the meta position, which enhanced its inhibition potential compared to p-chloro structure 14i. On the other hand, the bromo group in compound 14h reduced its inhibition potential, most probably due to its big size and steric hindrance. The para position of the methoxy group in 14n augmented inhibition activity compared to ortho methoxy derivative 14m.

2.4. Molecular Docking

In silico study is a modern technique used to predict the interactions between ligand and receptor proteins. It reduces laborious lab work and provides assistance to drug discovery [30]. Molecular docking studies of synthesized carboxamide derivatives were performed via Autodock vina 1.1.2 software pack and the results displayed their in silico interactions [5,31]. The docked conformations of all newly synthesized derivatives of benzotriazinones occupied the same region in the active site of the enzyme pocket where the standard drug acarbose’s structure was fitted. The binding modes and orientations of the two most potent compounds of the series were studied in detail to explore their binding interactions in the active site of the target enzyme. Figure S3 clearly depicts the binding mode of standard acarbose and shows superposition of the synthesized derivatives inside the enzyme cavity.
In the enzyme pocket, His279 residues were found to have hydrophobic pi–pi interactions with the benzene and the triazinone moiety of 14k. One hydrogen bond was established by amino acid residue Asn241 with the nitrogen atom of the triazinone ring, and another nitrogen atom of the same ring formed a carbon–hydrogen bond with amino acid residue His239 (Figure 3). Oxygen of the carboxamide was found to involve in hydrogen bonding with selected residue Arg312. The benzene ring of the carboxamide developed its pi–anion interaction with Asp349 of the enzyme site (Table 3). Another hydrophobic pi–alkyl binding was observed between Phe177 and methyl residue. These results clearly demonstrated tight packing of 14k in the selected enzyme pocket with a docking score of −9.9 Kcal/mol (Figure 3).
The ligand 14l was found to be fit at the catalytic sites of alpha-glucosidase with a binding energy of −9.8 kcal/mol. A strong H-bond appeared between nitrogen of triazinone and Arg312 with a distance of 2.75 Å. Another residue, Arg439, developed its H-bonding with oxygen of the carbonyl group at a distance of 2.53 Å. In the ligand, there were aromatic moieties that were found to be involved in hydrophobic π-stacking interactions with Phe300 and Phe177. Benzene of carboxamide also formed pi–anion electrostatic interactions with Asp439 in the wall of the enzyme. Hydrophobic pi–alkyl interactions were also seen between alkyl, aryl, and amino acid residues Phe177, Tyr71, and val303.

2.5. Computational Study

2.5.1. Frontier Molecular Orbital Analysis

Density frontier theory (DFT) calculations of all synthesized analogues were computed using Gaussian 09 software pack quantum chemical package. Geometrical structures were optimized at B3LYP/6-311 + G* level of theory, as reported in a previous manuscript [5,31,32]. Frontier molecular orbital (FMO) is a powerful computational tool for predicting the electrical, optical, and reactive properties of chemical structures. Quantum chemistry evaluation of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) is known as frontier molecular orbital analysis (FMO). When an electron enters a molecule, LUMO acts as an electron acceptor, and in case of ionization, the electron is removed from HOMO. The study of HOMO–LUMO provides reactivity parameters of chemical structures like EHOMO, ELUMO, electron affinity, ionization potential, electronegativity, electrophilicity, chemical potential, dipole moment, chemical softness, and chemical hardness (Table 4). The energy difference (Egap) between HOMO and LUMO illustrates compound reactivity and kinetic stability. Among all series, structures 14k and 14l were found to have small energy gap values, revealing that these compounds are more reactive compared to others.
Ionization potential (I) is the amount of energy required to remove an electron from an isolated atom or molecule, and its higher value indicates stability. In series, compound 14k showed the lowest ionization potential value, illustrating the reactive nature of these molecules [33]. The highest value of electron affinity in 14k and 14l depicts their tendency to accept electrons (Table 4). The chemical hardness (η) value shows the resistance of a molecule to changing its electronic distribution in a chemical reaction, and chemical softness (S) indicates an easily changeable electronic structure [33]. The lowest value of chemical hardness of 14k shows its reactive nature towards biomolecules whereas the highest value of 14d indicates poor inhibition potency.
The chemical potential (μ) presents the possibility of a chemical reaction in terms of negative value. A lower negative (more negative) value indicates the tendency of a molecule to accept electrons but 14k and 14l have comparable values to other analogues. Electrophilicity is a predictor of the electrophilic nature of a molecule, demonstrating its tendency to accepts electrons. The higher value of electrophilicity of 14k and 14l proved their high affinity towards other molecules. Least active compounds like 14b and 14d were found to be weak inhibitors with low electrophilicity values. Dipole moment presents charge delocalization on a molecule and the compounds 14k and 14l presented low values compared to other molecules [34].
From electronic parameters, it was observed that 14a–14g have the same ionization potential, electron affinity, and chemical hardness and softness. But in the case of 14g–14n, different groups are present on the benzene ring and their position affects electronic functions. Compound 14i has a chloro group at the para position and its chemical hardness is 2.17 eV, but 14j with m-chloro shows a value of 2.16 eV. The para-methylated compound 14k indicates chemical hardness of 2.14 eV and the meta-methylated hybrid 14l possesses a value of 2.16 eV. Among methoxy group derivatives, 14m and 14n both showed a value of 2.17 eV.
Hence, all electronic parameters clearly indicate that 14k and 14l are effective candidates for inhibiting alpha-glucosidase due to high electron affinity and their reactive nature to develop interactions inside the enzyme cavity. FMO analysis clearly illustrated HOMO delocalization on the triazinone ring and LUMO electron density was found to be constrained on entire benzotriazinone rings (Figure 4 and Figure S2).

2.5.2. Molecular Electrostatic Potential

MEP is an effective method for predicting the distribution of electron density over the surface of a designed drug and provides information about the reactivity of molecules [35]. It also facilitates understanding of the most reactive sites of molecules towards electrophilic or nucleophilic attack [36,37]. The MEP for all benzotriazinone carboxamides was computed at B3LYP/6-311 + G* and is shown in Figure 5 and Figure S5. Different atoms and rings in a molecule contain different surface electrostatic potential values in the order red < orange < yellow < green < blue. The red color represents the most negative potential and blue color shows the most positive potential. The green color indicates the zero potential site while the yellow area appears slightly positive. Compounds 14k and 14l exhibited electron density at the oxygen atom of the carbonyl group and the red portion is favorable for electrophilic reactivity. The blue side of the molecule cognates to nucleophilic reactivity [38,39]. Hence, MEP mapping illustrated negative and positive sides of the molecule and indicated the tendency of the molecule to develop different interactions under the enzyme cavity [40].

3. Materials and Methods

3.1. General

Cyanuric chloride and sulfonyl chlorides were purchased from Sigma Aldrich (Burlington, MA, USA) and 1,2,3-benzotriazole was obtained from Daejung chemicals. Melting points were noted using Fisher John apparatus and FTIR was recorded using Agilent 630. 1H-NMR was scanned in CDCl3 using Bruker 400 MHz and TMS was employed as internal standard. GC-MS was recorded on MAT312.

3.1.1. Isatoic Anhydride (11)

Isatoic anhydride was prepared using a previously developed method [29].

3.1.2. 4-(4-Oxobenzo[1,2,3]triazin-3(4H)-yl)butanoic Acid (12)

4-Aminobutyric acid (2.60 g, 20 mmol) and triethyl amine (3.06 g, 20 mmol) were taken in distilled water (50 mL) and isatoic anhydride 10 was added in small portions. Mixture was stirred for 10–15 min at 60 °C and then cooled to 0 °C. Dilute HCl (30%, 20 mL) and 5 mL aqueous solution of sodium nitrite (1.70 g, 24.8 mmol) was added, and temperature was maintained at 0–5 °C. Then, it was stirred for two hours at room temperature until yellow precipitates appeared, which were filtered, washed with excess water, and dried at 70 °C. Product was recrystallized from chloroform: n-hexane mixture. Off-white powder; yield: 3.77 g (81%) m.p. > 300 °C. FTIR (v-cm−1): 1030 (C-N), 1595 (C=O), 1666 (C=O), 3275 (N-H). 1H-NMR: (400 MHz, CDCl3): δH 2.25 (2H, sex, J = 7.1 Hz, CH2), 2.48 (2H, t, J = 7.4 Hz, CH2), 4.54 (2H, t, J = 6.8 Hz, CH2), 7.80 (1H, t, J = 7.6 Hz, Ar-H), 7.93 (1H, t, J = 7.2 Hz, Ar-H), 8.14 (1H, d, J = 8.0 Hz, Ar-H), 8.34 (1H, t, J = 7.6 Hz, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 23.32, 32.50, 48.75, 114.37, 119.75, 120.19, 125.13, 126.35, 128.35, 130.47, 131.07, 132.51, 134.43, 144.27, 146.16, 155.74, 171.37. EI-MS: m/z calcd. for C11H11N3O3 233 found [M-CO-H2O] 187.

3.1.3. 3-(4-(1H-Benzotriazol-1-yl)-4-oxobutyl)benzo[1,2,3]triazin-4(3H)-one (13)

1,2,3-Benzotriazole (2.4 g, 20 mmol) was taken in dried tetrahydrofuran (20 mL) and thionyl chloride (0.37 mL, 5 mmol) was added dropwise at room temperature. After 30 min stirring, compound 12 was dissolved in THF (50 mL) and poured down to reaction mixture that was stirred for 48 h. Precipitates were filtered and washed with excess tetrahydrofuran. Filtrate was evaporated under reduced pressure and chloroform was added, which was washed with sodium carbonate solution (3 × 10 mL) and dried over sodium sulfate. Solution was further filtered and evaporated to derive a crude product that was recrystallized from THF.
Beige brown powder; yield: 0.93 g (56%); m.p. 129–130 °C. FT-IR (v-cm−1): 1030 (C-N), 1681 (C=O), 1727 (C=O). 1H-NMR: (400 MHz, CDCl3): δH 2.47–2.57 (m, 2H, CH2), 3.57 (t, J = 7.0 Hz, 2H, CH2), 4.69 (t, J = 6.8 Hz, 2H, CH2), 7.48 (t, J = 7.6 Hz, 1H, ArH), 7.62 (t, J = 7.8 Hz, 1H, Ar-H), 7.78 (t, J = 7.6 Hz, 1H, Ar-H), 7.93 (t, J = 7.3 Hz, 1H, Ar-H), 8.07–8.12 (m, 2H, Ar-H), 8.22 (d, J = 8.4 Hz, 1H, Ar-H), 8.31 (d, J = 7.6 Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 23.32, 32.50, 48.75, 114.37, 119.75, 120.19, 125.13, 126.35, 128.35, 130.47, 131.07, 132.51, 134.43, 144.27, 146.16, 155.74, 171.37. GC-MS: m/z calcd. for C17H14N6O2 334 found 333.

3.1.4. N-alkyl/aryl-4-N-alkyl/aryl-4-(4-Oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide(14a–14n)

Compound 13 (0.5 g, 1.49 mmol), triethylamine (0.2 mL, 1.49 mmol), and alkyl/aryl amine (1.49 mmol) were taken in dichloromethane and stirred for 10 min to 24 h according to the time required for reaction completion. Dichloromethane was evaporated under reduced pressure and residue was washed with sodium carbonate solution to remove benzotriazole. All products were recrystallized from methanol.
N-propyl-4-(4-Oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14a). The compound 14a was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with propylamine (0.11 mL, 1.49 mmol). Off-white powder; yield: 0.41 g (91%); m.p. 127–128 °C. FTIR (v-cm−1): 1007 (C-N), 1640 (C=O), 1683 (C=O), 2960 (C-H), 3287 (N-H). 1H-NMR: (400 MHz, CDCl3): δH 0.76 (t, J = 7.4 Hz, 3H, CH3), 1.31 (sex, J = 6.5 Hz, 2H, CH2), 1.91 (quin, J = 7.16 Hz, 2H, CH2), 2.13 (t, J = 7.25 Hz, 2H, CH2), 2.88 (q, J = 6.5 Hz, 2H, CH2), 4.34 (t, J = 7.0 Hz, 2H, CH2), 7.76 (t, J = 5.0Hz, 1H, NH), 7.89 (dt, J = 6.7, 1.5 Hz, 1H, Ar-H), 8.04 (dt, J = 6.2, 1.25 Hz, 1H, Ar-H), 8.16 (d, J = 8.5 Hz, 1H, Ar-H), 8.22 (dd, J = 6.5, 1.5 Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 11.49, 22.39, 24.48, 32.47, 40.36, 49.05, 119.36, 124.67, 128.02, 132.97, 135.45, 143.81, 154.93, 171.23. GC-MS: m/z calcd. for C14H18N4O2 274 found [M + 1] 275.
N-Butyl-4-(4-oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14b). The compound 14b was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with butylamine (0.14 mL, 1.49 mmol). Lustrous buff brown powder; yield: 0.42 (85%); m.p. 123–124 °C. FT-IR (v-cm−1): 1008 (C-N), 1641 (C=O), 1685 (C=O), 2930 (C-H), 3304 (N-H). 1H-NMR: (400 MHz, CDCl3): δH 0.79 (t, J = 7.0 Hz, 3H, CH3), 1.17–1.26 (m, 6H, CH2), 2.00 (quin, J = 7.5 Hz, 2H, CH2), 2.11 (t, J = 7.25 Hz, 2H, CH2), 4.33 (t, J = 7.0 Hz, 2H, CH2), 7.75 (t, J = 6.0 Hz, 1H, N-H), 7.90 (t, J = 7.5 Hz, 1H, Ar-H), 8.05 (t, J = 7.3 Hz, 1H, Ar-H), 8.16 (d, J = 8.5 Hz, 1H, Ar-H), 8.22 (d, J = 7.5Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 14.03, 19.96 (2C), 30.95 (2C), 39.11, 48.68, 103.44, 121.87, 122.28, 130.37 (2C), 136.22, 153.63, 155.56. GC-MS: m/z calcd. for C15H20N4O2 288 found 288.
N-pentyl-4-(4-Oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14c). The compound 14c was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with pentylamine (0.17 mL, 1.49 mmol). Off-white powder; yield: 0.45 g (86%); m.p. 106–107 °C. FT-IR (v-cm−1): 1012 (C-N), 1644 (C=O), 1686 (C=O), 2922 (C-H), 3291 (N-H). 1H-NMR: (500 MHz, DMSO): δH 0.80 (t, J = 7.0 Hz, 3H, CH3), 1.11–1.25 (m, 4H, CH2), 1.29 (quin, J = 6.9 Hz, 2H, CH2), 2.01 (quin, J = 7.25 Hz, 2H, CH2), 2.12 (t, J = 7.3 Hz, 2H, CH2), 2.91 (q, J = 7.25 Hz, 2H, CH2), 4.34 (t, J = 6.9 Hz, 2H, CH2), 7.74 (t, J = 5.5 Hz, 1H, N-H), 7.88 (dt, J = 6.5, 1.0 Hz, 1H, Ar-H), 8.06 (dt, J = 6.3, 1.5 Hz, 1H, Ar-H), 8.15 (d, J = 8.0 Hz, 1H, Ar-H), 8.22 (dd, J = 7.0, 1.0 Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 14.42, 22.33, 24.87, 29.10, 29.21, 32.938, 38.93, 49.484, 119.77, 125.09, 128.44, 133.39, 135.87, 144.22, 155.35, 171.59. GC-MS: m/z calcd. for C16H22N4O2 302 found 302.
N-Hexyl-4-(4-oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14d). The compound 14d was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with hexyl amine (0.19 mL, 1.49 mmol). Lustrous off-white powder; yield: 0.047 g (85%); m.p. 102–103 °C. FT-IR (v-cm−1): 1010 (C-N), 1643 (C=O), 1685 (C=O), 2923 (C-H), 3307 (N-H), 1H-NMR: (400 MHz, CDCl3): δH 0.86 (t, J = 7.0 Hz, 3H, CH3), 1.27–1.34 (m, 6H, CH2), 1.48–1.51 (m, 2H, CH2), 2.80–2.83 (m, 4H, CH2), 3.23 (q, J = 5.9 Hz, 2H, CH2), 4.52 (t, J = 6.0 Hz, 2H, CH2), 6.07 (s, 1H, NH), 7.80 (dt, J = 6.4, 1.0 Hz, 1H, Ar-H), 7.96 (dt, J = 6.4, 1.2 Hz, 1H, Ar-H), 8.15 (d, J = 8.0 Hz, 1H, Ar-H), 8.34 (dd, J = 7.4, 1.0 Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 14.04, 22.57, 25.29, 26.62, 29.52, 31.48, 33.34, 39.83, 48.93, 119.71, 125.12, 128.37, 132.53, 134.98, 144.29, 155.95, 171.86. GC-MS: m/z calcd. for C17H24N4O2 316 found 316.
N-Heptyl-4-(4-oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14e). The compound 14e was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with heptylamine (0.22 mL, 1.49 mmol). Lustrous off powder; yield: 0.49 g (83%); m.p. 100–101 °C. FT-IR (v-cm−1): 1011 (C-N), 1643 (C=O), 1685 (C=O), 2925 (C-H), 3300 (N-H). 1H-NMR: (400 MHz, CDCl3): δH 0.851 (t, J = 6.6 Hz, 3H, CH3), 1.24–1.27 (m, 10H, CH2), 1.47–1.50 (m, 4H, CH2), 3.21 (q, J = 6.5 Hz, 2H, CH2), 4.52 (t, J = 6.0 Hz, 2H, CH2), 6.0 (s, 1H, N-H), 7.79 (t, J = 7.4 Hz, 1H, Ar-H), 7.94 (t, J = 7.8 Hz, 1H, Ar-H), 8.14 (d, J = 8.0 Hz, 1H, Ar-H), 8.33 (d, J = 7.6 Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 14.08, 22.60, 25.27, 26.91, 28.97, 29.58, 31.75, 33.41, 39.76, 48.95, 119.72, 125.11, 128.35, 132.50, 134.96, 144.28, 155.90, 171.75. GC-MS: m/z calcd. for C18H26N4O2 330 found 330.
N-Octyl-4-(4-oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14f). The compound 14f was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with octylamine (0.25 mL, 1.49 mmol). Off-white powder; yield: 0.51 g (84%); m.p. 95°–96 °C. FT-IR (v-cm−1): 1010 (C-N), 1643 (C=O), 1684 (C=O), 2921 (C-H), 3304 (N-H). 1H-NMR (400 MHz, CDCl3): δH 0.84 (t, J = 6.8 Hz, 3H, CH3), 1.23–1.26 (m, 10H, CH2), 1.47 (quin, J = 6.8 Hz, 2H, CH2), 2.24–2.27 (m, 4H, CH2), 3.20 (q, J = 7.0 Hz, 2H, CH2), 4.51 (t, J = 6.0 Hz, 2H, CH2), 5.92 (s, 1H, NH), 7.89 (dt, J = 7.0, 1.0Hz, 1H, Ar-H), 7.93 (dt, J = 7.0, 1.2 Hz, 1H, Ar-H), 8.13 (d, J = 8.4 Hz, 1H, Ar-H), 8.32 (dd, J = 7.6, 0.08 Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 14.10, 22.65, 25.25, 26.96, 29.22, 29.27, 29.61, 31.80, 33.50, 39.69, 48.98, 119.74, 125.11, 128.33, 132.48, 134.93, 144.28, 155.85, 171.61. GC-MS: m/z calcd. for C19H28N4O2 344 found 344.
N-(3,5-Dimethylphenyl)-4-(4-oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14g). The compound 14g was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with 3,5- dimethylaniline (0.18 g, 1.49 mmol). Beige brown powder; yield: 0.37 g (75%); m.p. 110–111 °C. FT-IR (v-cm−1): 1008 (C-N), 1656 (C=O), 1687 (C=O), 2909 (C-H), 3277 (N-H). 1H-NMR: (400 MHz, CDCl3): δH 2.25–2.44 (m, 10H, CH3, CH2), 4.60 (t, J = 5.6 Hz, 2H, CH2), 6.71 (s, 1H, Ar-H), 7.15 (s, 2H, Ar-H), 7.79 (dt, J = 7.2, 0.8 Hz, 1H, Ar-H), 7.94 (dt, J = 7.0, 1.4 Hz, 1H, Ar-H), 7.98 (s, 1H, N-H), 8.22 (d, J = 8.0 Hz, 1H, Ar-H), 8.35 (d, J = 6.8 Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 21.39 (2CH3), 25.54, 34.54, 48.80, 114.42, 117.62 (2CH), 119.64, 125.18, 126.00, 128.36, 132.57, 135.03, 137.82, 138.63 (2C), 156.20, 170.26. GC-MS: m/z calcd. for C19H20N4O2 336 found 338.
N-(4-Bromophenyl)-4-(4-oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14h). The compound 14h was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with 4-bromoaniline (0.25 g, 1.49 mmol). White powder; yield: 0.4 g (71%); m.p. 115–116 °C. FT-IR (v-cm−1): 1008 (C-N), 1656 (C=O), 1684 (C=O), 2946 (C-H), 3259 (N-H). 1H-NMR: (400 MHz, CDCl3): δH 2.16–2.54 (m, 4H, CH2), 4.60 (t, J = 6.0 Hz, 2H, CH2), 7.03–7.50 (m, 4H, Ar-H), 7.78–7.83 (m, 2H, Ar-H, N-H), 7.95 (dt, J = 7.0, 1.4 Hz, 1H, Ar-H), 8.15 (d, J = 8.0 Hz, 1H, Ar-H), 8.35 (d, J = 8.0 Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 25.58, 34.53, 48.78, 117.35, 119.69, 121.19, 125.20, 128.40, 129.92, 131.90, 132.55, 135.12 (2CH), 137.17, 144.30, 156.40, 170.35. GC-MS: m/z calcd. for C17H15BrN4O2 387 found 388.
N-(4-Chlorophenyl)-4-(4-oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14i). The compound 14i was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with 4- chloroaniline (0.19 g, 1.49 mmol). Off-white powder; yield: 0.34 g (69%); m.p. 144–145 °C. FT-IR (v-cm−1): 1009 (C-N), 1656 (C=O), 1687 (C=O), 2927 (C-H), 3281 (N-H). 1H-NMR: (400 MHz, CDCl3): δH 2.37–2.42 (m, 4H, CH2), 4.59 (t, J = 6.0 Hz, 2H, CH2), 7.23 (d, J = 6.8 Hz, Ar-H, 2H), 7.52 (d, J = 6.8 Hz, 2H, Ar-H), 7.81 (t, J = 7.2 Hz,1H, Ar-H), 7.94 (t, J = 7.2 Hz, 1H, Ar-H), 8.14 (d, J = 8.0 Hz, 1H, Ar-H), 8.26 (s, 1H, NH), 8.35 (d, J = 7.2Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 25.62, 34.65, 48.80, 119.55, 121.05 (2CH), 125.18, 128.45, 128.95 (2CH), 129.05, 132.74, 135.18, 136.70, 144.26, 156.39, 170.33. GC-MS: m/z calcd. for C17H15ClN4O2 342 found 341.
N-(3-Chlorophenyl)-4-(4-oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14j). The compound 14j was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with 3-chloroaniline (0.19 g, 1.49 mmol). Yellow crystals; yield: 0.36 g (71%); m.p. 209–210 °C. FT-IR (v-cm−1): 1012 (C-N), 1661 (C=O), 1689 (C=O), 2986 (C-H), 3332 (NH). 1H-NMR: (400 MHz, CDCl3): δH 2.37–2.44 (m, 4H, CH2), 4.60 (t, J = 5.8 Hz, 2H, CH2), 7.05 (dd, J = 1.0 Hz, J = 6.8Hz, 1H, Ar-H), 7.21 (t, J = 8.0 Hz, 2H, Ar-H), 7.41–7.43 (m, 1H, Ar-H), 7.67 (s, 1H, N-H,), 7.82 (dt, J = 7.0, 1.4 Hz, 1H, Ar-H), 7.96 (dt, J = 6.0, 1.4 Hz, 1H, Ar-H), 8.16 (d, J = 8.2 Hz, 1H, Ar-H), 8.37 (dd, J = 8.0, 1.2 Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 25.62, 34.52, 48.65, 117.66, 119.53, 119.77, 124.18, 125.19, 128.44, 129.93, 132.74, 134.59, 135.19, 139.24, 144.26, 156.46, 170.34. GC-MS: m/z calcd. for C17H15ClN4O2 342 found 341.
N-(4-methylphenyl)-4-(4-Oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14k). The compound 14k was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with 4-methylaniline (0.15 g, 1.49 mmol). Tan brown powder; yield: 0.35 g (73%); m.p. 175–176 °C. FT-IR (v-cm−1): 1012 (C-N), 1664 (C=O), 1673 (C=O), 2922 (C-H), 3482 (N-H). 1H-NMR: (400 MHz, CDCl3,): δH 2.30–2.42 (m, 7H, CH2), 4.57 (t, 2H, CH2), 7.08 (d, J = 6.0 Hz, 2H, Ar-H), 7.20 (d, J = 6.0 Hz, 2.0 Hz, 1H, Ar-H), 7.80 (t, J = 6.0 Hz, 1H, Ar-H), 7.94 (t, J = 6.0 Hz, 1H, Ar-H), 8.10 (s, 1H, N-H), 8.14 (d, J = 7.2 Hz, 1H, Ar-H), 8.35 (d, J = 5.6 Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 21.54, 25.53, 34.58, 48.83, 116.91, 119.63, 120.46, 125.00, 125.18, 128.39, 128.77, 132.62, 135.07, 137.96, 138.85, 144.27, 156.22, 170.19. GC-MS: m/z calcd. for C18H18N4O2 322 found 321.
N-(3-methylphenyl)-4-(4-Oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14l). The compound 14l was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with 3-methylaniline (0.15 g, 1.49 mmol). Light brown powder; yield: 0.34 g (72%); m.p. 105–106 °C. FT-IR (v-cm−1): 1008 (C-N), 1656 (C=O), 1687 (C=O), 2923 (C-H), 3281 (N-H). 1H-NMR: (400 MHz, CDCl3): δH 2.30–2.42 (m, 7H, CH2), 4.57–4.60 (m, CH2, 2H), 6.89 (s, 1H, Ar-H), 7.16–7.39 (m, Ar-H, 3H), 7.79- 8.03 (m, Ar-H, N-H, 3H), 8.13 (d, J = 6.8 Hz, Ar-H, 1H), 8.34 (d, J = 6.0 Hz, 1H, Ar-H) 13C-NMR: (100 MHz, CDCl3): δC 20.92, 25.59, 34.53, 48.89, 119.63, 120.03, 125.20, 128.41, 129.47, 132.50, 132.67, 133.89, 134.93, 135.10, 135.40, 144.27, 156.23, 170.26. GC-MS: m/z calcd. for C18H18N4O2 322 found 323.
N-(2-Methoxyphenyl)-4-(4-oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14m). The compound 14m was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with 2-methoxyaniline (0.18 g, 1.49 mmol). White powder; yield: 0.38 g (76%); m.p. 139–140 °C. FT-IR (v-cm−1): 1008 (C-N), 1656 (C=O), 1678 (C=O), 2835 (C-H), 3004 (N-H). 1H-NMR: (400 MHz, CDCl3): δH 2.36 (quin, J = 6.8 Hz, 2H, CH2), 2.51 (t, J = 7.4 Hz, 2H, CH2), 3.86 (s, 3H, CH3), 4.59 (t, J = 6.4 Hz, Ar-H, 2H), 6.83 (d, J = 8.0 Hz, 1H, Ar-H), 6.88 (t, J = 7.2 Hz, 1H, Ar-H), 6.99 (t, J = 7.4 Hz, 1H, Ar-H), 7.78 (t, J = 7.2 Hz, 1H, Ar-H), 7.89- 7.93 (m, 2H, NH, Ar-H), 8.11 (d, J = 8.0 Hz, 1H, Ar-H), 8.26 (d, J = 8.0 Hz, 1H, Ar-H), 8.32 (d, J = 8.0 Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 25.56, 34.32, 48.81, 55.52, 114.12 (2C), 119.63, 121.66 (2C), 125.17, 128.40, 131.18, 132.63, 135.08, 144.28, 156.23, 156.36, 170.00. GC-MS: m/z calcd. for C18H18N4O3 338 found 322.
N-(4-Methoxyphenyl)-4-(4-oxobenzo[1,2,3]triazin-3(4H)-yl)butanamide (14n). The compound 14n was obtained from the reaction of 13 (0.5 g, 1.49 mmol) with 4- methoxyaniline (0.18 g, 1.49 mmol). White powder; yield: 0.40 g (80%); m.p. 147–148 °C. FT-IR (v-cm−1): 1006 (C-N), 1656 (C=O), 1678 (C=O), 2938 (C-H), 3250 (N-H). 1H-NMR: (400 MHz, CDCl3): δH 2.36–2.42 (m, 4H, CH2), 3.77 (s, 3H, CH3), 4.60 (t, J = 5.4 Hz, 2H, CH2), 6.82 (d, J = 8.0 Hz, 2H, Ar-H), 7.45 (d, J = 8.0 Hz, 2H, Ar-H), 7.80 (t, J = 7.6 Hz, 1H, Ar-H), 7.95 (t, J = 8.0Hz, Ar-H, 1H), 7.99 (s, 1H, N-H), 8.14 (d, J = 8.0 Hz, 1H, Ar-H), 8.35 (d, J = 8.0 Hz, 1H, Ar-H). 13C-NMR: (100 MHz, CDCl3): δC 24.87, 34.75, 49.04, 55.70, 119.92, 119.80, 119.87, 121.05, 123.68, 125.10, 127.59, 128.30, 132.42, 134.86, 144.29, 147.83, 155.74, 169.81. GC-MS: m/z calcd. for C18H18N4O3 338 found 337.

3.2. Procedure for In Vitro Study

The alpha-glucosidase inhibitory assay was performed by adopting the method of Pierre et al., with minor modifications. Alpha-glucosidase (Cat No. 5003-1KU Type I) belonging to Saccharomyces cerevisiae was selected for this protocol because its structure and function were similar to yeast and mammalian enzymes. In test tubes, 10 μL of test compound (0.5 mM), 70 μL of saline phosphate buffer (50 mM) to adjust pH at 6.8, and 10 μL of α-glucosidase enzyme (0.0234 units) were added to prepare 100 μL assay mixture. A quantity of 10 μL p-nitrophenyl-α-D-glucopyranoside (0.5 mM, N1377 from Sigma, Burlington, MA, USA) was added to initiate the reaction and tubes were incubated for 30 min. Acarbose was used as a positive control. Free substrate change in absorbance was monitored at 400 nm. The alpha-glucosidase inhibitory activity was calculated as percentage inhibition by using formula:
% Inhibition = [(Abs. of control − Abs. of test)/Abs. of control] × 100
IC50 values were determined using EZ-Fit enzyme kinetics software version 5.03 (Perrella Scientific Inc. Amherst, Amherst, MA, USA).

3.3. Molecular Docking Protocol

In order to explore the most probable way of binding of synthesized hybrids inside active sites of α-glucosidase, Autodock Vina software (v.1.1.2.) along with its tools (Mgltools V.1.5.6) were employed to carry out molecular docking studies. The crystal structure of eukaryotic yeast (Saccharomyces cerevisiae) was not found in the Protein Data Bank; only some bacterial glucosidase structures were available. The sequence of Saccharomyces cerevisiae’s α-glucosidase is based on sequence of 584 amino acid residues (uniport ID: P53341). NCBI’s BLAST algorithm was used as a suitable template for homology modelling of target protein. For homology modeling, highest sequence similarity was observed in oligo-1,6-glucosidase (P53051) and selected to be used as a basic pattern. Sequence alignment was conducted by using Needleman–Wunsch Global Alignment Algorithm via Chimera (v.1.17.1). Structure modelling was processed using Modeller. Quality of the new structure was checked by using Ramachandran plot, which showed 97.3% of residues were in the favored region, and 99.7% of residues were in the allowed region. To review the quality of created homology model, molecular dynamics simulation was carried out using NAMD. Visualization of molecular dynamics trajectories was carried out using VMD. Protein molecule was solvated and equilibrated in a water box and modeled at physiological temperature of 310 K for 10 ps. Finally, optimized structure was applied for further molecular docking studies using Autodock Vina software pack. The optimized structure of all newly synthesized compounds at B3LYP/6-311 + G* level of theory were employed for docking studies using our previously reported molecular docking parameters [41]. The compounds having highest binding affinity were chosen to explore their binding interactions with amino acid residues of protein structure. Discovery Studio visualizer (v21.1.0.20298) was used for presentation of docked conformations and visualization of binding interactions inside the active pocket of receptor protein.

3.4. Computational Method

Becke’s three-parameter hybrid exchange functionals [42] and Lee–Yang–Parr correlation functionals (B3LYP) method along with 6-311 + G* basis set was employed for computational study [43,44]. Gaussian 09 software pack was used to perform all the computational calculations. To ensure that the optimized geometry corresponds to the equilibrium (minimum energy) structure, harmonic vibrational frequency analysis was applied at the same basic set level to detect any imaginary frequencies. All docked structures were utilized to explain QM descriptors. The Gauss view software package (v.5.0) was used to visualize the computed structures including HOMO, LUMO, and molecular electrostatic potential (MEP) representations. In addition, different electronic parameters were calculated by using Equations (1)–(7).
Ionization potential (I) = −EHOMO
Electron affinity (A) = −ELUMO
Chemical hardness (η) = (ELUMO − EHOMO)/2
Chemical softness (S) = 1/2η
Chemical potential (μ) = −[(EHOMO + ELUMO)/2]
Electronegativity (χ) = (EHOMO + ELUMO)/2
Electrophilicity (ω) = (EHOMO + ELUMO/2)2/2η

4. Conclusions

A series of benzotriazinone carboxamides was prepared at room temperature and characterized using different spectroscopic techniques including FTIR, 1H-NMR, 13C-NMR, and EI-MS. The alpha-glucosidase inhibition activity of all analogues was examined using an in vitro assay. Compounds 14k and 14l showed remarkable alpha-glucosidase inhibitory potential compared to the market drug acarbose. Molecular studies presented good docking scores with significant interactions between ligands and targeted receptor pockets. Frontier molecular orbital analysis of all hybrids predicted the reactive nature of 14k and 14l with lowest energy gap values between HOMO and LUMO. In electronic properties, lower values of chemical hardness and higher values of electron affinity and electrophilicity demonstrated the tendency of these compounds to develop interactions with biomolecules. An electrostatic potential map of all analogues was investigated to understand the electrophilic and nucleophilic regions of molecules. All in silico findings were found to be in good agreement with the in vitro results. Hopefully, this work may be useful for the optimization and structural modification of new antidiabetic drugs.

Supplementary Materials

The following supporting information are available online: FT-IR, 1H-NMR, 13CNMR, and MS spectra (Figures S6–S68) of all alpha-glucosidase inhibitors can be downloaded at https://www.mdpi.com/article/10.3390/molecules28186623/s1. Molecular docking 2D/3D maps (Figures S1–S3, Table S1), charge density distribution in molecular orbitals (Figure S4), and MEP mapping (Figure S5) of all synthesized compounds can be found in the Supplementary File.

Author Contributions

Conceptualization, M.A.M. and S.M.; methodology, Z.K. and H.A.A. software, S.M.E. and S.S.S.; validation, M.A.M.; formal analysis, S.R.S.; investigation, A.M.A.; resources; data curation, Z.K. and H.A.A.; writing—original draft preparation, R.H., H.A.A. and S.S.S.; writing—review and editing, Z.K. and R.H.; visualization; supervision, M.A.M.; project administration, S.M.E. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R145), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the Supplementary Materials. Samples of the compounds are available from the authors.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are available from the authors.

References

  1. Raut, S.K.; Khullar, M. Oxidative stress in metabolic diseases: Current scenario and therapeutic relevance. Mol. Cell. Biochem. 2023, 478, 185–196. [Google Scholar] [CrossRef]
  2. Borges, L.D.; Dias, H.H.; de Souza Ferreira, E.; Alves, P.M.; Silva, B.O.; de Paiva Santos, K.; da Costa, G.D.; Moreira, T.R.; Santos, D.S.; Cotta, R.M.M. Prevalence of Diabetes Mellitus among individuals with chronic kidney disease: Systematic review and meta-analysis. J. Evid. Based Healthc. 2023, 5, e4060. [Google Scholar] [CrossRef]
  3. Hussain, F.; Hafeez, J.; Khalifa, A.S.; Naeem, M.; Ali, T.; Eed, E.M. In vitro and in vivo study of inhibitory potentials of α-glucosidase and acetylcholinesterase and biochemical profiling of M. charantia in alloxan-induced diabetic rat models. Am. J. Transl. Res. 2022, 14, 3824. [Google Scholar]
  4. Farwa, U.; Raza, M.A. Heterocyclic compounds as a magic bullet for diabetes mellitus: A review. RSC Adv. 2022, 12, 22951–22973. [Google Scholar] [CrossRef]
  5. Khalid, Z.; Alnuwaiser, M.A.; Ahmad, H.A.; Shafqat, S.S.; Munawar, M.A.; Kamran, K.; Abbas, M.M.; Kalam, M.A.; Ewida, M.A. Experimental and Computational Analysis of Newly Synthesized Benzotriazinone Sulfonamides as Alpha-Glucosidase Inhibitors. Molecules 2022, 27, 6783. [Google Scholar] [CrossRef]
  6. Gondaliya, P.; Sayyed, A.A.; Bhat, P.; Mali, M.; Arya, N.; Khairnar, A.; Kalia, K. Mesenchymal stem cell-derived exosomes loaded with miR-155 inhibitor ameliorate diabetic wound healing. Mol. Pharm. 2022, 19, 1294–1308. [Google Scholar] [CrossRef]
  7. Elahabaadi, E.; Salarian, A.A.; Nassireslami, E. Design, synthesis, and molecular docking of novel hybrids of coumarin-dithiocarbamate alpha-glucosidase inhibitors targeting type 2 diabetes mellitus. Polycycl. Aromat. Compd. 2022, 42, 4317–4327. [Google Scholar] [CrossRef]
  8. Pałasz, A.; Cież, D.; Trzewik, B.; Miszczak, K.; Tynor, G.; Bazan, B. In the search of glycoside-based molecules as antidiabetic agents. Top. Curr. Chem. 2019, 377, 19. [Google Scholar] [CrossRef] [PubMed]
  9. Sepehri, N.; Asemanipoor, N.; Mousavianfard, S.A.; Hoseini, S.; Faramarzi, M.A.; Adib, M.; Biglar, M.; Larijani, B.; Hamedifar, H. New acridine-9-carboxamide linked to 1,2,3-triazole-N-phenylacetamide derivatives as potent α-glucosidase inhibitors: Design, synthesis, in vitro, and in silico biological evaluations. Med. Chem. Res. 2020, 29, 1836–1845. [Google Scholar] [CrossRef]
  10. Popović-Djordjević, J.B.; Jevtić, I.I.; Grozdanić, N.D.; Šegan, S.B.; Zlatović, M.V.; Ivanović, M.D.; Stanojković, T.P. α-Glucosidase inhibitory activity and cytotoxic effects of some cyclic urea and carbamate derivatives. J. Enzyme Inhib. Med. Chem. 2017, 32, 298–303. [Google Scholar] [CrossRef]
  11. Zhang, J.-P.; Li, Q.; Zhang, C.; Li, P.; Chen, L.-J.; Wang, Y.-H.; Ruan, X.H.; Xiao, W.; Xue, W. Synthesis, antibacterial, and antiviral activities of novel penta-1,4-dien-3-one derivatives containing a benzotriazin-4(3H)-one moiety. Chem. Pap. 2018, 72, 2193–2202. [Google Scholar] [CrossRef]
  12. Komet, M. Microwave synthesis and anticonvulsant activity of new 3-benzyl-1,2,3-benzotriazin-4(3H)-ones. J. Heterocycl. Chem. 1997, 34, 1391–1393. [Google Scholar] [CrossRef]
  13. Selim, Y.A.; Abd El-Azim, M.H.; El-Farargy, A.F. Synthesis and Anti-inflammatory Activity of Some New 1,2,3-Benzotriazine Derivatives Using 2-(4-Oxo-6-phenylbenzo[d][1,2,3]triazin-3(4H)-yl)acetohydrazide as a Starting Material. J. Heterocycl. Chem. 2018, 55, 1756–1764. [Google Scholar] [CrossRef]
  14. El Rayes, S.; Ali, I.; Fathalla, W.; Mahmoud, M. Synthesis and Biological Activities of Some New Benzotriazinone Derivatives Based on Molecular Docking; Promising HepG2 Liver Carcinoma Inhibitors. ACS Omega 2020, 5, 6781–6791. [Google Scholar] [CrossRef] [PubMed]
  15. Sorensen, E.S.; Macedo, A.B.; Resop, R.S.; Howard, J.N.; Nell, R.; Sarabia, I.; Newman, D.; Ren, Y.; Jones, R.B.; Planelles, V.; et al. Structure-activity relationship analysis of benzotriazine analogues as HIV-1 latency-reversing agents. Antimicrob. Agents Chemother. 2020, 64, e00888-20. [Google Scholar] [CrossRef]
  16. Zhang, F.; Wu, D.; Wang, G.-L.; Hou, S.; Ou-Yang, P.; Huang, J.; Xu, X.Y. Synthesis and biological evaluation of novel 1,2,3-benzotriazin-4-one derivatives as leukotriene A4 hydrolase aminopeptidase inhibitors. Chin. Chem. Lett. 2017, 28, 1044–1048. [Google Scholar] [CrossRef]
  17. Moghimi, S.; Goli-Garmroodi, F.; Pilali, H.; Mahdavi, M.; Firoozpour, L.; Nadri, H.; Moradi, A.; Asadipour, A.; Shafiee, A.; Foroumadi, A. Synthesis and anti-acetylcholinesterase activity of benzotriazinone-triazole systems. J. Chem. Sci. 2016, 128, 1445–1449. [Google Scholar] [CrossRef]
  18. Reddy, G.S.; Snehalatha, A.V.; Edwin, R.K.; Hossain, K.A.; Giliyaru, V.B.; Hariharapura, R.C.; Shenoy, G.G.; Misra, P.; Pal, M. Synthesis of 3-indolylmethyl substituted (pyrazolo/benzo) triazinone derivatives under Pd/Cu-catalysis: Identification of potent inhibitors of chorismate mutase (CM). Bioorg. Chem. 2019, 91, 103155. [Google Scholar] [CrossRef]
  19. Yan, Y.-C.; Wu, W.; Huang, G.-Y.; Yang, W.-C.; Chen, Q.; Qu, R.-Y.; Lin, H.Y.; Yang, G.F. Pharmacophore-Oriented Discovery of Novel 1,2,3-Benzotriazine-4-one Derivatives as Potent 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors. J. Agric. Food Chem. 2022, 70, 6644–6657. [Google Scholar] [CrossRef]
  20. Roca-Paixão, L.; Correia, N.T.; Danède, F.; Guerain, M.; Affouard, F. Carbamazepine/tartaric acid cocrystalline forms: When stoichiometry and synthesis method matter. Cryst. Growth Des. 2023, 23, 3. [Google Scholar] [CrossRef]
  21. Beydoun, A.; Kutluay, E. Oxcarbazepine. Expert Opin. Pharmacother. 2002, 3, 59–71. [Google Scholar] [CrossRef] [PubMed]
  22. Vendrame, M.; Loddenkemper, T.; Gooty, V.D.; Takeoka, M.; Rotenberg, A.; Bergin, A.M.; Eksioglu, Y.Z.; Poduri, A.; Duffy, F.H.; Libenson, M.; et al. Experience with rufinamide in a pediatric population: A single center’s experience. Pediatr. Neurol. 2010, 43, 155–158. [Google Scholar] [CrossRef]
  23. Fleischmann, R.; Iqbal, I.; Slobodin, G. Meloxicam. Expert Opin. Pharmacother. 2002, 3, 1501–1512. [Google Scholar] [CrossRef] [PubMed]
  24. Tanimoto, M.; Kanazawa, A.; Hirose, T.; Yoshihara, T.; Kobayashi-Kimura, S.; Nakanishi, R.; Tosaka, Y.; Sasaki-Omote, R.; Kudo-Fujimaki, K.; Komiya, K.; et al. Comparison of sitagliptin with nateglinide on postprandial glucose and related hormones in drug-naïve Japanese patients with type 2 diabetes mellitus: A pilot study. J. Diabetes Investig. 2015, 6, 560–566. [Google Scholar] [CrossRef] [PubMed]
  25. Nishio, S.; Abe, M.; Ito, H. Anagliptin in the treatment of type 2 diabetes: Safety, efficacy, and patient acceptability. Diabetes Metab. Syndr. Obes. 2015, 8, 163–171. [Google Scholar] [CrossRef]
  26. Peytam, F.; Adib, M.; Shourgeshty, R.; Firoozpour, L.; Rahmanian-Jazi, M.; Jahani, M.; Moghimi, S.; Divsalar, K.; Faramarzi, M.A.; Mojtabavi, S.; et al. An efficient and targeted synthetic approach towards new highly substituted 6-amino-pyrazolo [1,5-a]pyrimidines with α-glucosidase inhibitory activity. Sci. Rep. 2020, 10, 2595. [Google Scholar] [CrossRef]
  27. Fershtat, L.L.; Makhova, N.N. Molecular hybridization tools in the development of furoxan-based NO-donor prodrugs. Chem. Eur. J. 2017, 12, 622–638. [Google Scholar] [CrossRef]
  28. Harrison, J.R.; Brand, S.; Smith, V.; Robinson, D.A.; Thompson, S.; Smith, A.; Davies, K.; Mok, N.; Torrie, L.S.; Collie, I.; et al. A molecular hybridization approach for the design of potent, highly selective, and brain-penetrant N-myristoyltransferase inhibitors. J. Med. Chem. 2018, 61, 8374–8389. [Google Scholar] [CrossRef]
  29. Khalid, Z.; Shafqat, S.S.; Ahmad, H.A.; Rehman, H.M.; Munawar, M.A.; Ahmad, M.; Asiri, A.M.; Ashraf, M. Synthesis of 1,2,3-Benzotriazin-4(3H)-one derivatives as α-glucosidase inhibitor and their in-silico study. Med. Chem. Res. 2022, 31, 819–831. [Google Scholar] [CrossRef]
  30. Ekins, S.; Mestres, J.; Testa, B. In silico pharmacology for drug discovery: Methods for virtual ligand screening and profiling. Br. J. Pharmacol. 2007, 152, 9–20. [Google Scholar] [CrossRef]
  31. Batool, M.; Tajammal, A.; Farhat, F.; Verpoort, F.; Khattak, Z.A.; Shahid, M.; Ahmad, H.A.; Munawar, M.A.; Zia-ur-Rehman, M.; Asim Raza Basra, M. Molecular docking, computational, and antithrombotic studies of novel 1,3,4-oxadiazole derivatives. Int. J. Mol. Sci. 2018, 19, 3606. [Google Scholar] [CrossRef]
  32. Kazachenko, A.S.; Tomilin, F.N.; Pozdnyakova, A.A.; Vasilyeva, N.Y.; Malyar, Y.N.; Kuznetsova, S.A.; Avramov, P.V. Theoretical DFT interpretation of infrared spectra of biologically active arabinogalactan sulphated derivatives. Chem. Pap. 2020, 74, 4103–4113. [Google Scholar] [CrossRef]
  33. Reyes-Chaparro, A.; Flores-Lopez, N.; Quintanilla-Guerrero, F.; Nicolás-Álvarez, D.E.; Hernandez-Martinez, A. Design of new reversible and selective inhibitors of monoamine oxidase A and a comparison with drugs already approved. Bull. Natl. Res. Cent. 2023, 47, 46. [Google Scholar] [CrossRef]
  34. Sayed, D.S.E.; Abdelrehim, E.-S.M. Spectroscopic details on the molecular structure of pyrimidine-2-thiones heterocyclic compounds: Computational and antiviral activity against the main protease enzyme of SARS-CoV-2. BMC Chem. 2022, 16, 82. [Google Scholar] [CrossRef] [PubMed]
  35. Channar, P.A.; Arshad, N.; Larik, F.A.; Farooqi, S.I.; Saeed, A.; Hökelek, T.; Batool, B.; Ujan, R.; Ali, H.S.; Flörke, U. 4-(4-Bromophenyl)thiazol-2-amine: Crystal structure determination, DFT calculations, visualizing intermolecular interactions using Hirshfeld surface analysis, and DNA binding studies. J. Phys. Org. Chem. 2019, 32, e3968. [Google Scholar] [CrossRef]
  36. Khan, S.; Ullah, H.; Taha, M.; Rahim, F.; Sarfraz, M.; Iqbal, R.; Iqbal, N.; Hussain, R.; Ali Shah, S.A.; Ayub, K.; et al. Synthesis, DFT Studies, Molecular Docking and Biological Activity Evaluation of Thiazole-Sulfonamide Derivatives as Potent Alzheimer’s Inhibitors. Molecules 2023, 28, 559. [Google Scholar] [CrossRef] [PubMed]
  37. Elkolli, M.; Chafai, N.; Chafaa, S.; Kadi, I.; Bensouici, C.; Hellal, A.J. New phosphinic and phosphonic acids: Synthesis, antidiabetic, anti-Alzheimer, antioxidant activity, DFT study and SARS-CoV-2 inhibition. J. Mol. Struct. 2022, 1268, 133701. [Google Scholar] [CrossRef]
  38. Kanaani, A.; Ajloo, D.; Kiyani, H.; Ghasemian, H.; Vakili, M.; Feizabadi, M. Molecular structure, spectroscopic investigations and computational study on the potential molecular switch of (E)-1-(4-(2-hydroxybenzylideneamino)phenyl)ethanone. Mol. Phys. 2016, 114, 2081–2097. [Google Scholar] [CrossRef]
  39. Kosar, B.; Albayrak, C.; Spectroscopy, B. Spectroscopic investigations and quantum chemical computational study of (E)-4-methoxy-2-[(p-tolylimino)methyl]phenol. Spectrochim. Acta A Mol. Biomol. 2011, 78, 160–167. [Google Scholar] [CrossRef]
  40. Akram, N.; Mansha, A.; Premkumar, R.; Franklin Benial, A.M.; Asim, S.; Iqbal, S.Z.; Ali, H.S. Spectroscopic, quantum chemical and molecular docking studies on 2,4-dimethoxy-1,3,5-triazine: A potent inhibitor of protein kinase CK2 for the development of breast cancer drug. Mol. Simul. 2020, 46, 1340–1353. [Google Scholar] [CrossRef]
  41. Chaudhry, F.; Naureen, S.; Ashraf, M.; Al-Rashida, M.; Jahan, B.; Munawar, M.A.; Khan, M.A. Imidazole-pyrazole hybrids: Synthesis, characterization and in-vitro bioevaluation against α-glucosidase enzyme with molecular docking studies. Bioorg. Chem. 2019, 82, 267–273. [Google Scholar] [CrossRef] [PubMed]
  42. Becke, A.D. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 1993, 98, 5648. [Google Scholar] [CrossRef]
  43. Lee, C.; Yang, W.; Parr, R.G. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B 1988, 37, 785–789. [Google Scholar] [CrossRef] [PubMed]
  44. Miehlich, B.; Savin, A.; Stoll, H.; Preuss, H. Results obtained with the correlation energy density functionals of becke and Lee, Yang and Parr. Chem. Phys. Lett. 1989, 157, 200–206. [Google Scholar] [CrossRef]
Molecules 28 06623 g001
Figure 1. Reported benzotriazinone derivatives as bioactive compounds.
Figure 1. Reported benzotriazinone derivatives as bioactive compounds.
Molecules 28 06623 g001
Molecules 28 06623 g002
Figure 2. Design of present work.
Figure 2. Design of present work.
Molecules 28 06623 g002
Molecules 28 06623 sch001
Scheme 1. Oxobenzo[1,2,3]triazin-3(4H)-yl)-N-alkyl/phenyl butanamides.
Scheme 1. Oxobenzo[1,2,3]triazin-3(4H)-yl)-N-alkyl/phenyl butanamides.
Molecules 28 06623 sch001
Molecules 28 06623 g003
Figure 3. Two- and three-dimensional presentations of 14k and 14l inside alpha-glucosidase.
Figure 3. Two- and three-dimensional presentations of 14k and 14l inside alpha-glucosidase.
Molecules 28 06623 g003
Molecules 28 06623 g004
Figure 4. Pictorial illustration of charge density distribution in molecular orbitals of 14k–14l. HOMO is shown at the bottom and LUMO is at the top.
Figure 4. Pictorial illustration of charge density distribution in molecular orbitals of 14k–14l. HOMO is shown at the bottom and LUMO is at the top.
Molecules 28 06623 g004
Molecules 28 06623 g005
Figure 5. Molecular electrostatic potential mapping on the surface of 14k and 14l.
Figure 5. Molecular electrostatic potential mapping on the surface of 14k and 14l.
Molecules 28 06623 g005
Table 2. Results of alpha-glucosidase inhibition studies.
Table 2. Results of alpha-glucosidase inhibition studies.
Sr. No.CodesInhibition (%) at 0.5 mMIC50 (μM)
1. 14a39.13 ± 0.19-
2. 14b67.53 ± 0.14243.41 ± 0.18
3. 14c42.34 ± 0.25-
4. 14d66.20 ± 0.11335.04 ± 0.07
5. 14e40.23 ± 0.21-
6. 14f30.57 ± 0.16-
7. 14g78.32 ± 0.18196.52 ± 0.08
8. 14h74.25 ± 0.27238.15 ± 0.15
9. 14i82.77 ± 0.20163.29 ± 0.13
10. 14j89.35 ± 0.2152.01 ± 0.07
11. 14k94.18 ± 0.2327.13 ± 0.12
12. 14l92.63 ± 0.1232.14 ± 0.11
13. 14m84.31 ± 0.18121.26 ± 0.15
14. 14n87.46 ± 0.1575.37 ± 0.14
15. Acarbose92.23 ± 0.1637.38 ± 0.12
Table 3. Types of forces between ligand and enzyme pockets.
Table 3. Types of forces between ligand and enzyme pockets.
LigandAmino AcidDistance (Å)Attractive Forces
14kAsn2412.38Hydrogen bond
14kArg3122.57Hydrogen bond
14kHis2392.94Hydrogen bond
14kAsp3494.75Electrostatic
14kHis2794.14Hydrophobic
14kHis2795.15Hydrophobic
14kPhe1775.37Hydrophobic
14lArg3122.75Hydrogen bond
14lArg4392.53Hydrogen bond
14lAsp3494.14Electrostatic
14lPhe3004.44Hydrophobic
14lPhe3004.98Hydrophobic
14lPhe1774.99Hydrophobic
14lTyr714.19Hydrophobic
14lVal3035.04Hydrophobic
14lArg3135.37Hydrophobic
14lTyr715.11Hydrophobic
14lPhe1774.61Hydrophobic
Table 4. Electronic properties of all synthesized carboxamides (14a–14n).
Table 4. Electronic properties of all synthesized carboxamides (14a–14n).
CompoundsEHOMOELUMOEgapIAηSμχωD
14a−6.24−1.924.336.241.922.160.23−4.084.083.851.58
14b−6.24−1.914.336.241.912.160.23−4.084.083.841.52
14c−6.24−1.914.336.241.912.160.23−4.084.083.841.58
14d−6.24−1.914.336.241.912.160.23−4.074.073.841.54
14e−6.24−1.914.336.241.912.160.23−4.074.073.841.57
14f−6.24−1.914.336.241.912.160.23−4.074.073.841.51
14g−6.25−1.924.336.251.922.160.23−4.084.083.851.02
14h−6.25−1.924.346.251.922.170.23−4.084.083.852.99
14i−6.26−1.924.346.261.922.170.23−4.094.093.853.16
14j−6.25−1.924.336.251.922.160.23−4.094.093.862.80
14k−6.22−1.934.296.221.932.140.23−4.084.083.881.10
14l−6.25−1.934.326.251.932.160.23−4.094.093.871.20
14m−6.25−1.924.336.251.922.170.23−4.084.083.852.06
14n−6.24−1.914.336.241.912.170.23−4.074.073.832.77
All electronic parameters in eV units except dipole moment, measured in Debye.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Khalid, Z.; Shafqat, S.S.; Ahmad, H.A.; Munawar, M.A.; Mutahir, S.; Elkholi, S.M.; Shafqat, S.R.; Huma, R.; Asiri, A.M. A Combined Experimental and Computational Study of Novel Benzotriazinone Carboxamides as Alpha-Glucosidase Inhibitors. Molecules 2023, 28, 6623. https://doi.org/10.3390/molecules28186623

AMA Style

Khalid Z, Shafqat SS, Ahmad HA, Munawar MA, Mutahir S, Elkholi SM, Shafqat SR, Huma R, Asiri AM. A Combined Experimental and Computational Study of Novel Benzotriazinone Carboxamides as Alpha-Glucosidase Inhibitors. Molecules. 2023; 28(18):6623. https://doi.org/10.3390/molecules28186623

Chicago/Turabian Style

Khalid, Zunera, Syed Salman Shafqat, Hafiz Adnan Ahmad, Munawar Ali Munawar, Sadaf Mutahir, Safaa M. Elkholi, Syed Rizwan Shafqat, Rahila Huma, and Abdullah Mohammed Asiri. 2023. "A Combined Experimental and Computational Study of Novel Benzotriazinone Carboxamides as Alpha-Glucosidase Inhibitors" Molecules 28, no. 18: 6623. https://doi.org/10.3390/molecules28186623

Article Metrics

Back to TopTop