Efficient synthesis of promising antidiabetic triazinoindole analogues via a solvent-free method: investigating the reaction of 1,3-diketones and 2,5-dihydro-3H-[1,2,4]triazino[5,6-b]indole-3-thione

Abstract

Diabetes poses a significant global health challenge, driving the search for effective management strategies. In the past years, α-amylase inhibitors have emerged as promising candidates for regulating blood sugar levels. In this concern, we have synthesized a series of novel 3-methyl-2-aroylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole derivatives via the regioselective reaction of 2,5-dihydro-3H-[1,2,4]triazino[5,6-b]indole-3-thione and 1,3-diketones in the presence of NBS under solvent-free conditions. Subsequently, the inhibitory potential of the newly synthesized 3-methyl-2-aroylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole derivatives was assessed against the α-amylase enzyme to probe their antidiabetic properties. In vitro studies revealed moderate to excellent α-amylase inhibitory activity, with IC₅₀ values ranging from 16.14 ± 0.41 to 27.69 ± 0.58 μg ml⁻¹. Furthermore, SAR analysis showed that compounds containing halogen groups exhibited superior inhibition potential, surpassing the standard drug Acarbose (IC₅₀ = 18.64 ± 0.42 μg ml⁻¹), particularly derivatives substituted with 4-fluoro and 2,4-dichloro groups, with IC₅₀ values of 16.14 ± 0.41 μg ml⁻¹ and 17.21 ± 0.15 μg ml⁻¹, respectively. Additionally, molecular docking unveiled the binding modes of ligands with the active site of A. oryzae α-amylase. Encouragingly, the theoretical analyses closely mirrored the experimental findings, further underlining the promise of these synthetic molecules as potent α-amylase inhibitors.

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Introduction

Diabetes mellitus, a chronic metabolic disorder characterized by high blood sugar levels, presents a major global health challenge. In 2019, it affected an estimated 463 million people worldwide, projected to reach 700 million by 2045.^1,2 India faces a severe diabetes crisis, with approximately 77 million diagnosed cases and nearly 25 million prediabetics in 2019, expected to rise to over 134 million cases by 2045.³ Diabetes types include type-1, type-2, gestational diabetes and prediabetes, leading to serious health complications like blurred vision, increased hunger, hypertension, nephrotoxicity, polydipsia and renal disorders.^4,5 Innovative strategies and novel therapeutic agents are essential to address this growing burden.

α-Amylase inhibitors, recognized for their potential in managing glycemia, offer a promising avenue for diabetes treatment.^1,5,6 α-Amylase, an enzyme secreted by the pancreas and salivary glands, hydrolyzes starches into sugars, contributing to postprandial hyperglycemia in diabetics. Inhibition of α-amylase activity presents an attractive avenue for attenuating the postprandial glycemic surge, thereby aiding in glucose homeostasis.^7–10 α-Amylase inhibitors exert their effect by targeting the active site of the enzyme, restricting its ability to cleave complex carbohydrates into simpler sugars. Beyond glucose control, they may potentially impact insulin resistance, weight management and gut health. Studies explore inhibitors from natural (e.g. Phaseolus vulgaris extracts) and synthetic sources (e.g. Acarbose and Voglibose), with synthetic variants designed for enhanced efficacy and fewer side effects.^7–12 However, they often cause issues like abdominal pain, constipation and gastrointestinal problems, highlighting the need for new, low-toxicity inhibitors.

1,2,4-Triazino[5,6-b]indole derivatives are widely recognized as privileged structural motifs in drug discovery, showcasing a plethora of biological activities. These compounds exhibit efficacy across a spectrum of therapeutic domains including antidiabetic, antimicrobial, antiviral, anti-inflammatory, anti-leishmanial, antihypertensive, antitumor and enzyme inhibitory actions.^13–20Fig. 1 illustrates triazinoindole derivatives (1–9) with significant biological potential as reported in the literature.

Fig. 1 Various biologically active triazinoindole-based molecules.

The structural framework of 1,2,4-triazino[5,6-b]indole, characterized by a triazine ring fused with an indole moiety, embodies a balance of aromaticity and heterocyclic complexity. This fusion endows them with unique physicochemical attributes, developing intriguing opportunities for molecular design and synthesis. Their structural features, such as ring tension and electronic distribution, contribute to their potential reactivity and diverse interactions across biological systems, materials and catalytic processes.^18–22 Given their distinctive structural arrangement and versatile reactivity, [1,2,4]triazino[5,6-b]indoles stand as promising subjects for further exploration across various scientific disciplines.

The intricate landscape of 1,2,4-triazino[5,6-b]indoles reveals their potential as core scaffolds for designing complex molecules with tailored properties, offering promising avenues in drug discovery. Through collaborative efforts bridging synthetic chemistry, computational modeling and interdisciplinary applications, researchers aim to unravel the multifaceted nature of this intriguing heterocyclic entity. Despite their numerous attributes in the biological realm, these compounds exhibit promising potential as antidiabetic agents, supported by literature reports demonstrating their ability to modulate α-amylase activity and regulate glucose metabolism pathways.^6,23,24 Among these derivatives, thiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indoles represent a class of fused 1,2,4-triazino[5,6-b]indole compounds that have garnered significant interest in recent years due to their diverse biological activities and potential therapeutic applications.²⁵ These heterocyclic molecules possess a unique structural scaffold, combining thiazole, triazine and indole moieties, which contributes to their intriguing pharmacological profile. Numerous studies have highlighted the remarkable biological properties exhibited by thiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indoles across various therapeutic areas. Their pharmacological potential encompasses antitumor, antimicrobial, antiviral, anti-inflammatory and enzyme inhibitory activities.^25–28

Several synthetic approaches have been reported in the literature for the synthesis and characterization of thiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indoles; in particular, the synthon 2,5-dihydro-3H-[1,2,4]triazino[5,6-b]indole-3-thione has been employed for the synthesis of thiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indoles by reacting it with various substrates, such as tetracyanoethylene, α-halocarbonyl compounds, propargyl bromides, 1,2-dibromoalkenes and 2,3-dichloroquinoxaline^25,29–36 (Fig. 2). These methods, including cyclization reactions, heterocyclic condensations and cascade transformations, offer distinct advantages in efficiency and selectivity. However, they often involve hazardous conditions like highly reactive reagents and elevated temperatures, along with potentially toxic solvents, raising safety concerns.

Fig. 2 Synthetic routes for thiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indoles employing 2,5-dihydro-3H-[1,2,4]triazino[5,6-b]indole-3-thione as a key synthon.

The risks associated with these reaction conditions pose significant challenges for both chemists and the environment. Nevertheless, there is ongoing research interest in developing efficient and sustainable synthetic methods for synthesizing biologically active thiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indoles. Efforts are directed towards exploring alternative conditions that reduce the use of hazardous reagents and solvents, while also enhancing reaction efficiency and selectivity. Addressing these safety concerns and advancing synthetic chemistry techniques aim to facilitate wider exploration and application of thiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indoles across various scientific fields. Additionally, the introduction of an acyl group to this nucleus holds promise for enhancing its biological efficacy and offering various synthetic opportunities. However, incorporating an acyl group into the thiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole ring in an environmentally friendly manner has proven challenging.

Prompted by the abovementioned facts and continuing our research on synthesizing biologically active acylated azaheterocyclic compounds,^37–39 this study aims to explore the reactivity of unsymmetrical 2-bromo-1,3-diketones towards 2,5-dihydro-3H-[1,2,4]triazino[5,6-b]indole-3-thione for the regioselective synthesis of novel acyl functionalized thiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole derivatives using environment-friendly approaches. Additionally, we seek to evaluate these compounds for their potential as α-amylase inhibitors, which could have significant implications in the treatment of diseases such as diabetes, where regulation of amylase activity is crucial.

Results and discussion

In principle, the reaction between unsymmetrical 2-bromo-1,3-diketones 10, having two electrophilic carbonyl centres of different reactivity (with three electrophilic sites labelled as α₁, α₂ and α₃), and 1,2,4-triazino[5,6-b]indole-3-thione 11 (potential trinucleophilic sites labelled as β₁, β₂ and β₃) may lead to the formation of four possible regioisomers, namely 3-methyl-2-aroylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole 12, 3-aroyl-2-acetyllthiazolo[3′,2′:2,3] [1,2,4]triazino[5,6-b]indole 13, 1-methyl-2-aroylthiazolo[2′,3′:3,4][1,2,4]triazino[5,6-b]indole 14, and 2-aryl-1-acetylthiazolo[2′,3′:3,4][1,2,4]triazino[5,6-b]indole 15, depending on the reaction pathways (Scheme 1).

Scheme 1 Reaction of 2-bromo-1,3-diketones 10 with 1,2,4-triazino[5,6-b]indole-3-thione 11: possible regioisomers.

1,2,4-Triazino[5,6-b]indole-3-thione 11 was synthesized using a literature procedure,^25,28–30 involving the reaction of isatin 16 with thiosemicarbazide 17 under basic conditions at refluxing temperatures. The reaction proceeds through the formation of an intermediate thiosemicarbazone 18 followed by cyclization to yield the desired 1,2,4-triazino[5,6-b]indole-3-thione product 11 (Scheme 2).

Scheme 2 Synthesis of 1,2,4-triazino[5,6-b]indole-3-thione 11.

Initially, 2,5-dihydro-3H-[1,2,4]triazino[5,6-b]indole-3-thione 11 and 1-(4-methoxyphenyl)butane-1,3-dione 19a were selected as the model substrates for optimizing the reaction conditions. N-Bromosuccinimide (NBS) was chosen as the brominating reagent due to its wide availability, facile removal and, notably, commendable safety profile, rendering it suitable for the screening of reaction conditions. Inspired by our previous work related to visible-light photocatalysis for regioselective synthesis,^38–42 we explored the reaction of the model substrates in ethanol, by stirring the reaction mixture at room temperature under visible-light irradiation. Visible-light photocatalysis offers several advantages, including high yields, cost-effective and readily available energy sources, simple workup processes, eco-friendly conditions, and efficient, safe, and sustainable synthesis. However, despite continuous irradiation for 12 h, the reaction did not reach completion, yielding the product in only 34% yield. Furthermore, the reaction conditions were optimized in terms of reaction yields and time. We subsequently investigated the effect of various solvents, viz. water, DCM, DMF and mixtures of ethanol and water in different ratios. However, no significant improvement in the reaction yields was observed (entries 2–7, Table 1). Notably, varying the concentration of aqueous ethanol from 0.2 mL to 0.5 mL led to an increase in the reaction yield (entries 5 and 6, Table 1), underscoring the importance of solvent concentration in optimizing the reaction conditions. Additionally, the incorporation of the photoredox catalyst eosin Y also failed to complete the desired reaction (entry 7, Table 1). Pleasantly, the use of ethanol under refluxing conditions slightly improved the yield (entry 8, Table 1). Attempting to improve the yield, we evaluated other solvents under refluxing conditions (H₂O, DMF and DMSO), but the results were not promising (entries 9–11, Table 1). We acknowledge that the solubility of compound 11 is relatively low at room temperature across various solvents, which poses a significant challenge in the synthesis of compound 12a. Keeping this in mind and considering the importance of solvent-free grinding in terms of selectivity and productivity, we executed the planned reaction by grinding the mixture under solvent-free conditions at room temperature. Surprisingly, the reaction under solvent-free conditions afforded a single regioisomeric product in 94% yield in just 30 min at room temperature (entry 12, Table 1).

Table 1 Optimization of the reaction conditions^a for the synthesis of 12a

Entry	Reaction conditions^a	Energy source	Time	Yield^b (%)
a Reaction conditions: 2,5-dihydro-3H-[1,2,4]triazino[5,6-b]indole-3-thione 11 (1.0 equiv.), 1-(4-methoxyphenyl)butane-1,3-dione 19a (1.0 equiv.) and NBS (1.2 equiv.) were reacted under the indicated reaction conditions. b Isolated yields of 12a (%).
1	EtOH	CFL (27 W)	12 h	34
2	H2O	CFL (27 W)	12 h	28
3	DMF	CFL (27 W)	12 h	20
4	DCM	CFL (27 W)	12 h	15
5	EtOH/H2O (1 : 1)	CFL (27 W)	12 h	22
6	EtOH/H2O (4 : 1)	CFL (27 W)	12 h	30
7	EtOH/eosin Y	CFL (27 W)	12 h	40
8	EtOH	Reflux	4 h	52
9	H2O	Reflux	5 h	40
10	DMF	Reflux	5 h	18
11	DMSO	Reflux	5 h	20
12	Solvent-free	rt	30 min	94

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The obtained single regioisomeric product was identified using 1D and 2D NMR spectral analysis. The ¹H NMR spectrum of the obtained product displayed sharp singlets at δ 2.75 and 3.92 ppm, each corresponding to three protons, attributed to the methyl and methoxy groups, respectively. Additionally, the spectrum exhibited the characteristic aromatic proton signals associated with 1-(4-methoxyphenyl)butane-1,3-dione 19a and 1,2,4-triazino[5,6-b]indole-3-thione 11. Similarly, the ¹³C NMR spectrum exhibited two peaks in the aliphatic region at δ 13.8 and 55.9 ppm, accompanied by the appropriate number of signals corresponding to the condensed product, thereby confirming the successful condensation of the two reactants.

Moreover, to confirm the structure of the resulting regioisomer, comprehensive heteronuclear 2D NMR experiments [(¹H–¹³C) HMBC and (¹H–¹³C) HSQC] were meticulously conducted. The (¹H–¹³C) HMBC results unveiled noteworthy cross-peaks, particularly the correlation observed between the carbonyl carbon (δ 184.8 ppm) and the 2′/6′-H proton (δ 7.98–8.04 ppm) of the aryl ring, indicating the presence of a CO–Ar fragment rather than COCH₃. This finding effectively dismisses the possibility of forming 13a and 15a. Furthermore, the cross-peak between C-3 (δ 146.0 ppm) of the thiazole core and the methyl protons (δ 2.75 ppm) confirms the existence of a methyl group at the 3^rd position of the thiazole ring. Notably, no correlation was observed between the methyl protons and C-14, thereby excluding the possibility of 14a formation. The obtained correlations, illustrating the connectivity of atoms within the molecule, are depicted in Fig. 3. Through meticulous analysis of these correlations, the precise chemical constitution of the product was unequivocally determined as 2-(4-methoxybenzoyl)-3-methylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole 12a.

Fig. 3 2D correlation data of 2-(4-methoxybenzoyl)-3-methylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole 12a.

After identifying the correct regioisomeric structure of the obtained product, a systematic study of the substrate scope was carried out to assess the applicability of this solvent-free transformation by examining differently substituted unsymmetrical diketones 19(a–j) (Scheme 3) and the results are shown in Table 2. A wide range of 1,3-diketones incorporating electron-rich, electron-poor and heteroaromatic substituents underwent the desired cyclization under the optimal conditions, furnishing 3-methyl-2-aroylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indoles 12(a–j) as the final products with excellent yields. In general, electron-rich groups 12(a, g–i) afforded better yields than electron-poor groups 12(c–f).

Scheme 3 Solvent-free synthesis of 3-methyl-2-aroylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indoles 12(a–j).

Table 2 Substrate scope^a

a Reaction conditions: 2,5-dihydro-3H-[1,2,4]triazino[5,6-b]indole-3-thione 11 (1.0 equiv.), diketones 19(a–j) (1.0 equiv.) and NBS (1.2 equiv.) under solvent-free grinding for 30–45 min.

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Proposed mechanism

The proposed mechanistic pathway for the synthesis of 3-methyl-2-aroylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indoles 12(a–j) is elucidated in Scheme 4. The reaction initiates with the alkylation of 1,2,4-triazino[5,6-b]indole-3-thione 11, facilitated by the displacement of bromine from 2-bromo-1,3-diketone 10 by sulfur, yielding intermediate A. Subsequent intramolecular cyclization occurs through the attack of nitrogen on the less hindered and more electrophilic carbonyl centre adjacent to the CH₃ group, favouring the regioselective formation of intermediate B. Following this cyclization, the elimination of a water molecule ensues, yielding the desired product 12 exclusively.

Scheme 4 Proposed mechanistic pathway for the synthesis of 12.

Biological studies: antidiabetic activity

In vitro α-amylase inhibition assay. All the synthesized compounds 12(a–j) were screened for their inhibitory activity against an α-amylase enzyme derived from Aspergillus oryzae (A. oryzae), with Acarbose, a well-known starch blocker, employed as the standard drug for comparison.^1,2 The inhibitory activity of the compounds was determined through the calculation of IC₅₀ values at five different concentrations spanning a range of 12.5 to 200 μg ml⁻¹. The resultant IC₅₀ values in μg ml⁻¹ are tabulated and are presented in detail in Table 3.

Table 3 Inhibition of α-amylase at different concentrations and IC₅₀ values ± SD (three replicates) of synthesized compounds 12(a–j)

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Upon analysis, the tested derivatives exhibited a spectrum of effects on α-amylase inhibition. Notably, the IC₅₀ values ranged from 16.14 ± 0.41 to 27.69 ± 0.58 μg ml⁻¹ across the derivatives, indicating variable degrees of inhibitory activity. These values were compared to the IC₅₀ value of the standard drug Acarbose, which was determined to be 18.64 ± 0.42 μg ml⁻¹. In-depth structure–activity relationship (SAR) investigations provided intriguing insights into the efficacy of thiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole derivatives. Remarkably, derivatives incorporating halogen substituents 12(c–f) exhibited superior activity compared to the unsubstituted derivative 12b and those bearing methyl or methoxy substitutions 12(a, g–i) in the aroyl group. This observation underlines the significance of halogen substituents in enhancing the inhibitory potential of the synthesized compounds against the α-amylase enzyme. In particular, compounds 12c featuring a 4-fluoro substitution and 12f with a 2,4-dichloro substitution in the aroyl ring exhibited the most potent enzyme inhibitory activity, with IC₅₀ values of 16.14 ± 0.41 μg ml⁻¹ and 17.21 ± 0.15 μg ml⁻¹, respectively, surpassing that of Acarbose (IC₅₀ = 18.64 ± 0.42 μg ml⁻¹). However, compounds bearing 4-chloro 12d and 4-bromo 12e substitutions in the aroyl ring demonstrated comparable inhibition potential with IC₅₀ values of 19.25 ± 0.21 μg ml⁻¹ and 19.74 ± 0.34 μg ml⁻¹, respectively. A comparative analysis of the IC₅₀ values of the tested derivatives against Acarbose is illustrated in Fig. 4.

Fig. 4 Comparison of IC₅₀ values (α-amylase inhibition) between compounds 12(a–j) and Acarbose.

Molecular docking

Molecular docking is a vital computational technique used to predict the binding interactions between small molecules and their target receptors.⁴³ By analyzing key parameters such as root mean square deviations (RMSD) and receptor conformations, molecular docking offers a detailed understanding of how ligands interact with their target receptors at the atomic level. Herein, to gain comprehensive insights into the intricate binding mechanisms of the investigated ligands with the receptor site (α-amylase), molecular docking studies were conducted for all compounds with the receptor α-amylase (PDB ID: 7TAA), revealing binding energies ranging from −8.7 to −9.8 kcal mol⁻¹ (Table 4).

Table 4 Binding affinities of thiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indoles 12(a–j) for α-amylase

Compound	R^1	Binding energy (kcal mol^−1)
12a	4-OCH3C6H4	−9.4
12b	C6H5	−9.1
12c	4-FC6H4	−9.4
12d	4-ClC6H4	−9.3
12e	4-BrC6H4	−9.2
12f	2,4-Cl 2 C 6 H 4	−9.8
12g	4-CH3C6H4	−9.4
12h	3-OCH3C6H4	−9.4
12i	2-OCH3C6H4	−9.2
12j	Thiophen-2-yl	−8.7
11	—	−7.1
Acarbose	—	−8.5

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The most optimal docking configurations for compounds exhibiting varying degrees of activity are visually represented in Fig. 5. Compound 12c, demonstrating the highest in vitro inhibitory activity with a docking score of −9.4 kcal mol⁻¹, stands out alongside the compound showcasing the highest binding affinity in docking investigations, 12f, with a docking score of −9.8 kcal mol⁻¹. Conversely, 12j, displaying the least binding energy with a docking score of −8.7 kcal mol⁻¹, is also depicted, alongside the reference ligand Acarbose, which scored −8.5 kcal mol⁻¹. Notably, compound 11 exhibited a comparatively lower docking score of −7.1 kcal mol⁻¹, underscoring the significance of the newly constructed aroyl-functionalized thiazole moiety in compound 12. This structural modification appears to enhance amylase inhibition activity, highlighting the critical role of the aroyl group in improving the binding affinity and interaction with the target enzyme.

Fig. 5 Binding interactions of (A) compound 12c, (B) compound 12f, (C) compound 12j and (D) Acarbose with the receptor A. oryzae α-amylase (PDB: 7TAA).

Analysis of the docking results revealed that ligand 12f, featuring a 2,4-dichlorosubstitution, tightly occupies the active binding pocket of the α-amylase receptor. It engages in a diverse array of hydrogen bonding, electrostatic and hydrophobic interactions, surpassing those observed for compound 12j with a low docking score. Notably, the chlorine atoms in 12f establish interactions with HIS122, HIS296 and TYR82, enhancing binding, that were not seen in 12j (bearing a thiophenyl ring). Moreover, examination of the docking poses highlights the crucial role of the aroyl moiety in the synthesized compounds, emphasizing its significance in establishing robust binding within the enzyme's active site.

A comparative analysis of the docking poses for compound 12f and Acarbose with the active site of the enzyme revealed several common interacting amino acids, including HIS80, HIS296, ASP340, TRP83 and HIS296. Although compound 12c demonstrated a lower binding affinity than compound 12f, it displayed some shared interactions with the standard ligand Acarbose, such as HIS80, ASP206, ASP297 and ASP340. These common interactions suggest a potential mechanism contributing to the high amylase inhibition potency observed with compound 12c. The shared pattern of binding interactions suggests a potential explanation for the higher α-amylase inhibition potential exhibited by compounds 12c and 12f, as they share similar binding characteristics with the reference ligand Acarbose.

Conclusion

In the current research, a series of novel 3-methyl-2-aroylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indoles 12(a–j) was synthesized using environment friendly solvent-free conditions and meticulously characterized by spectral analysis. Biological evaluation of the synthesized derivatives revealed moderate to excellent α-amylase inhibition activity. Notably, the presence of different substituents on the attached aroyl moiety led to variations in inhibitory efficacy against the α-amylase enzyme, with compounds containing halogen groups 12(c–f) exhibiting excellent inhibition potential compared to others. All tested derivatives demonstrated α-amylase inhibitory activity with IC₅₀ values ranging from 16.14 ± 0.41 to 27.69 ± 0.58 μg ml⁻¹. Additionally, computational studies utilizing molecular docking were conducted to investigate the binding modes of ligands with the active site of A. oryzae α-amylase (PDB ID: 7TAA). Encouragingly, the results of these theoretical analyses aligned closely with the experimental findings, reinforcing the potential of the synthetic molecules as α-amylase inhibitors. This study underscores the promise of these synthetic compounds as potential candidates for further exploration in the development of novel bioactive molecules targeting diabetes treatment.

Experimental section

An electrical digital melting point apparatus (MEPA) was used to examine the melting points in open capillaries and these are not corrected. Analytical TLC was performed using Merck Kieselgel 60 F254 silica gel plates and visualized under UV light (254 nm). The IR spectra were recorded on a Buck Scientific IR M-500 spectrophotometer in KBr pellets (ν_max in cm⁻¹). ¹H NMR and ¹³C NMR spectra were recorded on a JEOL ECZS 400S (¹H: 400 MHz, ¹³C: 100 MHz, ¹⁹F: 376 MHz). 2D correlation spectroscopy, (¹H–¹³C) gs-HSQC, (¹H–¹³C) gs-HMBC, (¹H–¹⁵N) gs-HSQC and (¹H–¹⁵N) gs-HSQC of the samples were carried out at Kurukshetra University, Kurukshetra. 1,3-Diketones were synthesized using the method described in the literature.^40,44

General methods for the synthesis of 3-methyl-2-aroylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indoles 12(a–j)

1,3-Diketones 19(a–j) (1.0 equiv.) and NBS (1.2 equiv.) were thoroughly homogenized in a dry mortar until a thick paste was formed and then 2,5-dihydro-3H-[1,2,4]triazino[5,6-b]indole-3-thione 11 was added. The reaction mixture was ground for an additional 30–45 minutes under solvent-free conditions. The reaction progress was monitored by TLC with ethyl acetate–petroleum ether (30 : 70, v/v). After completion of the reaction, the reaction mixture was treated with distilled water and the resulting residue was filtered and recrystallized with ethanol. The solid obtained was dried to give pure compounds in high yields of 77–94%. The products were characterized by IR, 1D & 2D NMR and HRMS spectrometry.

2-(4-Methoxybenzoyl)-3-methylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole 12a. Reddish brown solid; m.p. 284 °C; yield 94%; IR (KBr) ν_max (cm⁻¹): 1682 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 8.54–8.52 (d, 1H, ³J = 7.6, 5-H), 8.04–7.98 (m, 3H, 2′,6′,7-H), 7.87–7.85 (d, 1H, ³J = 8.4, 8-H), 7.70–7.66 (t, 1H, ³J = 7.6, 6-H), 7.21–7.19 (d, 2H, ³J = 8.8, 3′,5′-H), 3.92 (s, 3H, OCH₃), 2.75 (s, 3H, CH₃); ¹³C NMR (101 MHz) δ (ppm) 184.8, 164.4, 159.9, 146.0, 145.2, 142.1, 140.3, 135.6, 132.5, 128.9, 125.1, 124.8, 124.3, 115.6, 114.7, 114.5, 55.9, 13.8; HRMS (ESI): m/z for C₂₀H₁₄N₄O₂S: 375.0845 [M + H]⁺.

2-Benzoyl-3-methylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole 12b. Brownish solid; m.p. 222 °C; yield 87%; IR (KBr) ν_max (cm⁻¹): 1688 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 8.54–8.52 (d, 1H, ³J = 7.6, 5-H), 8.05–7.97 (m, 3H, 2′,6′,7-H), 7.87–7.81 (m, 2H, 4′,8-H), 7.70–7.66 (m, 3H, 3′,5′,6-H), 2.75 (s, 3H, CH₃); ¹³C NMR (101 MHz) δ (ppm) 186.8, 160.0, 146.1, 145.2, 142.2, 141.5, 136.5, 135.7, 134.5, 129.6, 129.1, 124.9, 124.3, 115.6, 114.7, 13.8; HRMS (ESI): m/z for C₁₉H₁₂N₄OS: 345.0740 [M + H]⁺.

2-(4-Fluorobenzoyl)-3-methylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole 12c. Orange colour solid; m.p. 230 °C; yield 80%; IR (KBr) ν_max (cm⁻¹): 1692 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 8.54–8.52 (d, 1H, ³J = 8.0, 5-H), 8.10–8.01 (m, 3H, 2′,6′,7-H), 7.88–7.86 (d, 1H, ³J = 8.0, 8-H), 7.70–7.67 (t, 1H, ³J = 7.2. 6-H), 7.55–7.51 (m, 2H, 3′,5′-H), 2.74 (s, 3H, CH₃); ¹³C NMR (101 MHz) δ (ppm) 185.4, 166.9, 164.4, 160.1, 146.1, 145.1, 142.2, 141.3, 135.7, 133.2, 133.0, 132.9, 124.9, 124.8, 124.4, 116.5, 116.3, 115.6, 114.7, 13.8; HRMS (ESI): m/z for C₁₉H₁₁FN₄OS: 363.0642 [M + H]⁺.

2-(4-Chlorobenzoyl)-3-methylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole 12d. Brownish solid; m.p. 245 °C; yield 82%; IR (KBr) ν_max (cm⁻¹): 1690 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 8.55–8.53 (d, 1H, ³J = 8.0, 5-H), 8.05–7.97 (m, 3H, 2′,6′,7-H), 7.88–7.86 (m, 1H, 8-H), 7.79–7.74 (m, 2H, 3′,5′-H), 7.71–7.67 (m, 1H, 6-H), 2.74 (s, 3H, CH₃); ¹³C NMR (101 MHz) δ (ppm) 184.9, 160.1, 141.6, 139.6, 135.7, 135.2, 131.6, 130.6, 129.3, 125.0, 124.8, 124.4, 115.6, 114.7, 13.9; HRMS (ESI): m/z for C₁₉H₁₁ClN₄OS: 379.0350 [M + H]⁺.

2-(4-Bromobenzoyl)-3-methylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole 12e. Reddish brown solid; m.p. 262 °C; yield 80%; IR (KBr) ν_max (cm⁻¹): 1691 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 8.52–8.50 (d, 1H, ³J = 8.0, 5-H), 8.03–7.99 (m, 1H, 7-H), 7.91 (m, 4H, 2′, 3′,5′,6′-H), 7.86–7.84 (d, 1H, ³J = 8.0, 8-H), 7.68–7.66 (m, 1H, 6-H), 2.74 (s, 3H, CH₃); ¹³C NMR (101 MHz) δ (ppm) 184.0, 164.4, 161.2, 141.6, 140.9, 135.5, 132.1, 131.4, 128.5, 124.6, 122.9, 121.6, 118.2, 112.9, 13.8; HRMS (ESI): m/z for C₁₉H₁₁BrN₄OS: 422.9842 [M + H]⁺.

2-(2,4-Dichlorobenzoyl)-3-methylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole 12f. Dark grey solid; m.p. 276 °C; yield 78%; IR (KBr) ν_max (cm⁻¹): 1694 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 8.53–8.51 (d, 1H, ³J = 7.8, 5-H), 8.03–8.01 (m, 1H, 7-H), 7.92–7.85 (m, 2H, 6′,8-H), 7.80–7.78 (m, 1H, 6-H), 7.71–7.66 (m, 1H, 5′-H), 7.31–7.26 (m, 1H, 3′-H), 2.67 (s, 3H, CH₃); ¹³C NMR (101 MHz) δ (ppm) 185.3, 161.5, 146.4, 143.8, 142.7, 137.6, 135.8, 135.0, 131.2, 131.1, 130.0, 128.4, 125.9, 125.0, 124.4, 115.6, 114.9, 12.8; HRMS (ESI): m/z for C₁₉H₁₀Cl₂N₄OS: 412.9960 [M + H]⁺.

3-Methyl-2-(4-methylbenzoyl)thiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole 12g. Brownish solid; m.p. 268 °C; yield 89%; IR (KBr) ν_max (cm⁻¹): 1682 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 8.54–8.52 (d, 1H, ³J = 8.0, 5-H), 8.05–8.01 (m, 1H, 7-H), 7.91–7.85 (m, 3H, 2′,6′,8-H), 7.70–7.66 (m, 1H, 6-H), 7.50–7.48 (d, 2H, ³J = 8.0, 3′,5′-H), 2.75 (s, 3H, CH₃), 2.47 (s, 3H, 4′-CH₃); ¹³C NMR (101 MHz) δ (ppm) 186.3, 160.0, 146.1, 145.4, 145.2, 141.1, 135.6, 133.8, 129.9, 129.7, 125.0, 124.9, 124.3, 115.6, 114.7, 21.3, 13.8; HRMS (ESI): m/z for C₂₀H₁₄N₄OS: 359.0892 [M + H]⁺.

2-(3-Methoxybenzoyl)-3-methylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole 12h. Yellowish solid; m.p. 272 °C; yield 90%; IR (KBr) ν_max (cm⁻¹): 1684 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 8.54–8.52 (d, 1H, ³J = 7.6, 5-H), 8.05–8.01 (m, 1H, 7-H), 7.88–7.86 (d, 1H, ³J = 8.2, 8-H), 7.70–7.66 (m, 1H, 6-H), 7.62–7.54 (m, 2H, 5′,6′-H), 7.49–7.48 (m, 1H, 2′-H), 7.41–7.38 (m, 1H, 4′-H), 3.87 (s, 3H, OCH₃), 2.76 (s, 3H, CH₃); ¹³C NMR (101 MHz) δ (ppm) 187.2, 160.5, 160.0, 146.7, 145.9, 142.8, 142.2, 138.4, 136.2, 130.9, 125.5, 125.4, 124.9, 122.6, 121.1, 116.2, 115.3, 114.4, 56.1, 14.4; HRMS (ESI): m/z for C₂₀H₁₄N₄O₂S: 375.0097 [M + H]⁺.

2-(2-Methoxybenzoyl)-3-methylthiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole 12i. Brownish solid; m.p. 261 °C; yield 89%; IR (KBr) ν_max (cm⁻¹): 1686 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 8.29–8.27 (d, 1H, ³J = 7.8, 5-H), 8.02–8.00 (m, 1H, 7-H), 7.74–7.72 (d, 1H, ³J = 7.8, 8-H), 7.59–7.52 (m, 4H, 4′,5′,6′,6-H), 7.35–7.33 (m, 1H, 3′-H), 3.84 (s, 3H, OCH₃), 2.37 (s, 3H, CH₃); ¹³C NMR (101 MHz) δ (ppm) 193.6, 166.7, 159.9, 146.9, 143.5, 140.8, 137.8, 132.3, 130.5, 130.3, 123.5, 122.3, 121.9, 121.4, 120.1, 118.2, 118.0, 113.3, 55.9, 14.8; HRMS (ESI): m/z for C₂₀H₁₄N₄O₂S: 374.9984 [M + H]⁺.

3-Methyl-2-((2-thiophen)oyl)thiazolo[3′,2′:2,3][1,2,4]triazino[5,6-b]indole 12j. Brownish solid; m.p. 216 °C; yield 77%; IR (KBr) ν_max (cm⁻¹): 1680 (C=O); ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 8.54–8.53 (d, 1H, ³J = 7.8, 5-H), 8.37–8.36 (dd, 1H, J = 5.4 Hz, J = 1.2 Hz, 3′-H), 8.12–8.10 (m, 1H, 7-H), 8.05–8.00 (m, 1H, 5′-H), 7.88–7.86 (d, 1H, ³J = 8.2, 8-H,), 7.70–7.66 (m, 1H, 6-H), 7.43–7.41 (m, 1H, 4′-H), 2.91 (s, 3H, CH₃); ¹³C NMR (101 MHz) δ (ppm) 178.0, 160.1, 146.7, 142.8, 142.6, 141.6, 139.2, 137.9, 136.2, 130.0, 129.1, 125.4, 124.9, 123.7, 116.2, 115.3, 14.1; HRMS (ESI): m/z for C₁₇H₁₀N₄OS₂: 351.0301 [M + H]⁺.

Molecular docking

Molecular docking investigations were carried out utilizing the AutoDock Vina program to elucidate the molecular binding mechanisms of the compounds within the active site of the α-amylase receptor (Protein Data Bank ID [PDB ID]: 7TAA). The crystal structure of the receptor was obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank server (https://www.rcsb.org). Preprocessing of the protein structure was performed using Autodock MGL Tool 1.5.7 (https://www.mgltools.Scripps.Edu), involving the removal of extra cocrystallized ligands, water molecules and counter ions. Gasteiger charges and polar hydrogens were then added and the structure was saved in .pdbqt format. The structure of the inhibitors was sketched and optimized using MarvinSketch 22.1, and then saved in .pdb format. Conversion of .pdb files to .pdbqt format was achieved using MGL Tools 1.5.7. Grid dimensions were set to size_x = 34.0, size_y = 38.0 and size_z = 27.0, with center coordinates x = 37.717, y = 41.153 and z = 33.318. Docking results were analyzed using Discovery Studio Visualizer, version 21.1, to further elucidate the docking findings.

α-Amylase inhibition studies

The α-amylase inhibitory assay of the synthetic molecules 12(a–j) was conducted using a Systronics Spectrophotometer 169, following the DNSA assay method.^1,2 Acarbose, a well-known drug, served as the standard reference. Initially, stock solutions (1 mg ml⁻¹) of all compounds were prepared in DMSO, from which five different concentrations ranging between 12.5 and 200 μg ml⁻¹ were derived. The experimental procedure involved adding 1 ml of enzyme solution (50 μg ml⁻¹) and 1 ml of various concentrations of test samples (12.5, 25, 50, 100 and 200 μg ml⁻¹) to 20 ml test tubes, followed by incubation at 37 °C for 30 min. Subsequently, 1 ml of starch solution was added to each test tube and further incubated at 37 °C for an additional 30 min. The reaction was terminated by adding 1 ml of DNSA (96 mM) to the prepared test samples, which were then subjected to shaking and placed in a water bath at 80 °C for 10 min. After cooling, the absorbance at 540 nm was measured. Additionally, blank tests without enzymes were conducted and a control experiment without the test sample was carried out similarly, substituting the test sample with 1 ml of DMSO. The results were analyzed using the following mathematical equation:

Percentage inhibition = (A_control − A_test)/A_control × 100

where A_control is the absorbance value of the control experiment performed with DMSO alone and A_test is the absorbance value of different test samples.

Data availability

The ESI† consists of additional experimental data (¹H, ¹³C, HMBC, and HSQC NMR spectra) for the final compounds.

Conflicts of interest

The authors declare no competing interest.

Acknowledgements

We are highly thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for kindly providing financial assistance for JRF & SRF to Prince Kumar (Grant 09/105(0302)/2020-EMR-I).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ob01487a

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