Design, synthesis, and evaluation of novel substituted imidazo[1,2-c]quinazoline derivatives as potential α-glucosidase inhibitors with bioactivity and molecular docking insights

Abstract

α-Glucosidase inhibitors are important in the treatment of type 2 diabetes by regulating blood glucose levels and reducing carbohydrate absorption. The present study focuses on identifying new inhibitors bearing imidazo[1,2-c]quinazoline backbone through multi-step synthesis. The inhibitory potencies of the novel derivatives were tested against Saccharomyces cerevisiae α-glucosidase, revealing IC₅₀ values ranging from 50.0 ± 0.12 µM to 268.25 ± 0.09 µM. Among them, 2-(4-(((2,3-diphenylimidazo[1,2-c]quinazolin-5-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-methoxyphenyl)acetamide (19e) and 2-(4-((benzo[4,5]imidazo[1,2-c]quinazolin-6-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-methoxyphenyl)acetamide (27e) emerged as the most potent inhibitors and were further investigated in various assessments. Finally, molecular docking studies were performed to reveal the crucial binding interactions and to confirm the results obtained from structure-activity relationship (SAR) analysis.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-78878-2.

Introduction

Diabetes mellitus (DM) is a complex and chronic metabolic condition which requires continuous medical care alongside a range of multifactorial risk-reduction strategies, particularly those related to glucose regulation¹. Moreover, it is associated with various diseases, including cardiovascular disorders, kidney failure, neuropathy, lipid metabolism disorders, and other health-related complications². The global prevalence of diabetes has remarkably increased from 108 million in 1980 to 422 million in 2014, which is 8.5% of adults with different age. From 2000 to 2019, there was a 3% increase in diabetes-related mortality rates, and it had become the direct cause of 1.5 million deaths in 2019. Moreover, the raised blood glucose resulted from diabetes has been proved to indirectly contribute to mortality; for instance, almost 20% of cardiovascular-related deaths can be attributed to diabetes³.

Diabetes is classified into various groups, including type 1, type 2, maturity-onset diabetes of the young (MODY), gestational diabetes, neonatal diabetes, and steroid-induced diabetes. Among these types, diabetes mellitus type 2 (T2DM) is the most prevalent, accounting for over 95% of cases. In the past three decades, the global prevalence of T2DM has risen significantly. Historically, T2DM was predominantly observed in adults; however, there has been an increasing occurrence of this type of diabetes in children. Therefore, there is a globally acknowledged goal to decrease the rise in the rates of diabetes and obesity by 2025³.

A key strategy in managing hyperglycemia caused by T2DM involves disrupting the breakdown of dietary carbohydrates. α-Glucosidase, an enzyme located in the brush border of the intestine, plays a substantial role in breaking down complex sugars into monosaccharide units, which are subsequently released into the bloodstream. Therefore, an approved treatment for T2DM involves the inhibition of α-glucosidase to slow down glucose absorption, thereby reducing postprandial glucose levels. Additionally, according to the guidelines of International Diabetes Federation, α-glucosidase inhibitors, alongside insulin, metformin, and sulfonylureas, are considered optional treatments for managing uncontrolled hyperglycemia in patients with T2DM⁴.

All α-glucosidase inhibitors function in a dose-dependent and comparable mechanisms of action, although they exhibit some differences. These agents compete to bind to the active site of enzymes, resulting in interference with the cleavage of oligosaccharides and the subsequent formation of monosaccharides. Currently, acarbose, voglibose, miglitol, and emiglitate are examples of commercial drugs used to manage postprandial hyperglycemia. Among these drugs, acarbose has been the subject of the most extensive study. Despite its efficacy to inhibit α-glucosidase performance, its use is associated with various side effects, including diarrhea, vomiting, flatulence, severe stomach pain, and allergic reactions⁵. Therefore, discovery and development of novel α-glucosidase inhibitors that possess high potency and minimal side effects is highly demanding. In recent decades, numerous aza-heterocyclic compounds^6–14, particularly those bearing substituted quinazolines^15–30and substituted imidazoles^31–40, have been identified as exhibiting significant α-glucosidase inhibitory activities.

Hybridization strategy is a pivotal synthetic method in recent drug discovery and development process, wherein two or more pharmacophores are combined to prepare further bioactive small molecules. This approach aims not only to overcome the limitations of individual compounds but also to increase their potency and selectivity⁴¹. On the other hand, 1,2,3-triazoles have emerged as a noticeable pharmacophore in chemical biology and drug discovery in recent years. Their unique structural abilities to form pivotal protein-ligand interactions, including hydrogen bonding and pi-based interactions, alongside their facile and efficient synthesis through click reaction have made 1,2,3-triazoles a key building block for the preparation of small molecules focusing various therapeutic targets⁴². Regarding the significance of hybridization strategy alongside the remarkable features of 1,2,3-triazoles, this core has been widely used in numerous bioactive compounds. For example, several substituted quinazolines and imidazoles have been incorporated with 1,2,3-triazoles, leading to the identification of potential α-glucosidase inhibitors (Fig. 1)^43–48.

Fig. 1.

α-Glucosidase inhibitors bearing several substituted quinazolines and imidazoles incorporated with 1,2,3-triazoles.

As a part of our continuing investigation into the α-glucosidase inhibitors^49–53, we previously combined imidazole and quinazoline into one scaffold, resulting in the synthesis of substituted imidazo[1,2-c]quinazolines and introduction of this backbone as promising inhibitors⁵⁴. As previously described, the substituents on C-5 position of this backbone played a key role on the α-glucosidase inhibition. Consequently, we followed our investigation by incorporating the substituted 1,2,3-triazoles at this position to identify further potential inhibitors (Fig. 2).

Fig. 2.

Design strategy of novel imidazo[1,2-c]quinazoline derivatives as potential α-glucosidase inhibitors.

In the present study, we employed an efficient synthetic approach to prepare several series of imidazoquinazolines and evaluate their in-vitro activities against α-glucosidase. To provide a comprehensive structure-activity relationship analysis, our compounds were divided into two general categories: poly-substituted imidazo[1,2-c]quinazolines and benzo[4,5]imidazo[1,2-c]quinazolines. Each series was further subdivided into three groups: (1) compounds 15 and 24, bearing an amide functionality tail; (2) compounds 18 and 26, bearing a 1-aryl-1H-1,2,3-triazole tail; (3) compounds 19 and 27, bearing a (1H-1,2,3-triazol-1-yl)-N-arylacetamide tail. Based on the enzymatic inhibitory potencies, compounds 19e and 27e were selected for further evaluations, including kinetic study, circular dichroism measurement, fluorescence quenching measurements, and thermodynamic analysis of their binding to α-glucosidase. Finally, comprehensive molecular docking studies were performed to elucidate the mode of interactions between our compounds and enzyme binding site, as well as to highlight the role of different components of the scaffold.

Results and discussion

Chemistry

To synthesize the targeted multi-substituted imidazo[1,2-c]quinazolines 15, 18, 19, 24, 26, and 27, several efficient synthetic routes were conducted under the mild conditions (Fig. 3). Initially, a mixture of substituted anilines 1a-h and chloroacetyl chloride 2 in the presence of triethylamine (Et₃N) were stirred at ambient temperature in acetone within overnight to obtain substituted 2-chloro-N-arylacetamide 3a-h, followed by the bimolecular nucleophilic substitution (S_N2) with sodium azide (NaN₃) 4 to afford substituted 2-azido-N-arylacetamide 5a-h. Additionally, substituted benzylazides 7a-c were synthesized through S_N2 reaction between corresponding substituted benzylbromides 6a-c and NaN₃, which was performed in the presence of Et₃N in Dimethylformamide (DMF) within 2h.

Fig. 3.

Reaction conditions and reagents: (a) chloroacetyl chloride 2, Et₃N, acetone, r.t., overnight; (b) NaN₃ 4, DMF, r.t., overnight; (c) NaN₃ 4, DMF, r.t., 2h; (d) ammonium acetate 9, benzil 10, HOAc, reflux, 10h; (e) SnCl₂.2H₂O, HCl, MeOH, r.t., 6h; (f) CS₂ 13, KOH, EtOH, reflux, 3h; (g) 2-chloro-N-arylacetamide 3, K₂CO₃, DMF, 80 °C, 12h; (h) propargyl bromide 16, K₂CO₃, DMF, 80 °C, 12h; (i) 2-azido-N-arylacetamide 5, CuSO₄.5H₂O, sodium ascorbate, DMF, r.t., overnight; (j) substituted benzyl azides 7, CuSO₄.5H₂O, sodium ascorbate, DMF, r.t., overnight; (k) benzene-1,2-diamine 20, HOAc (20 mol%), EtOH, reflux, 10h.

The synthesis of first series of substituted imidazo[1,2-c]quinazolines, as illustrated in scheme 1-part B, was initiated with a cyclization reaction between 2-nitrobenzaldehyde 8, ammonium acetate 9, and benzil 10 under reflux in glacial acetic acid (HOAc) to obtain the 2-(2-nitrophenyl)-4,5-diphenyl-1H-imidazole 11. To prepare the similar compound from second series (2-(2-nitro,phenyl)-1H-benzo[d]imidazole 21), a cyclization reaction between 2-nitrobenzaldehyde 8 and benzene-1,2-diamine 20 was performed in the presence of catalytic amount of HOAc in ethanol (EtOH) under reflux. The conditions of subsequent steps for both parts B and C were common.

The nitro functionality in compounds 11 and 21 was reduced to amine moiety using stannous chloride dihydrate (SnCl₂. 2H₂O) and hydrochloric acid in methanol at ambient temperature within 6h to afford compounds 12 and 22, which subsequently went through the cyclization using carbon disulfide 13 and potassium hydroxide (KOH) in EtOH under reflux for approximately 3h to obtain the corresponding imidazo[1,2-c]quinazoline-5-thiol (compounds 14 and 23). Afterwards, they underwent two types of S_N2 reactions in the presence of potassium carbonate (K₂CO₃) in DMF at 80 °C for 12h. The first reaction involved derivatives of 2-chloro-N-arylacetamide (compound 3), leading to products 15a-c and 24a-c, which contained only the amide moiety. The second reaction utilized propargyl bromide (compound 16) to produce compounds 17 and 25 having acetylene moiety. These compounds then went through click reaction with previously prepared compounds 5a-h and 7a-c in the presence of CuSO₄.5H₂O and sodium ascorbate in DMF at room temperature within 12h. Consequently, the desired poly-substituted imidazo[1,2-c]quinazolines bearing 1,2,3-triazole ring (18a-c, 19a-h, 26a-c, and 27a-h) were prepared.

The structures of the isolated compounds 15a-c, 18a-c, 19a-h, 24a-c, 26a-c, and 27a-h were completely deduced on the basis of their IR, ¹H and ¹³C NMR spectroscopy, as well as high-resolution mass spectrometry (HRMS) and elemental analysis. Partial assignments of these resonances are given in the Experimental Part.

In vitro α-glucosidase inhibitory activity

Several series of substituted imidazo[1,2-c]quinazolines 15, 18, 19, 24, 26, and 27 were synthesized to assess their in vitro inhibitory activities against Saccharomyces cerevisiae α-glucosidase and compare their results with acarbose as a standard drug. Various derivatives were prepared to investigate the presence of one or two phenyl rings on the imidazoquinazoline backbone, the presence of amide functionality, the presence of triazole ring, and the role of substituents on the terminal phenyl rings. The results were summarized in Tables 1 and 2.

Table 1.

Substrate scope and in vitro α-glucosidase inhibitory activity of compounds 19a-h and 27a-h.

Table 2.

Substrate scope and in vitro α-glucosidase inhibitory activity of compounds 15 and 24.

As presented, all compounds demonstrated good to excellent inhibitory activities with IC₅₀ values ranging from 50.0 ± 0.12 µM to 268.25 ± 0.09 µM, showing better potencies in comparison to the acarbose with IC₅₀ value of 750.0 ± 1.5 µM. To describe the correlations between structures and observed activities, compounds were divided into three categories according to the substituents linked to the sulfur atom. Furthermore, each table’s compounds were subdivided into two groups, considering the substituents on the imidazole moiety of imidazoquinazoline backbone: derivatives bearing two phenyl rings were depicted on the right part, and derivatives bearing one fused phenyl ring were depicted on the left part. In both tables, compounds belonging to the first group exhibited superior inhibition potencies compared to their counterparts in the second group.

As illustrated in Table 1, compounds 19 and 27 featured both amide functionality and 1,2,3-triazole ring, and our goal was to examine the impact of substituents on the terminal phenyl ring. In the left series, compound 19a bearing unsubstituted phenyl ring displayed significant inhibitory activity (IC₅₀ = 118.25 ± 0.04 µM). Introducing a methyl group (CH₃) at C-4 position of this phenyl caused a decrease in potency; whereas incorporating a methoxy group (OCH₃) at same position yielded compound 19c with enhanced activity (IC₅₀ = 89.30 ± 0.34 µM). Further exploration involved the introduction of this group at other ring positions: C-3 position in compound 19d and C-2 position in compound 19e, both of which led to improved inhibitory activity. Notably, compound 19e, bearing 2- OCH₃, exhibited the greatest inhibition potency (IC₅₀ = 50.0 ± 0.12 µM), emerging as the most potent derivative in the present study and being 15 times more potent than acarbose, the standard drug for α-glucosidase inhibition. Subsequently, a chlorine atom was introduced at different positions on the terminal phenyl ring, resulting in compounds 19f, 19g, and 19h, which showed weaker inhibitory activities compared to their analogues bearing the OCH₃ group.

Within the right series, compounds 27, bearing benzo[4,5]imidazo[1,2-c]quinazoline backbone, showed great inhibitory activities. The presence of unsubstituted phenyl ring led to compound 27a with good potency (IC₅₀ = 122.25 ± 0.13 µM). Incorporating the C-4 position of terminal phenyl ring with any substituents (methyl in compound 27b, methoxy in compound 27c, and chlorine in compound 27f) caused the detrimental effects on the inhibition activities. Shifting the methoxy group to the C-3 position of this phenyl ring led to compound 27d with enhanced potency, while the presence of chlorine at this position (compound 27g) further decreased the potency. Regarding the C-2 position, introducing the methoxy group resulted in compound 27e exhibiting the best inhibitory activity in this series (IC₅₀ = 60.03 ± 0.82 µM). Notably, this compound was the second most potent compound in present study. However, substituting the terminal phenyl ring with 2-Cl in compound 27h, with IC₅₀ value of 123.39 ± 0.58 µM, did not yield to any improvement in comparison with compound 27a (IC₅₀ = 122.25 ± 0.13 µM).

After investigating the impact of substituents on the terminal phenyl ring, the substantial contributions of amide functional group and 1,2,3-triazole ring were examined, and compounds were synthesized bearing only one of these moieties. For example, compounds 15 and 24 were obtained by eliminating the triazole, while compounds 18 and 26 were synthesized by removing the amide functionality. Notably, all these compounds, with the exception of compound 18c, exhibited weaker inhibition potencies than their analogues listed in the Table 1. Within the subgroup of compounds bearing the amide group (15 and 24), compound 15b exhibited the highest inhibitory potency. Conversely, among the compounds containing the triazole moiety (18 and 26), compound 18c demonstrated the most potent inhibition.

In summary, our SAR analysis revealed that incorporating two phenyl rings connected to the imidazole moiety of imidazo[1,2-c]quinazoline significantly led to improved inhibitory activity. Moreover, the presence of both amide functionality and 1,2,3-triazole proved pivotal role for enhancing inhibitory efficacy. Regarding the role of substituents on the terminal phenyl ring, the introduction of methoxy group at C-2 position modified the inhibition potency. Compounds 19e and 27e emerged as the most potent inhibitors against α-glucosidase, showing IC₅₀ values of 50.0 ± 0.12 µM and 60.03 ± 0.82 µM, respectively. These compounds exhibited substantially greater potency compared to acarbose, the reference drug for α-glucosidase inhibition; therefore, they can be appropriate candidates for further evaluations in present study.

Enzyme kinetic study

To elucidate the inhibition mode of imidazoquinazolines 19 and 27, an enzyme kinetic study was conducted on the most active compounds, 19e and 27e (Fig. 4). This investigation involved varying concentrations of p-nitrophenyl α-D-glucopyranoside (1–16 mM) as the substrate, both in the absence and presence of compound 19e (0, 12.5, 25, and 50 µM) and compound 27e (0, 15, 30, and 60 µM). The Lineweaver–Burk plot was outlined in Fig. 3. As the concentrations of these inhibitors increased, the K_m value increased, whereas V_max remained constant. These findings revealed that imidazoquinazolines 19e and 27e bind to the active site of α-glucosidase, indicating their competitive inhibition and competing with acarbose to occupy this region. Furthermore, plotting K_m against various concentrations imidazoquinazolines 19e and 27e resulted into an estimated inhibition constant, yielding a K_i value of 25.0 µM for compound 19e and a K_i value of 12.0 µM for compound 27e.

Fig. 4.

Kinetics of α-glucosidase inhibition by imidazoquinazolines 19e and 27e: (A) The Lineweaver–Burk plot in the absence and presence of different concentrations of compound 19e; (B) The secondary plot between K_m and various concentrations of compound 19e; (C) The Lineweaver–Burk plot in the absence and presence of different concentrations of compound 27e; (D) The secondary plot between K_m and various concentrations of compound 27e.

Circular dichroism spectroscopy assessment

To indicate the chiral environment next to the enzyme amino acid residues of, an instrumental technique, named circular dichroism spectroscopy (CD), is employed by measuring the difference in absorption between right and left polarized light. There are various types of CD, one of which is measured in the far ultraviolet (UV) region ranging from 190 nm to 240 nm. This type offers crucial information about the arrangement of protein bonds and secondary structure of the proteins in dilute solutions. Considering characteristic CD signatures, proteins exhibit several principal conformations, including α-helix, extended β structure (or β-sheet), β-turn, and random coil (which are unordered structures). The type of conformation in the CD spectra is determined based on the specific wavelengths. For example, α-helix is characterized by negative CD bands observed at 222 nm and 208 nm, along with a positive CD band around 190 nm. The β-turn is identified by a negative CD band between 180 nm and 190 nm. Random coil is recognized by a characteristic negative CD band around region of 200 nm^55,56.

In present study, the CD spectra ranging from 180 nm to 250 nm were measured and analyzed using CDNN software in the presence of native α-glucosidase (Fig. 5a), enzyme exposed to imidazo[1,2-c]quinazole 19e (Fig. 5b), and enzyme exposed to benzo[4,5]imidazo[1,2-c]quinazoline 27e (Fig. 5c). Moreover, the percent of observed conformations are listed in Table 3. Comparing with native α-glucosidase, the percent of β-turn conformation increased for both compounds 19e and 27e; whereas the percent of α-helix conformation did not have any specific change, and the percent of random coil conformations decreased significantly. These results revealed that imidazoquinazolines 19e and 27e could determine the conformations of α-glucosidase and fix chiral side chains in the orientation of β-turn. Therefore, compounds 19e and 27e could inhibit α-glucosidase by altering the secondary structure of enzyme.

Fig. 5.

Circular dichroism (CD) spectra of the α-glucosidase: (A) in the absence of inhibitor (control); (B) in the presence of imidazo[1,2-c]quinazole 19e at the concentration of 50 µM; (C) in the presence of benzo[4,5]imidazo[1,2-c]quinazoline 27e at the concentration of 60 µM.

Table 3.

The secondary structure content of α-glucosidase.

Inhibitor	α-Helix (%)	β-Turn (%)	Random Coil (%)
Controla	28.8	28.8	42.4
imidazoquinazoline 19eb	28.9	46.9	24.2
imidazoquinazoline 27ec	32.1	54.9	13

^aControl is native enzyme in the absence of an inhibitor.

^bThe concentration of imidazoquinazoline 19e was 50 µM.

^cThe concentration of imidazoquinazoline 27e was 60 µM.

Fluorescence spectroscopy measurements

Fluorescence spectroscopy measurements are a traditional technique mainly used to explore the potential inhibitors and enzymes under the physiological conditions. As a consequence of binding the inhibitors to the active site of enzymes, the fluorescence characteristics and tertiary structure of the protein change, leading to fluorescence quenching which refers to any process which reduces the fluorescence intensity of an enzyme. This quenching caused by various reasons, including the collisional encounter between the fluorophore and the quencher (called dynamic quenching), formation of ground‐state complex between fluorophore and quencher (called static quenching), the reaction of the excited state, and transformation of energy, to name but a few.

In present study, the fluorescence quenching of α-glucosidase induced by imidazoquinazolines 19e and 27e was measured using a Synergy HTX multi-mode reader (Biotek Instruments, Winooski, VT, USA) equipped with a quartz cuvette of 10 mm. The excitation wavelength was set at 280 nm, and the emission spectra were reported at five different temperatures in the range from 300 to 450 nm with 10 accumulations for each collection point. The emission spectrum was corrected for the background fluorescence from the buffer solution and for the inner filter effect promoted by the inhibitors (Fig. 6). The active site of α-glucosidase contains tryptophan, tyrosine, and phenylalanine residues. As depicted in Fig. 6, fluorescence intensity of α-glucosidase increased to 340 nm, followed by a subsequent decrease. Considering that the maximum intensity of tryptophan fluorescence occurs around 340 nm, it suggests that both imidazoquinazolines 19e and 27e created pivotal interactions tryptophan in the binding site of α-glucosidase, changing the enzyme’s tertiary structure.

Fig. 6.

Fluorescence spectra of α-glucosidase at 20–60 °C: (A) in the absence of any inhibitor (control); (B) enzyme exposed to imidazo[1,2-c]quinazole 19e at the concentration of 50 µM; (C) enzyme exposed to benzo[4,5]imidazo[1,2-c]quinazoline 27e at the concentration of 60 µM.

In addition to predicting the enzyme’s tertiary structures in the presence of inhibitor, fluorescence spectroscopy assessments can provide insightful information about the binding constant, number of binding sites, and thermodynamic parameters of the studied interactions. Based on the results, imidazoquinazolines 19e and 27e proceeded static fluorescence quenching. Accordingly, the biding parameters were determined through the following equations:

The reaction is outlined as P + D → D_nP; where P, D, and D_nP represent the protein, drug (inhibitor), and resulting complex molecule, respectively. Using Equation 1, the binding constant of this complex, denoted as K_A, is calculated.

1

The number of binding sites is denoted as “n” remaining unchanged in the static quenching mechanism. The number of the binding site of protein and drug is n and 1, respectively. Therefore, the equivalent concentration of the complex D_nP is n[D_nP]. The equivalent concentration of the protein is n[P], and the equivalent concentration of the drug is [D].

The total concentration of protein is [P_t], and [P_t] is [P_f] + [D_nP]. The total concentration of the drug is [D_t], and [D_f] is [D_t]–n[D_nP]. Since protein (P) is the only fluorescence in present study; therefore,

2

The fluorescence intensity of protein in the presence and absence of drug is F and F₀. The correlation between these intensities and [D_t] is calculated in equation 3, by which the plot of F₀/F Vs. [D_t] F₀/(F₀–F) is outlined at 20 °C for both imidazoquinazolines 19e and 27e (Fig. 7). Moreover, using this equation, important parameters, including n and r at 20 °C, as well as K_A at 20 °C and 60 °C, are calculated, as listed in Table 4:

3

Fig. 7.

The plots F₀/F Vs. function of [D_t] F₀/(F₀–F) at 20 °C: (A) imidazo[1,2-c]quinazoline 19e; (B) benzo[4,5]imidazo[1,2-c]quinazoline 27e.

Table 4.

Binding constants and binding sites for imidazoquinazolines 19e and 27e.

Compound	KA (L mol–1 s–1) a	KA (L mol–1 s–1) b	n b	r b
19e	2.7 × 10 4	4.8 × 10 4	27	0.997
27e	5.0 × 10 4	6.8 × 10 4	16	0.999

^aTemperature is 60 °C.

^bTemperature is 20 °C.

The data in this Table 4 is graphed against temperature and binding constants, and important thermodynamic profile, including ΔG (free energy change), ΔH (enthalpy change), and ΔS (entropy change), could be computed through the equations as follow:

4

5

The obtained results are presented in Table 5:

Table 5.

Thermodynamic parameters of imidazoquinazolines 19e and 27e.

Compounds	KA (L mol–1 s–1) a	KA (L mol–1 s–1) b	ΔG (kJ mol–1)	ΔH (kJ mol–1)	ΔS (kJ mol–1)
19e	2.7 × 10 4	4.8 × 10 4	– 26.2	– 11.9	48.8
27e	5.0 × 10 4	6.8 × 10 4	– 27.0	– 6.4	70.5

^aTemperature is 60 °C.

^bTemperature is 20 °C.

These figures are of great significance to determine the type of non-covalent forces between drug and enzyme’s binding site, which are categorized into four groups: hydrophobic interaction, hydrogen bond, van der Waals forces, and electrostatic attraction. To identify the type of this interaction, the ΔH and ΔS values play a determining role, as follows: (1) ΔH > 0, ΔS > 0 indicating hydrophobic interactions; (2) ΔH < 0, ΔS < 0 indicating hydrogen bond and van der Waals interactions; (3) ΔH < 0, ΔS > 0 indicating van der Waals forces; and (4) ΔH < 0, ΔS > 0 indicating electrostatic interactions. Considering the signs of ΔH and ΔS as presented in Table 5, electrostatic forces were primarily formed between imidazoquinazolines 19e or 27e and the active site of α-glucosidase.

Molecular docking studies

Comprehensive molecular docking studies were performed on compounds 15, 18, 19, 24, 26, and 27 using AutoDock4 and AutoDockTools (version 1.5.6) to analyze the interactions between the compounds and the crystal structure of α-glucosidase enzyme from Saccharomyces cerevisiae(PDB ID: 3A4A)⁵⁷. As previously described in the SAR study, imidazo[1,2-c]quinazolines 15, 18, and 19 exhibited more favorable inhibitory potencies in comparison with their analog in benzo[4,5]imidazo[1,2-c]quinazoline 24, 26, and 27. Moreover, the presence of amide functionality and 1,2,3-triazole ring played a crucial role in the inhibitory activities. Therefore, these computational studies were conducted to rationalize these orders and find notable interactions.

To begin with the comparison between imidazo[1,2-c]quinazolines 19a-h and benzo[4,5]imidazo[1,2-c]quinazoline 27a-h, it was observed that most of the derivatives, particularly compounds 19, formed at least one hydrogen bond between Glu277 and one of the nitrogen atoms belonged to imidazole moiety of imidazoquinazoline core. Moreover, the imidazo[1,2-c]quinazolines 19 exhibited several interactions, including pi-lone pair, pi-cation/pi-anion, and pi-pi stacked bonds with the terminal phenyl moiety linked to the amide functionality of the compounds. However, these interactions were absent in the corresponding analogues from second series 27. As depicted in Fig. 8, compounds 19 were able to form several electrostatic and hydrophobic interactions through its phenyl rings linked to imidazo[1,2-c]quinazoline backbone, while the number of interactions in benzo[4,5]imidazo[1,2-c]quinazoline backbones 27 was reduced. The same trend was observed for other derivatives in both series.

Fig. 8.

2D models of interactions in (A) 19a; (B) 27a; (C) 19e; (D) 27e with 3A4A.

Up to this point, the superiority of compounds 19 over other series was established in both in vitro and in silico studies. Among them, compound 19e with an IC₅₀ value of 50 µM was the most potent derivative. Its binding energy affinity was -9.25 kcal/mol, remarkably better than that of acarbose (-5.01 kcal/mol) (Fig. 9). The binding mode of this compound was favorable for the formation of hydrogen bond with Arg315 and Asp352, a pi-sulfur bond with Phe178, as well as pi-pi bond between Tyr316 and the triazole ring. Further studies were also conducted on other compounds in the 19 series to find the key role of type of substituents and their positioning.

Fig. 9.

Acarbose and compound 19e are superimposed in the binding site of 3A4A.

The difference between compounds 19a and 19e could be associated with the ability of amide functionality to act as a hydrogen bond donor or acceptor. In compound 19a, the carbonyl group formed a hydrogen bond with Arg315; therefore, it was a hydrogen bond acceptor. However, in compound 19e, N-H moiety formed a hydrogen bond with Tyr158; therefore, it was a hydrogen bond donor, which might be resulted from the presence of methoxy at C-2 position. To investigate the role of positioning, the molecular docking studies were also performed for compounds 19c and 19d. In compound 19c, it was found that an unfavorable acceptor-acceptor bond was formed between carbonyl group and Gln353. Also, there were no interactions with the amide moiety in compound 19d. In a general trend among compounds 19, it was observed that chlorine caused a detrimental effect on the inhibitory activity (compounds 19f-19h). This may be related to the chlorine atom’s ability to hinder the amide functionality from acting as a hydrogen bond donor (Fig. 10).

Fig. 10.

2D models of interactions in (A) 19b; (B) 19c; (C) 19d; (D) 19e; (E) 19g; (F) 19h with 3A4A.

To investigate the substantial role of amide moiety, the molecular docking studies were carried out for compounds 19a, 19c, 19f, 27a, 27c, and 27f as well as their analogues (compounds 18a-c and 26a-c in Table 2, which lack amide functionality). The NH-bond and / or carbonyl group of the amide functionality formed important hydrogen bonds with different aminoacids, including Arg315 in 19a, Asp352 in 19c and 27a, Arg315 in 19f, Gln353 in 27c, and His280 in 27f. It was concluded that the absence of amide moiety in compounds 18a-c and 26a-c caused the lack of noticeable hydrogen bonds, which might be responsible for their lower inhibitory potency. Fig. 11 is exemplifying this noticeable comparison between compounds 18a and 19a.

Fig. 11.

2D models of interactions in (A) 19a; (B) 18a with 3A4A.

To investigate the differences between 19a, 19c, 19f, 27a, 27c, and 27f as well as their analogues (compounds 15a-c and 24a-c in Table 2, which lack 1,2,3-triazole ring), it was found that the removal of triazole ring caused the loss of a crucial hydrogen bond with Glu277 (Fig. 12). This loss could be related with the different pose of the compound in the enzyme pocket, leading to a different distance between this amino acid and the imidazoquinazoline core. As shown in Fig. 13, the binding mode of compound 15a was changed, resulting in an increased distance; consequently, the formation of the former hydrogen bond for compound 19a was not possible anymore.

Fig. 12.

2D models of interactions in (A) 19a; (B) 15a with 3A4A.

Fig. 13.

3D models of interactions of compound 19a (green) vs. 15a (blue) with 3A4A.

Conclusions

The study focused on the synthesis and evaluation of poly-substituted imidazo[1,2-c]quinazolines as potential α-glucosidase inhibitors. Among derivatives, compounds 19e and 27e were identified as the most effective inhibitors, significantly more potent than acarbose. Further analyses, including kinetic studies, circular dichroism, fluorescence spectroscopy, and thermodynamic profiling, revealed that these compounds inhibit α-glucosidase through competitive binding with the enzyme’s active site, altering its secondary and tertiary structures. Moreover, thermodynamic parameters indicated spontaneous electrostatic interactions between the compounds and the enzyme. There was a great agreement between SAR analysis and molecular docking studies, confirming necessity of the presence of amide functionality and a 1,2,3-triazole ring for the inhibitory activity. These findings suggest that substituted imidazo[1,2-c]quinazolines are promising candidates for development of new α-glucosidase inhibitors.

Experimental

All chemicals were purchased from Merck (Germany) and were used without further purification. The reaction progress and the purity of synthesized compounds were monitored by thin-layer chromatography (TLC) on silica gel 250-micron F254 plastic sheets; zones were detected visually under UV light (254 nm). Melting points were measured on an Electrothermal 9100 apparatus. IR spectra were recorded on a Shimadzu IR-460 spectrometer. ¹H and ¹³C NMR spectra were measured (DMSO-d₆ solution) with Bruker DRX-500 AVANCE (at 500.1 and 125.8 MHz) and Bruker DRX-400 AVANCE (at 400.1 and 100.1 MHz) instruments. Chemical shifts were reported in parts per million (ppm), downfield from tetramethylsilane. Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), and multiplet (m). HRMS analysis was performed using a Waters Synapt G1 HDMS High Definition mass spectrometer equipped with an electrospray ionization (ESI) source. The samples were prepared by diluting the isolated compounds in methanol to a final concentration of 10 µg/mL. The analysis was conducted mainly in positive ion mode with a mass range of m/z 50–1000. Elemental analyses for C, H and N were performed using a Heraeus CHN-O-Rapid analyzer.

General synthetic procedures

2-Chloro-N-arylacetamides 3, 2-azido-N-arylacetamides 5, arylazides 7, 2-(4,5-diphenyl-1H-imidazol-2-yl)aniline 12, and 2-(1H-benzo[d]imidazol-2-yl)aniline 22were synthesized using synthetic methods previously reported in the literature^54,58.

General procedure for the preparation of substituted imidazo[1,2-c]quinazoline-5-thiol (14 and 23)

A mixture of compound 12 or 22 (1 equiv.), CS₂ 13 (5 equiv.), KOH (2 equiv.) in EtOH was heated under reflux within 3h. After completion of the reaction as confirmed by TLC, the solvent was removed, and residue was poured into cool water. Subsequently, HCl was added gradually to the mixture to form precipitate. The resulting white powder was filtered and washed with great amount of water to yield in pure corresponding 14 and 23.

General procedure for the preparation of substituted 2-(imidazo[1,2-c]quinazolin-5-ylthio)-N-arylacetamides (15 and 24)

To a stirring solution of compound 14 or 23 (1 equiv.) and K₂CO₃ (1.5 equiv.) in DMF at 80 °C, 2-Chloro-N-arylacetamides 3 (1.2 equiv.) was added gradually and heated for 12h. After completion of the reaction as confirmed by TLC, the reaction mixture was cooled to the ambient temperature. Subsequently, water was added to the mixture and extracted three times with EtOAc. The combined organic extracts were washed with brine, dried over Na₂SO₄ and then concentrated. Finally, the residue was recrystallized in EtOH to obtain the desired compounds 15 and 24.

2-((2,3-diphenylimidazo[1,2-c]quinazolin-5-yl)thio)-N-phenylacetamide (15a):

Milky solid, mp 196–198 °C. IR (KBr) (ν_max/cm^–1): 3328 (NH), 1678 (C=O), 1597, 1473, 1339, 1278, 1198, 1106, 1082, 963, 855, 723, 635. ¹H NMR (400.1 MHz, DMSO-d₆): δ 10.50 (s, 1H, NH), 8.53 (d, J = 7.8 Hz, 1H, CH), 7.95 (d, J = 8.2 Hz, 1H, CH), 7.79 (t, J = 7.6 Hz, 1H, CH), 7.72–7.45 (m, 11H, 11CH), 7.38–7.14 (m, 5H, 5CH), 4.73 (s, 2H, CH₂). HRMS (ESI) m/z for C₃₀H₂₃N₄OS⁺ [M + H]⁺, calculated: 487.1593, found: 487.1622. Anal. Calcd. for C₃₀H₂₂N₄OS: C, 74.05; H, 4.56; N, 11.51.; found: C, 74.32; H, 4.28; N, 11.67 %.

2-((2,3-diphenylimidazo[1,2-c]quinazolin-5-yl)thio)-N-(4-methoxyphenyl)acetamide (15b):

Milky solid, mp 228–231 °C. IR (KBr) (ν_max/cm^–1): 3296 (NH), 1666 (C=O), 1562, 1499, 1453, 1398, 1342, 1293, 1297, 1083, 1011, 999, 865, 819, 767, 697, 637. ¹H NMR (400.1 MHz, DMSO-d₆): δ 10.11 (s, 1H, NH), 8.54 (d, J = 7.8 Hz, 1H, CH), 7.95 (d, J = 8.0 Hz, 1H, CH), 7.79 (t, J = 7.5 Hz, 1H, CH), 7.73–7.46 (m, 10H, 10CH), 7.38–7.27 (m, 3H, 3CH), 6.95 (d, J = 7.9 Hz, 2H, 2CH), 4.78 (s, 2H, CH₂), 3.86 (s, 3H, OCH₃). HRMS (ESI) m/z for C₃₁H₂₅N₄O₂S⁺ [M + H]⁺, calculated: 517.1698, found: 517.1673. Anal. Calcd. for C₃₁H₂₄N₄O₂S: C, 72.07; H, 4.68; N, 10.85.; found: C, 72.34; H, 4.84; N, 11.08 %.

N-(4-chlorophenyl)-2-((2,3-diphenylimidazo[1,2-c]quinazolin-5-yl)thio)acetamide (15c):

Milky solid, mp 242–245 °C. IR (KBr) (ν_max/cm^–1): 3278 (NH), 1686 (C=O), 1593, 1544, 1494, 1414, 1398, 1337, 1287, 1193, 1126, 1064, 994, 944, 788, 737, 699, 639. ¹H NMR (400.1 MHz, DMSO-d₆): δ 10.30 (s, 1H, NH), 8.55 (d, J = 7.4 Hz, 1H, CH), 7.97 (d, J = 7.6 Hz, 1H, CH), 7.78 (t, J = 7.4 Hz, 1H, CH), 7.70–7.45 (m, 10H, 10CH), 7.42–7.30 (m, 5H, 5CH), 4.75 (s, 2H, CH₂). HRMS (ESI) m/z for C₃₀H₂₂ClN₄OS⁺ [M + H]⁺, calculated: 521.1203, found: 521.1188. Anal. Calcd. for C₃₀H₂₁ClN₄OS: C, 69.16; H, 4.06; N, 10.75.; found: C, 68.98; H, 4.33; N, 10.92 %.

2-(benzo[4,5]imidazo[1,2-c]quinazolin-6-ylthio)-N-phenylacetamide (24a):

Yellow solid, mp 188–191 °C. IR (KBr) (ν_max/cm^–1): 3338 (NH), 1643 (C=O), 1523 1484, 1378, 1302, 1177, 1063, 966, 753, 685. ¹H NMR (500.1 MHz, DMSO-d₆): δ 10.94 (s, 1H, NH), 8.56 (d, J = 8.8 Hz, 1H, CH), 8.38 (d, J = 7.5 Hz, 1H, CH), 7.92–7.77 (m, 3H, 3CH), 7.61–7.52 (m, 3H, 3CH), 7.48–7.34 (m, 5H, 5CH), 4.80 (s, 2H, CH₂). HRMS (ESI) m/z for C₂₂H₁₇N₄OS⁺ [M + H]⁺, calculated: 385.1123, found: 385.1149. Anal. Calcd. for C₂₂H₁₆N₄OS: C, 68.73; H, 4.19; N, 14.57.; found: C, 68.56; H, 4.36; N, 14.79 %.

2-(benzo[4,5]imidazo[1,2-c]quinazolin-6-ylthio)-N-(4-methoxyphenyl)acetamide (24b):

Yellow solid, mp 206–208 °C. IR (KBr) (ν_max/cm^–1): 3298 (NH), 1677 (C=O), 1556, 1490, 1435, 1372, 1288, 1263, 1189, 1012, 988, 944, 899, 846, 793, 751, 622. ¹H NMR (500.1 MHz, DMSO-d₆): δ 10.63 (s, 1H, NH), 8.58 (d, J = 8.2 Hz, 1H, CH), 8.44 (d, J = 8.5 Hz, 1H, CH), 7.92 (d, J = 7.4 Hz, 1H, CH), 7.88 (d, J = 8.7 Hz, 1H, CH), 7.83 (t, J = 7.2 Hz, 1H, CH), 7.65–7.43 (m, 5H, 5CH), 7.00 (d, J = 8.7 Hz, 2H, 2CH), 4.75 (s, 2H, CH₂), 3.85 (s, 3H, OCH₃). HRMS (ESI) m/z for C₂₃H₁₉N₄O₂S⁺ [M + H]⁺, calculated: 415.1229, found: 415.1268. Anal. Calcd. for C₂₃H₁₈N₄O₂S: C, 66.65; H, 4.38; N, 13.52.; found: C, 66.83; H, 4.63; N, 13.36 %.

2-(benzo[4,5]imidazo[1,2-c]quinazolin-6-ylthio)-N-(4-chlorophenyl)acetamide (24c):

Yellow solid, mp 220–223 °C. IR (KBr) (ν_max/cm^–1): 3323 (NH), 1675 (C=O), 1608, 1584, 1522, 1477, 1453, 1388, 1257, 1227, 1166, 1076, 1029, 993, 875, 823, 783, 747, 623. ¹H NMR (500.1 MHz, DMSO-d₆): δ 10.98 (s, 1H, NH), 8.54 (d, J = 8.3 Hz, 1H, CH), 8.42 (d, J = 8.5 Hz, 1H, CH), 7.99–7.83 (m, 3H, 3CH), 7.67–7.51 (m, 5H, 5CH), 7.32 (d, J = 8.8 Hz, 2H, 2CH), 4.72 (s, 2H, CH₂). HRMS (ESI) m/z for C₂₂H₁₆ClN₄OS⁺ [M + H]⁺, calculated: 419.0733, found: 419.0698. Anal. Calcd. for C₂₂H₁₅ClN₄OS: C, 63.08; H, 3.61; N, 13.37.; found: C, 62.84; H, 3.83; N, 13.58 %.

General procedure for the preparation of substituted 5-(prop-2-yn-1-ylthio)imidazo[1,2-c]quinazoline (17 and 25)

A mixture of compound 14 or 23 (1 equiv.) and K₂CO₃ (1.5 equiv.) in DMF at ambient temperature was magnetically stirred for 30 min. Subsequently, propargyl bromide 16 (2 equiv.) was added, and then, the temperature was raised to 80 °C. After the reaction completed within an appropriate time, the reaction mixture was cooled to ambient temperature and introduced into the mixture of crushed ice and water. Over the next hour, the solid residue precipitated, which was then filtered and completely washed with water. Pure products 17 and 25 were obtained as a brown powder.

General procedure for the preparation of targeted compounds 18, 19, 26, and 27:

A mixture of compound 5 or 7 (1.2 equiv.), compound 17 or 25 (1 equiv.), CuSO₄.5H₂O (0.3 equiv.), and sodium ascorbate (0.3 equiv.) in DMF was magnetically stirred at ambient temperature for an appropriate time until the starting materials were completely consumed. Then, water was introduced to the reaction mixture, and stirring continued until complete precipitation occurred. The resultant precipitate was filtered and thoroughly washed with enough amount of water. Finally, the solid was recrystallized in EtOH to afford pure, desired products in the form of milky powder.

5-(((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)thio)-2,3-diphenylimidazo[1,2-c]quinazoline (18a):

Milky solid, mp 219–222 °C. IR (KBr) (ν_max/cm^–1): 1595, 1489, 1423, 1396, 1378, 1258, 1232, 1179, 1143, 1063, 996, 938, 848, 744, 650. ¹H NMR (500.1 MHz, DMSO-d₆): δ 8.50 (d, J = 7.7 Hz, 1H, CH), 8.13 (s, 1H, CH), 7.87 (d, J = 7.8 Hz, 1H, CH), 7.75 (t, J = 7.4 Hz, 1H, CH), 7.66 (t, J = 7.4 Hz, 1H, CH), 7.68–7.42 (m, 10H, 10CH), 7.35–7.14 (m, 5H, 5CH), 5.51 and 4.47 (2s, 4H, 2CH₂). HRMS (ESI) m/z for C₃₂H₂₅N₆S⁺ [M + H]⁺, calculated: 525.1861, found: 521.1879. Anal. Calcd. for C₃₂H₂₄N₆S: C, 73.26; H, 4.61; N, 16.02; found: C, 73.52; H, 4.78; N, 16.29 %.

5-(((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-2,3-diphenylimidazo[1,2-c]quinazoline (18b):

Milky solid, mp 251–253 °C. IR (KBr) (ν_max/cm^–1): 1598, 1492, 1432, 1388, 1278, 1245, 1191, 1128, 1086, 1023, 1008, 996, 944, 898, 795, 753, 636. ¹H NMR (500.1 MHz, DMSO-d₆): δ 8.51 (d, J = 7.6 Hz, 1H, CH), 8.16 (s, 1H, CH), 7.82 (d, J = 8.0 Hz, 1H, CH), 7.75 (t, J = 8.2 Hz, 1H, CH), 7.66 (t, J = 7.4 Hz, 1H, CH), 7.71–7.50 (m, 6H, 6CH), 7.33–7.20 (m, 6H, 6CH), 6.91 (d, J = 8.7 Hz, 2H, 2CH), 5.67 and 4.47 (2s, 4H, 2CH₂), 3.63 (s, 3H, OCH₃). HRMS (ESI) m/z for C₃₃H₂₇N₆OS⁺ [M + H]⁺, calculated: 555.1967, found: 555.1939. Anal. Calcd. for C₃₃H₂₆N₆OS: C, 71.46; H, 4.72; N, 15.15; found: C, 71.68; H, 4.50; N, 15.38 %.

5-(((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)-2,3-diphenylimidazo[1,2-c]quinazoline (18c):

Milky solid, mp 268–272 °C. IR (KBr) (ν_max/cm^–1): 1538, 1487, 1436, 1364, 1348, 1329, 1291, 1278, 1230, 1173, 1074, 1044, 931, 855, 839, 769, 688. ¹H NMR (400.1 MHz, DMSO-d₆): δ 8.48 (d, J = 8.7 Hz, 1H, CH), 8.13 (s, 1H, CH), 7.85 (d, J = 8.4 Hz, 1H, CH), 7.74 (t, J = 7.2 Hz, 1H, CH), 7.65 (t, J = 7.2 Hz, 1H, CH), 7.62–7.57 (m, 3H, 3CH), 7.56–7.45 (m, 4H, 4CH), 7.34 (d, J = 8.4 Hz, 2H, 2CH), 7.30–7.19 (m, 5H, 5CH), 5.52 and 4.47 (2s, 4H, 2CH₂). HRMS (ESI) m/z for C₃₃H₂₄ClN₆S⁺ [M + H]⁺, calculated: 559.1472, found: 559.1438. Anal. Calcd. for C₃₃H₂₃ClN₆S: C, 68.62; H, 4.32; N, 15.00; found: C, 68.79; H, 4.58; N, 15.24 %.

2-(4-(((2,3-diphenylimidazo[1,2-c]quinazolin-5-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)-N-phenylacetamide (19a):

Milky solid, mp 289–292 °C. IR (KBr) (ν_max/cm^–1): 3329 (NH), 1656 (C=O), 1528, 1484, 1377, 1327, 1289, 1233, 1188, 1069, 1022, 998, 845, 763, 695, 627. ¹H NMR (400.1 MHz, DMSO-d₆) δ 10.44 (s, 1H, NH-amid), 8.50 (d, J = 7.8 Hz, 1H, CH), 8.11 (s, 1H, CH), 7.92 (d, J = 8.1 Hz, 1H, CH), 7.74 (t, J = 7.5 Hz, 1H, CH), 7.69−7.45 (m, 10H, 10CH), 7.38−7.19 (m, 5H, 5CH), 7.07 (t, J = 7.3 Hz, 1H, CH), 5.27 and 4.55 (2s, 4H, 2CH₂). ¹³C NMR (100.1 MHz, DMSO-d₆) δ 164.59, 147.90, 142.56, 140.98, 140.56, 138.84, 133.77, 133.74, 130.88, 129.85, 129.35, 128.69, 128.08, 127.71, 127.03, 126.16, 124.20, 123.08, 122.83, 119.62, 117.60, 52.65, 26.89. HRMS (ESI) m/z for C₃₃H₂₆N₇OS⁺ [M + H]⁺, calculated: 568.1920, found: 568.1889. Anal. Calcd. for C₃₃H₂₅N₇OS: C, 69.82; H, 4.44; N, 17.27; found: C, 70.03; H, 4.68; N, 17.43 %.

2-(4-(((2,3-diphenylimidazo[1,2-c]quinazolin-5-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)-N-(p-tolyl)acetamide (19b):

Milky solid, mp 299–301 °C. IR (KBr) (ν_max/cm^–1): 3289 (NH), 1676 (C=O), 1577, 1533, 1485, 1453, 1378, 1329, 1267, 1174, 1075, 998, 893, 841, 762, 709, 688. ¹H NMR (400.1 MHz, DMSO-d₆) δ 10.35 (s, 1H, NH-amid), 8.50 (d, J = 7.8 Hz, 1H, CH), 8.11 (s, 1H, CH), 7.91 (d, J = 8.0 Hz, 1H, CH), 7.74 (t, J = 7.5 Hz, 1H, CH), 7.70−7.48 (m, 8H, 8CH), 7.43 (d, J = 8.2 Hz, 2H, 2CH), 7.32−7.18 (m, 3H, 3CH), 7.11 (d, J = 8.2 Hz, 2H, 2CH), 5.24 and 4.54 (2s, 4H, 2CH₂), 2.24 (s, 3H, CH₃). ¹³C NMR (100.1 MHz, DMSO-d₆) δ 164.30, 147.90, 142.56, 140.98, 140.56, 136.32, 133.76, 133.74, 133.17, 130.87, 129.84, 129.71, 128.69, 128.08, 127.71, 127.04, 126.19, 123.08, 122.83, 119.62, 117.59, 52.64, 26.90, 20.90. HRMS (ESI) m/z for C₃₄H₂₈N₇OS⁺ [M + H]⁺, calculated: 582.2076, found: 582.2108. Anal. Calcd. for C₃₄H₂₇N₇OS: C, 70.20; H, 4.68; N, 16.86; found: C, 70.38; H, 4.84; N, 16.71 %.

2-(4-(((2,3-diphenylimidazo[1,2-c]quinazolin-5-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-methoxyphenyl)acetamide (19c):

Milky solid, mp 342–345 °C. IR (KBr) (ν_max/cm^–1): 3348 (NH), 1668 (C=O), 1539, 1544, 1493, 1388, 1363, 1297, 1232, 1181, 1069, 1012, 978, 899, 833, 795, 744, 696, 682. ¹H NMR (400.1 MHz, DMSO-d₆) δ 10.30 (s, 1H, NH-amid), 8.50 (d, J = 7.8 Hz, 1H, CH), 8.10 (s, 1H, CH), 7.92 (d, J = 8.1 Hz, 1H, CH), 7.74 (t, J = 7.8 Hz, 1H, CH), 7.68−7.46 (m, 8H, 8CH), 7.46 (d, J = 8.1 Hz, 2H, 2CH), 7.34−7.16 (m, 3H, 3CH), 6.89 (d, J = 8.1 Hz, 2H, 2CH), 5.22 and 4.54 (2s, 4H, 2CH₂), 3.71 (s, 3H, OCH₃). ¹³C NMR (100.1 MHz, DMSO-d₆) δ 164.04, 155.95, 147.92, 142.56, 140.98, 140.56, 133.77, 133.74, 131.92, 130.87, 129.86, 128.69, 128.08, 127.71, 127.03, 126.15, 123.08, 122.83, 121.17, 117.61, 114.43, 55.60, 52.57, 26.90. HRMS (ESI) m/z for C₃₄H₂₈N₇O₂S⁺ [M + H]⁺, calculated: 598.2025, found: 598.1994. Anal. Calcd. for C₃₄H₂₇N₇O₂S: C, 68.32; H, 4.55; N, 16.40; found: C, 68.58; H, 4.73; N, 16.56 %.

2-(4-(((2,3-diphenylimidazo[1,2-c]quinazolin-5-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)-N-(3-methoxyphenyl)acetamide (19d):

Milky solid, mp 336–338 °C. IR (KBr) (ν_max/cm^–1): 3268 (NH), 1657 (C=O), 1535, 1505, 1474, 1422, 1358, 1299, 1232, 1183, 1096, 989, 923, 868, 835, 778, 726, 653. ¹H NMR (400.1 MHz, DMSO-d₆) δ 10.43 (s, 1H, NH-amid), 8.51 (d, J = 7.0 Hz, 1H, CH), 8.09 (s, 1H, CH), 7.93 (d, J = 7.2 Hz, 1H, CH), 7.76 (t, J = 7.1 Hz, 1H, CH), 7.70−7.47 (m, 8H, 8CH), 7.30−7.20 (m, 5H, 5CH), 6.66 (d, J = 7.3 Hz, 1H, CH), 5.25 and 4.55 (2s, 4H, 2CH₂), 3.71 (s, 3H, OCH₃). ¹³C NMR (100.1 MHz, DMSO-d₆) δ 164.60, 159.98, 147.84, 142.56, 140.98, 140.55, 140.00, 133.75, 133.74, 130.86, 130.16, 129.81, 128.69, 128.09, 127.72, 127.04, 126.32, 123.08, 122.81, 117.56, 111.85, 109.66, 105.38, 55.42, 52.72, 26.88. HRMS (ESI) m/z for C₃₄H₂₈N₇O₂S⁺ [M + H]⁺, calculated: 598.2025, found: 598.2058. Anal. Calcd. for C₃₄H₂₇N₇O₂S: C, 68.32; H, 4.55; N, 16.40; found: C, 68.63; H, 4.69; N, 16.26 %.

2-(4-(((2,3-diphenylimidazo[1,2-c]quinazolin-5-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-methoxyphenyl)acetamide (19e):

Milky solid, mp 318–321 °C. IR (KBr) (ν_max/cm^–1): 3269 (NH), 1678 (C=O), 1536, 1474, 1366, 1342, 1289, 1228, 1158, 1056, 1028, 918, 844, 795, 758, 656. ¹H NMR (400.1 MHz, DMSO-d₆) δ 9.70 (s, 1H, NH-amid), 8.49 (d, J = 7.7 Hz, 1H, CH), 8.10 (s, 1H, CH), 8.00−7.80 (m, 2H, 2CH), 7.73 (t, J = 7.3 Hz, 1H, CH), 7.70−7.42 (m, 8H, 8CH), 7.32−7.18 (m, 3H, 3CH), 7.09 (t, J = 7.6 Hz, 1H, CH), 7.05 (d, J = 7.0 Hz, 1H, CH), 6.89 (d, J = 7.8 Hz, 1H, CH), 5.36 and 4.54 (2s, 4H, 2CH₂), 3.84 (s, 3H, OCH₃). ¹³C NMR (100.1 MHz, DMSO-d₆) δ 164.86, 149.97, 147.90, 142.55, 140.97, 140.56, 133.77, 133.74, 130.87, 129.85, 128.69, 128.08, 127.71, 127.02, 126.96, 126.19, 125.31, 123.08, 122.82, 122.13, 120.73, 117.59, 111.70, 56.14, 52.64, 26.90. HRMS (ESI) m/z for C₃₄H₂₈N₇O₂S⁺ [M + H]⁺, calculated: 598.2025, found: 598.1976. Anal. Calcd. for C₃₄H₂₇N₇O₂S: C, 68.32; H, 4.55; N, 16.40; found: C, 68.08; H, 4.74; N, 16.28 %.

N-(4-chlorophenyl)-2-(4-(((2,3-diphenylimidazo[1,2-c]quinazolin-5-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)acetamide (19f):

Milky solid, mp 358–361 °C. IR (KBr) (ν_max/cm^–1): 3302 (NH), 1662 (C=O), 1548, 1499, 1432, 1387, 1343, 1268, 1183, 1048, 1023, 988, 848, 772, 724, 688, 624. ¹H NMR (400.1 MHz, DMSO-d₆) δ 10.58 (s, 1H, NH-amid), 8.49 (d, J = 7.7 Hz, 1H, CH), 8.10 (s, 1H, CH), 7.91 (d, J = 8.0 Hz, 1H, CH), 7.74 (d, J = 7.6 Hz, 1H, CH), 7.70−7.46 (m, 10H, 10CH), 7.38 (d, J = 8.7 Hz, 2H, 2CH), 7.30−7.15 (m, 3H, 3CH), 5.27 and 4.54 (s, 4H, 2CH₂). ¹³C NMR (100.1 MHz, DMSO-d₆) δ 164.81, 147.89, 142.55, 140.98, 140.56, 137.79, 133.76, 133.73, 130.87, 129.85, 129.28, 128.69, 128.08, 127.79, 127.71, 127.03, 126.15, 123.07, 122.83, 121.19, 117.60, 52.62, 26.88. HRMS (ESI) m/z for C₃₃H₂₅ClN₇OS⁺ [M + H]⁺, calculated: 602.1530, found: 602.1528. Anal. Calcd. for C₃₃H₂₄ClN₇OS: C, 65.83; H, 4.02; N, 16.28; found: C, 65.67; H, 4.28; N, 16.49 %.

N-(3-chlorophenyl)-2-(4-(((2,3-diphenylimidazo[1,2-c]quinazolin-5-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)acetamide (19g):

Milky solid, mp 339–342 °C. IR (KBr) (ν_max/cm^–1): 3278 (NH), 1674 (C=O), 1539, 1498, 1455, 1367, 1292, 1254, 1178, 1096, 1057, 1009, 932, 844, 787, 738, 727, 697, 623. ¹H NMR (400.1 MHz, DMSO-d₆) δ 10.64 (s, 1H, NH-amid), 8.49 (d, J = 7.3 Hz, 1H, CH), 8.13 (s, 1H, CH), 7.90 (d, J = 7.4 Hz, 1H, CH), 7.80−7.45 (m, 10H, 10CH), 7.40 (d, J = 7.3 Hz, 1H, CH), 7.35 (t, J = 7.6 Hz, 1H, CH), 7.30−7.18 (m, 3H, 3CH), 7.14 (d, J = 7.1 Hz, 1H, CH), 5.29 and 4.54 (2s, 4H, 2CH₂). ¹³C NMR (100.1 MHz, DMSO-d₆) δ 165.03, 147.83, 142.56, 140.99, 140.55, 140.24, 133.74, 133.64, 131.08, 130.87, 130.83, 129.82, 128.69, 128.08, 127.72, 127.04, 126.28, 123.95, 123.07, 122.81, 119.14, 118.04, 117.57, 52.68, 26.88. HRMS (ESI) m/z for C₃₃H₂₅ClN₇OS⁺ [M + H]⁺, calculated: 602.1530, found: 602.1486. Anal. Calcd. for C₃₃H₂₄ClN₇OS: C, 65.83; H, 4.02; N, 16.28; found: C, 66.02; H, 4.24; N, 16.52 %.

N-(2-chlorophenyl)-2-(4-(((2,3-diphenylimidazo[1,2-c]quinazolin-5-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)acetamide (19h):

Milky solid, mp 329–332 °C. IR (KBr) (ν_max/cm^–1): 3324 (NH), 1658 (C=O), 1552, 1438, 1366, 1297, 1256, 1158, 1107, 1088, 999, 903, 845, 754, 729, 624. ¹H NMR (400.1 MHz, DMSO-d₆) δ 10.03 (s, 1H, NH-amid), 8.49 (d, J = 7.6 Hz, 1H, CH), 8.14 (s, 1H, CH), 7.89 (d, J = 7.9 Hz, 1H, CH), 7.78−7.40 (m, 11H, 11CH), 7.31 (d, J = 7.6 Hz, 1H, CH), 7.30−7.15 (m, 4H, 4CH), 5.39 and 4.54 (2s, 4H, 2CH₂). ¹³C NMR (100.1 MHz, DMSO-d₆) δ 165.24, 147.82, 142.55, 140.98, 140.54, 134.55, 133.75, 133.73, 130.86, 130.82, 130.05, 129.81, 128.67, 128.08, 127.98, 127.72, 127.12, 127.00, 126.64, 126.23, 123.06, 122.81, 117.56, 52.46, 26.90. HRMS (ESI) m/z for C₃₃H₂₅ClN₇OS⁺ [M + H]⁺, calculated: 602.1530, found: 602.1563. Anal. Calcd. for C₃₃H₂₄ClN₇OS: C, 65.83; H, 4.02; N, 16.28; found: C, 66.04; H, 4.19; N, 16.11 %.

6-(((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)thio)benzo[4,5]imidazo[1,2-c]quinazoline (26a):

Milky solid, mp 209–211 °C. IR (KBr) (ν_max/cm^–1): 1595, 1531, 1473, 1436, 1407, 1338, 1308, 1224, 1158, 1069, 974, 853, 807, 744, 668. ¹H NMR (400.1 MHz, DMSO-d₆) δ 8.54 (d, J = 8.4 Hz, 1H, CH), 8.35 (d, J = 7.3 Hz, 1H, CH), 8.31 (s, 1H, CH), 7.93 (d, J = 8.0 Hz, 1H, CH), 7.84 (d, J = 7.7 Hz, 1H, CH), 7.77 (t, J = 7.1 Hz, 1H, CH), 7.64−7.26 (m, 8H, 8CH), 5.71 and 4.75 (2s, 4H, 2CH₂). HRMS (ESI) m/z for C₂₄H₁₉N₆S⁺ [M + H]⁺, calculated: 423.1392, found: 423.1421. Anal. Calcd. for C₂₄H₁₈N₆S: C, 68.23; H, 4.29; N, 19.89; found: C, 68.45; H, 4.42; N, 19.76 %.

6-(((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)benzo[4,5]imidazo[1,2-c]quinazoline (26b):

Milky solid, mp 244–247 °C. IR (KBr) (ν_max/cm^–1): 1608, 1572, 1538, 1488, 1414, 1323, 1285, 1273, 1218, 1142, 1105, 954, 908, 866, 747, 668, 629. ¹H NMR (400.1 MHz, DMSO-d₆) δ 8.50 (d, J = 7.9 Hz, 1H, CH), 8.35 (d, J = 8.2 Hz, 1H, CH), 8.31 (s, 1H, CH), 7.94 (d, J = 8.1 Hz, 1H, CH), 7.85 (d, J = 8.0 Hz, 1H, CH), 7.80 (t, J = 7.1 Hz, 1H, CH), 7.70−7.44 (m, 5H, 5CH), 5.58 and 4.87 (2s, 4H, 2CH₂), 3.74 (s, 3H, OCH₃). HRMS (ESI) m/z for C₂₅H₂₁N₆OS⁺ [M + H]⁺, calculated: 453.1498, found: 453.1526. Anal. Calcd. for C₂₅H₂₀N₆OS: C, 66.35; H, 4.45; N, 18.57; found: C, 66.58; H, 4.63; N, 18.82 %.

6-(((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)thio)benzo[4,5]imidazo[1,2-c]quinazoline (26c):

Milky solid, mp 252–254 °C. IR (KBr) (ν_max/cm^–1): 1591, 1501, 1475, 1336, 1296, 1224, 1158, 1104, 994, 905, 883, 828, 791, 627. ¹H NMR (400.1 MHz, DMSO-d₆) δ 8.50 (d, J = 7.9 Hz, 1H, CH), 8.38 (d, J = 8.3 Hz, 1H, CH), 8.31 (s, 1H, CH), 7.94 (d, J = 8.1 Hz, 1H, CH), 7.85 (d, J = 8.1 Hz, 1H, CH), 7.80 (t, J = 7.2 Hz, 1H, CH), 7.64 (t, J = 7.3 Hz, 1H, CH), 7.56 (t, J = 7.8 Hz, 1H, CH), 7.49 (t, J = 7.6 Hz, 1H, CH), 7.35 (d, J = 8.2 Hz, 2H, 2CH), 7.28 (d, J = 8.2 Hz, 2H, 2CH), 5.58 and 4.87 (2s, 4H, 2CH₂). HRMS (ESI) m/z for C₂₄H₁₈ClN₆S⁺ [M + H]⁺, calculated: 457.1002, found: 457.0968. Anal. Calcd. for C₂₄H₁₇ClN₆S: C, 63.08; H, 3.75; N, 18.39; found: C, 63.26; H, 3.99; N, 18.51 %.

2-(4-((benzo[4,5]imidazo[1,2-c]quinazolin-6-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-phenylacetamide (27a):

Milky solid, mp 273–276 °C. IR (KBr) (ν_max/cm^–1): 3284 (NH), 1659 (C=O), 1573, 1463, 1459, 1367, 1308, 1298, 1155, 1083, 998, 923, 848, 755, 727, 691, 634. ¹H NMR (500.1 MHz, DMSO-d₆) δ 10.46 (s, 1H, NH-amid), 8.52 (d, J = 7.8 Hz, 1H, CH), 8.36 (s, 1H, CH), 8.32 (d, J = 7.6 Hz, 1H, CH), 7.93 (d, J = 7.8 Hz, 1H, CH), 7.85 (d, J = 7.6 Hz, 1H, CH), 7.77 (t, J = 7.4 Hz, 1H, CH), 7.70−7.30 (m, 7H, 7CH), 7.08 (t, J = 7.8 Hz, 1H, CH), 5.35 and 4.94 (2s, 4H, 2CH₂). ¹³C NMR (125.1 MHz, DMSO-d₆) δ 164.52, 149.42, 147.15, 143.71, 142.11, 138.83, 132.55, 130.21, 129.35, 128.42, 127.59, 127.03, 126.82, 126.02, 124.23, 123.66, 120.00, 119.95, 119.66, 117.59, 115.25, 52.85, 26.40. HRMS (ESI) m/z for C₂₅H₂₀N₇OS⁺ [M + H]⁺, calculated: 466.1450, found: 466.1419. Anal. Calcd. for C₂₅H₁₉N₇OS: C, 64.50; H, 4.11; N, 21.06; found: C, 64.73; H, 4.28; N, 21.34 %.

2-(4-((benzo[4,5]imidazo[1,2-c]quinazolin-6-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(p-tolyl)acetamide (27b):

Milky solid, mp 284–286 °C. IR (KBr) (ν_max/cm^–1): 3228 (NH), 1656 (C=O), 1588, 1541, 1494, 1455, 1364, 1298, 1226, 1188, 1097, 1035, 941, 858, 802, 760, 724, 669, 635. ¹H NMR (500.1 MHz, DMSO-d₆) δ 10.35 (s, 1H, NH-amid), 8.52 (d, J = 7.6 Hz, 1H, CH), 8.40 (d, J = 7.8 Hz, 1H, CH), 8.30 (s, 1H, CH), 7.97−7.87 (m, 2H, 2CH), 7.80 (t, J = 7.4 Hz, 1H, CH), 7.64 (t, J = 7.5 Hz, 1H, CH), 7.56 (t, J = 7.4 Hz, 1H, CH), 7.50 (t, J = 7.2 Hz, 1H, CH), 7.43 (d, J = 7.6 Hz, 2H, 2CH), 7.10 (d, J = 7.6 Hz, 2H, 2CH), 5.30 and 4.93 (2s, 4H, 2CH₂), 2.24 (s, 3H, CH₃). ¹³C NMR (125.1 MHz, DMSO-d₆) δ 164.29, 149.47, 147.19, 143.88, 142.11, 136.32, 133.18, 132.51, 129.70, 128.93, 128.53, 127.58, 127.03, 126.43, 126.02, 124.24, 123.64, 120.04, 119.65, 117.06, 115.29, 52.74, 26.40, 20.90. HRMS (ESI) m/z for C₂₆H₂₂N₇OS⁺ [M + H]⁺, calculated: 480.1607, found: 480.1648. Anal. Calcd. for C₂₆H₂₁N₇OS: C, 65.12; H, 4.41; N, 20.45; found: C, 64.98; H, 4.62; N, 20.68 %.

2-(4-((benzo[4,5]imidazo[1,2-c]quinazolin-6-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-methoxyphenyl)acetamide (27c):

Milky solid, mp 299–302 °C. IR (KBr) (ν_max/cm^–1): 3266 (NH), 1678 (C=O), 1595, 1483, 1456, 1398, 1361, 1288, 1232, 1146, 1073, 1028, 999, 908, 761, 734, 695, 633. ¹H NMR (500.1 MHz, DMSO-d₆) δ 10.31 (s, 1H, NH-amid), 8.50 (d, J = 7.9 Hz, 1H, CH), 8.39 (d, J = 7.8 Hz, 1H, CH), 8.30 (s, 1H, CH), 7.95 (d, J = 7.0 Hz, 1H, CH), 7.88 (d, J = 7.6 Hz, 1H, CH), 7.79 (t, J = 7.8 Hz, 1H, CH), 7.70−7.40 (m, 5H, 5CH), 6.88 (d, J = 8.5 Hz, 2H, 2CH), 5.29 and 4.93 (2s, 4H, 2CH₂), 3.71 (s, 3H, OCH₃). ¹³C NMR (125.1 MHz, DMSO-d₆) δ 164.04, 155.99, 149.44, 147.14, 143.85, 142.11, 132.50, 131.93, 130.68, 128.89, 127.57, 127.01, 126.49, 126.00, 124.25, 123.63, 121.22, 120.03, 117.03, 115.27, 114.44, 55.61, 52.70, 26.38. HRMS (ESI) m/z for C₂₆H₂₂N₇O₂S⁺ [M + H]⁺, calculated: 496.1556, found: 496.1592. Anal. Calcd. for C₂₆H₂₁N₇O₂S: C, 63.02; H, 4.27; N, 19.79; found: C, 63.28; H, 4.09; N, 19.65 %.

2-(4-((benzo[4,5]imidazo[1,2-c]quinazolin-6-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(3-methoxyphenyl)acetamide (27d):

Milky solid, mp 272–275 °C. IR (KBr) (ν_max/cm^–1): 3281 (NH), 1663 (C=O), 1585, 1473, 1455, 1396, 1348, 1292, 1186, 1103, 1099, 1023, 975, 806, 774, 723, 689, 643. ¹H NMR (500.1 MHz, DMSO-d₆) δ 10.45 (s, 1H, NH-amid), 8.51 (d, J = 7.9 Hz, 1H, CH), 8.42−8.27 (m, 2H, 2CH), 7.93 (d, J = 7.7 Hz, 1H, CH), 7.86 (d, J = 7.2 Hz, 1H, CH), 7.78 (t, J = 7.8 Hz, 1H, CH), 7.61 (t, J = 7.6 Hz, 1H, CH), 7.54 (t, J = 7.7 Hz, 1H, CH), 7.46 (t, J = 7.4 Hz, 1H, CH), 7.27 (s, 1H, CH), 7.22 (t, J = 7.6 Hz, 1H, CH), 7.08 (d, J = 7.4 Hz, 1H, CH), 6.66 (d, J = 7.8 Hz, 1H, CH), 5.34 and 4.93 (2s, 4H, 2CH₂), 3.71 (s, 3H, OCH₃). ¹³C NMR (125.1 MHz, DMSO-d₆) δ 164.58, 160.00, 149.33, 147.11, 143.70, 142.08, 140.00, 132.50, 130.16, 128.78, 127.55, 127.01, 126.00, 125.26, 124.21, 123.63, 122.37, 119.97, 116.81, 115.22, 111.89, 109.71, 105.42, 55.43, 52.88, 26.38. HRMS (ESI) m/z for C₂₆H₂₂N₇O₂S⁺ [M + H]⁺, calculated: 496.1556, found: 496.1526. Anal. Calcd. for C₂₆H₂₁N₇O₂S: C, 63.02; H, 4.27; N, 19.79; found: C, 62.86; H, 4.39; N, 20.02 %.

2-(4-((benzo[4,5]imidazo[1,2-c]quinazolin-6-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-methoxyphenyl)acetamide (27e):

Milky solid, mp 258–261 °C. IR (KBr) (ν_max/cm^–1): 3314 (NH), 1675 (C=O), 1577, 1468, 1432, 1396, 1275, 1212, 1180, 1145, 1073, 1043, 1010, 913, 819, 761, 734, 695. ¹H NMR (500.1 MHz, DMSO-d₆) δ 9.69 (s, 1H, NH-amid), 8.53 (d, J = 8.1 Hz, 1H, CH), 8.40 (d, J = 7.7 Hz, 1H, CH), 8.31 (s, 1H, CH), 8.00−7.40 (m, 8H, 8CH), 7.22 (t, J = 7.6 Hz, 1H, CH), 7.07 (d, J = 7.4 Hz, 1H, CH), 6.89 (d, J = 7.6 Hz, 1H, CH), 5.42 and 4.93 (2s, 4H, 2CH₂), 3.83 (s, 3H, OCH₃). ¹³C NMR (125.1 MHz, DMSO-d₆) δ 164.77, 156.65, 149.98, 147.55, 143.70, 142.05, 140.00, 132.52, 130.81, 128.71, 127.56, 126.98, 126.01, 125.32, 124.19, 123.83, 123.65, 122.13, 120.73, 119.97, 117.95, 115.23, 111.73, 56.16, 52.84, 26.40. HRMS (ESI) m/z for C₂₆H₂₂N₇O₂S⁺ [M + H]⁺, calculated: 496.1556, found: 496.1586. Anal. Calcd. for C₂₆H₂₁N₇O₂S: C, 63.02; H, 4.27; N, 19.79; found: C, 63.22; H, 4.42; N, 19.99 %.

2-(4-((benzo[4,5]imidazo[1,2-c]quinazolin-6-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(4-chlorophenyl)acetamide (27f):

Milky solid, mp 309–312 °C. IR (KBr) (ν_max/cm^–1): 3268 (NH), 1654 (C=O), 1532, 1457, 1404, 1348, 1273, 1178, 1078, 1011, 962, 833, 786, 740, 655, 629. ¹H NMR (500.1 MHz, DMSO-d₆) δ 10.58 (s, 1H, NH-amid), 8.53 (d, J = 7.9 Hz, 1H, CH), 8.38 (d, J = 7.4 Hz, 1H, CH), 8.32 (s, 1H, CH), 7.94 (d, J = 7.8 Hz, 1H, CH), 7.88 (d, J = 7.6 Hz, 1H, CH), 7.79 (t, J = 7.7 Hz, 1H, CH), 7.70−7.50 (m, 5H, 5CH), 7.37 (d, J = 8.2 Hz, 2H, 2CH), 5.34 and 4.93 (2s, 4H, 2CH₂). ¹³C NMR (125.1 MHz, DMSO-d₆) δ 164.75, 149.39, 148.79, 143.75, 142.10, 137.80, 132.53, 130.70, 129.27, 128.86, 127.83, 127.60, 127.04, 126.61, 126.01, 124.25, 123.65, 121.22, 120.01, 116.98, 115.24, 52.78, 26.37. HRMS (ESI) m/z for C₂₅H₁₉ClN₇OS⁺ [M + H]⁺, calculated: 500.1060, found: 500.1091. Anal. Calcd. for C₂₅H₁₈ClN₇OS: C, 60.06; H, 3.63; N, 19.61; found: C, 60.18; H, 3.86; N, 19.48 %.

2-(4-((benzo[4,5]imidazo[1,2-c]quinazolin-6-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(3-chlorophenyl)acetamide (27g):

Milky solid, mp 292–294 °C. IR (KBr) (ν_max/cm^–1): 3268 (NH), 1654 (C=O), ¹H NMR (500.1 MHz, DMSO-d₆) δ 10.87 (s, 1H, NH-amid), 8.49 (d, J = 8.2 Hz, 1H, CH), 8.32 (d, J = 7.3 Hz, 1H, CH), 8.26 (s, 1H, CH), 7.96 (d, J = 8.4 Hz, 1H, CH), 7.90 (d, J = 7.2 Hz, 1H, CH), 7.85−7.28 (m, 8H, 8CH), 5.40 and 4.91 (2s, 4H, 2CH₂). ¹³C NMR (125.1 MHz, DMSO-d₆) δ 164.73, 149.86, 147.24, 143.80, 142.43, 134.89, 132.67, 130.78, 128.74, 128.12, 127.94, 127.65, 127.53, 127.36, 126.96, 126.57, 126.28, 125.81, 124.58, 123.00, 119.72, 117.41, 115.22, 52.10, 26.42. HRMS (ESI) m/z for C₂₅H₁₉ClN₇OS⁺ [M + H]⁺, calculated: 500.1060, found: 500.1076. Anal. Calcd. for C₂₅H₁₈ClN₇OS: C, 60.06; H, 3.63; N, 19.61; found: C, 60.24; H, 3.87; N, 19.47 %.

2-(4-((benzo[4,5]imidazo[1,2-c]quinazolin-6-ylthio)methyl)-1H-1,2,3-triazol-1-yl)-N-(2-chlorophenyl)acetamide (27h):

Milky solid, mp 279–282 °C. IR (KBr) (ν_max/cm^–1): 3342 (NH), 1666 (C=O), 1588, 1492, 1414, 1368, 1238, 1177, 1099, 1010, 956, 821, 785, 736, 686, 625. ¹H NMR (500.1 MHz, DMSO-d₆) δ 10.03 (s, 1H, NH-amid), 8.52 (d, J = 8.2 Hz, 1H, CH), 8.40 (d, J = 7.9 Hz, 1H, CH), 8.32 (s, 1H, CH), 7.96 (d, J = 7.8 Hz, 1H, CH), 7.90 (d, J = 7.9 Hz, 1H, CH), 7.81 (t, J = 7.6 Hz, 1H, CH), 7.75−7.45 (m, 5H, 5CH), 7.31 (d, J = 7.4 Hz, 1H, CH), 7.21 (t, J = 7.8 Hz, 1H, CH), 5.44 and 4.94 (2s, 4H, 2CH₂). ¹³C NMR (125.1 MHz, DMSO-d₆) δ 165.27, 149.45, 147.21, 143.79, 142.10, 134.57, 132.53, 130.06, 128.91, 128.00, 127.59, 127.14, 127.04, 127.03, 126.65, 126.28, 126.25, 126.03, 124.24, 123.67, 120.01, 117.00, 115.27, 52.53, 26.38. HRMS (ESI) m/z for C₂₅H₁₉ClN₇OS⁺ [M + H]⁺, calculated: 500.1060, found: 500.1108. Anal. Calcd. for C₂₅H₁₈ClN₇OS: C, 60.06; H, 3.63; N, 19.61; found: C, 60.31; H, 3.44; N, 19.86 %.

α-Glucosidase inhibition assay

α-Glucosidase enzyme ((EC3.2.1.20, Saccharomyces cerevisiae, 20 U/mg) and substrate (p-nitrophenyl glucopyranoside) were purchased from Sigma-Aldrich. Enzyme was prepared in potassium phosphate buffer (pH 6.8, 50 mM), as well as substituted imidazo[1,2-c]quinazolines 15a-c, 18a-c, 19a-h, 24a-c, 26a-c, and 27a-h were dissolved in DMSO (10% final concentration). The various concentrations of these compounds (20 mL), enzyme solution (20 mL) and potassium phosphate buffer (135 mL) were added in the 96-well plate and incubated at 37 °C for 10 min. Afterwards, the substrate (25 mL, 4 mM) was added to the mentioned mixture and allowed to incubate at 37 °C for 20 min. Finally, the change in absorbance was measured at 405 nm by using spectrophotometer (Gen5, Power wave xs2, BioTek, America). The percentage of enzyme inhibition was calculated using equation 1and IC₅₀ values were obtained from non-linear regression curve using the Logit method.

6

Kinetic studies

The kinetic analysis was performed for the most potent compounds (19e and 27e) to reveal the inhibition mode against α-glucosidase. The 20 mL of enzyme solution (1U/mL) was incubated with different concentrations of compound 19e (0, 12.5, 25, and 50 µM) and compound 27e (0, 15, 30, and 60 µM) for 15 min at 30 °C. Afterwards, various concentrations of substrate (p-nitrophenyl glucopyranoside, 1 to 10 mM) was added to measure the change of absorbance for 20 min at 405 nm by using spectrophotometer (Gen5, Power wave xs2, BioTek, America).

In the presence of a competitive inhibitor, K_m increases while V_max does not change. Michaelis–Menten saturation curve for an enzyme reaction shows the relation between the substrate concentration and reaction rate as bellow:

7

According to Michaelis–Menten graph, Km_app is also defined as:

8

[I] is the concentration of inhibitor.

Lineweaver Burk plot that provides a useful graphical method for analysis of the Michaelis–Menten is represented as:

9

Therefore, the slope of Lineweaver Burk plot is equal to:

10

The Km_app value is calculated by equation 6:

11

Therefore, from replot of Km_app Vs. [I], equation 7 can be used for the calculation of K_I^59,60:

12

Fluorescence spectroscopy measurements

This assay was carried out for the most potent derivative 19e and 27e to measure the fluorescence intensity. To this aim, different solutions containing different concentrations (0 to 1.0 µM) of the inhibitor and α-glucosidase (3 mL, 0.1 U/mL) were held for 10 min to equilibrate before measurements. Moreover, the fluorescence of the buffer containing compound 19e and 27ein the absence of the enzyme were subtracted as the background fluorescence. Afterwards, at the excitation wavelength of 280 nm, the fluorescence emission spectra were measured from 300 to 450 nm using a Synergy HTX multi-mode reader (Biotek Instruments, Winooski, VT, USA) equipped with a 1.0 cm quartz cell holder⁶¹.

Molecular docking studies

Molecular docking study using AutoDock4 and Auto Dock Tools (version 1.5.6) was performed on substituted imidazo[1,2-c]quinazolines 15, 18, 19, 24, 26, and 27 to elucidate the patterns of their interactions in the active site of α-glucosidase enzyme from Saccharomyces cerevisiae (PDB ID: 3A4A). Receptor was prepared by removing water molecules and computing Kollman charges with BIOVIA Discovery Studio visualizer and Auto Dock Tools. To investigate the optimal docking grid, redocking process was performed with Acarbose as ligand, and RMSD value of 1.57 was achieved. Afterwards, ligands 15, 18, 19, 24, 26, and 27 were prepared by adding Gasteiger Charges using Auto Dock Tools, and the docking procedure was conducted with 100 genetic algorithm runs using AutoDock4 and AutoGrid4. The interactions were visualized by PLIP online service and PyMOL Molecular Graphics System, Version 2.5.2 Schrödinger, LLC.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

A.F., L.F., and F.P. designed the study and conducted the experiments. F.P., F.S.H., R.F.M., and M.J.D.N. synthesized the targeted compounds. F.P., M.S.M., and B.B. wrote the manuscript, analyzed the characterization data, prepared the Supporting Information File, and carried out the docking studies. S.M. and M.A.F. performed the in vitro enzymatic analysis, kinetic study, circular dichroism spectroscopy, fluorescence spectroscopy measurements, and thermodynamic analysis. F.B., R.D., and M.B.T. revised the manuscript. M.N. and R.F. prepared the revised version of manuscript.

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.