Quinoline-based α-glucosidase inhibitors: synthesis, in vitro potency of inhibition, enzyme kinetics and in silico studies

doi:10.21203/rs.3.rs-3240719/v1

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Quinoline-based α-glucosidase inhibitors: synthesis, in vitro potency of inhibition, enzyme kinetics and in silico studies

https://doi.org/10.21203/rs.3.rs-3240719/v1

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published 04 Jan, 2024

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In an attempt to find potential α-glucosidase inhibitors, a series of twenty new quinoline linked benzothiazole hybrids (8a-t) were synthesized in good yields from suitable reaction procedures and their chemical structures were analyzed by ¹HNMR, ¹³CNMR, IR, and ESI-MS analysis. The synthesized derivatives further screened for their activity against α-glucosidase. Among them, compounds 8b, 8h, 8n and 8o exhibited remarkable α-glucosidase inhibitory activity with IC₅₀values ranging from 38.2 ± 0.3 to 79.9 ± 1.2 µM compared with standard drug acarbose (IC₅₀ = 750.0 ± 2.0 µM). Enzyme kinetic studies of the most active compound (8h) indicated a non-competitive inhibition with K_i value of 38.2 µM. Moreover, the homology modeling, molecular docking and molecular dynamics simulation studies were conducted to reveal key interactions between the most active compound 8h and the targeted enzyme. These results are complementary to the experimental observations. In order to predict the druggability of the novel derivatives, the pharmacokinetic properties were also applied. These findings could be useful for the design and development of new α-glucosidase inhibitors.

Biological sciences/Computational biology and bioinformatics

Biological sciences/Drug discovery

Health sciences/Endocrinology

Physical sciences/Chemistry

Benzothiazole

Quinoline

α-glucosidase

Diabetes

Enzyme kinetic

Synthetic and natural products containing quinoline structure representing diverse pharmacological activities such as anti-biotic [1], anti-cancer [2], anti-fungal [3], anti-malarial [4], anti-bacterial [5], anti-HIV [6], anti-rheumatoid arthritis [7], anti-tuberculosis [8], anti-diabetic [9], anti-oxidant [10], anti-inflammatory [11]. Introducing chloroquine as anti-malaria began a new era of quickly developing drugs like pamaquine, ciprofloxacin, camptothecin, topotecan. On the other hand, thiazole moiety consisting both nitrogen and sulfur is also well known due to their wide spectrum biological potential including anti-inflammatory [12], antitumor [13], antiviral [14], antifungal [15] and antibacterial activities [16]. Moreover, abafungin, ritonavir, tiazofurin, niridazole and nitazoxanide possess this widely used scaffold. Nowadays, as physical activity reduction and obesity increase lead to change lifestyle, the worldwide outbreak of diabetes is taking on pandemic dimensions, and the number of people were suffering from diabetes is estimated to extend to 439 million by 2030 [17]. Recent researches proposed that vascular damage is induced by acute blood glucose elevations via direct action on the vascular endothelium. Finally, myocardial infarctions, coronary heart disease, and cerebral apoplexy are happening [18]. Thus, holding back or protecting at-risk individuals from developing cardiovascular diseases is possible by the careful control of blood glucose levels [19]. The currently anti-diabetic drugs are classified based on distinct mechanistic groups such as insulin secretagogues (sulfonylureas), insulin sensitizers (biguanides and thiazolidinediones), insulin mimetics (glucagon-like peptide analogues and agonists), α-glucosidase inhibitors (miglitol, acarbose, voglibose), and DPP-4 inhibitors [20]. So, due to undesirable side effects of these drugs in some patients or fail their effectiveness over time, looking for novel and safe anti-diabetics is still an urgent clinical need and challenging project. Among these classes, α-glucosidase inhibitors indicated substantial potential for preventing an association between postprandial hyperglycemia and macrovascular complications in diabetic subjects [21]. Moreover, α-glucosidase removes viral glycoproteins and its inhibitors may be beneficial in the viral infection treatment. α-Glucosidase, an exo-type glycosidase enzyme which found on the brush borders of the small intestine, converts carbohydrates into monosaccharides in order to prepare energy for regular function of human body [22]. α-Glucosidase inhibition not only defers the absorption of carbohydrates but also reduces the summit of postprandial blood glucose. Quinoline pharmacophore, which has strong interactions with the active site of α-glucosidase, is well known for its inhibitory activities (Compounds A-D). Also, thiazoles and benzothiazoles (Compounds E, F) has been represented as α-glucosidase inhibitors [10, 23-27] for their ability of providing leads with easy synthetic protocol and structural diversity that make ideal framework in antidiabetic drug discovery (pioglitazone and rosiglitazone) (Figure 1).

In continuing our research to find new pharmacophore for the potent α-glucosidase inhibitors and due to develop more effective α-glucosidase inhibitors by combining the quinoline and benzothiazole moieties in one molecule, we report the synthesis of novel quinolone tagged benzothiazole derivatives, the in vitro α-glucosidase inhibition and a molecular dynamic simulation study was also performed to represent a structural rational of the obtained inhibitory potencies.

The title compounds, 2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-benzylacetamide derivatives (8a-v), were synthesized according to Scheme 1. As shown, the key intermediate 5, 3-(benzo[d]thiazol-2-yl)quinoline-2-thiol, was prepared in three steps. Firstly, to the stirred phosphoryl chloride in DMF at 0-5 °C, acetanilide was added after warming to the room temperature and the resulting mixture was heated for 12 h at 80-90 °C. The obtained 2-chloroquinoline-3-carbaldehyde (2), was entered to the next stage of the reaction without any purification. Secondly, the mixture of compound 2 and sodium sulfide was left to stir for 2 hours at room temperature in DMF. Then, the reaction mixture was poured into crushed ice and acidified with acetic acid. The obtained 2-mercaptoquinoline-3-carbaldehyde (3) was further recrystallized in ethanol. In the third step, the latter intermediate (3) and 2-aminobenzenethiol 4 were dissolved and stirred at room temperature. Along with adding sodium metabisulfite, the resulted mixture is allowed to reflux for about 12 hours. After reaction completion, the mixture was precipitated in ice water, filtered, and purified by recrystallization in ethanol to afford compound 5. On the other hand, the chloroacetyl chloride was added to amine derivatives 6 in DMF, while they were cooled to 0°C. Then the mixture was left to stir at room temperature for 12 hours, which was resulted acetamide derivatives 7. Finally, the 2-chloro-N-substituted acetamide derivatives (7) were reacted with 3-(benzo[d]thiazol-2-yl)quinoline-2-thiol (3) in dry acetone and K₂CO₃ at room temperature for 4 h, filtered and the solid product was recrystallized by ethanol to give title products (8).

In order to discover potent α-glucosidase inhibitors and to investigate the structure-activity relationships, all synthesized derivatives 8a-t were screened to assess their potential inhibitory activities. Half maximal inhibitory concentration of the potency of the compounds in inhibiting α-glucosidase, in comparison with acarbose as the reference drug, are shown in Table 1. Based on the obtained IC₅₀s, all of tested compounds demonstrated potent inhibitory activities (IC₅₀s = 38.2 ± 0.3 - 384.3 ± 0.3 µM) against α-glucosidase compared with acarbose (IC₅₀ = 750.0 ± 2.0 µM). The para-fluorophenyl (8h) and para-tolyl (8c) derivatives are the most active and weakest ones among all synthesized compounds, respectively.

In general, series A (compounds 8a-p) containing no CH₂ unit in linker (n= 0) showed better α-glucosidase inhibitory activity than the corresponding congener with one or two CH₂ unit (n= 1, 2). Interestingly, the most potent compounds were in series A, including 8b, 8h, 8n and 8o with IC₅₀values ranging from 38.2 ± 0.3 to 79.9 ± 1.2 µM. Most of the latter derivatives bear an electron-withdrawing groups and also substitution in ortho and para positions. Although the weakest compound had a para-toluene moiety (8c), compound 8b with ortho-toluene group showed 5-fold more potency. However, the ethyl group in para position (8d) was better tolerated compared to para-methyl group.

Interestingly, the unsubstituted analogs (8a, 8q and 8t) showed weak activity α-glucosidase. This finding indicated that the general backbone has not enough intrinsic potential for the α-glucosidase inhibitory activity. Comparison between halogenated derivatives (8g-l) revealed that the presence of halogen on the para position significantly improved the activity. Moreover, introduction of 4-fluoro substituent on the phenyl ring (8h) increased the inhibitory activity between different 4-halogenated derivatives. The 4-fluorobenzyl derivative 8s with the corresponding non-benzylated analog 8h showed that the presence of benzyl ring significantly decreased the activity about 2-fold in case of electron-withdrawing group. The presence of benzyl ring had positive effect on the α-glucosidase activity, based on the obtained IC₅₀ values for the corresponding analogs including electron-donating moieties (8c, 8r). The activity of 4-methylbenzyl derivative 8r was about 2-fold more than that of 8c with 4-methylphenyl, indicating the negative effect of phenyl substituent.

Introducing the second methyl group to compound 8b (IC₅₀ = 79.9 ± 1.2 µM) with ortho-phenyl moiety decreased the activity in the obtained compounds; 8e (IC₅₀ = 130.4 ± 0.5 µM) and 8f (IC₅₀ = 231.6 ± 0.7 µM). The better results were observed with 2,3-dimethyl substituent. While the unsubstituted phenyl analog 8a showed low activity, introduction of α-naphthyl group can restore and also increase the activity with more than 2-fold improvement in compound 8p. Compounds 8m-o containing nitro group were remarkably potent. Among them, the derivatives 8n bearing ortho-nitrotoluene substituent (IC₅₀ = 65.9 ± 1.0 µM) and 8o containing 5-nitrothiazolyl moiety (IC₅₀ = 47.2 ± 0.6 µM) exhibited superior activity compared to the 8m with para-nitrophenyl substitution (IC₅₀ = 198.9 ± 1.5 µM). Furthermore, although the para-toluene derivative 8c was the weakest compound, insertion of the nitro group on the ortho position of phenyl ring resulted in compound 8n with very potent activity (IC₅₀= 65.9 ± 1.0 µM).

Moreover, as stated in Table 1, the phenethylacetamide derivative 8t exhibited superior activity compared to the related benzylacetamide and phenylacetamide analogs 8q and 8a, respectively. Overall, the compounds containing electron-withdrawing groups were better than the compounds bearing electron-donating ones. However, substitution in para position with electron-withdrawing and small size groups is more favorable, and electron-donating in ortho position has more positive inhibitory effect. The SAR analysis demonstrated that the different chloro-substituted derivatives were potent and also the meta derivative was the most one among them.

In general, the obtained biological results revealed that the effect of halogen substituent dramatically impacts on the α-glucosidase inhibition. Importantly, our study demonstrates that the aryl part should be optimized by varying the halogen substituent, and the flour atom presence in the structure is rational. The summary of SAR is presented in Figure 2.

Regarding to Fig. 3, the Lineweaver–Burk plot indicated a non-competitive inhibition for compound 8h. The results show that decreased efficacy of the α-glucosidase enzyme was obtained by binding of 8h to an allosteric site, which is differ from the active site; where the acarbose binds. Furthermore, the plot of the K_m versus different concentrations of inhibitor gave an estimate of the inhibition constant, K_i value of 38.2 μM.

In order to conduct the enzyme assay, the Saccharomyces cerevisiae α-glucosidase enzyme (EC. 3. 2. 1. 20) have been used. However, since the 3-D crystallographic structure of this enzyme is not available in public databases, a new homology model generated using the protein sequence obtained from uniport.org [28]. To create the model, the structure of S. cerevisiae isomaltose (PDB: 3A47) utilized as a template due to its high sequence similarity with the Saccharomyces cerevisiae α-glucosidase (85% similarity). The alignment of the sequences is presented in Figure 4.

To identify potential active sites of the modelled enzyme, a docking study was conducted on the protein model. The site mapping tool was utilized to identify five potential active sites, based on the site map score and overall surface area of the active sites [29]. Based on the scores and surface areas, the most probable active site was chosen as the grid box area for further docking studies. The active site selected for further studies was demonstrated in Figure 5, and was found to contain a plausible surface area of hydrogen bond acceptor/donor and hydrophobic sites.

The predicted active site was utilized for the docking study with compound 8h, which is the most biologically active compound that has been synthesized. The interactions between the compound 8h and the active site pocket residues of the enzyme were observed, as shown in Figure 5b. These interactions included a hydrogen bond between the amine hydrogen of the amide group and PRO 309, dual pi-pi stacking interactions between the benzothiazole system and PHE 157 residue, and several hydrophobic interactions with PRO 240, PHE 158, PHE 231, PHE 300, VAL 303, PHE 310, PHE 311, and LEU 176 residues [30].

The stability of the enzyme-inhibitor complex and enzyme were compared by evaluating the backbone root mean square deviation (RMSD) over 1000 trajectories during a 100 ns MD simulation (Figure 6). The RMSD value of the α-glucosidase Enzyme stabilized after 5 ns, reaching an average value of 3 Å and remaining relatively constant with fewer fluctuations until 40 ns, after which the RMSD value increased significantly and continued to rise until the end of the simulation, with an average RMSD value of 4.5 Å [31, 32].

As shown in Figure 6, the RMSD plot of the α-glucosidase and α-glucosidase-compound 8h complex revealed that the complex stabilized after 20 ns, reaching an average value of 3 Å and maintaining the same level with an amplitude of 1.5 Å until the end of the simulation [33]. The overall RMSD values of the systems exhibited a significant difference, which can be attributed to the stabilizing effect of compound 8h as an inhibitor on the enzyme.

Figure 8 illustrates the interactions between compound 8h and the enzyme's active site pocket, which were observed to occur for over 20% of the simulation duration. The interactions included hydrogen bonding between the amide group and residues PHE 310 and THR 307, a π-cation interaction between the benzothiazole group and ARG 312, as well as two dual π-π stack interactions: one between the quinoline and phenyl rings and HIS 279, and the other between the benzothiazole and phenyl rings with PHE 310. Additionally, hydrophobic interactions were observed with PHE 300 and VAL 303 [35].

As part of preclinical drug development studies, the physicochemical properties and pharmacokinetic profile of the most potent α-glucosidase inhibitors (8h, 8n and 8o) were executed from the SwissADME website and Osiris DataWarrior. As illustrated in Table 2, the data obtained for the compounds are within the limits of Lipinski’s drug-likeness rule and have no violations. Accordingly, P-gp inhibition, log P and bioavailability score have favorable values and also there is no mutagenicity and tumorigenicity predicted for these compounds. Therefore, all information for are within the accepted range for drug-like molecules.

Enzyme inhibition studies

The mode of inhibition of the most active compound 8h, with the lowest IC₅₀, was investigated against the α-glucosidase with different concentrations of p-nitrophenyl-α-D-glucopyranoside (1–16 mM) as substrate in the absence and presence of sample 8h at different concentrations (0, 9.5, 19.1, and 38.2 µM). A Lineweaver–Burk plot was generated to recognize the type of inhibition and the Michaelis–Menten constant (K_m) value was defined from the plot between the reciprocal of the substrate concentration (1/[S]) and reciprocal of enzyme rate (1/V) over different concentrations of inhibitor.

Homology modeling

Homology modeling was performed using the amino acid sequence FASTA file of the saccharomyces cerevisiae α-glucosidase enzyme (EC. 3. 2. 1. 20), which was downloaded from uniprot.org (1) using the uniprot code P38158. The saccharomyces cerevisiae isomaltase enzyme (PDB ID: 3A47) was chosen as the template enzyme based on previous reports (3). The homology modeling was conducted using Maestro Prime (4).

Molecular docking

Molecular modeling investigations were carried out using the Maestro Molecular Modeling platform (version 10.5) by Schrödinger, LLC (2).

For molecular docking, the modeled protein from the previous stage was prepared using the Protein Preparation Wizard (5), and missing sidechains and loops were filled using the Prime tool (4). H-bonds were assigned by PROPKA at PH: 7.4. The 2D structure of the ligand was drawn in ChemDraw (ver. 16) and exported as an SDF file for use in the next step. The ligand was prepared using the OPLS_2005 forcefield and EPIK (7) at a target PH of 7.0 ± 2, using the LigPrep module (6). The SiteMap tool (8) was utilized to identify potential binding sites of the enzyme-substrate complex, with the SiteMap report including 5 potential binding sites with at least 15 site points per site and a more restrictive definition of hydrophobicity. A grid box was generated for each binding site using the reported sites as entries, with a box size of 25 Å. Compound 8h was docked onto the binding sites using Glide (9) with standard precision and flexible ligand sampling, with 20 poses reported per ligand.

Molecular dynamic:

Molecular dynamics simulations were performed using Desmond from the Schrodinger Maestro interface (10), based on the results of the previous docking stage. An orthorhombic cell was defined and filled with TIP3P model water molecules, and adequate Na ions were added to the system to neutralize the overall charge of the complex. The simulation time was 100 ns, and the NPT ensemble was applied with a constant number of atoms, constant pressure (1.01325 bar), and constant temperature (300 K), using the 1.0-ps interval Nose-Hoover chain method as the default thermostat and the 2.0-ps interval Martyna-Tobias-Klein as the default barostat. The results of the molecular dynamics simulation were analyzed using the Maestro graphical interface (11).

General procedure for the preparation of 2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-arylacetamide (8a-t)

A mixture of 3-(benzo[d]thiazol-2-yl)quinoline-2-thiol (1 mmol) 7, 2-chloro-N-substituted acetamide derivatives 5a-t (1.2 mmol) in dry acetone (10 mL) and anhydrous K₂CO₃ (1 mmol) was stirred at room temperature for 4 h, filtered and the solid product formed was crystallized in ethanol to give final products 8a-t.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-phenylacetamide (8a)

Cream solid;Yield:71%; mp: 193-195 ^oC; IR (KBr, v_max) 3213 (NH), 3020 (CH Aromatic), 2965 (CHAliphatic), 1671 (C=O) Cm^-1; ¹H NMR (500 MHz,DMSO-d6) δ 10.41 (s, 1H, NH_Amid), 8.85 (s, 1H, H₄), 8.24 (d, J=8.00 Hz, 1H, H_Ar), 8.17 (d, J=8.20 Hz, 1H, H_Ar), 8.11 (d, J=8.20 Hz, 1H, H_Ar), 7.91 (d, J=8.40 Hz, 2H, H_Ar), 7.80 (t, J=7.70 Hz, 1H, H_Ar), 7.61 (d, J=7.80 Hz, 2H, H_Ar), 8.57-8.53 (m, 1H, H_Ar), 7.30 (t, J=7.70 Hz, 2H, H_Ar), 7.03 (t, J=7.40 Hz, 1H, H_Ar), 4.21 (s, 2H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 167.55, 164.32, 156.93, 153.53, 147.44, 139.75, 138.74, 135.34, 132.28, 129.24, 127.55, 127.37, 126.89, 126.61, 125.61, 125.18, 123.70, 122.82, 119.54, 36.64, ppm ; ESI-MS (C₂₄H₁₇N₃OS₂): calculated m/z 427.08 [M+H]⁺, observed m/z 427.24 [M+H]⁺; Anal. Calcd : C₂₄H₁₇N₃OS₂ ; C, 67.42; H, 4.01; N, 9.83; Found; C, 67.59; H, 4.38; N, 9.98.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(o-tolyl)acetamide (8b)

Cream solid;Yield:68%; mp: 211-213 ^oC; IR (KBr, v_max) 3221 (NH), 3025 (CH Aromatic), 2960 (CHAliphatic), 1679 (C=O) Cm^-1; ¹H NMR (500 MHz,DMSO-d6) δ 9.67 (s, 1H, NH_Amid), 8.87 (s, 1H, H₄), 8.24 (d, J=8.20 Hz, 1H, H_Ar), 8.17 (d, J=8.20 Hz, 1H, H_Ar), 8.13 (d, J=8.20 Hz, 1H, H_Ar), 7.98 (d, J=8.40 Hz, 1H, H_Ar), 7.84 (t, J=7.70 Hz, 1H, H_Ar), 7.61 (d, J=8.10 Hz, 2H, H_Ar), 7.55 (t, J=7.70 Hz, 1H, H_Ar), 7.38 (d, J=8.10 Hz, 1H, H_Ar), 7.17 (d, J=7.50 Hz, 1H, H_Ar), 7.11 (t, J=7.40 Hz, 1H, H_Ar), 7.06 (d, J=7.60 Hz, 1H, H_Ar), 4.26 (s, 2H, CH₂), 2.14 (s, 3H, CH₃) ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 167.44, 164.32, 156.92, 153.53, 147.50, 138.82, 136.85, 135.38, 132.28, 132.05, 130.72, 129.29, 127.65, 127.36, 126.92, 126.60, 126.39, 125.77, 125.60, 125.24, 123.69, 122.83, 35.90, ppm ; ESI-MS (C₂₅H₁₉N₃OS₂): calculated m/z 441.10 [M+H]⁺, observed m/z 441.21 [M+H]⁺; Anal. Calcd : C₂₅H₁₉N₃OS₂ ; C, 68.00; H, 4.34; N, 9.52; Found; C, 68.21; H, 4.58; N, 9.76.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(p-tolyl)acetamide (8c)

Cream solid;Yield:68%; mp: 217-219 ^oC; IR (KBr, v_max) 3234 (NH), 3040 (CH Aromatic), 2975 (CHAliphatic), 1664 (C=O) Cm^-1; ¹H NMR (500 MHz,DMSO-d6) δ 10.32 (s, 1H, NH_Amid), 8.84 (s, 1H, H₄), 8.23 (d, J=7.90 Hz, 1H, H_Ar), 8.16 (d, J=8.40 Hz, 1H, H_Ar), 8.10 (d, J=8.10 Hz, 1H, H_Ar), 7.91 (d, J=8.40 Hz, 1H, H_Ar), 7.80 (t, J=7.80 Hz, 1H, H_Ar), 7.62 (d, J=7.60 Hz, 1H, H_Ar), 7.55 (d, J=7.70 Hz, 1H, H_Ar), 7.51 (d, J=7.70 Hz, 2H, H_Ar), 7.47 (d, J=7.90 Hz, 1H, H_Ar), 7.09 (d, J=7.90 Hz, 1H, H_Ar), 4.19 (s, 2H, CH₂), 2.23 (s, 3H, CH₃) ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 167.27, 164.31, 156.95, 153.53, 147.45, 138.72, 137.24, 135.34, 132.59, 132.28, 129.60, 129.25, 127.57, 127.36, 126.89, 126.60, 125.63, 125. 18., 123.70, 122.82, 119.67, 119.55, 36.60, 20.89, ppm ; ESI-MS (C₂₅H₁₉N₃OS₂): calculated m/z 441.10 [M+H]⁺, observed m/z 441.12 [M+H]⁺; Anal. Calcd : C₂₅H₁₉N₃OS₂ ; C, 68.00; H, 4.34; N, 9.52; Found; C, 68.16; H, 4.49; N, 9.68.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(4-ethylphenyl)acetamide (8d)

Cream solid;Yield:81%; mp: 195-197 ^oC; IR (KBr, v_max) 3401 (NH), 3065(CH Aromatic), 2950(CHAliphatic), 1670 (C=O) Cm^-1; ¹H NMR (500 MHz,DMSO-d6) δ 10.33 (s, 1H, NH_Amid), 8.85 (s, 1H, H₄), 8.24 (d, J=8.00 Hz, 1H, H_Ar), 8.17 (d, J=8.20 Hz, 1H, H_Ar), 8.11 (d, J=8.10 Hz, 1H, H_Ar), 7.92 (d, J=8.40 Hz, 1H, H_Ar), 7.81 (t, J=7.80 Hz, 1H, H_Ar), 7.62 (t, J=7.80 Hz, 1H, H_Ar), 7.60-7.54 (m, 2H, H_Ar), 7.52 (d, J=8.50 Hz, 2H, H_Ar), 7.12 (t, J=8.00 Hz, 2H, H_Ar), 4.19 (s, 2H, CH₂), 3.63 (d, J=7.60 Hz, 2H, CH_2Ethyl), 1.13 (t, J=7.60 Hz, 3H, CH_3Ethyl), ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 167.34, 164.31, 156.90, 153.51, 147.44, 139.15, 138.70, 137.36, 135.32, 132.29, 129.23, 128.41, 127.57, 127.38, 126.91, 126.63, 125.61, 125.16, 123.69, 122.79, 119.80, 119.68, 36.54, 28.03, 16.13, ppm ; ESI-MS (C₂₆H₂₁N₃OS₂): calculated m/z 455.11 [M+H]⁺, observed m/z 455.19[M+H]⁺; Anal. Calcd : C₂₆H₂₁N₃OS₂ ; C, 68.54; H, 4.65; N, 9.22; Found; C, 68.69; H, 4.84; N, 9.41.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(2,3-dimethylphenyl)acetamide (8e)

Cream solid;Yield:66%; mp: 210-212 ^oC; IR (KBr, v_max) 3227 (NH), 3020 (CH Aromatic), 2965 (CHAliphatic), 1678 (C=O) Cm^-1; ¹H NMR (500 MHz,DMSO-d6) δ 9.73 (s, 1H, NH_Amid), 8.87 (s, 1H, H₄), 8.24 (d, J=8.10 Hz, 1H, H_Ar), 8.17 (d, J=8.20 Hz, 1H, H_Ar), 8.13 (d, J=8.30 Hz, 1H, H_Ar), 7.99 (d, J=8.40 Hz, 1H, H_Ar), 7.84 (t, J=7.80 Hz, 1H, H_Ar), 8.61 (d, J=7.90 Hz, 2H, H_Ar), 7.56 (d, J=7.60 Hz, 1H, H_Ar), 7.12 (d, J=7.60 Hz, 1H, H_Ar), 7.05-6.95 (m, 2H, H_Ar), 4.25 (s, 2H, CH₂), 2.20 (s, 3H, CH₃), 2.01 (s, 3H, CH₃), ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 167.45, 164.33, 156.97, 153.53, 147.52, 138.80, 137.38, 136.64, 135.39, 132.27, 131.59, 129.29, 127.68, 127.36, 126.91, 126.60, 125.77, 125.61, 125.24, 123.78, 123.70, 122.83, 35.83, 20.56, 14.46, ppm ; ESI-MS (C₂₆H₂₁N₃OS₂): calculated m/z 455.11 [M+H]⁺, observed m/z 455.24[M+H]⁺; Anal. Calcd : C₂₆H₂₁N₃OS₂ ; C, 68.54; H, 4.65; N, 9.22; Found; C, 68.69; H, 4.83; N, 9.51.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(2,6-dimethylphenyl)acetamide (8f)

Cream solid;Yield:68%; mp: 215-217 ^oC; IR (KBr, v_max) 3235 (NH), 3030 (CH Aromatic), 2970 (CHAliphatic), 1684 (C=O) Cm^-1; ¹H NMR (500 MHz,DMSO-d6) δ 9.56 (s, 1H, NH_Amid), 8.87 (s, 1H, H₄), 8.25 (d, J=8.00 Hz, 1H, H_Ar), 8.18-8.11(m, 2H, H_Ar), 8.02 (d, J=8.50 Hz, 1H, H_Ar), 7.85 (t, J=7.60 Hz, 1H, H_Ar), 7.62 (d, J=7.40 Hz, 2H, H_Ar), 7.55 (t, J=7.60 Hz, 1H, H_Ar), 7.02-6.96 (m, 3H, H_Ar), 4.28 (s, 2H, CH₂), 2.06 (s, 6H, 2xCH₃) ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 168.77, 160.08, 155.46, 150.11, 144.22, 138.82, 136.14, 135.73, 135.56, 135.41, 133.67, 132.21, 131.24, 130.18, 129.27, 128.18, 128.03, 127.73, 127.38, 126.93, 126.88, 126.60, 123.68, 122.83, 121.62, 37.82, 18.48, ppm ; ESI-MS (C₂₆H₂₁N₃OS₂): calculated m/z 455.11 [M+H]⁺, observed m/z 455.19[M+H]⁺; Anal. Calcd : C₂₆H₂₁N₃OS₂ ; C, 68.54; H, 4.65; N, 9.22; Found; C, 68.72; H, 4.87; N, 9.45.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(2-fluorophenyl)acetamide (8g)

Brown solid;Yield:65%; mp: 201-203 ^oC; IR (KBr, v_max) 3343(NH), 3045 (C-H Aromatic), 2970 (CHAliphatic), 1654 (C=O) Cm^-1;¹H NMR (500 MHz,DMSO-d6) δ 10.47 (s, 1H, NH_Amid), 8.86 (s, 1H, H₄), 8.25 (d, J=8.10 Hz, 1H, H_Ar), 8.17 (d, J=8.20 Hz, 1H, H_Ar), 8.12 (d, J=8.10 Hz, 1H, H_Ar), 7.90 (d, J=8.40 Hz ,1H, H_Ar), 7.80 (t, J=7.70 Hz, 1H, H_Ar), 7.63 (d, J=6.70 Hz, 2H, H_Ar), 7.59-7.50 (m, 2H, H_Ar), 7.14 (t, J=8.80 Hz, 3H, H_Ar), 4.20 (s, 2H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 167.48, 164.32, 156.91, 147.44, 138.76, 135.33, 132.31, 129.28, 127.54, 127.37, 126.90, 126.62, 125.61, 125.19, 123.70, 122.84, 121.30, 121.23, 115.88, 115.71, 36.55 ppm ; ESI-MS (C₂₄H₁₆FN₃OS₂): calculated m/z 445.07 [M+H]⁺, observed m/z 445.20 [M+H]⁺; Anal. Calcd : C₂₄H₁₆FN₃OS₂ ; C, 64.70; H, 3.62; N, 9.43; Found; C, 64.86; H, 3.71;N, 9.68.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(4-fluorophenyl)acetamide (8h)

Brown solid;Yield:68%; mp: 219-221 ^oC; IR (KBr, v_max) 3354(NH), 3030 (C-H Aromatic), 2980 (CHAliphatic), 1659 (C=O) Cm^-1;¹H NMR (500 MHz,DMSO-d6) δ 9.91 (s, 1H, NH_Amid), 8.88 (s, 1H, H₄), 8.23 (d, J=8.10 Hz, 1H, H_Ar), 8.16 (d, J=8.00 Hz, 1H, H_Ar), 8.12 (d, J=8.10 Hz, 1H, H_Ar), 7.95 (d, J=8.40 Hz ,1H, H_Ar), 7.83 (d, J=8.40 Hz, 2H, H_Ar), 7.65-7.56 (m, 4H, H_Ar), 7.54 (t, J=7.90 Hz, 1H, H_Ar), 7.37 (d, J=8.40 Hz, 1H, H_Ar), 4.29 (s, 2H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 168.32, 164.29, 156.56, 153.52, 147.44, 138.93, 135.31, 134.64, 132.32, 129.30, 129.26, 128.07, 127.67, 127.39, 127.03, 126.65, 126.38, 125.65, 125.28, 123.70, 122.83, 35.87, ppm ; ESI-MS (C₂₄H₁₆FN₃OS₂): calculated m/z 445.07 [M+H]⁺, observed m/z 445.18 [M+H]⁺; Anal. Calcd : C₂₄H₁₆FN₃OS₂ ; C, 64.70; H, 3.62; N, 9.43; Found; C, 64.91; H, 3.76;N, 9.59.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(2-chlorophenyl)acetamide (8i)

Brown solid;Yield:71%; mp: 221-223 ^oC; IR (KBr, v_max) 3327(NH), 3015 (C-H Aromatic), 2940 (CHAliphatic), 1657(C=O) Cm^-1;¹H NMR (500 MHz,DMSO-d6) δ 10.33 (s, 1H, NH_Amid), 8.88 (s, 1H, H₄), 8.24 (d, J=8.00 Hz, 1H, H_Ar), 8.18 (d, J=8.10 Hz, 1H, H_Ar), 8.15-8.09 (m, 2H, H_Ar), 8..03 (d, J=8.40 Hz, 1H, H_Ar), 7.91 (d, J=8.50 Hz ,1H, H_Ar), 7.82 (t, J=7.90 Hz, 1H, H_Ar), 7.75 (d, J=8.30 Hz, 1H, H_Ar), 7.62 (t, J=8.10 Hz, 1H, H_Ar), 7.55-7.46 (m, 1H, H_Ar), 7.38 (t, J=8.00 Hz, 2H, H_Ar), 4.40 (s, 2H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 166.98, 164.31, 156.97, 155.66, 153.53, 147.46, 138.73, 135.35, 132.89, 132.29, 129.26, 127.58, 127.37, 126.89, 126.61, 125.65, 125.18, 123.70, 122.83, 121.10, 114.35, 36.50, ppm ; ESI-MS (C₂₄H₁₆ClN₃OS₂): calculated m/z 461.04 [M+H]⁺, observed m/z 461.23 [M+H]⁺; Anal. Calcd : C₂₄H₁₆ClN₃OS₂ ; C, 62.40; H, 3.49; N, 9.10; Found; C, 62.49; H, 3.69; N, 9.32.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(3-chlorophenyl)acetamide (8j)

Brown solid; Yield:65%; mp: 226-228 ^oC; IR (KBr, v_max) 3336(NH), 3030 (C-H Aromatic), 2965 (CHAliphatic), 1651(C=O) Cm^-1;¹H NMR (500 MHz,DMSO-d6) δ 10.62 (s, 1H, NH_Amid), 8.87(s, 1H, H₄), 8.25 (d, J=8.00 Hz, 1H, H_Ar), 8.17 (d, J=8.10 Hz, 1H, H_Ar), 8.12 (d, J=8.10 Hz, 1H, H_Ar), 7.86 (d, J=8.40 Hz ,1H, H_Ar), 7.83-7.78 (m, 2H, H_Ar), 7.63 (t, J=7.70 Hz, 1H, H_Ar), 7.59-7.54 (m, 2H, H_Ar), 7.50 (d, J=8.20 Hz, 1H, H_Ar), 7.34 (t, J=8.30 Hz, 1H, H_Ar), 7.09 (d, J=7.90 Hz, 1H, H_Ar), 4.20 (s, 2H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 168.07, 164.33, 156.83, 147.41, 142.73, 141.20, 138.77, 135.32, 133.57, 132.33, 130.98, 129.30, 127.39, 126.94, 126.64, 125.57, 125.20, 123.71, 123.38, 122.85, 118.95, 117.87, 36.65, ppm ; ESI-MS (C₂₄H₁₆ClN₃OS₂): calculated m/z 461.04 [M+H]⁺, observed m/z 461.11 [M+H]⁺; Anal. Calcd : C₂₄H₁₆ClN₃OS₂ ; C, 62.40; H, 3.49; N, 9.10; Found; C, 62.53; H, 3.72; N, 9.38.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(4-chlorophenyl)acetamide (8k)

Brown solid; Yield:73%; mp: 2230-228 ^oC; IR (KBr, v_max) 3328(NH), 3040 (C-H Aromatic), 2950 (CHAliphatic), 1668 (C=O) Cm^-1;¹H NMR (500 MHz,DMSO-d6) δ 10.56 (s, 1H, NH_Amid), 8.85 (s, 1H, H₄), 8.23 (d, J=8.00 Hz, 1H, H_Ar), 8.16 (d, J=8.20 Hz, 1H, H_Ar), 8.11 (d, J=8.20 Hz, 1H, H_Ar), 7.87 (d, J=8.40 Hz ,1H, H_Ar), 7.79 (t, J=8.00 Hz, 1H, H_Ar), 6.67 (d, J=8.40 Hz, 2H, H_Ar), 7.62 (t, J=8.20 Hz, 1H, H_Ar), 7.58-7.50 (m, 2H, H_Ar), 7.35 (d, J=8.50 Hz, 2H, H_Ar), 4.20 (s, 2H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 167.80, 164.32, 156.86, 153.53, 147.42, 138.72, 135.32, 132.31, 129.27, 129.15, 129.10, 128.93, 127.50, 127.37, 126.91, 126.62, 125.57, 125.18, 123.70, 123.39, 122.99, 122.82, 121.18, 121.05, 120.96, 36.52, ppm ; ESI-MS (C₂₄H₁₆ClN₃OS₂): calculated m/z 461.04 [M+H]⁺, observed m/z 461.11 [M+H]⁺; Anal. Calcd : C₂₄H₁₆ClN₃OS₂ ; C, 62.40; H, 3.49; N, 9.10; Found; C, 62.61; H, 3.64; N, 9.27.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(4-bromophenyl)acetamide (8l)

Brown solid; Yield:70%; mp: 187-189^oC; IR (KBr, v_max) 3320(NH), 3030 (C-H Aromatic), 2960 (CHAliphatic), 1650 (C=O) Cm^-1;¹H NMR (500 MHz,DMSO-d6) δ 10.56 (s, 1H, NH_Amid), 8.85 (s, 1H, H₄), 8.23 (d, J=8.00 Hz, 1H, H_Ar), 8.16 (d, J=8.10 Hz, 1H, H_Ar), 8.10 (d, J=8.20 Hz, 1H, H_Ar), 7.87 (d, J=8.40 Hz ,1H, H_Ar), 7.80 (t, J=7.60 Hz, 1H, H_Ar), 7.65-7.58 (m, 3H, H_Ar), 7.48 (d, J=8.50 Hz, 2H, H_Ar), 4.20 (s, 2H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 167.81, 159.50, 156.87, 153.53, 143.57, 137.40, 135.32, 133.55, 132.07, 131.15, 129.29, 127.74, 127.39, 126.93, 126.64, 125.57, 125.19, 124.28, 123.71, 122.85, 121.92, 121.75, 121.43, 38.76, ppm ; ESI-MS (C₂₄H₁₆BrN₃OS₂): calculated m/z 504.99 [M+H]⁺, observed m/z 505.08 [M+H]⁺; Anal. Calcd : C₂₄H₁₆BrN₃OS₂ ; C, 56.92; H, 3.18; N, 8.30; Found; C, 57.12; H, 3.34; N, 8.49.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(4-nitrophenyl)acetamide (8m)

Cream solid;Yield:71%;mp: 209-211 ^oC; IR (KBr, v_max) 3375 (NH), 3045(CH Aromatic), 2960(CHAliphatic), 1665 (C=O) Cm^-1; ¹H NMR (500 MHz,DMSO-d6) δ 11.07 (s, 1H, NH_Amid), 8.87 (s, 1H, H₄), 8.25 (d, J=8.10 Hz, 1H, H_Ar), 8.23 (d, J=8.80 Hz, 2H, H_Ar), 8.11 (d, J=8.20 Hz, 1H, H_Ar), 7.89 (d, J=8.80 Hz, 2H, H_Ar), 7.80 (t, J=8.60 Hz, 1H, H_Ar), 7.76 (d, J=8.10 Hz, 1H, H_Ar), 7.62 (t, J=7.60 Hz, 1H, H_Ar), 7.58-7.53 (m, 2H, H_Ar), 4.25 (s, 2H, CH₂), ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 168.84, 164.34, 156.73, 153.53, 147.36, 145.92, 142.61, 138.78, 135.27, 132.35, 129.28, 127.39, 126.94, 126.65, 125.56, 125.49, 125.19, 123.69, 122.83, 119.13, 36.84, ppm ; ESI-MS (C₂₄H₁₆N₄O₃S₂): calculated m/z 479.02 [M+H]⁺, observed m/z 479.23 [M+H]⁺; Anal. Calcd : C₂₄H₁₆N₄O₃S₂; C, 52.60; H, 2.73; N, 14.60; Found; C, 52.78; H, 2.90; N, 14.82.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(4-methyl-2-nitrophenyl)acetamide (8n)

Cream solid;Yield:71%;mp: 221-223 ^oC; IR (KBr, v_max) 3364 (NH), 3035(CH Aromatic), 2950(CHAliphatic), 1661 (C=O) Cm^-1; ¹H NMR (500 MHz,DMSO-d6) δ 9.94 (s, 1H, NH_Amid), 8.88 (s, 1H, H₄), 8.24 (d, J=8.10 Hz, 1H, H_Ar), 8.17 (d, J=8.10 Hz, 1H, H_Ar), 8.14 (d, J=8.20 Hz, 1H, H_Ar), 7.11-7.09 (m, 1H, H_Ar), 8.03 (d, J=8.20 Hz, 1H, H_Ar), 7.92 (t, J=8.00 Hz, 1H, H_Ar), 7.89 (d, J=8.10 Hz, 1H, H_Ar), 7.81 (t, J=7.80 Hz, 1H, H_Ar), 7.63-7.57 (m, 2H, H_Ar), 7.56-7.53 (m, 1H, H_Ar), 4.25 (s, 2H, CH₂), 2.34 (s, 3H, CH₂) ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 168.40, 164.32, 156.73, 153.53, 147.42, 143.58, 143.47, 138.89, 135.32, 132.37, 129.32, 127.50, 127.39, 126.99, 126.64, 125.92, 125.85, 125.65, 125.27, 123.69, 123.45, 122.84, 122.29, 122.22, 36.21, 18.27, ppm ; ESI-MS (C₂₅H₁₈N₄O₃S₂): calculated m/z 486.08 [M+H]⁺, observed m/z 486.19 [M+H]⁺; Anal. Calcd : C₂₅H₁₈N₄O₃S₂; C, 61.71; H, 3.73; N, 11.51; Found; C, 61.87; H, 3.95; N, 11.70.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(5-nitrothiazol-2-yl)acetamide (8o)

Cream solid;Yield:68%;mp: 199-201 ^oC; IR (KBr, v_max) 3355 (NH), 3070(CH Aromatic), 2965(CHAliphatic), 1670 (C=O) Cm^-1; ¹H NMR (500 MHz,DMSO-d6) δ 10.93 (s, 1H, NH_Amid), 8.81 (s, 1H, H_Ar), 8.49 (s, 1H, H₄), 8.23 (d, J=8.00 Hz, 1H, H_Ar), 8.18 (d, J=8.20 Hz, 1H, H_Ar), 8.09 (d, J=8.10 Hz, 1H, H_Ar), 7.85 (d, J=8.50 Hz, 1H, H_Ar), 7.79 (t, J=7.90 Hz, 1H, H_Ar), 7.61 (t, J=7.70 Hz, 1H, H_Ar), 7.55 (d, J=8.00 Hz, 1H, H_Ar), 4.32 (s, 2H, CH₂), ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 164.31, 157.00, 153.47, 147.41, 145.39, 138.64, 135.37, 132.26, 129.21, 127.43, 127.34, 126.82, 126.58, 125.71, 125.11, 123.69, 122.78, 37.62, ppm ; ESI-MS (C₂₁H₁₃N₅O₃S₃): calculated m/z 479.02 [M+H]⁺, observed m/z 479.23 [M+H]⁺; Anal. Calcd : C₂₆H₂₁N₃OS₂ C, 52.60; H, 2.73; N, 14.60; Found; C, 52.78; H, 2.90; N, 14.82.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(naphthalen-1-yl)acetamide (8p)

Cream solid;Yield:62%; mp: 198-200 ^oC; IR (KBr, v_max) 3224 (NH), 3025 (CH Aromatic), 2980 (CHAliphatic), 1673 (C=O) Cm^-1; ¹H NMR (500 MHz,DMSO-d6) δ 10.33 (s, 1H, NH_Amid), 8.88 (s, 1H, H₄), 8.24 (d, J=8.00 Hz, 1H, H_Ar), 8.18 (d, J=8.40 Hz, 1H, H_Ar), 8.16-8.08 (m, 2H, H_Ar), 8.03 (d, J=8.30 Hz, 1H, H_Ar), 7.91 (t, J=8.50 Hz, 1H, H_Ar), 7.82 (t, J=8.00 Hz, 1H, H_Ar), 7.75 (d, J=8.20 Hz, 1H, H_Ar), 7.62 (t, J=8.00 Hz, 2H, H_Ar), 7.56-7.45 (m, 3H, H_Ar), 7.38 (t, J=8.00 Hz, 2H, H_Ar), 4.41 (s, 2H, CH₂), 2.06 (s, 6H, 2xCH₃) ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 168.26, 159.21, 153.55, 149.33, 144.49, 134.15, 132.28, 130.26, 129.32, 128.54, 128.42, 127.70, 127.38, 126.95, 126.62, 126.47, 126.11, 126.04, 125.88, 123.71, 123.37, 123.01, 122.84, 122.22, 122.01, 121.86, 119.67, 38.87, ppm ; ESI-MS (C₂₈H₁₉N₃OS₂): calculated m/z 477.10 [M+H]⁺, observed m/z 477.32 [M+H]⁺; Anal. Calcd : C₂₈H₁₉N₃OS₂ ; C, 70.41; H, 4.01; N, 8.80; Found; C, 70.53; H, 4.31; N, 8.97.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-benzylacetamide (8q)

Brown solid; Yield: 65%; mp: 180-183 ^o C; IR (KBr, v_max) 3320(NH), 3040 (C-H Aromatic), 2980(CHAliphatic), 1680(C=O) Cm^-1;¹H NMR (500 MHz,DMSO-d6) δ 8.84 (s, 1H, H₄), 8.70 (t, J=6.30 Hz, 1H, NH_Amid), 8.23 (d, J=8.20 Hz, 1H, H_Ar), 8.16 (d, J=8.20 Hz, 1H, H_Ar), 8.11 (d, J=8.10 Hz, 1H, H_Ar), 7.88 (d, J=8.50 Hz, 1H, H_Ar), 7.82 (t, J=7.80 Hz, 1H, H_Ar), 7.61 (d, J=7.50 Hz, 2H, H_Ar), 7.54 (t, J=7.80 Hz, 1H, H_Ar), 7.23-6.12 (m, 5H, H_Ar), 4.31 (d, J=6.10 Hz, 2H, CH_2Benzyl), 4.10 (s, 2H, CH₂), ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 168.43, 164.33, 156.84, 153.53, 147.49, 139.80, 138.74, 135.39, 132.16, 129.20, 128.56, 127.82, 127.50, 127.35, 127.08, 126.87, 126.58, 125.78, 125.21, 123.68, 122.82, 42.94, 35.28, ppm ; ESI-MS (C₂₅H₁₉N₃OS₂): calculated m/z 441.10 [M+H]⁺, observed m/z 441.18 [M+H]⁺; Anal. Calcd : C₂₅H₁₉N₃OS₂ ; C, 68.00; H, 4.34; N, 9.52; Found; C, 68.17; H, 4.58; N, 9.69.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(4-methylbenzyl)acetamide (8r)

Brown solid; Yield: 74%; mp: 182-184 ^o C; IR (KBr, v_max) 3326(NH), 3045 (C-H Aromatic), 2990(CHAliphatic), 1679 (C=O) Cm^-1;¹H NMR (500 MHz,DMSO-d6) δ 8.84 (s, 1H, H₄), 8.64 (t, J=6.00 Hz, 1H, NH_Amid), 8.24 (d, J=8.00 Hz, 1H, H_Ar), 8.16 (d, J=8.10 Hz, 1H, H_Ar), 8.12 (d, J=8.20 Hz, 1H, H_Ar), 7.85 (d, J=8.10 Hz, 1H, H_Ar), 7.80 (t, J=7.90 Hz, 1H, H_Ar), 7.61 (d, J=7.50 Hz, 2H, H_Ar), 7.55 (t, J=7.80 Hz, 1H, H_Ar), 7.08 (d, J=8.10 Hz, 2H, H_Ar), 6.97 (d, J=8.10 Hz, 2H, H_Ar), 4.25 (d, J=5.90 Hz, 1H, CH_2Benzyl), 4.07 (s, 2H, CH₂), 2.22 (s, 23H, CH₃), ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 168.40, 164.32, 156.79, 153.51, 147.47, 138.69, 136.70, 136.11, 135.38, 132.10, 129.44, 129.30, 129.27, 129.21, 129.15, 129.12, 128.94, 127.81, 127.69, 127.54, 127.35, 126.85, 126.58, 125.75, 125.17, 123.67, 122.78, 42.73, 35.30, 21.11, ppm ; ESI-MS (C₂₆H₂₁N₃OS₂): calculated m/z 455.11 [M+H]⁺, observed m/z 455.36 [M+H]⁺; Anal. Calcd : C₂₆H₂₁N₃OS₂ ; C, 68.54; H, 4.65; N, 9.22; Found; C, 68.71; H, 4.91; N, 9.39.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-(4-fluorobenzyl)acetamide (8s)

Brown solid; Yield: 74%; mp: 213-215 ^o C; IR (KBr, v_max) 3337(NH), 3030 (C-H Aromatic), 2980(CHAliphatic), 1662 (C=O) Cm^-1;¹H NMR (500 MHz,DMSO-d6) δ 8.84 (s, 1H, H₄), 8.70 (t, J=6.40 Hz, 1H, NH_Amid), 8.23 (d, J=8.00 Hz, 1H, H_Ar), 8.16 (d, J=8.10 Hz, 1H, H_Ar), 8.11 (d, J=8.10 Hz, 1H, H_Ar), 7.85-7.77 (m, 2H, H_Ar), 7.60 (d, J=7.70 Hz, 2H, H_Ar), 7.54 (t, J=7.70 Hz, 1H, H_Ar), 7.23 (d, J=6.90 Hz, 2H, H_Ar), 6.96 (t, J=8.60 Hz, 2H, H_Ar), 4.28 (d, J=6.00 Hz, 2H, CH_2Benzyl), 4.08 (s, 2H, CH₂), ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 168.52, 164.33, 160.53, 156.77, 153.51, 147.44, 138.72, 136.01, 135.37, 132.13, 129.53, 129.47, 129.19, 127.75, 127.36, 126.88, 126.60, 125.76, 125.17, 123.67, 122.81, 115.30, 115.13, 42.25, 35.27, ppm ; ESI-MS (C₂₅H₁₈FN₃OS₂): calculated m/z 459.09 [M+H]⁺, observed m/z 459.21 [M+H]⁺; Anal. Calcd : C₂₅H₁₈FN₃OS₂ ; C, 65.34; H, 3.95; N, 9.14; Found; C, 65.51; H, 4.09; N, 9.33.

2-((3-(benzo[d]thiazol-2-yl)quinolin-2-yl)thio)-N-phenethylacetamide (8t)

Cream solid;Yield:66%; mp: 225-227 ^oC; IR (KBr, v_max) 3243 (NH), 3050 (CH Aromatic), 2975 (CHAliphatic), 1679 (C=O) Cm^-1; ¹H NMR (500 MHz,DMSO-d6) δ 8.84 (s, 1H, H₄), 8.30-8.22 (m, 2H, NH_Amid, H_Ar), 8.16 (d, J=8.10 Hz, 1H, H_Ar), 8.12 (d, J=8.10 Hz, 1H, H_Ar), 8.90 (d, J=8.50 Hz, 1H, H_Ar), 7.83 (t, J=7.70 Hz, 1H, H_Ar), 7.61 (d, J=7.90 Hz, 2H, H_Ar), 7.55 (t, J=7.60 Hz, 1H, H_Ar), 7.16 (d, J=6.90 Hz, 2H, H_Ar) 7.12 (d, J=7.20 Hz, 2H, H_Ar), 3.99 (s, 2H, CH₂), 3.32-3.25(m, 2H, CH₂), 2.69 (t, J=7.40 Hz, 3H, CH_3Ethyl), ppm. ¹³C NMR (125 MHz, DMSO-d6): δ 168.37, 164.28, 156.82, 153.52, 147.47, 139.79, 138.74, 135.40, 132.23, 129.21, 128.97, 128.67, 127.68, 127.34, 126.87, 126.58, 126.45, 125.75, 125.21, 123.69, 122.82, 41.09, 41.08, 35.51, ppm ; ESI-MS (C₂₆H₂₁N₃OS₂): calculated m/z 455.11 [M+H]⁺, observed m/z 455.27[M+H]⁺; Anal. Calcd : C₂₆H₂₁N₃OS₂ ; C, 68.54; H, 4.65; N, 9.22; Found; C, 68.69; H, 4.90; N, 9.41.

In conclusion, we have developed a new series of quinolone-benzothiazole hybrid including twenty derivatives. All synthesized compounds 8a-t displayed promising α-glucosidase inhibitory activity in comparison to the standard drug acarbose. Three derivatives; 8h, 8n and 8o, were represented significantly higher inhibitory potency than the others as they possess at least one electron-withdrawing group (F, NO₂). Among the synthesized compounds, compound 8h with fluorophenyl moiety on para-position, was found to be the most potent one with an IC₅₀ value of 38.2 ± 0.3 μM. A kinetic study revealed that compound 8h acts through a non-competitive inhibition mechanism with a K_i value of 38.2 μM. Moreover, a docking study and molecular dynamics simulation were conducted on the new protein which was generated via homology modeling. Results showed that the most active compounds 8h interacted with important amino acids in the active site of the enzyme.

Author contribution statement

MKG and ND contributed in the synthesis of compounds. MNM performed the computational studies. MN analyzed the spectroscopic data. MAF and SM carried out the pharmacological experiments. MM supervised the pharmacologic part and analyzed biological data. SMH supervised the work, drafted the manuscript and critically revised the manuscript.

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability No data was used for the research described in the article.

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Scheme 1 is available in the Supplementary Files section.

Tables 1 and 2 are available in the Supplementary Files section.

No competing interests reported.

Scheme1.jpg
Scheme 1. (i) a. POCl₃, DMF, 0 °C, b. 80 °C, 12h; (ii) Na₂S, DMF, room temperature; (iii) sodium metabisulfite, EtOH, reflux; (iv) chloroacetyl chloride, DMF, room temperature, 12h; (v) Acetone, K₂CO₃, room temperature, 4h.
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Quinoline-based α-glucosidase inhibitors: synthesis, in vitro potency of inhibition, enzyme kinetics and in silico studies

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