Facile synthesis and in silico studies of benzothiazole-linked hydroxypyrazolones targeting α-amylase and α-glucosidase 

Abstract

Aim: The objective of the present investigation was to design and synthesize new heterocyclic hybrids comprising benzothiazole and indenopyrazolone pharmacophoric units in a single molecular framework targeting α-amylase and α-glucosidase enzymatic inhibition. Materials & methods: 20 new benzothiazole-appended indenopyrazoles, 3a–t, were synthesized in good yields under environment-friendly conditions via cycloaddition reaction, and assessed for antidiabetic activity against α-amylase and α-glucosidase, using acarbose as the standard reference. Results: Among all the hydroxypyrazolones, 3p and 3r showed the best inhibition against α-amylase and α-glucosidase, which finds support from molecular docking and dynamic studies. Conclusion: Compounds 3p and 3r have been identified as promising antidiabetic agents against α-amylase and α-glucosidase and could be considered valuable leads for further optimization of antidiabetic agents.

GRAPHICAL ABSTRACT

Plain language summary

Summary points.

• A series of 20 benzothiazole-appended indenopyrazoles were synthesized in good yields.

• Structural confirmation of the newly synthesized compounds was ascertained by spectral analysis data and finally by single-crystal x-ray crystallography.

• The titled compounds were screened for their in vitro antidiabetic activity.

• Among all the derivatives, 3p and 3r exhibited promising inhibitory activity against both enzymes, α-amylase and α-glucosidase.

• Enzymatic inhibition was supported by docking studies of compounds 3p with binding energies of -10.3 and -10.4 kcal/mol and 3r with binding energies of -10.3 and -10.7 kcal/mol for α-amylase and α-glucosidase, respectively.

• The docked complexes of the most active compound, 3r, with α-amylase and α-glucosidase were subjected to molecular dynamics study to check the complex stability and the ligand–target interactions at the active sites of the enzymes.

Diabetes mellitus, including Type 1 and Type 2, is one of the worldwide, chronic, persistent, metabolic and epidemic diseases that cause hyperglycemia [1,2]. It has affected approximately 529 million people worldwide in 2021, while it is projected that by 2050, the count may elevate to nearly 1.31 billion [3], which is associated with the increase in blood glucose levels, more cases of obesity, stress and decreased lifespan. The common cause of uncontrolled diabetes, the delay of time to go for treatment, may lead to severe consequences such as liver damage, cardiovascular symptoms, retinopathy, ophthalmic infections and sexual dysfunction [4,5]. Because diabetes distresses about 5% of the global population, it continues to present a formidable challenge for the medical community due to the need for effective treatment with minimal adverse effects. Frequently, research is being directed toward suitable treatments for Type 2 diabetes mellitus by investigators worldwide. Literature reports reveal that a specific set of receptors – such as G protein-coupled receptors, peroxisome proliferator-activated receptor-γ, dipeptidyl peptidase-4, phosphoenolpyruvate carboxykinase, protein tyrosine phosphatase 1B, α-amylase, α-glucosidase, fructose-1,6-bisphosphatase, free fatty acid receptors 1, glucagon receptor, glycogen phosphorylase, aldose reductase and sodium-glucose co-transporter-2 – have been discovered for the progress of antidiabetic medicines and chemotherapies [6,7]. Antihyperglycemic medicines such as acarbose, voglibose, biguanides, insulin, sulfonylureas and miglitol are used to treat Type 2 diabetes mellitus, as they block α-amylase or α-glucosidase and are functioning at regulatory postprandial blood glucose levels. However, these drugs have several drawbacks: oral unavailability, short half-lives, severe gastrointestinal disorders (flatulence, abdominal pain, diarrhea etc.) and an expensive cost [8,9]. Consequently, there is a strong need to develop novel pharmaceutical agents with better selectivity and a reduced incidence of adverse effects.

In recent years, 1,3-benzothiazole, a privileged template in the thiazole family, has attracted considerable attention from synthetic organic chemists due to its existence in natural products, food additives, industrial chemical products, vulcanization inducers, agrochemicals and materials [10–12] as well as showing immense pharmacological activities such as antimicrobial, antidiabetic, anti-inflammatory, anti-HIV, anticancer, antioxidant and antitubercular [13–20]. The importance of the 1,3-benzothiazole scaffold lies in the fact that it has been found to be a crucial component of several commercially available drugs such as zopolrestat, riluzole and sibenadet hydrochloride (Viozan) [10,21]. The numerous applications of benzothiazoles trigger researchers to develop different synthetic routes for their preparation [22,23].

On the other hand, pyrazole skeletons are ubiquitous, five-membered N-heterocyclic compounds that have gained considerable attention due to their versatile applicability as natural products, pharmaceuticals, crop protection chemicals, chemical industrial products and are building blocks for organic and inorganic chemistry [24–28]. Pyrazole and its derivatives exhibit a myriad spectrum of biological activities such as antidepressant, antimicrobial, antiviral, antitumor, analgesic, antitubercular, anti-inflammatory, antihypertensive, antiarthritic, anticonvulsant, antihyperglycemic and anti-HIV [29–33]. Pyrazoles are considered to be the main structural units in several drugs of importance such as deracoxin, celecoxib, pyrazomycin, ramifenazone, phenylbutazone, sildenafil, betazole, fomepizole, crizotinib, tetrazine, lonazolac, difenamizole, tepoxalin, antipyrine, mepirizole, novalgin, zaleplon, indiplon, suzinabant, apixaban, rimonabant and fezolamine [34,35]. Among the pyrazoles, in particular, indenopyrazoles are fused pyrazoles that have gained considerable global research interest owing to their multifaceted pharmacological activities such as anticancer, cannabinoid receptor affinity, anti-inflammatory, antimicrobial, antidiabetic, antidepressive and tubulin polymerization inhibitor [29,36–39]. Due to these facts, various strategies for their preparation remain an area of interest among chemists throughout the world and methods have been reported frequently for their synthesis [40–43]. Considering these facts and in continuation of the authors' interest in the synthesis of heterocycles with diverse biological activities, they herein report the synthesis of 20 benzothiazole-appended hydroxypyrazolones, 3a–t, to show the cumulative effect of these pharmacophores toward α-amylase and α-glucosidase inhibition. Furthermore, in silico molecular docking and dynamic studies of 3a–t were also supported by in vitro antidiabetic results.

The novelty of this work targeting the synthesis of fused hydroxypyrazolone 3 by the reaction of 2-benzoyl/substituted benzoyl-(1H)-indene-1,3(2H)-dione 1 with 2-hydrazinyl-4/6-substituted benzo[d]thiazole 2 lies in the fact that hydroxypyrazolines are very difficult to form from other substrates under these reaction conditions. However, the authors are the first to isolate these hydroxypyrazolones 3 in good yields under environmentally-friendly reaction conditions. Additionally, to the best of their knowledge, they are among the first to report their antidiabetic activity.

Materials & methods

All chemicals were procured from commercial sources and used as such without further purification unless otherwise specified. The solvents were purified and dried before use. Melting points (mps) were determined using open capillary tubes on an electrothermal apparatus (Labco Co., Bengaluru, India) and are uncorrected. Progress of reactions was monitored by TLC analysis using Merck UV-254 plates. Fourier-transform infrared spectroscopy (FTIR) spectra were recorded on an IR Affinity-1 FTIR (Shimadzu, Tokyo, Japan) spectrophotometer in the range of 400–4000 cm^-1 using KBr as a standard. ¹H and ¹³C NMR spectra were recorded on a Bruker Avance III NMR spectrometer (MA, USA) at the operating frequencies 400 MHz and 100 MHz, respectively, using tetramethylsilane as an internal standard. Chemical shift (δ) values are given in parts per million (ppm) and the coupling constant (J) is reported in Hz. D₂O was added to confirm the exchangeable protons. High-resolution mass spectra (HRMS) were recorded under SCIEX-QTOF mass analyzer conditions (Singapore). The ChemDraw ULTRA 12.0.2 (Cambridge Soft, PerkinElmer, Inc., MA, USA) was used for naming the synthesized compounds.

General procedure for the synthesis of 2-(benzoyl/substituted benzoyl)-(1H)-indene-1,3(2H)-diones (1a–j)

An equimolar mixture of diethylphthalate and appropriate ketone (acetophenone/substituted acetophenone) in the presence of freshly prepared sodium methoxide and dry methanol under refluxing underwent Claisen condensation to give 2-(benzoyl/substituted benzoyl)-(1H)-indene-1,3(2H)-diones (1a–j) in good yields in accord with the procedure described in the literature [44].

General procedure for the synthesis of 2-hydrazinyl-4/6-substituted benzo[d]thiazoles (2a & 2b)

Equimolar quantities of appropriate 2/4-substituted aniline and potassium thiocyanate in the presence of glacial acetic acid were added to a solution of bromine in glacial acetic acid dropwise while stirring and maintaining the temperature of the reaction mixture below 10°C by keeping it in an ice bath to give the corresponding 4/6-substituted benzo[d]thiazol-2-amines, which, upon subsequent treatment with hydrazine hydrate in the presence of ethylene glycol as solvent under refluxed conditions, gave the desired 2-hydrazinyl-4/6-substituted benzo[d]thiazoles (2a and b) in high yields [45].

General procedure for the synthesis of 8b-hydroxy-1-(4/6-substituted benzo[d]thiazol-2-yl)-3-(3-phenyl/substituted phenyl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-ones (3a–t)

A mixture of equimolar quantities of 2-(benzoyl/substituted benzoyl)-(1H)-indene-1,3(2H)-dione (1; 1 mmol) and 2-hydrazinyl-4/6-substituted benzo[d]thiazole (2; 1 mmol) was taken in a round-bottomed flask equipped with a reflux condenser with a guard tube. To this, freshly dried ethanol (30 ml) was added and the resultant mixture was refluxed on a water bath until completion of the reaction (2–4.5 h) as monitored by TLC. The solid that was separated on cooling, filtered and crystallized from dry ethanol afforded the corresponding 8b-hydroxy-1-(4/6-substituted benzo[d]thiazol-2-yl)-3-(3-phenyl/substituted phenyl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-ones (3a–t) in good yields (61–75%).

The physical and spectral data of the synthesized hydroxypyrazolones 3a–t follow.

8b-Hydroxy-1-(4-methylbenzo[d]thiazol-2-yl)-3-phenyl-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3a)

Amounts of reactants taken: 1a = 250 mg and 2a = 179 mg; reaction time = 2.5 h; yield 296 mg, 72%; yellow solid, mp 190–192°C; R_f = 0.30 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3412 (br, O–H and N–H str.), 2925, 2855, 1731 (C=O str.), 1601 (C=N str.), 1527, 1457, 1302, 1097 and 765; ¹H NMR (400 MHz, CDCl₃): δ 8.65 (d, 1H, ³J = 7.84 Hz, C₈-H), 8.12–8.08 (m, 2H, C₅-H, C₇-H), 7.84–7.75 (m, 2H, C₂″-H, C₆″-H), 7.60–7.47 (m, 5H, C₆-H, C₇′-H, C₃″-H, C₄″-H, C₅″-H), 7.27 (d, 1H, ³J = 7.40 Hz, C₅′-H), 7.15 (t, 1H, ³J = 7.68 Hz, C₆′-H), 6.70 (brs, 1H, NH, exchangeable with D₂O), 4.83 (s, 1H, OH, exchangeable with D₂O), 2.81 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 194.05 (C=O), 161.97 (C₂′), 150.73, 150.44, 148.62 (C_8a), 136.62, 135.63, 130.93, 130.45, 130.41, 129.90, 129.49, 128.57, 127.97, 126.97, 125.80, 124.28, 122.80, 118.81, 99.39 (C_3a), 67.03 (C_8b), 18.55 (C₄′-CH₃). HRMS (ESI⁺) m/z calcd for C₂₄H₁₇N₃O₂S [M+H]⁺ 412.1113, found 412.1115.

8b-Hydroxy-1-(4-methylbenzo[d]thiazol-2-yl)-3-(m-tolyl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3b)

Amounts of reactants taken: 1b = 264 mg and 2a = 179 mg; reaction time = 2.0 h; yield 259 mg, 61%; pale yellow solid, mp 180–182°C; R_f = 0.31 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3434 (br, O–H and N–H str.), 2918, 2852, 1727 (C=O str.), 1594 (C=N str.), 1533, 1463, 1302, 1145 and 768; ¹H NMR (400 MHz, CDCl₃): δ 8.64 (d, 1H, ³J = 7.84 Hz, C₈-H), 7.94–7.86 (m, 2H, C₅-H, C₇-H), 7.82–7.75 (m, 2H, C₂″-H, C₆″-H), 7.59–7.54 (m, 2H, C₆-H, C₇′-H), 7.43–7.37 (m, 1H, C₄″-H), 7.32–7.23 (m, 2H, C₅′-H, C₅″-H), 7.15 (t, 1H, ³J = 7.68 Hz, C₆′-H), 6.69 (brs, 1H, NH, exchangeable with D₂O), 4.84 (s, 1H, OH, exchangeable with D₂O), 2.81 (s, 3H, CH₃), 2.48 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 194.09 (C=O), 161.95 (C₂′), 150.75, 150.38, 148.94 (C_8a), 138.24, 136.62, 135.64, 131.34, 130.87, 130.45, 129.78, 129.49, 128.48, 128.35, 126.98, 125.72, 125.35, 124.26, 122.77, 118.79, 99.35 (C_3a), 67.13 (C_8b), 21.54 (C₃″-CH₃), 18.61 (C₄′-CH₃). HRMS (ESI⁺) m/z calcd for C₂₅H₁₉N₃O₂S [M+H]⁺ 426.1273, found 426.1272.

8b-Hydroxy-3-(3-methoxyphenyl)-1-(4-methylbenzo[d]thiazol-2-yl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3c)

Amounts of reactants taken: 1c = 280 mg and 2a = 179 mg; reaction time = 2.0 h; yield 278 mg, 63%; pale yellow solid, mp 183–185°C; R_f = 0.27 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3438 (br, O–H and N–H str.), 2929, 2845, 1724 (C=O str.), 1588 (C=N str.), 1533, 1456, 1305, 1144 and 772; ¹H NMR (400 MHz, CDCl₃): δ 8.63 (d, 1H, ³J = 7.88 Hz, C₈-H), 7.86–7.75 (m, 2H, C₅-H, C₇-H), 7.73–7.64 (m, 2H, C₂″-H, C₆″-H), 7.59–7.54 (m, 2H, C₆-H, C₇′-H), 7.45–7.39 (m, 1H, C₄″-H), 7.27 (d, 1H, ³J = 7.44 Hz, C₅′-H), 7.15 (t, 1H, ³J = 7.68 Hz, C₆′-H), 7.04 (ddd, 1H, ³J = 8.28, ⁴J = 2.60, ⁵J = 0.8 Hz, C₅″-H), 6.66 (brs, 1H, NH, exchangeable with D₂O), 4.82 (s, 1H, OH, exchangeable with D₂O), 3.94 (s, 3H, OCH₃), 2.81 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 193.93 (C=O), 161.90 (C₂′), 159.55 (C₃″), 150.75 (C₃″), 150.45 (C₃′_a), 148.50 (C_8a), 136.62 (C₇), 135.64 (C_4a), 131.21 (C₁″), 130.94 (C₆), 130.45 (C₇′_a), 129.60 (C₄″), 129.54 (C₄′), 126.96 (C₅′), 125.71 (C₈), 124.28 (C₅), 122.80 (C₄′), 120.75 (C₆″), 118.78 (C₇′), 116.72 (C₄″), 112.64 (C₂″), 99.66 (C_3a), 67.14 (C_8b), 55.61 (C₃″-OCH₃), 18.64 (C₄′-CH₃). HRMS (ESI⁺) m/z calcd for C₂₅H₁₉N₃O₃S [M+H]⁺ 442.1223, found 442.1222.

3-(3-Chlorophenyl)-8b-hydroxy-1-(4-methylbenzo[d]thiazol-2-yl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3d)

Amounts of reactants taken: 1d = 285 mg and 2a = 179 mg; reaction time = 3.0 h; yield 289 mg, 65%; pale yellow solid, mp 201–203°C; R_f = 0.31 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3424 (br, O–H and N–H str.), 2922, 2855, 1708 (C=O str.), 1591 (C=N str.), 1538, 1461, 1309, 1156 and 760; ¹H NMR (400 MHz, CDCl₃): δ 8.65 (d, 1H, ³J = 7.64 Hz, C₈-H), 7.99–7.92 (m, 2H, C₅-H, C₇-H), 7.87–7.74 (m, 3H), 7.58 (d, ³J = 6.96 Hz, 1H), 7.50–7.39 (m, 3H, C₅′-H, C₄″-H, C₅″-H), 7.17 (t, 1H, ³J = 7.56 Hz, C₆′-H), 6.69 (brs, 1H, NH, exchangeable with D₂O), 4.80 (s, 1H, OH, exchangeable with D₂O), 2.82 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 193.95 (C=O), 161.72 (C₂′), 150.60, 150.15, 147.19 (C_8a), 136.78, 135.57, 134.67, 131.69, 130.81, 130.58, 130.33, 129.86, 129.64, 127.52, 127.09, 126.32, 125.78, 124.38, 123.03, 118.84, 99.64 (C_3a), 66.83 (C_8b), 18.62 (C₄′-CH₃). HRMS (ESI⁺) m/z calcd for C₂₄H₁₆ClN₃O₂S [M+H]⁺ 446.0723, found 446.0722.

3-(3-Bromophenyl)-8b-hydroxy-1-(4-methylbenzo[d]thiazol-2-yl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3e)

Amounts of reactants taken: 1e = 329 mg and 2a = 179 mg; reaction time = 4.0 h; yield 328 mg, 67%; orange solid, mp 204–206°C; R_f = 0.34 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3421 (br, O–H and N–H str.), 2919, 2850, 1707 (C=O str.), 1589 (C=N str.), 1537, 1459, 1308, 1100 and 759; ¹H NMR (400 MHz, CDCl₃): δ 8.63 (d, 1H, ³J = 7.84 Hz, C₈-H), 8.23 (t, 1H, ⁴J = 1.68 Hz, C₂″-H), 8.06–7.99 (m, 1H, C₆″-H), 7.84–7.76 (m, 2H, C₅-H, C₇-H), 7.62–7.53 (m, 3H), 7.38 (t, ³J = 7.92 Hz, 1H), 7.27 (d, 1H, ³J = 7.60 Hz, C₅′-H), 7.15 (t, 1H, ³J = 7.28 Hz, C₆′-H), 6.63 (brs, 1H, NH, exchangeable with D₂O), 4.78 (s, 1H, OH, exchangeable with D₂O), 2.81 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 193.94 (C=O), 161.71 (C₂′), 151.18, 150.61, 150.19 (C_8a), 146.80, 136.75, 135.56, 133.15, 131.94, 130.93, 130.56, 130.38, 130.07, 129.63, 127.03, 126.73, 125.84, 124.34, 122.99, 122.72, 118.82, 99.66 (C_3a), 67.13 (C_8b), 18.60 (C₄′-CH₃). HRMS (ESI⁺) m/z calcd for C₂₄H₁₆BrN₃O₂S [M+H]⁺ 490.0223, found 490.0224.

8b-Hydroxy-1-(4-methylbenzo[d]thiazol-2-yl)-3-(p-tolyl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3f)

Amounts of reactants taken: 1f = 264 mg and 2a = 179 mg; reaction time = 2.5 h; yield 281 mg, 66%; pale yellow solid, mp 184–186°C; R_f = 0.33 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3420 (br, O–H and N–H str.), 2917, 2855, 1708 (C=O str.), 1593 (C=N str.), 1540, 1462, 1317, 1079 and 765; ¹H NMR (400 MHz, CDCl₃): δ 8.64 (d, 1H, ³J = 7.52 Hz, C₈-H), 8.28 (d, ³J = 8.16 Hz, 1H), 7.99 (d, ³J = 8.24 Hz, 1H), 7.79 (dd, ³J = 7.84, ³J = 7.40 Hz, 1H), 7.54 (ddd, ³J = 7.64, ⁴J = 5.16, ⁵J = 1.12 Hz, 2H), 7.34–7.29 (m, 3H), 7.27 (d, 1H, ³J = 7.36 Hz, C₅′-H), 7.15 (t, 1H, ³J = 7.64 Hz, C₆′-H), 6.75 (brs, 1H, NH, exchangeable with D₂O), 4.84 (s, 1H, OH, exchangeable with D₂O), 2.81 (s, 3H, CH₃), 2.44 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 193.93 (C=O), 162.07 (C₂′), 150.66 (C_8a), 140.94, 136.63, 135.62, 133.54, 130.70, 130.46, 129.44, 129.34, 127.99, 127.74, 125.76, 124.28, 122.81, 119.12, 118.79, 99.40 (C_3a), 67.13 (C_8b), 21.72 (C₄″-CH₃), 18.67 (C₄′-CH₃). HRMS (ESI⁺) m/z calcd for C₂₅H₁₉N₃O₂S [M+H]⁺ 426.1273, found 426.1272.

8b-Hydroxy-3-(4-methoxyphenyl)-1-(4-methylbenzo[d]thiazol-2-yl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3g)

Amounts of reactants taken: 1g = 280 mg and 2a = 179 mg; reaction time = 2.5 h; yield 291 mg, 66%; pale yellow solid, mp 187–189°C; R_f = 0.29 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3441 (br, O–H and N–H str.), 2926, 2855, 1698 (C=O str.), 1606 (C=N str.), 1561, 1459, 1335, 1104 and 754; ¹H NMR (400 MHz, CDCl₃): δ 8.64 (d, 1H, ³J = 7.84 Hz, C₈-H), 8.06 (d, ³J = 8.76 Hz, 2H), 7.79 (dd, ³J = 7.84, ³J = 7.60 Hz, 2H), 7.59–7.53 (m, 2H), 7.26 (d, 1H, ³J = 7.48 Hz, C₅′-H), 7.14 (t, 1H, ³J = 7.64 Hz, C₆′-H), 7.02 (d, ³J = 8.76 Hz, 2H), 6.72 (brs, 1H, NH, exchangeable with D₂O), 4.81 (s, 1H, OH, exchangeable with D₂O), 3.91 (s, 3H, OCH₃), 2.80 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 194.21 (C=O), 162.05 (C₂′), 161.50, 150.85, 150.43, 148.57 (C_8a), 136.64, 135.56, 130.81, 130.41, 129.69, 129.37, 126.94, 125.72, 124.24, 122.62, 118.77, 114.03, 99.35 (C_3a), 67.51 (C_8b), 55.30 (C₄″-OCH₃), 18.61 (C₄′-CH₃). HRMS (ESI⁺) m/z calcd for C₂₅H₁₉N₃O₃S [M+H]⁺ 442.1223, found 442.1222.

3-(4-Fluorophenyl)-8b-hydroxy-1-(4-methylbenzo[d]thiazol-2-yl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3h)

Amounts of reactants taken: 1h = 268 mg and 2a = 179 mg; reaction time = 3.0 h; yield 283 mg, 66%; yellow solid, mp 200–202°C; R_f = 0.32 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3425 (br, O–H and N–H str.), 2918, 2849, 1709 (C=O str.), 1600 (C=N str.), 1531, 1464, 1305, 1158 and 766; ¹H NMR (400 MHz, CDCl₃): δ 8.63 (d, 1H, ³J = 7.80 Hz, C₈-H), 8.14–8.08 (m, 1H), 8.06 (d, ³J = 8.80 Hz, 1H), 7.80 (dt, ³J = 7.40, ⁴J = 4.36 Hz, 2H), 7.59–7.55 (m, 2H), 7.26 (d, 1H, ³J = 8.32 Hz, C₅′-H), 7.22–7.11 (m, 2H), 7.02 (d, ³J = 8.76 Hz, 1H), 6.63 (brs, 1H, NH, exchangeable with D₂O), 4.80 (s, 1H, OH, exchangeable with D₂O), 2.80 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 194.24 (C=O), 161.96 (C₂′), 161.84, 161.45, 150.51, 150.42, 148.52 (C_8a), 147.62, 136.73, 136.60, 130.43 (d, J_C–F = 11.13 Hz), 129.68, 127.00, 125.63 (d, J_C–F = 255.90 Hz, C₄″), 124.22, 122.83, 122.60, 118.78, 115.75 (d, J_C–F = 21.97 Hz), 114.04, 99.36 (C_3a), 67.14 (C_8b), 18.33 (C₄′-CH₃). HRMS (ESI⁺) m/z calcd for C₂₄H₁₆FN₃O₂S [M+H]⁺ 430.1023, found 430.1021.

3-(4-Chlorophenyl)-8b-hydroxy-1-(4-methylbenzo[d]thiazol-2-yl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3i)

Amounts of reactants taken: 1i = 285 mg and 2a = 179 mg; reaction time = 3.5 h; yield 303 mg, 68%; pale yellow solid, mp 205–207°C; R_f = 0.33 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3416 (br, O–H and N–H str.), 2919, 2852, 1708 (C=O str.), 1591 (C=N str.), 1536, 1463, 1305, 1087 and 766; ¹H NMR (400 MHz, CDCl₃): δ 8.63 (d, 1H, ³J = 7.56 Hz, C₈-H), 8.04 (d, ³J = 7.92 Hz, 2H), 7.84–7.76 (m, 2H), 7.59–7.55 (d, ³J = 7.36 Hz, 2H), 7.47 (d, ³J = 7.92 Hz, 2H), 7.27 (d, 1H, ³J = 7.48 Hz, C₅′-H), 7.16 (t, 1H, ³J = 7.48 Hz, C₆′-H), 6.66 (brs, 1H, NH, exchangeable with D₂O), 4.78 (s, 1H, OH, exchangeable with D₂O), 2.81 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 193.94 (C=O), 161.80 (C₂′), 150.72, 150.36, 147.53 (C_8a), 136.78, 136.43, 135.54, 130.86, 130.54, 129.61, 129.17, 128.88, 128.44, 127.04, 125.74, 124.36, 122.92, 118.82, 99.65 (C_3a), 67.12 (C_8b), 18.60 (C₄′-CH₃). HRMS (ESI⁺) m/z calcd for C₂₄H₁₆ClN₃O₂S [M+H]⁺ 446.0723, found 446.0721.

3-(4-Bromophenyl)-8b-hydroxy-1-(4-methylbenzo[d]thiazol-2-yl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3j)

Amounts of reactants taken: 1j = 329 mg and 2a = 179 mg; reaction time = 4.0 h; yield 343 mg, 70%; orange solid, mp 211–213°C; R_f = 0.36 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3416 (br, O–H and N–H str.), 2923, 2852, 1707 (C=O str.), 1592 (C=N str.), 1536, 1462, 1304, 1149 and 766; ¹H NMR (400 MHz, CDCl₃): δ 8.63 (d, 1H, ³J = 7.52 Hz, C₈-H), 7.97 (d, ³J = 8.20 Hz, 2H), 7.84–7.76 (m, 2H), 7.68–7.53 (m, 5H), 7.27 (d, 1H, ³J = 8.52 Hz, C₅′-H), 7.16 (t, 1H, ³J = 7.48 Hz, C₆′-H), 6.69 (brs, 1H, NH, exchangeable with D₂O), 4.79 (s, 1H, OH, exchangeable with D₂O), 2.80 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 193.91 (C=O), 161.69 (C₂′), 150.66, 150.27, 147.65 (C_8a), 136.77, 135.55, 131.82, 130.82, 130.55, 129.61, 129.36, 128.86, 127.06, 125.77, 124.90, 124.36, 122.95, 118.81, 99.34 (C_3a), 66.83 (C_8b), 18.61 (C₄′-CH₃). HRMS (ESI⁺) m/z calcd for C₂₄H₁₆BrN₃O₂S [M+H]⁺ 490.0223, found 490.0221.

1-(6-Fluorobenzo[d]thiazol-2-yl)-8b-hydroxy-3-phenyl-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3k)

Amounts of reactants taken: 1a = 250 mg and 2b = 183 mg; reaction time = 1.5 h; yield 311 mg, 75%; yellow solid, mp 196–198°C; R_f = 0.34 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3430 (br, O–H and N–H str.), 2925, 2854, 1721 (C=O str.), 1603 (C=N str.), 1542, 1459, 1315, 1110 and 757; ¹H NMR (400 MHz, CDCl₃): δ 8.56 (d, 1H, J = 7.88 Hz, C₈-H), 8.15–8.04 (m, 2H), 7.81 (dd, ³J = 7.68 Hz, 2H), 7.74 (dd, ³J = 8.8, ⁴J = 4.16 Hz, 1H), 7.58 (t, 1H, ³J = 7.64 Hz, C₆-H), 7.54–7.47 (m, 3H), 7.44 (dd, ³J = 8.08, ⁴J = 2.56 Hz, 1H), 7.17 (td, ³J = 8.88, ⁴J = 2.64 Hz, 1H), 6.37 (brs, 1H, NH, exchangeable with D₂O), 4.84 (s, 1H, OH, exchangeable with D₂O). ¹³C NMR (100 MHz, CDCl₃): δ 193.82 (C=O), 162.24, 162.22, 158.98 (d, J_C–F = 240.86 Hz, C₆′), 150.47, 148.94 (C_8a), 147.95, 147.94, 136.69, 135.67, 132.22 (d, J_C–F = 10.65 Hz), 130.51, 129.80, 128.59, 127.97, 125.77, 124.33, 120.61, 120.52, 114.18, 113.94 (d, J_C–F = 23.93 Hz), 108.13, 107.86, 99.32 (C_3a), 67.30 (C_8b). HRMS (ESI⁺) m/z calcd for C₂₃H₁₄FN₃O₂S [M+H]⁺ 416.0863, found 416.0862.

1-(6-Fluorobenzo[d]thiazol-2-yl)-8b-hydroxy-3-(m-tolyl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3l)

Amounts of reactants taken: 1b = 264 mg and 2b = 183 mg; reaction time = 2.5 h; yield 270 mg, 63%; pale yellow solid, mp 198–200°C; R_f = 0.31 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3423 (br, O–H and N–H str.), 2965, 2853, 1702 (C=O str.), 1618 (C=N str.), 1572, 1467, 1356, 1120 and 761; ¹H NMR (400 MHz, CDCl₃): δ 8.56 (d, 1H, ³J = 7.84 Hz, C₈-H), 7.89 (d, ³J = 6.56 Hz, 2H), 7.80 (dd, ³J = 7.52 Hz, 2H), 7.74 (dd, ³J = 8.84, ⁴J = 4.16 Hz, 1H), 7.57 (t, ³J = 7.32 Hz, 1H, C₆-H), 7.46–7.37 (m, 2H), 7.30 (d, ³J = 9.84 Hz, 1H), 7.17 (td, ³J = 8.92, ⁴J = 2.64 Hz, 1H), 6.37 (brs, 1H, NH, exchangeable with D₂O), 4.83 (s, 1H, OH, exchangeable with D₂O), 2.48 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 193.93 (C=O), 162.21 (C₂′), 158.93 (d, J_C–F = 244.26 Hz, C₆′), 150.46, 149.13 (C_8a), 147.98, 138.45, 138.23, 136.66, 135.68, 132.28, 132.17, 131.40, 130.48, 129.70, 128.42 (d, J_C–F = 12.24 Hz), 125.79, 125.34, 124.28, 120.58, 120.49, 114.03 (d, J_C–F = 23.96 Hz), 108.11, 107.84, 98.99 (C_3a), 67.13 (C_8b), 21.36 (C₃″-CH₃). HRMS (ESI⁺) m/z calcd for C₂₄H₁₆FN₃O₂S [M+H]⁺ 430.1023, found 430.1021.

1-(6-Fluorobenzo[d]thiazol-2-yl)-8b-hydroxy-3-(3-methoxyphenyl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3m)

Amounts of reactants taken: 1c = 280 mg and 2b = 183 mg; reaction time = 2.0 h; yield 285 mg, 64%; pale yellow solid, mp 208–210°C; R_f = 0.30 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3416 (br, O–H and N–H str.), 2963, 2929, 1717 (C=O str.), 1603 (C=N str.), 1543, 1463, 1318, 1112 and 759; ¹H NMR (400 MHz, CDCl₃): δ 8.58 (d, 1H, ³J = 7.84 Hz, C₈-H), 7.81 (t, ³J = 7.68 Hz, 2H), 7.76 (dd, ³J = 8.88, ⁴J = 4.16 Hz, 1H), 7.68 (d, ³J = 7.68 Hz, 1H), 7.66–7.62 (m, 1H), 7.57 (t, 1H, ³J = 7.32 Hz, C₆-H), 7.44–7.39 (m, 2H), 7.17 (td, ³J = 8.92, ⁴J = 2.60 Hz, 1H), 7.04 (dd, ³J = 8.24, 1.88 Hz, 1H), 6.50 (brs, 1H, NH, exchangeable with D₂O), 4.80 (s, 1H, OH, exchangeable with D₂O), 3.93 (s, 3H, OCH₃). ¹³C NMR (100 MHz, CDCl₃): δ 193.76 (C=O), 162.18 (C₂′), 159.60, 159.01 (d, J_C–F = 241.10 Hz, C₆′), 150.24, 148.98 (C_8a), 147.45, 136.74, 135.64, 131.95 (d, J_C–F = 10.99 Hz), 130.96, 130.59, 129.64, 125.89, 124.48 (d, J_C–F = 26.10 Hz), 120.77, 120.52, 120.43, 116.79, 114.29, 114.05, 112.72, 108.17, 107.90, 99.47 (C_3a), 67.41 (C_8b), 55.45 (C₃″-OCH₃). HRMS (ESI⁺) m/z calcd for C₂₄H₁₆FN₃O₃S [M+H]⁺ 446.0973, found 446.0972.

3-(3-Chlorophenyl)-1-(6-fluorobenzo[d]thiazol-2-yl)-8b-hydroxy-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3n)

Amounts of reactants taken: 1d = 285 mg and 2b = 183 mg; reaction time = 2.5 h; yield 292 mg, 65%; pale yellow solid, mp 206–208°C; R_f = 0.33 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3420 (br, O–H and N–H str.), 2926, 2852, 1717 (C=O str.), 1604 (C=N str.), 1558, 1484, 1316, 1112 and 767; ¹H NMR (400 MHz, CDCl₃): δ 8.57 (d, 1H, ³J = 7.84 Hz, C₈-H), 8.08 (dd, ⁴J = 2.64 Hz, 1H), 8.00–7.93 (m, 1H), 7.86–7.73 (m, 3H), 7.59 (t, 1H, ³J = 7.52 Hz, C₆-H), 7.48–7.38 (m, 3H), 7.18 (td, ³J = 8.88, ⁴J = 2.60 Hz, 1H), 6.46 (brs, 1H, NH, exchangeable with D₂O), 4.77 (s, 1H, OH, exchangeable with D₂O). ¹³C NMR (100 MHz, CDCl₃): δ 193.58 (C=O), 162.06 (C₂′), 159.09 (d, J_C–F = 241.40 Hz, C₆′), 150.23, 147.70 (C_8a), 147.48, 136.85, 135.59, 134.70, 131.98, 131.51, 130.55 (d, J_C–F = 21.45 Hz), 129.87, 127.52, 126.35, 125.88, 124.73, 124.69, 120.63 (d, J_C–F = 8.79 Hz), 120.59, 114.37, 114.13, 108.20, 107.94, 99.58 (C_3a), 67.12 (C_8b). HRMS (ESI⁺) m/z calcd for C₂₄H₁₃ClFN₃O₂S [M+H]⁺ 450.0473, found 450.0475.

3-(3-Bromophenyl)-1-(6-fluorobenzo[d]thiazol-2-yl)-8b-hydroxy-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3o)

Amounts of reactants taken: 1e = 329 mg and 2b = 183 mg; reaction time = 3.5 h; yield 326 mg, 66%; orange solid, mp 216–218°C; R_f = 0.33 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3418 (br, O–H and N–H str.), 2926, 2855, 1717 (C=O str.), 1604 (C=N str.), 1541, 1461, 1315, 1111 and 765; ¹H NMR (400 MHz, CDCl₃): δ 8.59 (d, 1H, ³J = 7.92 Hz, C₈-H), 8.24 (t, ⁴J = 1.76 Hz, 1H), 8.03 (dd, ³J = 6.88, ⁴J = 1.40 Hz, 1H), 7.87–7.73 (m, 3H), 7.63–7.55 (m, 2H), 7.44 (dd, ³J = 8.08, ⁴J = 2.56 Hz, 1H), 7.39 (t, ³J = 7.84 Hz, 1H), 7.19 (td, 1H, ³J = 8.92, ⁴J = 2.60 Hz,), 6.50 (brs, 1H, NH, exchangeable with D₂O), 4.79 (s, 1H, OH, exchangeable with D₂O). ¹³C NMR (100 MHz, CDCl₃): δ 193.60 (C=O), 161.98 (C₂′), 159.09 (d, J_C–F = 241.40 Hz, C₆′), 150.23, 147.53 (C_8a), 136.83, 135.60, 133.90, 133.31, 132.11, 131.81, 130.52 (d, J_C–F = 24.55 Hz), 130.11, 126.79, 125.95, 124.74, 124.70, 124.39, 122.76, 120.67 (d, J_C–F = 8.85 Hz), 114.34, 114.10, 108.18, 107.91, 99.58 (C_3a), 67.14 (C_8b). HRMS (ESI⁺) m/z calcd for C₂₄H₁₃BrFN₃O₂S [M+H]⁺ 493.9973, found 493.9971.

1-(6-Fluorobenzo[d]thiazol-2-yl)-8b-hydroxy-3-(p-tolyl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3p)

Amounts of reactants taken: 1f = 264 mg and 2b = 183 mg; reaction time = 3.0 h; yield 292 mg, 68%; pale yellow solid, mp 203–205°C; R_f = 0.34 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3396 (br, O–H and N–H str.), 2926, 2855, 1706 (C=O str.), 1608 (C=N str.), 1552, 1459, 1316, 1112 and 736; ¹H NMR (400 MHz, CDCl₃): δ 8.54 (d, 1H, ³J = 7.40 Hz, C₈-H), 8.26 (d, ³J = 8.12 Hz, 1H), 8.03–7.93 (m, 1H), 7.81 (t, ³J = 7.64 Hz, 1H), 7.66 (d, ³J = 7.16 Hz, 1H), 7.61–7.49 (m, 2H), 7.47–7.36 (m, 1H), 7.31 (d, ³J = 7.88 Hz, 2H), 7.19 (td, ³J = 8.92, ⁴J = 2.60 Hz, 1H), 6.78 (brs, 1H, NH, exchangeable with D₂O), 4.85 (s, 1H, OH, exchangeable with D₂O), 2.44 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃): δ 183.72 (C=O), 159.20 (C₂′), 150.89, 147.46 (C_8a), 141.19, 140.57, 139.89, 137.29, 136.76, 135.63, 133.67, 132.25, 130.83, 130.59, 129.41 (d, J_C–F = 9.36 Hz), 129.32 (d, J_C–F = 253.71 Hz, C₆′), 127.75, 124.52, 124.50, 123.93, 123.84, 115.29 (d, J_C–F = 24.65 Hz), 108.29, 108.03, 99.36 (C_3a), 67.48 (C_8b), 21.63 (C₄″-CH₃). HRMS (ESI⁺) m/z calcd for C₂₄H₁₆FN₃O₂S [M+H]⁺ 430.1023, found 430.1021.

1-(6-Fluorobenzo[d]thiazol-2-yl)-8b-hydroxy-3-(4-methoxyphenyl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3q)

Amounts of reactants taken: 1f = 280 mg and 2b = 183 mg; reaction time = 2.5 h; yield 307 mg, 69%; pale yellow solid, mp 212–214°C; R_f = 0.32 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3430 (br, O–H and N–H str.), 2925, 2853, 1724 (C=O str.), 1606 (C=N str.), 1541, 1460, 1313, 1135 and 755; ¹H NMR (400 MHz, CDCl₃): δ 8.56 (d, 1H, ³J = 7.84 Hz, C₈-H), 8.05 (d, ³J = 8.96 Hz, 2H), 7.83–7.75 (m, 2H), 7.72 (dd, ³J = 8.80, ⁴J = 4.64 Hz, 1H), 7.57 (t, 1H, ³J = 7.32 Hz, C₆-H), 7.42 (dd, ³J = 8.08, ⁴J = 2.56 Hz, 1H), 7.16 (td, ³J = 8.92, ⁴J = 2.60 Hz, 1H), 7.02 (d, ³J = 8.96 Hz, 2H), 6.41 (brs, 1H, NH, exchangeable with D₂O), 4.79 (s, 1H, OH, exchangeable with D₂O), 3.91 (s, 3H, OCH₃). ¹³C NMR (100 MHz, CDCl₃): δ 194.04 (C=O), 162.28, 162.26, 161.53 (C₂′), 158.87 (d, J_C–F = 240.50 Hz, C₆′), 150.56, 148.74 (C_8a), 148.03, 148.02, 136.66, 135.60, 132.18 (d, J_C–F = 10.63 Hz), 130.44, 129.70, 125.78, 124.26, 122.48, 120.43, 120.34, 114.06, 113.85, 107.96 (d, J_C–F = 26.85 Hz), 99.34 (C_3a), 67.50 (C_8b), 55.32 (C₄″-OCH₃). HRMS (ESI⁺) m/z calcd for C₂₄H₁₆FN₃O₃S [M+H]⁺ 446.0973, found 446.0971.

1-(6-Fluorobenzo[d]thiazol-2-yl)-3-(4-fluorophenyl)-8b-hydroxy-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3r)

Amounts of reactants taken: 1h = 268 mg and 2b = 183 mg; reaction time = 2.5 h; yield 295 mg, 68%; yellow solid, mp 213–215°C; R_f = 0.35 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3426 (br, O–H and N–H str.), 2973, 2855, 1721 (C=O str.), 1605 (C=N str.), 1546, 1460, 1315, 1119 and 759; ¹H NMR (400 MHz, CDCl₃): δ 8.59 (d, 1H, ³J = 7.92 Hz, C₈-H), 8.14–8.04 (m, 2H), 7.87–7.69 (m, 3H), 7.59 (t, 1H, ³J = 7.52 Hz, C₆-H), 7.45 (dd, ³J = 8.00, ⁴J = 2.60 Hz, 1H), 7.26–7.13 (m, 3H), 6.51 (brs, 1H, NH, exchangeable with D₂O), 4.81 (s, 1H, OH, exchangeable with D₂O). ¹³C NMR (100 MHz, CDCl₃): δ 193.93 (C=O), 165.41, 162.90, 162.10, 159.02 (d, J_C–F = 235.51 Hz, C₆′), 150.37, 148.12 (C_8a), 147.52, 136.87, 135.56, 130.61, 130.17, 130.08, 126.03, 125.95 (d, J_C–F = 24.91 Hz), 120.59, 120.50, 115.94, 115.72, 114.32, 114.08, 108.14 (d, J_C–F = 8.20 Hz), 107.91, 99.43 (C_3a), 67.33 (C_8b). HRMS (ESI⁺) m/z calcd for C₂₄H₁₃F₂N₃O₂S [M+H]⁺ 434.0773, found 434.0772.

3-(4-Chlorophenyl)-1-(6-fluorobenzo[d]thiazol-2-yl)-8b-hydroxy-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3s)

Amounts of reactants taken: 1i = 285 mg and 2b = 183 mg; reaction time = 3.0 h; yield 315 mg, 70%; pale yellow solid, mp 210–212°C; R_f = 0.36 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3428 (br, O–H and N–H str.), 2966, 2927, 1702 (C=O str.), 1603 (C=N str.), 1551, 1460, 1346, 1133 and 750; ¹H NMR (400 MHz, CDCl₃): δ 8.62 (d, 1H, ³J = 8.96 Hz, C₈-H), 8.36 (d, ³J = 8.36 Hz, 1H), 8.15–8.00 (m, 2H), 7.95–7.78 (m, 2H), 7.74–7.68 (m, 1H), 7.64–7.58 (m, 1H), 7.54 (d, ³J = 8.44 Hz, 1H), 7.52–7.45 (m, 1H), 7.42 (d, ³J = 6.24 Hz, 1H), 7.15 (brs, 1H, NH, exchangeable with D₂O), 4.94 (s, 1H, OH, exchangeable with D₂O). ¹³C NMR (100 MHz, CDCl₃): δ 193.25 (C=O), 159.71 (C₂′), 159.43, 148.43 (C_8a), 136.97, 135.52, 132.69 (d, J_C–F = 236.52 Hz, C₆′), 132.47, 131.05, 130.84, 129.28 (d, J_C–F = 24.49 Hz), 129.11 (d, J_C–F = 8.73 Hz), 128.96, 125.95, 124.67, 124.52, 120.30, 118.53, 118.09, 114.74, 114.49, 108.31, 108.04, 107.85, 99.68 (C_3a), 67.48 (C_8b). HRMS (ESI⁺) m/z calcd for C₂₄H₁₃ClFN₃O₂S [M+H]⁺ 450.0473, found 450.0472.

3-(4-Bromophenyl)-1-(6-fluorobenzo[d]thiazol-2-yl)-8b-hydroxy-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-one (3t)

Amounts of reactants taken: 1j = 329 mg and 2b = 183 mg; reaction time = 4.0 h; yield 351 mg, 71%; orange solid, mp 220–222°C; R_f = 0.38 [hexane:ethylacetate = 75:25 (v/v)]; FTIR spectrum (KBr, ν_max, cm^-1): 3427 (br, O–H and N–H str.), 2924, 2852, 1704 (C=O str.), 1599 (C=N str.), 1554, 1460, 1318, 1071 and 734; ¹H NMR (400 MHz, CDCl₃): δ 8.58 (d, 1H, ³J = 7.44 Hz, C₈-H), 7.97 (d, ³J = 8.40 Hz, 2H), 7.87–7.74 (m, 3H), 7.68–7.55 (m, 3H), 7.44 (dd, ³J = 7.80, ⁴J = 2.16 Hz, 1H), 7.19 (td, ³J = 8.80, ⁴J = 2.16 Hz, 1H), 6.49 (brs, 1H, NH, exchangeable with D₂O), 4.80 (s, 1H, OH, exchangeable with D₂O). ¹³C NMR (100 MHz, CDCl₃): δ 193.94 (C=O), 162.05 (C₂′), 151.91, 150.28 (d, J_C–F = 22.56 Hz), 147.88 (C_8a), 147.83, 147.81, 136.84, 135.58, 135.30, 130.71, 130.62 (d, J_C–F = 248.74 Hz, C₆′), 130.57, 128.75, 125.84, 124.86 (d, J_C–F = 22.56 Hz), 124.42, 120.73, 120.64, 114.26, 114.07, 108.19, 107.90, 99.37 (C_3a), 66.82 (C_8b). HRMS (ESI⁺) m/z calcd for C₂₄H₁₃BrFN₃O₂S [M+H]⁺ 493.9973, found 493.9971.

Pharmacological assay

In vitro α-amylase & α-glucosidase inhibitory activity

The synthesized hydroxypyrazolones 3a–t were assayed for their in vitro α-amylase and α-glucosidase inhibitory activity by employing the protocol described in the literature by Wickramaratne et al. [46] and Kim et al. [47] with slight modifications. In the case of α-amylase, different concentrations of a sample (1 mg/ml in DMSO) were mixed with α-amylase (5 mg/ml in phosphate buffer) and 1 ml of phosphate buffer of pH 6.8 in test tubes, which were then incubated for 10 min at 35°C. After incubation, 500 μl starch solution (1%) was added to each of the test tubes and they were again incubated for 10 min at 35°C. After the incubation was over, 500 μl 3,5-dinitrosalicylic acid reagent (12 g of sodium potassium tartrate tetrahydrate in 8.0 ml of 2 M NaOH and 20 ml of 96 mM of 3,5-dinitrosalicylic acid solution) was added to each test tube to stop the enzyme reaction and then placed in a boiling water bath for 10 min until red color appeared. Once the color appeared, the test tubes were cooled to room temperature by adding distilled water to make the volume of each test tube 4 ml; in the case of α-glucosidase, a sample (100 μl of 2–20 mg/ml in DMSO) was added to α-glucosidase (1 U/ml) prepared in 0.1 M phosphate buffer (pH 6.9), and 250 μl of 0.1 M phosphate buffer was added to get 0.5–5.0 mg/ml final concentration. The mixture was preincubated at 37°C for 20 min. After preincubation, 10 μl of 10 mM 4-nitrophenyl-β-D-glucopyranoside prepared in 0.1 M phosphate buffer (pH 6.9) was added and incubated at 37°C for 30 min and the reactions were stopped by adding 650 μl 1 M sodium carbonate solution. Acarbose was taken as the standard drug for both enzymes and was tested under similar conditions to compare the results of tested samples. The reaction system without α-amylase and α-glucosidase was used as a blank and a control was performed with the test medium without test samples. Absorbance was measured at 545 and 405 nm using a UV-visible spectrophotometer. Each experiment was performed in triplicate. The enzyme inhibitory activity of each test sample was expressed as percentage (%) inhibition, which was determined by Equation 1:

$$\text{Enzyme} \text{inhibitory} \text{activity} (\% \text{Inhibition})=\left(\frac{{\text{A}}_{\text{control}}-{\text{A}}_{\text{sample}}}{{\text{A}}_{\text{control}}}\right)\times 100$$ (Equation 1)

where A_control = absorbance of control and A_sample = absorbance of sample.

The % α-amylase and α-glucosidase inhibition was plotted against the sample concentration and the IC₅₀ values were obtained from the graph; the results are depicted in Table 1.

Table 1.

IC₅₀ (μg/ml) values of pyrazolones 3a–t for in vitro α-amylase and α-glucosidase inhibitory activity.

Compound	R	R1	α-Amylase	α-Glucosidase
			IC50 (μg/ml)	IC50 (μg/ml)
3a	H	4-CH3	196.00 ± 1.85	802.21 ± 5.26
3b	3-CH3	4-CH3	414.11 ± 23.34	936.91 ± 15.67
3c	3-OCH3	4-CH3	309.73 ± 3.57	876.78 ± 4.59
3d	3-Cl	4-CH3	316.09 ± 9.17	880.10 ± 10.65
3e	3-Br	4-CH3	297.00 ± 2.50	960.89 ± 5.50
3f	4-CH3	4-CH3	237.79 ± 3.40	867.87 ± 5.30
3g	4-OCH3	4-CH3	420.08 ± 6.24	976.09 ± 8.94
3h	4-F	4-CH3	299.82 ± 3.20	968.92 ± 5.20
3i	4-Cl	4-CH3	280.52 ± 3.25	890.12 ± 6.29
3j	4-Br	4-CH3	316.46 ± 2.85	890.14 ± 4.80
3k	H	6-F	295.98 ± 5.13	900.98 ± 8.45
3l	3-CH3	6-F	344.60 ± 18.81	915.98 ± 19.45
3m	3-OCH3	6-F	352.29 ± 6.30	957.78 ± 7.35
3n	3-Cl	6-F	204.64 ± 3.04	825.28 ± 8.45
3o	3-Br	6-F	261.59 ± 2.22	861.59 ± 5.67
3p	4-CH3	6-F	171.00 ± 1.51	750.00 ± 3.65
3q	4-OCH3	6-F	383.52 ± 4.62	998.89 ± 4.87
3r	4-F	6-F	155.10 ± 1.32	657.10 ± 6.78
3s	4-Cl	6-F	236.30 ± 5.56	855.89 ± 7.67
3t	4-Br	6-F	337.23 ± 17.77	945.67 ± 19.80
Acarbose			125.53 ± 4.10	510.13 ± 4.82

In silico studies

Molecular docking studies

Selection of target proteins & protein preparation

The two protein targets – namely, α-amylase and α-glucosidase – were used for this study. Out of these targets, the 3D crystallographic structure of α-amylase (Protein Data Bank [PDB] ID: 1B2Y) was selected and retrieved using the PDB in PDB format and the homology model of α-glucosidase was used (due to the unavailability of 3D x-ray crystallographic structure derived from Saccharomyces cerevisiae in the PDB). So, it was built up from oligo-1,6-glucosidase (isomaltase, PDB ID: 3A4A), which has high sequence similarity [48]. The two protein preparation steps were carried out using BIOVIA Discovery Studio. At physiological pH 7.4, hetero atoms and water molecules were removed and polar hydrogens were added, which are essential to perform the docking. After these steps, some charges (e.g., Kollman charges) were added to the protein PDB structures with the help of docking software called AutoDockTools 1.5.6 and then saved in the PDBQT format, which is a required format for further docking analysis. The identification of the binding sites of the two targets was done through a literature survey and with the help of protein alignment steps by Schrödinger Maestro.

Preparation of ligands

A series of 20 compounds, 3a–t, with acarbose as standard were drawn using ChemDraw software, and energy minimization was carried out using Chem 3D and saved in PDB format. Then polar hydrogen charges were added to the ligand structures and saved in PDBQT format using AutoDockTools 1.5.6 for a further docking process [49]. The visualization of the ligands was done using PyMOL and BIOVIA Discovery Studio.

Molecular dynamics studies

Molecular dynamics (MD) simulations were performed using GROMACS 2020.3 [50]. The CHARMM-GUI webserver was used for the construction of the simulation systems with the CHARMM-36M force fields for both targets. The above force field was used to describe the motions of the amino acid atoms of the protein targets [51,52]. The simulation analyses were carried out in a rectangular box of dimensions 115 Å × 75 Å × 50 Å, where the complexes were solvated and the Monte Carlo ion placing method was followed for adding some ions such as Na⁺ and Cl^- for neutralization [53].

X-ray crystallography of 3c

Chloroform was chosen as a crystallizing solvent to obtain single crystals of compound 3c. A well-suited crystal was selected and affixed to a SuperNova, Dual, Cu at 0, Eos diffractometer, maintaining the crystal's temperature at 298 K during data acquisition. The crystal structures were elucidated using olex2 [54] employing the Charge Flipping method. The refinement of the crystal structures was conducted through the ShelXL [55] refinement package, applying the least squares minimization technique [56].

Results & discussion

Chemistry

The 1,3-diketones – 2-benzoyl/substituted benzoyl-(1H)-indene-1,3(2H)-diones 1 – needed for the purpose were synthesized by reacting equimolar quantities of diethylphthalate and an appropriate ketone (acetophenone/substituted acetophenone) in the presence of freshly prepared sodium methoxide and dry methanol following the procedure reported in the literature [44]. The required hydrazines – 2-hydrazinyl-4/6-substituted benzo[d]thiazoles 2 – were prepared reacting 4/6-substituted benzo[d]thiazol-2-amines with hydrazine hydrate using ethylene glycol as a solvent, which in turn were prepared by reaction of 2/4-substituted aniline and potassium thiocyanate/bromine in glacial acetic acid with stirring in good yields following the protocol described in the literature [45]. The target compounds 8b-hydroxy-1-(4/6-substituted benzo[d]thiazol-2-yl)-3-(3-phenyl/substituted phenyl)-1,8b-dihydroindeno[1,2-c]pyrazol-4(2H)-ones 3a–t were prepared by refluxing an equimolar mixture of 2-benzoyl/substituted benzoyl-(1H)-indene-1,3(2H)-diones 1 with 2-hydrazinyl-4/6-substituted benzo[d]thiazoles 2 in the presence of freshly dried ethanol in good yields (Figure 1).

Figure 1.

Synthetic route for the preparation of benzothiazole-tethered fused hydroxypyrazolones 3a–t

.

The structures of all the newly synthesized hydroxypyrazolones, 3a–t, were evident by employing different physical and spectral (IR, ¹H and ¹³C NMR, 2D NMR), HRMS and single-crystal x-ray crystallography techniques. In their IR spectra, strong absorption bands appeared in the regions at 1731–1698 and 1618–1588 cm^-1, which were  assigned to C=O and C=N stretchings, respectively. The O–H absorption band, in each case, was exhibited in the region at 3441–3396 cm^-1. The most salient feature of their ¹H NMR spectra, in each case, is the obtention of a doublet (³J = 7.52–8.96 Hz) in the most downfield region at δ 8.65–8.54, which was safely attributed to C₈-H owing to the anisotropic–diamagnetic effect of lone pair of electrons located on nitrogen/sulfur of benzothiazole moiety. Another prominent feature of the ¹H NMR spectra of 3a–t, in each case, is the resonance due to the OH proton (exchangeable with D₂O) displayed as a one-proton broad singlet in the region at δ 4.94–4.77 and the NH proton (exchangeable with D₂O) as a broad singlet obtained in the region at δ 7.15–6.37, thereby supporting the formation of desired hydroxypyrazolones. The 2D NMR spectral analysis of one of the derivatives (3c) proved helpful for assignments of proton and carbon signals in ¹H and ¹³C NMR spectra, respectively, of all the synthesized derivatives, 3a–t. In ¹H–¹H COSY of 3c, correlations were found between δ 8.63 (C₈-H) and δ 7.86–7.75 (C₇-H); δ 7.86–7.75 (C₇-H) and δ 7.59–7.54 (C₆-H); δ 7.86–7.75 (C₇′-H) and δ 7.15 (C₆′-H); δ 7.27 (C₅′-H) and δ 7.15 (C₆′-H); δ 7.73–7.64 (C₂″-H and C₆″-H) and δ 7.45–7.39 (C₄″-H); and δ 7.45–7.39 (C₄″-H) and δ 7.04 (C₅″-H). The ¹H and ¹³C correlation was determined by heteronuclear single quantum coherence experiments. Heteronuclear single quantum coherence showed correlation as follows: δ 8.63 (C₈-H) → δ 125.72 (C₈); δ 7.86–7.75 (C₇-H and C₅-H) → δ 136.62 (C₇), 124.28 (C₅); δ 7.59–7.54 (C₆-H and C₇′-H) → δ 130.94 (C₆), 118.78 (C₇′); δ 7.27 (C₅′-H) → δ 126.96 (C₅′); δ 7.15 (C₆′-H) → δ 122.80 (C₆′); δ 7.73–7.64 (C₂″-H and C₆″-H) → δ 120.75 (C₂″), 112.64 (C₆″); δ 7.45–7.39 (C₄″-H) → δ 129.60 (C₄″), δ 7.04 (C₅″-H) → δ 116.72 (C₅″) δ 4.82 (OH)→ δ 67.27 (C_8b); δ 3.93 (OCH₃) → δ 55.61; and δ 2.81 (CH₃) → δ 18.64. Additionally, heteronuclear multiple bond correlation analysis results proved fruitful for the chemical shift assignment of the remaining protons and carbons.

The ¹³C NMR spectra of 3a–t, in each case, demonstrated a signal in the most downfield region in the range of δ 194.24–183.72, which was safely ascribed to the carbonyl carbon atom (C₄). The chemical shift that appeared in the next downfield regions at δ 165.41–159.20 was safely assigned to C₂′ of the benzothiazole ring. The signal exhibited in the region at δ 67.83–66.83 was undoubtedly dispensed to the sp³ carbon atom (C_8b), which supports the formation of hydroxypyrazolones. The signal displayed at δ 99.82–99.68 was ascribed to C_3a of the pyrazolone ring. Furthermore, the results of mass spectral analysis (HRMS) of 3a–t showed full agreement with their calculated molecular weights (vide experimentally).

The structural confirmation of one of the derivatives (3c) was achieved by single-crystal x-ray crystallography. The pale yellow-colored single crystals of 3c were obtained from a solution of chloroform at room temperature. Subsequently, these crystals were meticulously analyzed using a SuperNova, Dual, Cu at home/near, Eos diffractometer to determine their molecular structure. The obtained x-ray analysis data have been deposited with the Cambridge Crystallographic Data Centre and they have been assigned the unique CCDC number 2302166. Figure 2 depicts the x-ray crystal structure of 3c, including its ORTEP diagram. Comprehensive crystallographic data of 3c are provided in Table 2 & 3 [54,55,56].

Figure 2.

X-ray crystal structure (ORTEP diagram) of derivative 3c.

Table 2.

Selected bond distances, bond angles and standard uncertainties of 3c are given in parentheses.

Bond	Bond distance (Å)	Bond	Bond distance (Å)
S1-C18	1.744 (2)	C8-C9	1.549 (3)
S1-C24	1.738 (3)	C1-C9	1.474 (3)
O1-C7	1.401 (3)	C1-C6	1.387 (3)
O2-C9	1.199 (3)	C1-C2	1.392 (4)
O3-C13	1.357 (3)	C19-C24	1.391 (4)
O3-C17	1.426 (3)	C19-C20	1.404 (3)
N3-C18	1.300 (3)	C6-C5	1.388 (4)
N3-C19	1.397 (3)	C13-C14	1.384 (4)
N2-N1	1.379 (2)	C24-C23	1.393 (4)
N2-C10	1.286 (3)	C16-C15	1.375 (4)
N1-C18	1.351 (3)	C2-C3	1.369 (4)
N1-C7	1.480 (3)	C20-C21	1.375 (4)
C11-C10	1.464 (3)	C20-C25	1.505 (5)
C11-C12	1.384 (3)	C14-C15	1.381 (4)
C11-C16	1.393 (3)	C5-C4	1.377 (4)
C10-C8	1.515 (3)	C23-C22	1.377 (4)
C12-C13	1.389 (3)	C21-C22	1.375 (5)
C7-C8	1.540 (3)	C3-C4	1.385 (5)
C7-C6	1.520 (3)

Bond	Bond angle	Bond	Bond angle
C24-S1-C18	87.82 (11)	O2-C9-C8	125.3 (2)
C13-O3-C17	118.4 (2)	O2-C9-C1	127.1 (2)
C18-N3-C19	109.2 (2)	C1-C9-C8	107.5 (2)
C10-N2-N1	109.25 (18)	N3-C19-C20	124.7 (2)
N2-N1-C7	113.10 (17)	C24-C19-N3	115.1 (2)
C18-N1-N2	119.66 (18)	C24-C19-C20	120.2 (2)
C18-N1-C7	123.85 (18)	C1-C6-C7	111.1 (2)
C12-C11-C10	119.6 (2)	C1-C6-C5	119.8 (2)
C12-C11-C16	119.4 (2)	C5-C6-C7	129.1 (2)
C16-C11-C10	121.0 (2)	O3-C13-C12	115.4 (2)
N3-C18-S1	117.55 (17)	O3-C13-C14	124.8 (2)
N3-C18-N1	122.7 (2)	C14-C13-C12	119.8 (2)
N1-C18-S1	119.71 (16)	C19-C24-S1	110.31 (18)
N2-C10-C11	121.7 (2)	C19-C24-C23	121.7 (2)
N2-C10-C8	112.95 (19)	C23-C24-S1	128.0 (2)
C11-C10-C8	125.4 (2)	C15-C16-C11	119.7 (3)
C11-C12-C13	120.5 (2)	C3-C2-C1	117.7 (3)
O1-C7-N1	111.30 (19)	C19-C20-C25	120.4 (3)
O1-C7-C8	111.94 (19)	C21-C20-C19	117.2 (3)
O1-C7-C6	113.77 (19)	C21-C20-C25	122.3 (3)
N1-C7-C8	101.17 (17)	C15-C14-C13	119.4 (2)
N1-C7-C6	112.34 (19)	C4-C5-C6	118.2 (3)
C6-C7-C8	105.47 (19)	C16-C15-C14	121.2 (3)
C10-C8-C7	103.07 (18)	C22-C23-C24	117.1 (3)
C10-C8-C9	113.05 (19)	C22-C21-C20	122.2 (3)
C7-C8-C9	105.16 (19)	C21-C22-C23	121.6 (3)
C6-C1-C9	110.6 (2)	C2-C3-C4	120.9 (3)
C6-C1-C2	121.8 (2)	C5-C4-C3	121.6 (3)
C2-C1-C9	127.6 (2)

Table 3.

X-ray crystallographic data of 3c.

Chemical formula	C25H19N3O3S
Formula weight	441.49
Crystal system, space group	Triclinic, P-1
Temperature (K)	293 (2)
Radiation type, wavelength (A0)	CuKα, 1.54184
a, b, c (A°)	8.5976(8), 9.8154(8), 13.3751(10)
α, β, γ (°)	73.692(7), 88.597(7), 80.832(7)
V(A°3)	1069.21(16)
Z	2
Density (calc.) (g/cm3)	1.371
F(000)	460.0
μ (mm-1)	1.620
Crystal size (mm3)	0.234 × 0.207 × 0.135
2θ range for data collection (°)	6.888–138.21
Completeness to θ = 66.97	98.98%
θmin, θmax (°)	4.7510, 69.0890
Measured reflections	5741
Independent reflections	3890
Reflections with I≥2σ (I)	2745
Rint, Rsigma	0.0262, 0.0386
Index ranges	-10≤ h ≤10, -9≤ k ≤11, -14 ≤ l ≤16
Absorption correction	Multifaceted crystal model
Tmin, Tmax	0.666, 1.000
Refinement method	ShelXL refinement package using least squares minimization
N of reflections	3890
N of parameters	292
N of restraints	0.0
Goodness-of-fit on F2	1.032
Max. shift, av. shift	0.000, 0.000
Δρmin, Δρmax (e A°-3)	-0.590, 0.595

In the present investigation, targeted for the synthesis of fused hydroxypyrazolones by the reaction of 2-benzoyl/substituted benzoyl-(1H)-indene-1,3(2H)-diones 1 with 2-hydrazinyl-4/6-substituted benzo[d]thiazoles 2, the authors were delighted to get the respective intermediate hydroxypyrazolines 3, which are highly difficult to synthesize from other substrates under these reaction conditions. Therefore, it is evident that the substrates used in the present investigation are suitable contenders for the formation of hydroxypyrazolines. Earlier, the authors reported the formation of analogous hydroxypyrazolines and are the first to isolate them in good yields [57,58].

Biological studies

In vitro α-amylase & α-glucosidase inhibitory activity evaluation

The α-amylase inhibitory activity of 3a–t was assessed by following the procedure reported in the literature by Wickramaratne et al. [46] and the α-glucosidase enzymatic inhibitory activity of 3a–t was screened by following the protocol described in the literature by Kim et al. [47] using acarbose as the standard reference in both protocols. The results of the α-amylase and α-glucosidase inhibitory activity of 3a–t with their IC₅₀ values are depicted in Table 1.

It is inferred from the data presented in Table 1 that the derivatives 3a, 3p and 3r were found to be good α-amylase inhibitors with IC₅₀ values of 196.00 ± 1.85, 171.00 ± 1.51 and 155.10 ± 1.32 μg/ml, respectively. Further, the compounds 3e, 3f, 3h, 3i, 3k, 3n, 3o and 3s with IC₅₀ values in the range of 204.64 ± 3.04 to 297.00 ± 2.50 μg/ml exhibited moderate inhibitory activity, while 3b, 3c, 3d, 3g, 3j, 3l, 3m, 3q and 3t with IC₅₀ values ranging from 309.73 ± 3.57 to 420.08 ± 6.24 μg/ml showed poor inhibition as compared with the standard reference, acarbose, with an IC₅₀ value of 125.53 ± 4.10 μg/ml. The results of enzymatic inhibitory activity, shown in Table 3, revealed that derivative 3r showed considerable α-glucosidase inhibitory activity IC₅₀ value of 657.10 ± 6.78 μg/ml, whereas the remaining compounds 3a– q, 3s and 3t with IC₅₀ values in the range of 750.00 ± 3.65 to 998.89 ± 4.87 μg/ml – were found to be moderately active as compared with the standard reference, acarbose, with an IC₅₀ value of 510.13 ± 4.82 μg/ml.

According to the results of the α-amylase and α-glucosidase inhibitory activity depicted in Table 1, the following structure–activity relationships of indenopyrazolones 3a–t may be established:

• 1.The derivatives containing substituents as R¹ = 6–F on the benzothiazole ring and R = 4–F and 4–CH₃ on the phenyl ring attached to indenopyrazolone displayed good α-amylase and α-glucosidase inhibitory activity in comparison with other substituents present on these moieties of their respective derivatives.
• 2.In the case of α-amylase inhibitory action, the derivatives having substituents as R¹ = 4–CH₃ on the benzothiazole ring and differently substituted phenyl group appended with indenopyrazolones showed the order as R = H <4–CH₃ <4–Cl <3–Br <4–F <3–OCH₃ <3–Cl <4–Br <4–OCH₃ <3–CH₃, whereas the compounds having substituents as R¹ = 6–F on the benzothiazole ring and substituents present on the phenyl ring exhibited the order as R = 4–F <4–CH₃ <3–Cl <4–Cl <3–Br < H <4–Br <3–CH₃ <3–OCH₃ <4–OCH₃.
• 3.In the case of α-glucosidase inhibitory activity, the derivatives bearing substituents as R¹ = 4–CH₃ on the benzothiazole ring linked with indenopyrazolones having differently substituted phenyl group demonstrated the order as R = H <4–CH₃ <3–OCH₃ <3–Cl <4–Cl <4–Br <3–CH₃ <3–Br <4–F <4–OCH₃ while the compounds having substituents as R¹ = 6–F on benzothiazole ring and substituents present on phenyl ring revealed the order as R = 4–F <4–CH₃ <3–Cl <4–Br <3–Br < H <3–CH₃ <4–Cl <3–OCH₃ <4–OCH₃.

The above-mentioned findings are summarized in Figure 3 and are given in the supplementary material.

Figure 3.

Structure–activity relationships of hydroxypyrazolones

3a–t.

In silico studies

Molecular docking analysis & visualization

Molecular docking analysis was performed against both targets to determine the molecular interactions between ligands and protein targets against acarbose used as standard. Vina as well as AutoDockTools 1.5.6 were used for the estimation of the binding energies. A grid box was set around the binding site of the target proteins. The grid box for α-amylase of dimensions X, Y and Z is 18.9093, 5.7903 and 47.0061, and the grid box for α-glucosidase is -6.3706, 0.7451 and 34.9015. Nine different docked poses for each ligand were generated as the output files with different binding energies (kcal/mol). The outcomes of docking results were analyzed and ranked according to their dock scores. The docking scores of ligands against α-amylase and α-glucosidase range from -9.5 to -10.5 kcal/mol and -9.5 to -11.2 kcal/mol, respectively, as depicted in Supplementary Table l.

The structural conformations of all nine docked poses at binding sites against both targets were estimated using the software PyMOL and BIOVIA Discovery Studio. Finally, the highly stable docked poses of all ligands with their generated output files were taken and further proceeded for 2D and 3D visualization studies against α-amylase and α-glucosidase. The 2D docking interactions were visualized using Maestro and 3D docking interactions were analyzed using BIOVIA Discovery Studio. Out of the total 20 derivatives, five potent compounds along with the standard acarbose were taken for the detailed molecular docking studies to predict the type of interactions in the active site of the α-amylase and α-glucosidase by using the 2D and 3D visualization diagrams with the help of Maestro and Discovery Studio Visualizer.

Docking against α-amylase

The docked poses of derivatives 3r, 3p, 3a, 3n and 3s and standard reference acarbose against α-amylase are depicted in Figures 4–6 and their 2D and 3D binding interactions with α-amylase are described as follows.

Figure 4.

Molecular docking analysis of 3r and 3p. (A)

  2D and 3D binding interactions of 3r and (B) 2D and 3D binding interactions of 3p against α-amylase.

Figure 5.

Molecular docking analysis of 3n and 3a.

(A) 2D and 3D binding interactions of 3n and (B) 2D and 3D binding interactions of 3a against α-amylase.

Figure 6.

Molecular docking analysis of 3s and acarbose. (A)

2D and 3D binding interactions of 3s and (B) 2D and 3D binding interactions of the standard, acarbose, against α-amylase.

It is demonstrated in Figure 4A that the dihydroindenone moiety plays an integral role in the binding of the most potent compound, 3r, showing charged (negative) interactions with the catalytic residues (Asp197, Glu233 and Asp300), which help penetrate the molecule into the active site as well as charged (positive) interaction with the Arg195 residue. The 4-fluorophenyl moiety also showed a lot of integral interactions, as the fluorine interacted with the Gln63 residue through polar interactions, and the Tyr62 residue interacted hydrophobically with the phenyl ring. The pyrazole moiety showed two types of interactions that could help increase binding affinity with the active site of the target in which residues Trp58, Leu162 and Leu165 interacted hydrophobically and Thr163 interacted polarly. Additionally, glycinic and hydrophobic interactions were found to occur between Gly306 and Tyr151 of α-amylase and benzothiazole scaffold.

Figure 4B describes that 3p showed four different types of interactions with different scaffolds. The two negatively charged interactions were shown at the anionic active site by two important acid catalytic residues, Glu233 with 4-methylphenyl and Asp300 with benzene of the indenone ring. The seven amino acid residues Ile235, Ala198, Leu162, Tyr62, Leu165, Trp59 and Trp58 of the hydrophobic region residing near the catalytic site showed hydrophobic interactions. Out of the above amino acids, the first two amino acids interacted with the 4-methylphenyl ring, and the remaining five amino acids interacted with the indenone moiety. The glycinic interactions with Gly306 were shown by the benzothiazole ring and His305 showed polar interactions with the nitrogen atom of the benzothiazole ring.

Figure 5A & B illustrate that compounds 3n and 3a displayed a similar pattern of interactions between the catalytic residues Asp300, Asp197 and Glu233 with the indenone ring through anionic interactions, and the same moiety demonstrated polar interactions with two residues, His299 and His201; however, Arg195 showed charged positive interactions. The indenone and 4-chlorophenyl ring of 3n exhibited polar interactions with His305, Gln63 and Thr163, and Trp58, Trp59, Tyr62, Leu165 and Leu162 showed hydrophobic interaction, whereas Tyr151 showed hydrophobic interaction with fluorine of the benzothiazole ring. The indenone and phenyl ring of 3n demonstrated polar interactions with Thr163 and Gln63, while Trp58, Tyr62, Leu165 and Leu162 showed hydrophobic interactions. The π system of indenopyrazole interacted with Trp59, which is shown in Figure 5B.

It is shown in Figure 6A that 3s showed anionic interactions with the active site residues Glu233 and Asp300, and the indenone ring was responsible for the polar interaction with residues Gln63, His101 and Thr163. Also, the benzene ring of the indenone moiety displayed hydrophobic interactions with Trp58, Trp59, Tyr62, Leu162 and Leu165. The amino acid residues of the target protein i.e. Ala198 and Ile235, interacted hydrophobically, and His201 showed polar interaction with the 4-chlorobenzene ring. The thiazole ring of the benzothiazole moiety exhibited a specific interaction with Gly306 and a polar interaction with His305. The hydrogen bond interaction was shown Figure 6A between His305 and the lone pair of nitrogen of the benzothiazole scaffold, which is significant for the ideal binding.

The standard reference, acarbose (Figure 6B), demonstrated the essential interactions with the triad residues  i.e. Asp300, Glu233 and Asp197 that could be responsible for effective fitness at the carbohydrate-binding site, which tends to make the molecule more potent with different patterns of interactions as compared with the test compounds. Asp197 not only showed the anionic interaction but also interacted with the OH group through hydrogen bonding. Another electrostatic hydrogen bonding occurred between the OH group and the Tyr59 residue of the target. The hydrophobic interaction extended between the Ala106 residue, while polar interactions of His305 amino acid residue made the molecule have a snug fit into an active site of the α-amylase, which makes the acarbose more potent because of the absence of these additional interactions in the compounds, as mentioned above.

Docking against α-glucosidase

The docked poses of derivatives 3r, 3p, 3n, 3o and 3j and standard reference acarbose against α-glucosidase are shown in Figure 7, whereas Figures 8 and 9 are depicted in the supplementary material. Their 2D and 3D binding interactions with α-glucosidase are ascribed as follows.

Figure 7.

Molecular docking analysis of 3r and 3o. (A)

2D and 3D binding interactions of 3r and (B) 2D and 3D binding interactions of 3o against α-glucosidase.

Figure 8.

Molecular docking analysis of 3p and 3j. (A)

 2D and 3D binding interactions of 3p and (B) 2D and 3D binding interactions of 3j against α-glucosidase.

Figure 9.

Molecular docking analysis of 3n and acarbose. (A)

 2D and 3D binding interactions of 3n and (B) 2D and 3D binding interactions of the standard reference, acarbose, against α-glucosidase.

The comparatively potent compounds 3r and 3o displayed binding interactions to the active site of α-glucosidase and showed the best fit to bind to the active site residues of the target. Figure 7A and B exhibits that the main residues of the catalytic site are Asp214, Glu276 and Asp349. The common interactions for both derivatives included negatively charged interactions of the benzothiazole moiety with Asp214 and Glu276, but the residues Asp349 and Asp408 interacted negatively with the indenone portion of the respective compounds. Three polar interactions were illustrated between the 4-fluorophenyl ring and Asn241 and His239 residues. Another amino acid that interacted polarly with the benzothiazole group was Thr215, and the same ring showed cationic interactions with Arg312 and Arg439. The hydrophobic interactions of the pyrazole ring were found to occur with Phe157, Phe158 and Phe177. The amino acid residues Val303, Phe300 and Tyr313 expressed hydrophobic interactions with the indenone ring, whereas hydrogen bonding was displayed between Phe157 and –NH of the pyrazole ring. Additionally, 3o exhibited two π–π stacking interactions between Phe157 with π electrons of the thiazole ring of the benzothiazole moiety and the 3-bromophenyl ring.

It is demonstrated in Supplementary Figure 2A & B that compounds 3p and 3j showed some common integral interactions with the target. Both these derivatives displayed an anionic interaction between Asp349 and indenone and 4-methylbenzothiazole rings. In both cases, the indenone ring was responsible for the interaction with Tyr313 hydrophobically and with Arg312 for positively charged interactions, while the benzothiazole ring system had a common interaction with Asp408. His239 and Asn241 exhibited polar interactions at the different parts of these derivatives (i.e., 3p with the methyl substituent of the phenyl ring and 3j with the central hydroxypyrazoline ring). 6-Fluorobenzothiazole of 3p showed some additional anionic interactions with catalytic residues Asp214 and Glu276; the indenone ring showed three hydrophobic interactions with Tyr313, Phe300 and Val303; and the pyrazole-tethered benzothiazole portion interacted hydrophobically with residues Phe177, Phe158 and Phe157. However, the indenone ring of 3j demonstrated hydrophobic interactions with different residues of the enzyme, such as Phe311, Phe310, Pro309 and Phe157. The 4-methylbenzothiazole moiety showed two hydrophobic interactions with Phe300 and Val303. The main hydrogen interaction of the ligand 3p was observed between –NH of the pyrazole ring and the Phe157 residue of the enzyme.

It is shown in Supplementary Figure 3A that 3n exhibited four anionic interactions between three residues – Asp349, Asp214 and Glu276 – with the 6-fluorobenzothiazole ring, and Glu304 interacted with the indenone ring. The different parts of 3n displayed cationic interactions such as the 3-chlorophenyl ring with Lys155, 6-fluorobenzothiazole with Arg439 and Arg312 with the indenone moiety. The polar interactions were observed between the chlorobenzene moiety and His239 and Ser156, the fluorine of the benzothiazole ring with Thr215 and the indenone ring with the His279 residue. The central core π system of the indenone ring interacted cationically with Arg312.

The standard reference, acarbose Supplementary Figure 3B, showed three negatively charged interactions between the cyclohexene ring and Asp349, Glu304 and Asp408. Three cationic interactions were observed; one was between the cyclohexene ring and Arg439, and the remaining two interactions were between different pyranose rings with Arg312 and Lys155 residues. Acarbose contains more hydroxyl groups and exhibits more polar interactions with the residues Ser308, Thr307, Gln350, His239, Asn241 and His279. All the remaining residues of acarbose showed the hydrophobic interactions along with other interactions. The OH group of the pyranose ring demonstrated hydrogen bonding with the Ser156 residue, and Asp349 showed hydrogen bonding with the hydroxyl group of the cyclohexene ring. These bonded interactions of acarbose help tightly fit into the active site of α-glucosidase enzyme, thereby making it more active against the target (Table 4).

Table 4.

Binding scores of ligands 3a–t against α-amylase and α-glucosidase.

Compound	Binding energy (kcal/mol) against α-amylase	Binding energy (kcal/mol) against α-glucosidase
3a	-10.3	-10.4
3b	-9.9	-11.2
3c	-10.3	-10.9
3d	-10.5	-11.1
3e	-10.5	-11.2
3f	-10.5	-10.4
3g	-10.0	-10.3
3h	-10.5	-10.8
3i	-10.4	-10.3
3j	-10.1	-9.5
3k	-10.2	-10.2
3l	-10.5	-11.0
3m	-10.1	-10.4
3n	-10.2	-10.9
3o	-9.7	-11.0
3p	-10.3	-10.4
3q	-9.9	-9.7
3r	-10.3	-10.7
3s	-9.5	-10.2
3t	-10.2	-10.1
Acarbose	-7.8	-9.0

Molecular dynamics simulations

MD is a computer simulation method for analyzing the physical movements of atoms and molecules. The stability of the complexes, binding affinity, conformational changes and atomic-level interactions of the molecule with the active site residues can be assessed easily by simulating the dynamic behavior of the molecule. MD simulations have various merits, as they help analyze biomolecules in a well-defined system that has well-established conditions (which can be predefined by the user) with certain factors such as pH, ionic concentration and temperature. It also helps analyze how a molecule behaves (e.g., of the biological environment) using methodologies of classical mechanics [53]. GROMACS is a very fast program used for MD simulations, so, because of its rapidness, all MD simulations were performed using the GROMACS 2020.3 version [50].

Derivative 3r showed good potency against two targets in in vitro assay, so it was selected for further molecular dynamics simulation studies. The docked complexes of 3r (top scoring poses were selected for further study) with the two enzymes α-amylase and α-glucosidase were subjected to MD study to check the complex stability and the ligand–target interactions at the active site of the enzyme.

The protein–ligand complexes of both targets, α-amylase and α-glucosidase, were used to build system topology. The webserver CHARMM-GUI was used for construction of the simulation systems with the CHARMM-36M force fields for both targets and the above force fields were used to describe the motions of the amino acid atoms of the protein targets [51]. It is a very highly advanced and flexible computer program to identify the dynamic, equilibrium and structural properties of molecules [52]. The simulation analyses were carried out in a rectangular box of dimensions 115 Å × 75 Å × 50 Å, where the complexes were solvated and the Monte Carlo ion placing method was followed for adding some ions such as Na⁺ and Cl^- for neutralization [53]. After completion of the simulation, the trajectory was analyzed for root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (R_g) and several hydrogen bonds. Donor–acceptor distance is a crucial parameter dealing with the hydrogen interactions between a molecule and a target. This study also deals with the estimation of the donor–acceptor distances in nm. It refers to the separation between the donor atom (commonly hydrogen) and the acceptor atom (often oxygen or nitrogen) involved in the formation of a hydrogen bond. It is a major protein–ligand interaction involved in stabilizing a complex [59]. The formation of these bonds is dependent on the distance between the atoms. The radius of gyration can also be defined as the compactness or structural changes of a molecular system over time and it also describes the conformational patterns occupied by the protein. If the R_g value is small, it means the complex is tighter and vice versa [52]. MD simulations of 3r against α-amylase and α-glucosidase are depicted in Figures 10 & 11.

Figure 10.

Analysis of molecular dynamics trajectories of 3r α-amylase complex.

(A) Root mean square deviation (nm) of the 3r α-amylase complex system. (B) Root mean square fluctuation (nm) of the ligand 3r. (C) Root mean square fluctuation (nm) of the protein (α-amylase). (D) Radius of gyration of the protein α-amylase. (E) Hydrogen bond distribution with donor–acceptor distance (nm). (F) Number of hydrogen bond estimations.

Rg: Radium of gyration; RMSD: Root mean square deviation; RMSF: Root mean square fluctuation.

Figure 11.

Analysis of molecular dynamics trajectories of 3r α-glucosidase complex.

(A) Root mean square deviation (nm) of the 3r α-glucosidase complex system. (B) Root mean square fluctuation (nm) of the ligand 3r. (C) Root mean square fluctuation (nm) of the protein (α-glucosidase). (D) Radius of gyration of the protein α-glucosidase. (E) Hydrogen bond distribution with donor–acceptor distance (nm). (F) Number of hydrogen bond estimations.

Rg: Radius of gyration; RMSD: Root mean square deviation; RMSF: Root mean square fluctuation.

The trajectory analysis was done for 100 ns. Supplementary Figure 4A demonstrates that the RMSD value of the protein was stable and did not show any fluctuations throughout the experiment. The overall RMSD value of the ligand 3r was between 0.5 and 2 nm, and the ligand was unstable at the start of the experiment (0–40 nm), which had more fluctuations and then it decreased and became stable (with fewer fluctuations after 40 nm). The overall RMSF value of the ligand was between 0.05 and 0.2 nm and the terminal part of ligand 3r showed more fluctuation (Figure 10B). The higher RMSF value lay in the range of 0.1–0.6 nm between 100 and 150 residues of the protein, indicating the stability of the complex, which is given in Supplementary Figure 4C.

The duration of the experiment was 100 ns. As shown in Supplementary Figure 4D, the radius of gyration was constant (straight line) without any fluctuations and the value lay between 2 and 2.5 ns, which describes that the protein structure is highly compacted. Supplementary Figure 4E shows that the hydrogen bonds obtained in the crystal structure were retained throughout the 100 ns simulation. The complex displayed a major fluctuation in hydrogen bond numbers of 20–27.5 and minimum fluctuation in hydrogen bond numbers of 12.5–20 throughout the 100 ns simulation. Hydrogen bond interactions play an integral role in the formation of tight ligand–protein complexes. It is revealed in Figure 10F that a maximum of five number of hydrogen bonds are formed between the protein and ligand 3r in the range of 60–70 ns.

It is shown in Supplementary Figure 5A that the RMSD value of the protein was at 0.2 nm initially and it increased to 0.4 nm at 100 ns, which was stable (not showing any fluctuations). The overall RMSD value of the ligand 3r was between 0.2 and 0.8 nm and the ligand's RMSD value started at 0.2 nm and showed fluctuations until 40 nm and became stable (with fewer fluctuations after 40 nm). The overall RMSF value of the ligand lay between 0.05 and 0.2 nm and the terminal part of the ligand 3r showed high RMSF values (Supplementary Figure 5B). The higher RMSF value lay in the range of 0.1–0.6 nm between 400 and 450 residues of the protein, indicating the stability of the complex, which is presented in the Supplementary Figure 5C.

If the R_g value is small, it means the complex is tighter, and vice versa (1.1). The duration of the experiment was 100 ns. From Supplementary Figure 5D, it is inferred that the radius of gyration was constant (straight line) without any fluctuations and the value lay between 2 and 2.5 nm, describing a highly compacted protein structure. Supplementary Figure 5E shows that hydrogen bonds obtained in the crystal structure were retained throughout the 100 ns simulation. The complex showed a major fluctuation in hydrogen bonds up to 40 at the distance of 0.3 nm and the minimum fluctuation in hydrogen bond number at ten throughout the 100 ns simulation. Hydrogen bond interactions play an integral role in the formation of tight ligand–protein complexes. Figure 11F  demonstrates that a maximum three number of hydrogen bonds are formed between protein–ligand complex systems in the range of 65–85 ns.

Conclusion

In the present study, 20 new benzothiazole-tethered indenopyrazole heterocyclic hybrids, 3a–t, were efficiently synthesized. The synthesis of 3a–t was achieved by the cycloaddition reaction of 2-benzoyl/substituted benzoyl-(1H)-indene-1,3(2H)-diones (1) with 2-hydrazinyl-4/6-substituted benzo[d]thiazoles (2) in the presence of freshly dried ethanol. The structures of all the newly synthesized derivatives were elucidated using different physical and spectral analysis data such as FTIR, NMR (¹H and ¹³C), 2D NMR, HRMS and single-crystal x-ray crystallography. All the compounds and the standard drug, acarbose, were tested for their in vitro α-amylase and α-glucosidase inhibitory activity. Antidiabetic results revealed that most of the derivatives exhibited good-to-moderate inhibition toward α-amylase and α-glucosidase. Among all the tested compounds, 3p and 3r demonstrated good inhibitory potential with IC₅₀ values of 171.00 ± 1.51 μg/ml and 155.10 ± 1.32 μg/ml toward α-amylase and with IC₅₀ values of 750.00 ± 3.65 μg/ml and 657.10 ± 6.78 μg/ml toward α-glucosidase, respectively, in comparison with the standard rug, acarbose (IC₅₀ value of 125.53 ± 4.10 μg/ml for α-amylase and 510.13 ± 4.82 μg/ml for α-glucosidase). Furthermore, to find out the probable mechanism of action and the binding conformation responsible for α-amylase and α-glucosidase inhibitory activity, MD studies of compounds 3p (binding energies -10.3 and -10.4 kcal/mol) and 3r (binding energies -10.3 and -10.7 kcal/mol) in comparison with the standard, acarbose (binding energies -7.8 and -9.0 kcal/mol) were performed. MD simulations of docked complexes of 3r against α-amylase and α-glucosidase were conducted to assess the stability of the complexes and to investigate the interactions between the ligand and active site of these enzymes. Overall, the enzyme inhibitory results of the titled compounds revealed that out of 20 newly synthesized hydroxypyrazolones, 3p and 3r have the potential to serve as promising antidiabetic agents. It is worth considering structural amendments to these compounds as well as their in vivo pharmacological assay; they might lead to the development of potent antidiabetic drug candidates.

Keeping in mind that the development of novel, potent and selectively targeted agents with a precisely defined mechanism of action is critically significant for endocrinologists to mitigate the substantial side effects and resistance issues associated with currently available antidiabetic agents, our findings revealed that benzothiazole-linked hydroxypyrazolones are favorable antidiabetic agents and open several future perspectives on the designs of new, potent antidiabetic agents acting against α-amylase and α-glucosidase.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.