The Synthesis, In Vitro Bio-Evaluation, and In Silico Molecular Docking Studies of Pyrazoline–Thiazole Hybrid Analogues as Promising Anti-α-Glucosidase and Anti-Urease Agents

In the present work, a concise library of benzothiazole-derived pyrazoline-based thiazole (1–17) was designed and synthesized by employing a multistep reaction strategy. The newly synthesized compounds were screened for their α-glucosidase and urease inhibitory activities. The scaffolds (1–17) were characterized using a combination of several spectroscopic techniques, including FT-IR, 1H-NMR, 13C-NMR, and EI-MS. The majority of the synthesized compounds demonstrated a notable potency against α-glucosidase and urease enzymes. These analogues disclosed varying degrees of α-glucosidase and urease inhibitory activities, with their IC50 values ranging from 2.50 to 17.50 μM (α-glucosidase) and 14.30 to 41.50 (urease). Compounds 6, 7, 14, and 12, with IC50 values of 2.50, 3.20, 3.40, and 3.50 μM as compared to standard acarbose (IC50 = 5.30 µM), while the same compounds showed 14.30, 19.20, 21.80, and 22.30 comparable with thiourea (IC50 = 31.40 μM), respectively, showed excellent inhibitory activity. The structure−activity relationship revealed that the size and electron-donating or electron-withdrawing effects of substituents influenced the enzymatic activities such as α-glucosidase and urease. Compound 6 was a dual potent inhibitor against α-glucosidase and urease due to the presence of -CF3 electron-withdrawing functionality on the phenyl ring. To the best of our knowledge, these synthetic compounds were found to be the most potent dual inhibitors of α-glucosidase and urease with minimum IC50 values. Moreover, in silico studies on most active compounds, i.e., 6, 7, 14, and 12, were also performed to understand the binding interaction of most active compounds with active sites of α-glucosidase and urease enzymes.


Introduction
The discovery of new drugs is often facilitated by the inhibition of enzymes.Urease and α-glucosidase play important roles in a variety of medical applications.There are vital enzymes that serve an integral role in various clinical fields [1,2].There are millions of people worldwide affected by diabetes mellitus (DM), a metabolic disorder caused by high blood sugar levels [3].A number of complications are associated with diabetes mellitus metabolic disease, such as cardiovascular disorders, reduced wound healing ability, and cancer, as well as severe health problems such as retinopathy, neuropathy, and amputations.Diabetes mellitus can be cured largely through the control of glycemia [4][5][6].There is a significant amount of α-glucosidase present on the epithelial walls of the small intestines.Carbohydrate metabolism is regulated by it.It is also beneficial to treat diabetes by inhibiting this enzyme, since it reduces the blood's glucose level [7,8].Miglitol, acarbose, and voglibose are a few of the medications that can cause flatulence, diarrhoea, and gastrointestinal disease [9,10].Additionally, it contributes to the conversion of oligosaccharides into simple sugar via hydrolysis in the salivary glands and pancreas.The treatment of diabetes is therefore enhanced by its ability to lower the postprandial blood sugar levels.The high bioavailability of pyrazoline in the organic realm has led to the development of quick, efficient, and environmentally friendly methods for synthesizing it [11].
Urease belongs to the super family of phosphodiesterase and amidohydrolase, which initiate the degradation of urea into ammonia and carbon dioxide.Eukaryotes and prokaryotes both produce urease enzymes.In response to the hyperactivity of the urease enzyme, excessive amounts of ammonia are released into the stomach, causing alkalinity and helping to improve mucosal permeability [12][13][14][15].Even though humans lack urease enzymes, they synthesize urea as an end product of protein metabolism and eliminate it through urine [16][17][18].Urease enzymes facilitate monitoring and regulating the nitrogen metabolism in cattle and many other animals.In addition to causing bacterial infections, high levels of these enzymes can cause various pathogenic conditions.In humans, low stomach pH eases the growth of Helicobacter pylori (HP).In the long run, it causes ulcers in the stomach and intestines, and ultimately may lead to cancer [19][20][21][22][23].
Individuals who concurrently have diabetes mellitus and peptic ulcers are advised to employ a variety of medical therapies.The coexistence of a peptic ulcer can potentially amplify health concerns in patients diagnosed with diabetes due to the commonly seen phenomenon of delayed wound healing.A proposed approach has been formulated to address the management of individuals who have both diabetes mellitus and peptic ulcers.This technique aims to simultaneously target the activities of α-glucosidase and urease.This technique holds significant potential as a therapeutic alternative for people with DM and peptic ulcers [24,25].
Pyrazole-based compounds have garnered significant attention owing to their diverse antineoplastic properties, including chemotherapy.The presence of a pyrazole core has been recognized as a significant contributor to the augmented cytotoxicity of chalcones [26].
In recent times, there has been a notable surge in interest surrounding pyrazoline as a promising heterocyclic framework within the realm of medicine.This heightened attention can be attributed to the widespread use of pyrazoline in the development of therapeutic medications.The compound has a significant role in various fields, such as synthetic and medicinal chemistry, serving as a crucial foundational component for the production of pharmaceuticals.Heterocyclic compounds have been found to have an affinity towards a diverse range of biotargets across several fields [27][28][29].Both agribusiness and pharmacological research have derived advantages from the utilization of these scaffolds [30].
There have been numerous reports of thiazole and pyrazoline scaffolds exhibiting a broad range of chemical reactivity and pharmacological activities for decades [31,32].Numerous studies have demonstrated that these five-membered heterocyclic compounds delocalize free radicals and inhibit bacteria by producing stable DPPH fragments [33].Thiazole-based pyrazoline scaffolds also exhibit anti-inflammatory [34], antidiabetic [35], Alzheimer's disease [36], anti-HIV [37], antiviral [38], anticonvulsant, antitumor activities [39], and antimicrobial properties [40].The pyrazoline skeleton has been extensively studied using a variety of structural manipulations and it has been concluded that the steric and electronic properties of substitutes at the N-1, C-3, and C-5 positions, as well as their chirality, have significant effects on their biological activity [41].A pyrazoline's N-N bonds are said to play an important role in its pharmacological properties [42].
Pyrazoline structures have been found to possess noteworthy pharmacological properties, encompassing anti-inflammatory, antioxidant, analgesic, antifungal, antipyretic, antiangiogenic, antibacterial, antitumor, and antiviral activities [43,44].Our research group is presently involved in the development of novel and more effective synthetic routes for small compounds.This endeavour aims to enable the investigation of their potential biological activities, specifically their inhibitory effects on enzymes such as α-glucosidase and urease, which have been observed to be significant.
As pyrazoline structural patterns play an important role in medicinal chemistry, we present our findings concerning the synthesis, characterization, and assessment of novel pyrazoline derivatives.As dual inhibitors, these derivatives inhibit both α-glucosidase and urease enzymes.A variety of pyrazoline derivatives [45,46] containing different substitution arrangements were evaluated for their ability to inhibit both α-glucosidase and urease enzymes.Among heterocyclic compounds, these analogues have shown a considerable potency, providing a greater pool of potential of α-glucosidase and urease inhibitors than previously known (Figure 1).their chirality, have significant effects on their biological activity [41].A pyrazoline's N-N bonds are said to play an important role in its pharmacological properties [42].
Pyrazoline structures have been found to possess noteworthy pharmacological properties, encompassing anti-inflammatory, antioxidant, analgesic, antifungal, antipyretic, antiangiogenic, antibacterial, antitumor, and antiviral activities [43,44].Our research group is presently involved in the development of novel and more effective synthetic routes for small compounds.This endeavour aims to enable the investigation of their potential biological activities, specifically their inhibitory effects on enzymes such as α-glucosidase and urease, which have been observed to be significant.
As pyrazoline structural patterns play an important role in medicinal chemistry, we present our findings concerning the synthesis, characterization, and assessment of novel pyrazoline derivatives.As dual inhibitors, these derivatives inhibit both α-glucosidase and urease enzymes.A variety of pyrazoline derivatives [45,46] containing different substitution arrangements were evaluated for their ability to inhibit both α-glucosidase and urease enzymes.Among heterocyclic compounds, these analogues have shown a considerable potency, providing a greater pool of potential of α-glucosidase and urease inhibitors than previously known (Figure 1).

Chemistry
In order to synthesize pyrazoline-based thiazoles, a multi-step reaction procedure was involved.The desired pyrazoline-based thiazoles derivatives (1−17) were synthesized using the method outlined in Scheme 1.In the first step, 1-(2,5-dichloropyridin-3-yl)ethan-1-one (I) was reacted with benzo[d]thiazole-2-carbaldehyde (II) to produce a substrate chalcone (III) [47].This reaction was carried out in the presence of a 50% sodium hydroxide solution in absolute ethanol.It was refluxed for 8-10 h continuously with constant stirring in an ice bath.The substrate (III) was neutralized with dilute hydrochloric acid and washed with pet-ether.The substrate (III) endured cyclization by reacting with thiosemicarbazide that was stirred in acetic acid, and heated under reflux conditions for 5-7 h, enabling the formation of an intermediate pyrazoline (IV).Finally, the substrate (IV) was reacted with differently substituted phenacyl bromide in the presence of triethyl amine (Et 3 N) in solvent absolute ethanol, affording the targeted pyrazoline-based thiazole derivatives (V) in an appropriate yield (Scheme 1).

Chemistry
In order to synthesize pyrazoline-based thiazoles, a multi-step reaction procedure was involved.The desired pyrazoline-based thiazoles derivatives (1−17) were synthesized using the method outlined in scheme 1.In the first step, 1-(2,5-dichloropyridin-3-yl)ethan-1-one (I) was reacted with benzo[d]thiazole-2-carbaldehyde (II) to produce a substrate chalcone (III) [47].This reaction was carried out in the presence of a 50% sodium hydroxide solution in absolute ethanol.It was refluxed for 8-10 h continuously with constant stirring in an ice bath.The substrate (III) was neutralized with dilute hydrochloric acid and washed with pet-ether.The substrate (III) endured cyclization by reacting with thiosemicarbazide that was stirred in acetic acid, and heated under reflux conditions for 5-7 h, enabling the formation of an intermediate pyrazoline (IV).Finally, the substrate (IV) was reacted with differently substituted phenacyl bromide in the presence of triethyl amine (Et3N) in solvent absolute ethanol, affording the targeted pyrazoline-based thiazole derivatives (V) in an appropriate yield (Scheme 1).Scheme 1.General scheme for the synthesis of benzothiazole-derived pyrazoline-based thiazole (1-17).
Compound 6, with an IC 50 of 2.50 ± 0.30 M for α-glucosidase and IC 50 = 14.30± 3.90 µM for urease inhibition, demonstrated an impressive profile, which was partly attributed to its trifluoromethyl group [48].Trifluoromethyl groups have a strong affinity for the enzyme's active sites, primarily through hydrogen bonding interactions.Additionally, the -CF 3 group reduces the electronic density of the aromatic ring, increasing its acidity and affecting the electrostatic interactions with the substrate.This accounts for the compound's significant inhibitory activities against α-glucosidase and urease.Furthermore, compound 7 (IC 50 = 3.20 ± 0.10 µM and IC 50 = 19.20 ± 0.10 µM) contains para-fluoro and ortho-hydroxy groups, while compound 12 (IC 50 = 3.50 ± 0.10 µM and IC 50 = 22.30 ± 0.80 µM) features a para-fluoro group attached to the aromatic ring.Due to the nucleophilicity, metabolic stability, and hydrogen bonding capabilities of the hydroxy and fluoro groups, these structural features enhance the enzyme inhibitory potential.Thus, these compounds have notable α-glucosidase and urease inhibitory properties.Furthermore, compounds 11 and 12, which both contain fluorine in metaand para-positions, enhance their biological potential via their elevated electronegativity and ability to form hydrogen bonds with enzyme active sites.Compound 12 (IC 50 = 3.50 ± 0.10 µM and 22.30 ± 0.80µM), with a para-fluoro group, exhibits a higher inhibitory potential compared to compound 11 (IC 50 = 3.80 ± 0.30 µM and 25.30 ± 0.20µM), which has a meta-fluoro group.Due to the presence of meta-fluoro, the inhibitory potential may be decreased.Consequently, substituents play a significant role in determining α-glucosidase and urease inhibitory profiles (Table 1).
Compounds 14 (IC 50 = 3.40 ± 0.10 µM and 21.80 ± 2.90), 9 (IC 50 = 4.40 ± 0.30 µM and 29.40 ± 0.10), and 17 (IC 50 = 6.80 ± 0.20 µM and 31.30± 0.40) exhibited notably higher α-glucosidase and urease inhibitory potential.The enhanced activity is attributed to the presence of an ortho-hydroxy group attached to the aromatic ring, as well as a di-chloro group in the ortho-meta position.By increasing the binding affinity with the substrate and forming hydrogen bonds with the enzyme's active sites, the hydroxy group plays a crucial role in the inhibitory profile.Furthermore, the presence of two chlorine groups that are electron-donating for a resonance effect makes the hydroxyl group much more willing to form hydrogen bonds with the biological target.Further, compound 9 demonstrated a higher potency than compound 17, primarily due to the presence of a nitro group, which enhances the inhibitory potential of the scaffolds.As a toxicophore and a pharmacophore, the nitro group induces charge density by withdrawing electrons from the aromatic system.The -Cl group also influences the electronic cloud through an inductive effect on the aromatic system.As a result, the presence of -NO 2 and -Cl groups significantly enhances the biological profile of the molecule.As a result of the ortho-meta interaction within the aromatic system, compound 14 exhibited a superior inhibitory potential compared to both 9 and 17.The position of substituents on the phenyl ring of the pyrazoline-thiazole skeleton plays a crucial role in determining the α-glucosidase and urease inhibitory potential shown below in Table 1.Compounds 14 (IC50 = 3.40 ± 0.10 μM and 21.80 ± 2.90), 9 (IC50 29.40 ± 0.10), and 17 (IC50 = 6.80 ± 0.20 μM and 31.30± 0.40) exhi α-glucosidase and urease inhibitory potential.The enhanced activit presence of an ortho-hydroxy group attached to the aromatic ring, a group in the ortho-meta position.By increasing the binding affinity w forming hydrogen bonds with the enzyme's active sites, the hyd crucial role in the inhibitory profile.Furthermore, the presence of that are electron-donating for a resonance effect makes the hydrox willing to form hydrogen bonds with the biological target.Fu demonstrated a higher potency than compound 17, primarily due nitro group, which enhances the inhibitory potential of the scaffol and a pharmacophore, the nitro group induces charge density by w from the aromatic system.The -Cl group also influences the electro inductive effect on the aromatic system.As a result, the presence of significantly enhances the biological profile of the molecule.As a re interaction within the aromatic system, compound 14 exhibited a potential compared to both 9 and 17.The position of substituents on t pyrazoline-thiazole skeleton plays a crucial role in determining th By increasing the binding affinity w forming hydrogen bonds with the enzyme's active sites, the hyd crucial role in the inhibitory profile.Furthermore, the presence of that are electron-donating for a resonance effect makes the hydrox willing to form hydrogen bonds with the biological target.Fu demonstrated a higher potency than compound 17, primarily due nitro group, which enhances the inhibitory potential of the scaffol and a pharmacophore, the nitro group induces charge density by w from the aromatic system.The -Cl group also influences the electro inductive effect on the aromatic system.As a result, the presence of significantly enhances the biological profile of the molecule.As a re interaction within the aromatic system, compound 14 exhibited a potential compared to both 9 and 17.The position of substituents on t By increasing the binding affinity w forming hydrogen bonds with the enzyme's active sites, the hyd crucial role in the inhibitory profile.Furthermore, the presence of that are electron-donating for a resonance effect makes the hydrox willing to form hydrogen bonds with the biological target.Fu demonstrated a higher potency than compound 17, primarily due nitro group, which enhances the inhibitory potential of the scaffol and a pharmacophore, the nitro group induces charge density by w from the aromatic system.The -Cl group also influences the electro inductive effect on the aromatic system.As a result, the presence of significantly enhances the biological profile of the molecule.As a re interaction within the aromatic system, compound 14 exhibited a potential compared to both 9 and 17.The position of substituents on t By increasing the binding affinity w forming hydrogen bonds with the enzyme's active sites, the hyd crucial role in the inhibitory profile.Furthermore, the presence of that are electron-donating for a resonance effect makes the hydrox willing to form hydrogen bonds with the biological target.Fu demonstrated a higher potency than compound 17, primarily due nitro group, which enhances the inhibitory potential of the scaffol and a pharmacophore, the nitro group induces charge density by w from the aromatic system.The -Cl group also influences the electro inductive effect on the aromatic system.As a result, the presence of significantly enhances the biological profile of the molecule.As a re interaction within the aromatic system, compound 14 exhibited a presence of an ortho-hydroxy group attached to the aromatic ring, a group in the ortho-meta position.By increasing the binding affinity w forming hydrogen bonds with the enzyme's active sites, the hyd crucial role in the inhibitory profile.Furthermore, the presence of that are electron-donating for a resonance effect makes the hydrox willing to form hydrogen bonds with the biological target.Fu demonstrated a higher potency than compound 17, primarily due nitro group, which enhances the inhibitory potential of the scaffol and a pharmacophore, the nitro group induces charge density by w from the aromatic system.The -Cl group also influences the electro inductive effect on the aromatic system.As a result, the presence of significantly enhances the biological profile of the molecule.As a re interaction within the aromatic system, compound 14 exhibited a Compounds 14 (IC50 = 3.40 ± 0.10 μM and 21.80 ± 2.90), 9 (IC50 29.40 ± 0.10), and 17 (IC50 = 6.80 ± 0.20 μM and 31.30± 0.40) exhi α-glucosidase and urease inhibitory potential.The enhanced activi presence of an ortho-hydroxy group attached to the aromatic ring, group in the ortho-meta position.By increasing the binding affinity w forming hydrogen bonds with the enzyme's active sites, the hyd crucial role in the inhibitory profile.Furthermore, the presence of that are electron-donating for a resonance effect makes the hydrox willing to form hydrogen bonds with the biological target.Fu demonstrated a higher potency than compound 17, primarily due nitro group, which enhances the inhibitory potential of the scaffol and a pharmacophore, the nitro group induces charge density by w from the aromatic system.The -Cl group also influences the electro inductive effect on the aromatic system.As a result, the presence of significantly enhances the biological profile of the molecule.As a re Compound 4 (IC 50 = 4.10 ± 0.30 µM and 23.30 ± 2.40) possesses a para-nitro group that exhibits strong binding with enzyme active sites, resulting in an increase in potency.The electron-withdrawing properties of nitro groups cause charge density, which is explained by the nitro group's electron-withdrawing properties.However, compound 1 (IC 50 = 13.10 ± 0.40 µM and 41.50 ± 1.70) has a para-bromo group and an ortho-nitro group, with the para-bromo group causing steric hindrance, resulting in a reduced potency in comparison to compound 4, as well as acarbose and thiourea (IC 50 = 5.30 ± 0.30 µM and IC 50 = 31.40± 2.50 µM, respectively).A substituent at the ortho-nitro position of compound 5 (IC 50 = 6.70 ± 0.30 µM and 32.50 ± 2.10) also exhibited a lower potency than 4 because of the position of the substituent of the -NO 2 group on the meta-position.Hence, these scaf-folds have different inhibitory potentials against alpha-glucosidase and urease depending on the position of the substituent, as shown in Table 1.
Additionally, compounds 8 and 13 (IC 50 = 13.10 ± 0.10 µM and 39.20 ± 0.60), 2 (IC 50 = 14.20 ± 0.10 µM and 37.20 ± 1.90), and 16 (N.A.) exhibited poor inhibitory activity due to the presence of bulky groups such as N,N-dimethyl, ortho-meta-dimethyl, benzyloxyphenyl, and naphthalene, which cause steric hindrance.These compounds are less potent when compared to the standard drugs acarbose and thiourea (IC 50 = 5.30 ± 0.30 µM and IC 50 = 31.40± 2.50 µM, respectively).Accordingly, bulky groups lead to a lower efficacy as compared to standard drugs.As such, the biological significance of scaffolds can also vary depending on the position and nature of their substituents, either through hydrogen bonding or Van der Waals interactions.Accordingly, the synthesized analogues have electron-withdrawing properties (-CF 3 , -NO 2 , and -Cl) and strong hydrogen bonding substituents, such as the -OH and -F groups, that make them excellent α-glucosidase and urease inhibitors.Accordingly, the inhibitory profile of the synthesized compounds depends on the position, nature, and number of substituents attached to the aromatic ring of the benzothiazole-derived pyrazoline-based thiazole moiety (1-17).

General Information
This study used chemicals and solvents that were obtained from reputable suppliers, namely Merck and Sigma-Aldrich, without any further purification.An electrothermal digital device was used to determine the melting points.A Bio-Rad spectrophotometer was used to acquire the infrared spectra.Bruker spectrometers with proton frequencies of 600 MHz and carbon frequencies of 126 MHz were used to acquire the NMR spectra.Nuclear magnetic resonance (NMR) chemical shift values were first established using the δ (parts per million) scale.Thin-layer chromatography (TLC) was employed as a means of assessing and overseeing the advancement and culmination of the reaction.The spots on the TLC plate were visualized by exposing it to 254 nm ultraviolet light (UV).In addition to DMSO-d6, a reference compound was used to calculate chemical shift values.Moreover, the coupling constants are expressed in hertz (Hz).We acquired mass spectra using the mass spectrometers MAT 113D, JEOL JMS-600H, and MAT 312 (high-resolution electron ionization mass spectrometry (HR-EI-MS).

General Information
This study used chemicals and solvents that were obtained from reputable suppliers, namely Merck and Sigma-Aldrich, without any further purification.An electrothermal digital device was used to determine the melting points.A Bio-Rad spectrophotometer was used to acquire the infrared spectra.Bruker spectrometers with proton frequencies of 600 MHz and carbon frequencies of 126 MHz were used to acquire the NMR spectra.Nuclear magnetic resonance (NMR) chemical shift values were first established using the δ (parts per million) scale.Thin-layer chromatography (TLC) was employed as a means of assessing and overseeing the advancement and culmination of the reaction.The spots on the TLC plate were visualized by exposing it to 254 nm ultraviolet light (UV).In addition to DMSO-d 6 , a reference compound was used to calculate chemical shift values.Moreover, the coupling constants are expressed in hertz (Hz).We acquired mass spectra using the mass spectrometers MAT 113D, JEOL JMS-600H, and MAT 312 (high-resolution electron ionization mass spectrometry (HR-EI-MS).

Synthesis of Chalcone Derivatives
In the first step, chalcone was synthesized using the Claisen-Schmidt condensation reaction, in which 1 mmol of 1-(2,5-dichloropyridin-3-yl)ethan-1-one was taken in a roundbottom flask and sodium hydroxide (40% NaOH) was added as a catalyst in solvent ethanol and stirred in the solution for 30 min in an ice bath, followed by the addition of 1 mmol of benzo[d]thiazole-2-carbaldehyde dropwise into it.The reaction was refluxed and stirred for from 8 to 10 h to form targeted product chalcone derivatives in a good yield.The obtained product was neutralized with dilute hydrochloric acid (10%), washed with chilled pet-ether and n-hexane, recrystallized from ethanol, and then dried as final products.

Synthesis of Pyrazoline
In this step, chalcone (1 mmol) was reacted with 1 mmol of thiosemicarbazide in the presence of 5 mL of acetic acid in ethanol.The reaction was refluxed for 7-10 h, yielding pyrazoline analogues.The reaction progress was monitored through TLC.The product was washed with chilled pet-ether, recrystallized with ethanol, and dried as a final product for further reaction.

Synthesis of Pyrazoline Derivatives with Phenacyl Bromide
In the last step, the 1 mmol of pyrazoline substrate was reacted with 1 mmol of differently substituted phenacyl bromide in the presence of triethyl amine (2-4 drops) in solvent absolute ethanol, affording the targeted benzothiazole-derived pyrazoline-based thiazole derivatives in an appropriate yield.The progress of the reaction was monitored through TLC.The solvent was evaporated through a rotary evaporator.The synthesized products were washed with n-hexane and recrystallized in chloroform, yielding pure products.The synthesized product was characterized through different spectroscopic techniques, including 1 H-NMR, 13 C-NMR, and HREI-MS (Scheme 1).

α-Glucosidase Inhibitory Assay
A procedure based on the previously reported procedure with minor modifications was used to determine the inhibitory capacity of the newly designed derivatives of benzothiazole-derived pyrazoline-based thiazole (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).For 20 min at 37 • C, 250 mL of 1.0 U/mL carbose was incubated with 500 mL of 1.0 U/mL glucose in 100 mM of phosphate buffer (pH 6.8) for 20 min.The reaction mixture was then incubated for 1 h at 37 • C with 250 mL of 4-nitrophenyl-b-D-glucopyranoside solution and 250 mL of 1% starch dissolved in 100 mM of phosphate buffer (pH 6.8), respectively.Following this, the reaction mixture was heated for 10 min with 1.0 mL of 3,5-dinitrosalicylic acid colouring reagent.The absorbance of the final reaction mixture was determined for α-glucosidase at 405 nm.To correct the absorbance, a blank was prepared.The positive control used was acarbose solution.A percentage of the inhibitory activity was calculated using a control sample without the inhibitors [53].

Urease Inhibitory Assay
In this study, newly synthesized pyrazoline-based thiazole derivatives (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) were evaluated for their ability to inhibit urease activity.During the experiment, 55 L of buffer solution containing 100 mM of urea was combined with 25 L of enzyme solution to prepare reaction mixtures.A volume of 5 L containing 1 mM concentrations of recently produced compounds was then added to these mixtures.After 15 min of incubation at 30 • C, the reactions were allowed to occur in 96-well plates.The detection of ammonia generation by urease was carried out by using the Indophenol technique.A phenol reagent containing 1% w/v phenol and 0.005% w/v sodium nitroprusside was added to each well along with 70 lL of an alkali reagent containing 0.5% w/v NaOH and 0.1% active chloride NaOCl.Utilizing a microplate reader manufactured by Molecular Device in the United States, the increased absorbance was measured at 630 nm after another 50 min.In triplicate, the aforementioned procedures were performed using a constant end volume of 200 mL.The final data, representing the rate at which the absorbance changed per minute, were analysed with the SoftMax Pro program by Molecular Device, New York, NY, USA.In alkaline conditions with a pH of 8.2, the experiments were conducted using 0.01 M K 2 HPO 4 •3H 2 O, 1 mM EDTA, and 0.01 M LiCl.A conventional inhibitor of urease, thiourea, was used in this study to determine the percentage of inhibition [54].

Conclusions
A series of newly benzothiazole-derived pyrazoline-based thiazole derivatives (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) were designed and synthesized.The synthesized benzothiazole-derived pyrazoline-based thiazole compounds were characterized through different spectroscopic techniques such as 1 H-NMR, 13 C-NMR, and HRMS.After that, their inhibitory activity against α-glucosidase and urease enzymes was evaluated.The compounds (1-17) exhibited remarkable inhibitory potential against targeted α-glucosidase and urease enzymes under the positive control of acarbose and thiourea as standard drugs, respectively.Among the synthesized series, the compounds 6, 7, 12, and 14 demonstrated were found to be significantly active, as seen by their very low IC 50 values, indicating their notable inhibitory activity.Furthermore, the structure-activity relationship (SAR) study was carried out based on various functional groups linked to the aryl group, the nature of the substituent, and positions of the substituent, which play a significant role in determining the effectiveness of these compounds as inhibitors of α-glucosidase and urease enzymes.In addition, to correlate the in vitro study well with the in silico study, a molecular docking study was conducted for the most active compounds and the result obtained corroborated that these active compounds established several key interactions with the active sites of targeted enzymes.

Figure 2 .
Summary of SAR studies of pyrazoline-thiazole hybrids analogues against α-glucosidase and urease enzymes.

Figure 4 .
Figure 4. Protein-ligand interaction 2D and 3D profile of an active analogue 6 against urease.

Figure 4 .
Figure 4. Protein-ligand interaction 2D and 3D profile of an active analogue 6 against urease.

Figure 4 .
Figure 4. Protein-ligand interaction 2D and 3D profile of an active analogue 6 against urease.

Figure 6 .
Figure 6.Protein-ligand interaction 2D and 3D profile of an active analogue 7 against urease.Figure 6. Protein-ligand interaction 2D and 3D profile of an active analogue 7 against urease.

Figure 8 .
Figure 8. Protein-ligand interaction 2D and 3D profile of an active analogue 12 urease.Figure 8. Protein-ligand interaction 2D and 3D profile of an active analogue 12 urease.

Figure 10 .
Figure 10.Protein-ligand interaction 2D and 3D profile of an active analogue 7 against urease.

Figure 10 .
Figure 10.Protein-ligand interaction 2D and 3D profile of an active analogue 7 against urease.