INTRODUCTION

Copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions are the preliminary examples of click-chemistry [1]. CuAAC reactions are designed to use eco-friendly solvents in the presence of copper as a catalyst, but a complementary solvent is also used due to the limited solubility of organic compounds [2]. Mechanochemistry is a solvent-free method and is used for synthesizing metal alloys [3]. J. Mack and co-workers proved that the surface of the milling vail could be used instead of organometallic catalysts to perform catalytic reactions [4]. Many researchers used a mechanochemical pathway to synthesize organic compounds using sodium ascorbate and copper sulfate [5]. It is widely accepted that the surface of a modified flask/ container/milling vail can serve as a catalytic surface [6]. Recently L. Teresa and co-workers have used the copper vail instead of the copper catalyst for the synthesis of CuAAC reactions [4], and it has been noted that the separation of products is much more workable than the solution-based methods.

It has recently been seen that COVID-19 patients with diabetes are more vulnerable to fatalities and complications than COVID-19 patients without diabetes [7]. Diabetic complications arise due to hyperglycemia which triggers the polyol pathway holding the reduction of glucose to sorbitol in the presence of the aldose reductase enzyme called ALR2 [8].

The enzymes reducing the aldehydes are called aldehyde reductase (ALR1), which are accurately responsible for reducing the aldehyde functionality of glucose in the polyol pathway [9]. ALR1 is also engaged in the metabolism of methylglyoxal and 3-deoxyglucosone, producing lethal glycation products [10]. ALR1 and ALR2 inhibitors are in dire need, which can normalize the polyol pathway and fight secondary diabetic problems [11].

Thiazole-containing benzylidene triazoles exhibit several biological activities [12, 13] such as sedative, bactericidal, fungicidal, anesthetic, cardiotonic, anti-inflammatory [14], immunosuppressant, antibiotic, anticancer [15], and anthelmintic activity. Considering the potential of nitrogen-containing heterocycles, we synthesized the thiazolidinone-triazole derivatives via surface scratching approach and determined the antidiabetic potential. In addition, pharmacokinetic and molecular docking studies were also carried out.

RESULTS AND DISCUSSION

Synthesis of thiazolidinone-triazole derivatives. The synthesis of thiazolidinone-triazole derivatives 9a–9c was completed in various steps. In the first step, 2-(phenylamino)thiazol-4(5H)-one 3 was synthesized by a reported method [16] (Scheme 1). The melting point value of 3 agreed with the literature. The N–CS–N and C=S stretching appeared at 1270 and 1220 cm–1. Similarly, in the case of 1H NMR, one proton of NH moiety appeared at 10.03 ppm, while two protons of CSNH2 group appeared at 8.02 ppm, confirming the formation of compound 3.

Scheme
scheme 1

1.

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In the second step, 2-(prop-2-yn-1-yloxy)benzaldehyde 5 was synthesized by the reaction of benzaldehyde 4 with propargyl bromide in the presence of potassium carbonate by known protocol. The melting point value of 5 agreed with the literature. A high field singlet appears at 2.9 ppm due to hydrogen of the acetylenic group. The CH2 protons of the propargyl group appeared at 4.96 ppm. Similarly, 13C signals of the propargyl group appeared upfield between 70.31 ppm and 90.22 ppm, confirming the synthesis of compound 5.

In third step, 2-(phenylamino)thiazol-4(5H)-one 3 and 2-(prop-2-yn-1-yloxy)benzaldehyde 5 were refluxed the presence of malononitrile to form dipolarophile (E)-2-(phenylamino)-5-[2-(prop-2-yn-1-yloxy)benzylidene]thiazol-4(5H)-one 6. In this reaction, active methylene present in thiazole 3 passes through a nucleophilic addition reaction giving rise to a double bond in arylidene malononitriles via a Michael-type reaction to produce compound 6. In addition to all other peaks, a characteristic proton signal of ethylenic CH2 appears at 3.77 ppm while the 13C signal appears toward low-field at 157.43 ppm to rectify the formation of compound 6.

As presented in Scheme 1, (Z)-2-(phenylamino)-5-[2-(prop-2-yn-1-yloxy)benzylidene]thiazol-4(5H)-one 6 and benzyl azides 8a–8c were reacted in copper vail via mechanochemical approach. Many studies have reported that elemental copper has been used in CuAAC reactions. It is considered that a comproportionation reaction takes place, and copper(0) gives active copper(I) catalyst. We mechanically synthesized thiazolidinone-triazole derivatives by grinding the alkyne 6 and substituting benzyl azide 8a–8c in a custom-made copper flask. It is a ball milling-type reaction. We believe that the surface of the flask and the hanging ball provides the catalytic site and start the reaction with copper metal in a zero-oxidation state. The coupling results in a good yield (73–80%) of thiazolidinone-triazole derivatives 9a–9c.

The 1H NMR of 9b showed a singlet at δ 7.79 ppm for the triazolic proton, δ 8.32 ppm for the methine proton, and two singlets at δ 10.44 and 12.10 ppm, standing for a tautomeric form of 9b. Two protons of the CH2 group between the triazole ring and phenyl ring appeared at δ 5.20 ppm, while two protons of OCH2 appeared at 5.64 ppm. The 13C NMR spectrum of 9b exhibited a peak at 57.44 ppm linked to the CH2 group between the triazole and phenyl ring. The peak at 62.45 ppm is related to the carbon of the OCH2 group. The theoretical CHN values were in good agreement with the experimental ones and confirmed the formation of 9b. The same interpretation way has been adopted for 9a and 9c. The spectral data of 9a–9c justify the successful synthesis of thiazolidinone-triazole derivatives.

Biological activity assay. The thiazolidinone-triazole derivatives 9a–9c were estimated for in vitro enzyme inhibitory potential on aldose and aldehyde reductase enzymes (Table 1). For the aldose reductase (ALR1) enzyme, sulindac was used as a reference drug, and D- and L-glyceraldehyde were used as substrates. While for the aldehyde reductase (ALR2) enzyme, valproic acid was used as a reference drug and D-glucuronic acid as a substrate. The activity of compounds showing more than 50% inhibition is expressed as IC50±SEM (µM).

Table 1. In vitro inhibitory activity of thiazolidinone-triazole derivatives 9a–9c against aldehyde (ALR1) and aldose (ALR2) reductase enzymes
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All three compounds 9a–9c shows good activity against ALR1 and ALR2. However, compound 9c was more active against ARL2, showing only 90±0.56 IC50 against ALR1. Furthermore, an improved inhibitor potency of compound 9c was also seen against ALR1 (IC50 54±1.98 mM) and ALR2 (IC50 10±1.56 mM).

Molecular docking studies of ALR1 and ALR2 inhibitors. In 9a (Fig. 1), the bromine atom of the benzene ring next to the triazole ring made a conventional hydrogen bond with Lys263. Many hydrophobic interactions were also seen. SER211 made a donor hydrogen bond with the π ring of benzene. TYR210 was making π–π stacked interactions with the phenolic benzene. LEU213 and ARG218 made π-alkyl interactions with the triazole and thiazole rings, respectively. SER215 was pi-lone-pair interactions with the thiazole ring. Docking of compound 9b revealed hydrogen-bonded interactions between the oxygen atom of the thiazole ring and GLN184 (Figs. S1a–S1c, see Supplementary Materials). Some hydrophobic interactions were also seen. TYR50 made π-donor hydrogen bond interactions with the thiazole ring, and LYS263 made π-alkyl interactions with the benzene ring attached to the nitrogen atom. Compound 9c was making maximum interactions in docking (Figs. S2a–S2c, see Supplementary Materials). LEU213 was making π-sigma interactions with the benzene ring attached to chlorine. TRP 22 was making interactions with the oxygen of thiazole. LYS263 was making a carbon-hydrogen bond with the thiazole, triazole ring, and benzene ring present between them. The hydrophobic interactions, LEU 213 and LEU 229, made pi-sigma and π-alkyl interactions with benzene of chlorine atom. The electrostatic (π-cation) interaction was seen in the benzene ring and ARG269.

Fig. 1.
figure 1

Representative 3D (a) and 2D depictions (b) of docking of 9a in the active site of ALR1.

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Docking studies of ALR2 inhibitors were also carried out for 9a–9c. In compound 9a, CYS 298 made a pi-donor hydrogen bond with the benzene ring attached to the nitrogen. Hydrophobic interactions were also seen that are believed necessary for efficient binding. TYR209 was making π–π stacked interactions with a benzene ring attached to a bromine atom. VAL 47 made π-alkyl interactions with the triazole and benzene ring attached to an oxygen atom (Fig. S3a–S3c, see Supplementary Materials). Docking of 9b revealed several hydrophobic interactions (Fig. 2). TYR 209 was making π–π T-shaped interactions with a benzene ring attached to chlorine. While PHE 121 was making π–π stacked interactions with the benzene ring attached to an oxygen atom. PHE 122 and CYS 298 interact through π-sulfur interactions. π-donor hydrogen bond interaction was seen in TYR 48 and a triazole ring. Several hydrophobic interactions were seen in the docking study of 9c (Fig. S4a–S4c, see Supplementary Materials). LEU 300, ILE 260, and TYR 209 made π-alkyl and alkyl interactions with the chlorine atom. PHE 122 and CYS 298 made π-sulfur interactions with benzene, thiazole, and triazole ring, while π-donor hydrogen bond was seen among the benzene, nitrogen atom of triazole, and TYR 48.

Fig. 2.
figure 2

Representative 3D (a) and 2D depictions (b) of docking of 9b in the active site of ALR2.

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ADME properties. The most critical and challenging part of the drug discovery process is optimizing ADME properties. These properties were estimated by the Swiss ADME tool and helped discover the drug by evaluating the drug-likeness of compounds. These properties recommended that compounds 9a–9c are better to use as a drug and have a high possibility of absorption and blood-brain penetration. ADME prediction scores for the synthesized compounds and reference drugs are given in Table 2. Overall, the results proved that compound 9c having chloro group para to the attachment of triazole to benzene ring has the most promising activity among all the derivatives. The compound 9b (with m-chloro substituent) among 9a–9c was moderate active against both ALR1 and ARL2 with an IC50 value of 90±0.56 and 28±1.23. The decreasing order of inhibitory activity is 9a > 9b > 9c.

Table 2. Pharmacokinetic properties of ALR1 and ALR2 inhibitors 9a–9c
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CONCLUSION

Thiazolidinone-triazole derivatives have been successfully prepared using green technology and were characterized by NMR spectroscopy and CHN analysis. The antidiabetic potential of synthesized compounds has been tested against aldehyde reductase (ALR1) and aldose reductase (ALR2) enzymes. In silico molecular docking, the study was also performed to further study the putative binding of active compounds with the target enzyme to lead compound for further drug development.

EXPERIMENTAL

All the reagents were commercially available (Sigma Aldrich) and used without further purification. Melting points were determined by electrothermal 9100 apparatus. 1H and 13C NMR spectra were measured on a Bruker 300 spectrometer operating at 300 and 75 MHz, respectively, using CDCl3 as a solvent and TMS as the internal standard. Elemental analysis was carried out on a PerkinElmer 2400 II CHNS/O analyzer.

1-Phenylthiourea 2, 2-(phenylamino)thiazol-4(5H)-one 3, 2-(prop-2-yn-1-yloxy)benzaldehyde 5, (E)-2-(phenylamino)-5-(2-(prop-2-yn-1-yloxy)benzylidene)thiazol-4(5H)-one 6, and substituted benzyl azides 8a–8c were prepared according to the reported protocols [16].

1-Phenylthiourea (2). Yield 85%, white solid, mp 153–154°C. IR spectrum, ν, cm–1: 3480, 2921, 1632, 1270, 1220. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm: 7.07 m (2H, ArH), 7.20 m (1H, ArH), 7.41–7.44 m (2H, ArH), 8.02 s (2H, NH2), 10.03 s (1H, NH). 13C NMR spectrum (75 MHz, CDCl3), δC, ppm: 116.08, 122.22, 123.41, 128.78, 137.31. Found, %: C 55.50; H 5.13, N 18.42. C7H8N2S. Calculated, %: C 55.24; H 5.30; N 18.40.

2-(Phenylamino)thiazol-4(5H)-one (3). Yield 91%, beige crystals, mp 197–198°C. IR spectrum, ν, cm–1: 1190, 1640, 2844, 3008, 3336. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm: 4.98 s (2H, CH2), 7.18 d (2H, ArH), 7.64 d (2H, ArH), 12.54 s (1H, NH). 13C NMR spectrum (75 MHz, CDCl3), δC, ppm: 75.03, 135.71, 149.20, 150.31, 160.44, 165.56, 180.01. Found, %: C 56.49; H 4.33, N 14.42. C9H8N2OS. Calculated, %: C 56.23; H 4.19; N 14.57.

2-(Prop-2-yn-1-yloxy)benzaldehyde (5). Yield 93%, white solid, mp 72–74°C; IR spectrum, ν, cm–1: 1135, 1201, 1622, 2933, 3471. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm: 2.90 s (1H), 4.96 d (2H, CH2), 7.15 d (2H, ArH), 7.16 d (2H, ArH), 7.21 d (2H, ArH), 10.5 d (1H, CHO). 13C NMR spectrum (75 MHz, CDCl3), δC, ppm: 70.31, 90.22, 101.45, 120.32, 121.76, 123.34, 124.56, 124.87, 144.65, 171.67. Found, %: C 75.20; H 4.93. C10H8O2. Calculated, %: C 74.99; H 5.03.

(Z)-2-(Phenylamino)-5-(2-(prop-2-yn-1-yloxy)benzylidene)thiazol-4(5H)-one (6). Yield 97%, cream solid, mp 201–202°C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm: 3.77 s (1H, CH), 4.61 s (1H, OCH2), 4.69 s (1H, OCH2), 7.07 m (2H, ArH), 7.16 d (2H, ArH), 7.22 m (1H, ArH), 7.29–7.61 m (4H, ArH), 7.83 s (1H, CH), 12.19 s (1H, NH). 13C NMR spectrum (75 MHz, CDCl3), δC, ppm: 57.31, 76.18, 81.13, 117.75, 121.04, 121.74, 123.35, 125.07, 125.91, 128.41, 128.62, 129.01, 129.64, 129.91, 136.44, 143.50, 157.43, 160.33, 164.21. Found, %: C 68.13; H 4.33, N 8.42. C19H14N2O2S. Calculated, %: C 68.25; H 4.22; N 8.38.

General procedure for synthesis of thiazolidinone-triazole derivatives 9a–9c. 1.6 mmol of (Z)-2-(phenylamino)-5-[2-(prop-2-yn-1-yloxy)benzylidene]thiazol-4(5H)-one 6 and 1.5 mmol of benzyl azide 8a–8c were added to a custom-made mortar having pestle hanged with the cap. The cap-bearing pestle was inserted with a perfluoroelastomer O-ring and tightly screwed. The closed mortar and pestle were placed on a shaker for 2 h, so that pestle could move and supply the milling function. The resulting mixture was collected using ethyl acetate. The organic layer was removed under reduced pressure to give the following thiazolidinone-triazole derivatives 9a–c in 73 to 80% yield.

(Z)-5-(2-{[1-(2-Bromobenzyl)-1H-1,2,3-triazol-4-yl]methoxy}benzylidene)-2-(phenylamino)thiazol-4(5H)-one (9a). Yield 80%, yellow solid, mp 209.4–211.2°C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm: 5.25– 5.30 m (2H, CH2), 5.62 s (2H, OCH2), 7.03 m (2H, ArH), 7.15–7.51 m (9H, ArH), 7.58 d (1H, ArH, J = 5.0 Hz,), 7.77 d (1H, J = 6.2 Hz), 7.90 s (1H, H), 8.31–8.35 m (1H, H), 11.54–12.35 m (1H, NH). 13C NMR spectrum (75 MHz, CDCl3), δC, ppm: 52.58, 62.13, 113.06, 113.68, 116.17, 121.90, 125.54, 129.69, 130.64, 132.17, 135.84, 137.14, 143.17, 158.05, 172.05. Found, %: C 56.96; H 3.93; N 12.62. C26H20ClN5O2S. Calculated, %: C 57.15; H 3.69; N 12.82.

(Z)-5-(2-{[1-(3-Chlorobenzyl)-1H-1,2,3-triazol-4-yl]methoxy}benzylidene)-2-(phenylamino)thiazol-4(5H)-one (9b). Yield 78%, creamy solid, mp 210–222°C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm: 5.20 s (2H, CH2), 5.64 s (2H, OCH2), 7.07 m (3H, ArH), 7.20 m (1H, ArH), 7.32–7.39 m (7H, ArH), 7.53 d (1H, ArH, J = 6.4 Hz), 7.79 s (1H, H), 8.32 s (1H, H), 12.10 m (1H, NH). 13C NMR spectrum (75 MHz, CDCl3), δC, ppm: 57.44, 62.45, 116.09, 116.15, 120.98, 121.81, 121.89, 125.53, 127.02, 127.49, 129.64, 129.75, 129.96, 130.82, 131.94, 138.94, 158.80, 171.13. Found, %: C 62.47; H 4.19; N 13.72. C26H20ClN5O2S. Calculated, %: C 62.21; H 4.02; N 13.95.

(Z)-5-(2-{[1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl]methoxy}benzylidene)-2-(phenylamino)thiazol-4(5H)-one (9c). Yield 85%, creamy solid, mp 246–248°C. 1H NMR spectrum (300 MHz, CDCl3), δ, ppm: 5.27 s (2H, CH2), 5.74 s (2H, OCH2), 7.08 s (3H, ArH), 7.27 d (2H, ArH, J = 5.0 Hz), 7.33 m (1H, ArH), 7.43 s (3H, ArH), 7.58 d (2H, ArH, J = 4.0 Hz), 7.86 s (2H, ArH), 8.33 s (1H, H), 12.56 s (1H, NH). 13C NMR spectrum (75 MHz, CDCl3), δC, ppm: 51.48, 62.47, 113.75, 121.03, 121.81, 125.26, 125.66, 128.35, 128.74, 129.27, 129.48, 130.35, 131.48, 131.77, 132.24, 137.64, 143.61, 157.47, 171.07. Found, %: C 62.48; H 3.97; N 14.83. C26H20ClN5O2S. Calculated, %: C 62.21; H 4.02; N 13.95.

Biological activity. The analytical grade chemicals are used. An ELISA microplate reader (EL×800 BioTek Instruments, Inc., VT, USA) was used for the sample analysis in the at 340 nm UV range. Gilson micropipettes were used for the sample loading. Substrates used in enzymatic activity were D-, L-glyceraldehyde (ALR2), and sodium D-glucuronate (ALR1). Sulindac and valproic acid were standard inhibitors for ALR2 and ALR1, respectively. NADPH (nicotinamide adenine dinucleotide phosphate, Sigma-Aldrich) was used as a cofactor.

ALR1 and ALR2 inhibitory activity. Both the reductases’ enzymes ALR2 and ALR1 were separated and refined according to the reported methods [17, 18]. The inhibitory activity of the synthesized compounds 9a–9c was figured out by developing an earlier protocol with minor modifications [19, 20].

In each well, 20 µL of synthesized compound (1 mM) was mixed with 20 µL buffer (100 mM potassium dihydrogen phosphate pH 6.2) 70 µL enzyme either ALR1 (2.19 μg/well) or ALR2 (1.85 μg/well). The mixture was incubated at 37°C for 10 min followed by adding 40 µL substrate (50 mM) and 50 µL NADPH (0.5 mM) as a cofactor. Valproic acid (for ALR1) and Sulindac (for ALR2) were used as positive control. The optical density was measured at 340 nm after 30 min of incubation. IC50 values were calculated by graph pad prism® software, and the following formula calculated percentage inhibition:

$${\rm{Inhibition}} = 100 - {{{\rm{Absorbance}}\;{\rm{test}}\;{\rm{well}}} \over {{\rm{Absorbance}}\;{\rm{test}}\;{\rm{control}}}} \times 100.$$

Molecular docking. Molecular docking studies were performed to explain the binding mode and interactions of the binding site. Autodock vina software was used [21]. The crystal structures of human ALR2 (1US0 at 0.66 Å) and porcine ALR1 (3FX4 at 1.99 Å) were downloaded from the Protein Data Bank [22].

The binding sites of ALR2 and ALR1 were used as the docking site. The 2D chemical structures of ligands were sketched using Chem Draw Professional 13.0 to draw the 2D structure of ligands, and Chem 3D 13.0 was used for energy minimization and 3D structure.

Pharmacokinetic properties. ADME (Distribution, Excretion, Metabolism, and Absorption) properties of synthesized compounds 9a–9c were calculated using Swiss ADME tools [23, 24].