Synthesis, in-vivo anti-diabetic & anticancer activities and molecular modelling studies of tetrahydrobenzo[d]thiazole tethered nicotinohydrazide derivatives

2.1 Chemistry methods

Melting points were determined using the Shital-digital programmable melting point apparatus. The IR spectra were recorded on a VERTEX 70 Brucker by using KBr. A Bruker spectrometer 400 and 100 MHz was employed for recording ¹H NMR and ¹³C NMR spectra respectively and CDCl₃ and DMSO‑d₆ were used as solvent and TMS as an internal standard. Mass spectra were recorded on an Agilent-LCMS instrument.

Synthesis of 2,2,5,5-tetramethyl cyclohexane-1,3-dione (2): To a solution of 5,5-dimethyl cyclohexane-1,3-dione 1 (300 g, 2142 mmol) in acetonitrile (2.4 L) solvent and added K₂CO₃ (888.0 g, 6428.5 mmol) at 0 °C and stirred 20 min. Then, to the reaction mass added methyl iodide (760 g, 5357.14 mmol) and heated at 50 °C for l0 h. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mixture was concentrated, diluted with water, and extracted into ethyl acetate. The combined organic layer was back washed with water, brine solution, dried over Na₂SO_4, and concentrated below 40 °C under reduced pressure. The crude product was treated with n-hexane and the solid thus precipitated was filtrated to give the title compound as a white solid 2 (180.0 g, yield: 50%).

Synthesis of 3-hydroxy-2, 2, 5, 5-tetramethyl cyclohexanone (3): To a solution of 2 (200 g, 1190.4 mmol) in MeOH (2 L) at room temperature, cooled to 0 °C followed by addition of NaBH₄ (16.6 g, 440.4 mmol. 0.3 eq). The reaction mixture was stirred under nitrogen for 5 h at 0–10 °C. The reaction progress was monitored by TLC. After completion of the reaction, the mixture was quenched with saturated NH₄Cl solution. Then, the solvent was evaporated under reduced pressure and the residue was extracted with EtOAc. The combined organic layer was washed with water, brine solution, dried over Na₂SO₄ and concentrated under vacuum. The crude product obtained was purified by silica column chromatography to give compound 3 as a white solid (l2l g. yield: 60%).

Synthesis of 2,2,5,5-tetramethyl cyclohex-3-enone (4): The solution of 3 (95 g, 558.82 mmol) in pyridine (300 mL) at room temperature, was cooled to 0 °C followed by addition of POCl₃ (427.5 g, 2794.11 mmol) and stirred at 100 °C for 3 h. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mixture was cooled to room temperature and poured into crushed ice (highly exothermic), and diluted with diethyl ether. The separated ether layer was neutralized with l N HCI and washed with water, brine solution. The ether layer was dried over Na₂SO₄ and concentrated below 30 °C in a vacuum. The crude product was purified by silica column chromatography using 100% hexane as eluent to give compound 4 as a light green liquid (37 g, 42%).

Synthesis of 2,2,5,5-tetramethyl cyclohexanone (5): The solution of 4 (35 g, 30.263 mmol) was taken in ethyl acetate (250 mL) and methanol (250 mL) solvent mixture in Parr shaker vessel. The above solution was purged with nitrogen for l0 min. Prior to the addition 10% Pd/C (10 g) to the reaction mixture was kept under hydrogen atmosphere (60 psi pressure) for 16 h. The reaction progress was monitored by TLC. After completion of the reaction, the reaction mixture was filtered through celite pad and dried over anhydrous Na₂SO₄ and concentrated below 30 °C in a vacuum to give a green liquid 5. The crude product was used in the next step without further purification (27 g. yield-76%).

Synthesis of 4,4,7,7-tetramethyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine (6): To a solution of 5 (33 g, 213.9 mmol) in ethanol followed by taken into a sealed tube and added iodine (65 g, 256.7 mmol). Then, the reaction mixture was maintained at 60 °C for 2 h. The reaction mixture was cooled to room temperature. Then, added thiourea (40 g, 525.97 mmol) and maintained at 110 °C for 12 h. The reaction mixture was cooled to room temperature and poured into a cold aqueous NaOH solution and extracted with ethyl acetate. The organic layer was washed with water and brine and dried over anhydrous Na₂SO₄ and concentrated under a vacuum to get the crude product. The crude product was purified by silica column chromatography (EtOAc/ Pet ether) to give the compound 6 as a yellow solid (15.5 g, yield: 34.5%).

Synthesis of ethyl 6-((4,4,7,7-tetramethyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)amino)nicotinate (7): To a solution of 6 (10 g, 47.61 mmol) in toluene (100 mL) and added ethyl 6-chloronicotinate (8.8 g, 47.619 mmol), Na₂CO₃ (10.6 g, 99.9 mmol) and Xantphos (1 0.4 g, 2.38 mmol) under argon over 10 min. Pd₂(dba)₃ (0.87 g, 0.95 mmol) was added to the reaction mixture and stirred at 110 °C for 8 h. 'I'he reaction mixture was cooled to room temperature and concentrated under vacuum and the residue was poured into water and extracted with dichloromethane. The combined extracts were washed with water and brine solution. The organic layer was dried over anhydrous Na₂SO₄ and concentrated under a vacuum. The obtained solid was purified with diethyl ether/hexane to give the title compound as a yellow solid (10 g, yield: 50–53 %). M.P: 220–224 °C.

Synthesis of ethyl 6-((cyclopropylmethyl)(4,4,7,7-tetramethyl-4,5,6,7-tetrahydrobenzo[d]thiazol −2-yl) amino)nicotinate (8): To a solution of 7 (10 g, 27.85 mmol) in DMF (100 mL) followed by addition of cesium carbonate (27.2 g, 83.56 mmol) and methyl cyclopropyl bromide (5.8 g, 43.17 mmol) at room temperature. The reaction mixture was maintained at 100 °C for 3 h. The reaction mixture was cooled to room temperature, poured into water, and extracted with ethyl acetate. The organic layer was washed with water and brine solution, dried over anhydrous Na₂SO_4, and concentrated under vacuum to get a crude compound. The crude product was purified by silica column chromatography to give compound 8 as a light yellow liquid (10.0 g, yield: 87%).

Procedure for the synthesis of 9 & 10: Charged ethyl ester of carboxylic acid 7 & 8 (0.01 mol.) in ethanol (100 mL) and added hydrazine monohydrate (0.011 mol). Then refluxed the reaction mixture for 2 h. After that, the solution was cooled to room temperature and the precipitate formed was then filtered off, dried, and recrystallized from ethanol. (yield: 65–80%).

General procedure for the synthesis of series-1 (11a-11o) & Series 2 (12a-12o): 0.01 mol of previously obtained carboxylic acid hydrazides (9&10)were dissolved in 10–20 mL of ethanol and then 0.011 mol of aldehyde was added. The mixture was heated under reflux for 3 h. After that, the solution was allowed to cool at room temperature and filtered off and recrystallized from ethanol. (yield: 65–70%).

3.1 Experimental animals

Swiss albino mice weighing 25–30 g were used for this study. Forty-eight animals were grouped into eight groups with series 11 derivatives while fifty-four mice were grouped into nine groups with series 12 derivatives and housed for one week to acclimatize to laboratory conditions before starting the experiment. Animals were fed with a standard diet and water ad libitum, but 12 h prior to inducing diabetes to the mice; the animals were deprived of food but not water. All the experimental procedures were carried out in accordance with CPCSEA guidelines (IAEC approved number is I/IAEC/AGI/027/2017 SM). The Institutional Animal Ethics Committee approved the experimental protocol.

3.2 Induction of diabetes

Overnight-fasted Swiss albino mice were injected with alloxan (150 mg/kg) in saline buffer intraperitoneally (Sekiou et al., 2021). To prevent hypoglycemia animals were fed with 20% (w/v) glucose solution. After 72 h fasting blood glucose levels (FBG) were determined by using ONETOUCH select simple (J&J) Glucometer strips on Day one, day seven, and day 21, and day 28. Glibenclamide was used as the standard 10 mg/kg BW.

3.3 Group design

Mice with < 250 mg/dl were excluded from the experiment and the rest were divided into eight groups with series 11 derivatives and into nine groups with series twelve derivatives. In series eleven, Group 1 (control) was administered with distilled water orally. Group II was treated as diabetic control (Alloxan 150 mg/kg, i.p), Group III,IV,V,VI,VII were treated with 11d, 11f, 11g, 11j, and 11o compounds respectively and group VIII was treated with glibenclamide 10 mg/kg. In the same manner for series twelve compounds, Group I (control) was administered with distilled water orally. Group II was treated as diabetic control (Alloxan 150 mg/kg, i.p), Group III,IV,V,VI,VII,VIII were treated with 12g, 12h, 12i, 12j, 12n, 12o compounds respectively, and group IX was treated with glibenclamide 10 mg/kg.

3.4 Evaluation of cytotoxicity by MTT assay

In 96 well microtiter plates, MCF7 cells were seeded at 1x105 cells/mL in minimum essential medium with fetal bovine serum. For adhesion, the cells were cultured overnight. Drug concentrations were added in triplicates in repeated three-fold dilutions and incubated for 48 h at 5 percent CO₂ at 37 °C (see list of drugs and corresponding cell line used in respective results Table). After that, the cells were given 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) treatment (Sigma Chemical Co., St. Louis, MO). All of the medium, including the MTT solution (5 mg/mL), was evacuated from the wells four hours later. The absorbance was measured at 570 nm using a 96 well microplate reader after the residual formazan crystals were dissolved in DMSO (SynergyTM HT, Bio-Tek Instruments, Inc.). As a negative control, untreated cells were used to calculate the cytotoxicity index. The background-corrected absorbance was used to compute the percentage of cytotoxicity (Datta et al., 2014). The IC₅₀ was calculated using the dose–response curve as a guide. By visualizing duplicate data points across a concentration range and computing values using the PRISM program's regression analysis, the drug concentration that reduced cell viability by 50% (IC₅₀) was determined. Based on the reduction of the yellow-colored water-soluble tetrazolium dye MTT to formazan crystals, the MTT assay is a colorimetric assay used to determine cell growth and cytotoxicity. MTT is reduced to insoluble formazan crystals by mitochondrial lactate dehydrogenase, which when dissolved in a suitable solvent exhibits a purple hue whose intensity is proportional to the number of viable cells and can be quantified spectrophotometrically at 570 nm (Alley et al., 1988; Mosmann, 1983). A linear regression equation, Y = Mx + C, was used to calculate the IC₅₀ value. M, and C values were taken from the viability graph (Ganesh et al., 2015), with Y = 50.

Table 1 Yield and Melting point details of compounds obtained in 11(a-o)series.

Entry	Aldehydes	Product	M.P (°C)	Yield %
1		11a	240–244	77
2		11b	254–258	68
3		11c	309–312	67
4		11d	262–264	70
5		11e	324–326	69
6		11f	320–322	65
7		11g	333–335	69
8		11h	229–232	70
9		11i	128–132	70
10		11j	280–284	66
11		11k	245–249	67
12		11l	270–274	61
13		11m	306–310	65
14		11n	268–272	68
15		11o	271–274	66

3.5 Molecular docking study

Molecular docking studies of the synthesized molecules 11f and 11 g with human PPAR-γ protein complexed with RXR alpha Nuclear Receptor (PDB ID: 3DZY) were carried out using AutoDock Tools (Ghanbari-Ardestani et al., 2019). Chem3D Ultra 16.0 software was used for the generation of ligand structures. Further, the ligand energies were minimized by using MOPAC (semi-empirical quantum mechanics) tool. The PDB structure of hPPAR-γ protein-RXR alpha nuclear receptor complex (PDB ID: 3DZY) with co-crystal ligand Rosiglitazone was downloaded and imported to the workspace. The protein was processed, and the grid box was generated in a rectangular shape with the value of X: 2.97; Y: 32.82; Z: 11.6 coordination. PyMol visualization tool was employed for the analysis of output files generated from docking. Later, the docking results were validated with the docked co-crystal ligand Rosiglitazone BRL(Expand this for the first time). Each pose was examined manually, and the best pose was taken for per docked ligand. The hydrogen and hydrophobic interactions of the docked ligands with target protein were studied using LIGPLOT (Wallace et al., 1995).

4.1 Chemistry

The synthesis of title hybrid compounds 11 and 12 were designed and synthesized by adopting an easy synthetic route as established in Schemes 1&2. In brief, the commercially accessible starting compound 5,5-dimethylcyclohexane-1,3-dione (1) could react with methyl iodide in the presence of potassium carbonate as a catalyst in acetonitrile as solvent at 50 °C for 10 h to form 2,2,5,5-tetramethylcyclohexane-1,3-dione (2). Further, compound 2 has undergone selective reduction with sodium borohydride in methanol at room temperature for 5 h to give 3-hydroxy-2,2,5,5-tetramethylcyclohexanone (3). The resulting compound 3 was treated with POCl₃ in pyridine at 100 °C for chlorination followed by dehydrochlorination to afford 2,2,5,5-tetramethylcyclohex-3-enone (4). Later, compound 4 has undergone hydrogenation with Pd/C and H₂ at 60 psi at room temperature for 16 h to get 2,2,5,5-tetramethylcyclohexanone (5). The second part of the synthesis deals with the development of structural fragments required for the generation of diversity to the core scaffold. This step corresponds to the synthesis of 4,4,7,7-tetramethyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-amine (6) with good yield by reaction of thiourea with 5 in the presence of I₂ in ethanol at 110 °C for 16 h with help of a sealed tube. Further, 6 was reacted with ethyl 6-chloronicotinate in the presence of Na₂CO₃, Xantphos and Pd₂(dba)₃ under argon at 110 °C for 16 h to afford ethyl 6-((4,4,7,7-tetramethyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)amino)nicotinate (7) key intermediate.

Compound 7 was reacted with (bromomethyl)cyclopropane in the presence of Cs₂CO₃ as base in DMF as solvent for 3 h at 110 °C to form ethyl 6-((cyclopropylmethyl)(4,4,7,7-tetramethyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)amino)nicotinate (8). Compound 7 and 8 were condensed with hydrazine hydrate in refluxing ethanol for 1 h to form 6-((4,4,7,7-tetramethyl-4,5,6,7-tetrahydro-benzo[d]thiazol-2-yl)amino)nicotinohydrazide (9) and 6-((cyclopropylmethyl)(4,4,7,7-tetramethyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)amino)nicotinohydrazide (10) respectively. The various substituted benzaldehydes were further allowed to treat with the previously prepared 9 and 10 in ethanol at 60 °C for 2 h to afford desired (Z)-N'-benzylidene-6-((4,4,7,7-tetramethyl-4,5,6,7-tetrahydrobenzo[d]thiazol-2-yl)amino)nicotinohydrazide (11) and (Z)-N'-benzylidene-6-((cyclopropylmethyl) (4,4,7,7–tetramethyl-4,5,6,7-tetrahydrobenzo [d] thiazol-2-yl) amino)nicotinohydrazide (12) derivatives respectively in an efficient manner with good yields. The details of the analogs of 11(a-o) series were included in Table 1 and 12(a-o) are given in Table 2.

Table 2 Yield and Melting point details of compounds obtained in 12(a-o)series.

Entry	Aldehydes	Product	M.P (°C)	Yield %
1		12a	149–151	77
2		12b	186–188	69
3		12c	130–132	68
4		12d	199–203	68
5		12e	132–136	69
6		12f	174–178	69
7		12g	137–141	68
8		12h	190–194	70
9		12i	174–178	74
10		12j	177–180	69
11		12k	222–224	68
12		12l	228–232	65
13		12m	188–192	65
14		12n	185–189	68
15		12o	214–216	68

In 11a-o series, the compounds were obtained in the yield range 61–77%. The compound 11a with no substitution on the aromatic ring that is the product with benzaldehyde was found to be obtained in the highest yield in the series. Further, followed by 70% yields of compounds 11d with para-fluoro and 11h with m-CF₃ along with para-fluoro substitutions. The compound 11l with para-methoxy substitution was obtained with the lowest yield (61%).

In 12a-o series, the compounds were obtained in the yield range 65–77%. The compound 12a with no substitution on the aromatic ring that is the product with benzaldehyde was found to be again obtained in the highest yield in the series. Further, followed by 74% yields of compounds 12i with para-CF₃ and 11h with m-CF₃ along with para-fluoro substitutions were obtained with 70% yield. The compounds 12l and 12m with para-methoxy substitution and p-methoxy with Ortho-fluoro substitutions respectively were obtained with the lowest yield (65%).

IR, NMR, and mass spectroscopy were used to determine the structure of the produced title compounds. Spectral data were used to describe all of the tetrahydro thiazolyl conjugated nicotinyl hydrazide analogs (11a-11o) and (12a-12o). Compounds (11a–11o) had a significant C=O stretching band at around 1728 cm⁻¹ and a strong –NH stretching band at around 3326 cm⁻¹, according to IR spectral data. IR studies revealed a strong C=O stretching band of about 1739 cm⁻¹ and a strong –NH stretching band at 3256–3544 cm⁻¹ for additional isomers (12a-12o). The presence of certain functional groups in the molecules was also confirmed by the IR spectra. The ¹H NMR values of 11a in the aromatic area are δ_H 7.0–8.9 and two –NH protons region are 11.5–12.0 in the first step. Similarly, 12a proton NMR values range from 7.2 to 8.9, with one NH proton at 10.3. The cyclopropylmethyl group removed one –NH proton. The authors confirmed the substitution reaction of the cyclopropylmethyl group based on this reason.

5.1 In-vivo antidiabetic studies

Effect of Chemically Synthetic Compounds on blood Glucose Levels: Alloxan administration leads to a significant increase in blood glucose levels (295.3 ± 6.40 mg/dL) when compared to normal control mice. Treatment with chemically synthetic compounds for seven days significantly reduced the glucose levels when compared to diabetic mice. Among the synthetic compounds, compounds 11f and 11g showed significant reduction in the blood glucose levels from 214.3 ± 2.92 to 118.8 ± 6.21 mg/dL and 226.8 ± 5.10 to 115.5 ± 3.09 mg/dL, respectively. Whereas the compounds 11d, 11j and 11o showed a moderate decrease in the blood glucose level as compared to standard glibenclamide treated mice (Table 3, Table 4). The antidiabetic action of these molecules was significant and the mechanism needs to be known clearly as the mechanistic studies could be through insulin pathways or through the biochemical alterations in the mice which leads to lower blood glucose levels in the mice. For 28 days, the change in the blood glucose levels was clearly evident and the raised blood glucose levels were normalized with the use of the tested compounds. All the results were compared with the diabetic control to see the change in the parameters.

5.2 In-vitro anti-cancer studies

The results of the MTT test Fig. 1 demonstrated that all the compounds did not inhibit the proliferation of MCF7 cells effectively (Table 5). Compounds 11f, 11h, and 11n showed the potential inhibitory effects based on the IC₅₀ values. In the 12 series the compounds 12f, 12k, and 12n have shown the potential activity. The anticancer activity (IC₅₀) of compound 7 and comparison with the standard drug oxaliplatin has been shown in Fig. 2.

Close

Fig. 2

The apoptosis stages tested with Oxaliplatin (OXP) and COMP-7.

Export to PPT

Close

Fig. 3

Comparison of the dose–response of selected compounds of series1 showing lower IC₅₀ with MCF 7cell lines and response of increasing dose concentration of the compounds of series1 added to the cells in culture.

Export to PPT

MTT measures cell respiration and the amount of formazan produced is proportional to the number of living cells present in the culture. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the drug. IC₅₀ is the concentration of the tested drug able to cause the death of 50% of the cells and can be predictive of the degree of cytotoxic effect. The lower the value, the more cytotoxic is the substance. Fig. 3 shows the comparison of the selected compounds of series 11 showing the lowest IC₅₀ value against MCF7 cancer cell lines. Our results revealed that these selected compounds of series 11f, 11h, 11n, 12f, 12k, 12n are potential anticancer drug properties compared to compound 7 (Ulukaya et al., 2004).

Close

Fig. 4

(A) Binding interactions and best poses for the synthesized compounds against hPPAR-γ protein active site (PDB ID: 3DZY). The best docked pose for active compounds 11f and 11g was represented in the image. The co-crystal ligand Rosiglitazone (BRL) was used as positive control.

Export to PPT

5.3 Molecular docking

The active compounds (11f and 11g) from the preliminary results were studied for in silico binding analysis with human PPAR-γ protein complexed with RXR alpha Nuclear Receptor (Fig. 4). The glitazone-based anti-diabetics which are derivatives of Thiazolidinediones have been widely used as PPAR-γ agonists given their ability to induce activation of the said protein (Staels and Fruchart, 2005). Since the core scaffold of the newly synthesized (thiazole) is like that of thiazolidinediones, a representative drug from that category Rosiglitazone is taken as a reference to the perform docking studies. The analysis of molecular docking results revealed that all the compounds are bound to the active site of proteins by hydrogen bonds and hydrophobic bonds. Ten different conformations per each ligand were generated and examined. The docked ligands showed good binding energies and RMSD scores with the target protein (Table 6). It was determined that the compound 11g was the best docked ligand as compared to other ligands. The binding energies for the compounds 11f and 11g in the protein active site were −8.4 and −8.2 kcal/mol, respectively. The co-crystal ligand rosiglitazone (BRL) showed −6.1 kcal/mol. Both the compounds formed hydrogen bonds with the target protein. PPAR gamma is the main target of thiazolidinediones used in diabetes mellitus characterized by insulin resistance. Thiazolidinediones, acting via PPARγ, influence free fatty acid flux and thus reduce insulin resistance and blood glucose levels [28/33]. The binding interactions among protein and ligands were majorly influenced by hydrogen bonds and hydrophobic interactions (Fig. 4). The binding energies, RMSD score and the amino acids of the target protein interacted with the ligands were shown in Table 6.