INTRODUCTION:

G protein-coupled receptor 119 (GPR119) has arisen as a prospective target for diabetes mellitus (DM) treatment owing to its involvement in regulating glucose levels. When activated, GPR119 promotes insulin secretion and boosts the release of incretin hormones, improving blood sugar control. Importantly, this mechanism does not cause hypoglycaemia. As a result, the development of effective and selective GPR119 agonists has become a key focus in diabetes research.¹ The 4-nitro-N-(piperidin-4-yl)benzamide scaffold has shown promise as a potential GPR119 agonist due to its strong receptor-binding affinity and metabolic stability. The design of GPR119 agonists focuses on making structural modifications to enhance receptor interactions, improve bioavailability, and optimize pharmacokinetic properties.^{2, 3} Molecular modeling studies, including docking simulations and pharmacophore modeling, play a crucial role in identifying structural features essential for receptor activation.^{4, 5} Incorporating electron-withdrawing groups like the nitro (-NO₂) group enhances binding affinity by facilitating strong interactions with crucial residues within the receptor's binding site. Meanwhile, the piperidinyl unit aids in maintaining conformational stability and increases lipophilicity, thereby improving the compound's drug-like characteristics.⁶ GPR119 was recognized as a probable therapeutic target for type 2 diabetes in the early 2000s. Early synthetic agonists, including AR231453, exhibited notable glucose-lowering effects in preclinical studies. Later, clinical trials with compounds such as MBX-2982 and GSK1292263 highlighted their ability to enhance glucose metabolism.⁷ BMS-903452, a potent GPR119 agonist with a pyridone core connected to a piperidine moiety, demonstrated strong glucose-lowering effects in rodent diabetes models. This study emphasized the crucial role of the 4-nitro-N-(piperidin-4-yl)benzamide scaffold in enhancing receptor activation and optimizing pharmacokinetic properties.⁸ Development of aryl N-methoxyamide derivatives with potent GPR119 agonistic activities, demonstrating their potential in lowering glucose excursion in vivo.⁹ Computational studies were conducted to design novel GPR119 agonists featuring the N-(piperidin-4-yl)benzamide moiety. Researchers employed 3D-QSAR modeling and pharmacophore analysis to identify crucial molecular features for receptor activation. Virtual screening and molecular docking further revealed promising candidates with strong binding affinity.¹⁰ A series of 7-azaspiro[3.5]nonane derivatives incorporating the N-(piperidin-4-yl)benzamide framework were synthesized and assessed for GPR119 agonistic activity.¹¹ In 2021, researchers identified MK-8282, a potent GPR119 agonist with a fluoro-pyrimidine and bridged piperidine structure. This compound demonstrated enhanced glucose tolerance across various animal models and showed minimal off-target effects, making it a promising oral treatment for type 2 diabetes.³ A 2021 study investigated the development and biological assessment of N-(piperidin-4-yl)-N-(trifluoromethyl) pyrimidin-4-amine derivatives as GPR119 agonists.¹² In one research 4-nitro-N-(piperidin-4-yl)benzamide and its derivatives have been explored as sigma receptor ligands, suggesting possible applications in neurological disorders. Sigma receptor modulation has shown promise in enhancing neuroprotection and cognitive function.¹³ Benzamide scaffolds have demonstrated potential in targeting enzymes such as α-glucosidase and prolyl endopeptidase, which are linked to metabolic and neurological disorders.¹⁴ These studies highlight the effective combination of computational modeling, chemical synthesis, and biological assessment in developing 4-nitro-N-(piperidin-4-yl)benzamide-based GPR119 agonists.

This study focuses on designing, synthesizing, and characterizing novel 4-nitro-N-(piperidin-4-yl)benzamide derivatives as potential GPR119 agonists. Including nitro groups and piperidinyl moieties is anticipated to improve receptor binding, metabolic stability, and pharmacokinetics. By integrating molecular modeling, synthetic refinement, and characterization, this research aims to design and develop highly effective and specific GPR119 agonists for diabetes treatment.

MATERIALS AND METHODS:

Tert-butyl 4-aminopiperidine-1-carboxylate, along with various benzoic acid and nicotinic acid/chlorides derivatives, and other chemicals were procured from BLD Pharma, Hyderabad, India, and S.K. Traders, Indore. The synthesized compounds' reaction progress and purity were monitored frequently using ALUGRAM Xtra SIL G/UV₂₅₄ pre-coated thin-layer chromatography (TLC) plates. The spots for various chemical compounds were observed using an iodine chamber or a UV lamp. The synthesized intermediates and final compounds' infrared (IR) spectra were analyzed using a BRUKER FT-IR spectrometer at SGSITS, Indore. ¹H and ¹³C NMR spectra were recorded on an AVANCE NEO Ascend 500 Bruker BioSpin International AG spectrometer, using DMSO/CDCl₃ as solvents at the Indian Institute of Technology, Indore. All ¹H NMR chemical shift measurements are given in parts per million (ppm) on the δ scale, using either the residual CDCl₃ signal (7.26 ppm) or the central peak of DMSO-d₆ (2.50 ppm) as an internal reference. Liquid Chromatography–Mass Spectrometry (LC-MS) analysis was executed on an LCMS Single Quad, Agilent mass spectrometer at IISER Bhopal. Melting points (uncorrected) were assessed using an ANALAB (µThermoCal50) melting point apparatus at SGSITS, Indore.

The synthesis of 4-nitro-N-(piperidin-4-yl)benzamide derivatives typically follows a two-step strategy. Initially, 4-nitrobenzoic acid undergoes amidation with tert-butyl 4-aminopiperidine-1-carboxylate, resulting in a protected intermediate. This intermediate is then subjected to deprotection using trifluoroacetic acid (TFA) to yield the desired benzamide core. Further derivatization is achieved through coupling reactions with various substituted benzoic and nicotinic acids or their acyl chlorides. These reactions utilize standard peptide coupling reagents such as EDC, and HOBT/HATU, with TEA in DMF, facilitating efficient amide bond formation. Structural modifications at different positions enable optimization of the pharmacological properties of the synthesized analogs. Founded on pharmacophore design, the reaction scheme for synthesizing 4-nitro-N-(piperidin-4-yl)benzamide derivatives¹⁵ is shown in Scheme 1.

Synthesis of tert-butyl 4-(4-nitrobenzamido) piperidine-1-carboxylate, intermediate-1 (S5I1)

tert-Butyl 4-aminopiperidine-1-carboxylate (2) (01gm, 5.98 mmol) was solubilized in dimethylformamide (DMF) (10 mL) in a reaction flask. HATU (4.55 gm, 11.96 mmol) was incorporated into the reaction mixture with stirring, followed by triethylamine (TEA) (4.9 mL, 35.90 mmol) which was introduced dropwise by maintaining the cool temperature with the help of an ice bath. 4-nitrobenzoic acid (1) (01 gm, 5.98 mmol) was introduced with continuous stirring at atmospheric temperature for 12 hours. The reaction's advancement was tracked using TLC at frequent intervals. Upon the accomplishment of the reaction, the reaction mixture was transferred into ice-cold water with stirring to quench the reaction. The yellowish-brown sticky precipitate was found, which was strained and rinsed, and yielded tert-butyl 4-(4-nitrobenzamido)piperidine-1-carboxylate (S5I1).

Appearance: Solid Yellowish brown sticky, mp 155-157℃, Yield 1.75 gm (52%), molecular formula: C₁₇H₂₃N₃O₅

IR (KBr, in cm-1): 3352.53 (Amide N-H) (str.), 3076.05 (Ar. C-H), 2942.29 (Ali. C-H), 2979.39 2852.04 (Boc C-H), 1690.03 (Boc C=O), 1644.89 (Amide C=O) (str.), 1601.06 (N=O) (str.); ¹H NMR (400 MHz, DMSO) δ 8.59 (d, J = 7.7 Hz, 1H), 8.28 (d, J = 8.3 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 4.02 – 3.87 (m, 3H), 2.87 (s, 2H), 2.82 (s, 2H), 1.77 (dd, J = 12.5, 4.5 Hz, 2H), 1.46 – 1.32 (m, 9H); ¹³C NMR (101 MHz, DMSO) δ 163.98, 153.96, 148.98, 140.25, 128.83, 123.48, 78.86, 78.71, 46.87, 40.15, 39.94, 39.73, 39.64, 39.52, 39.31, 39.10, 38.89, 31.19, 28.11, 28.08; LC/MS (ESI) m/z: 350.21.

Synthesis of 4-nitro-N-(piperidin-4-yl)benzamide, intermediate-2 (S5I2)

Intermediate-1 (S5I1) (1.75 gm, 5.01 mmol) was solubilized in dichloromethane (15 mL), and trifluoroacetic acid (2.30 mL, 30.08 mmol) was slowly introduced with continuous mixing at cool temperature. Stirring was maintained at environmental temperature for 08 hours, with simultaneous assessment of the advancement of the reaction by thin-layer chromatography at regular intervals. Once the reaction was complete, the blend was transferred slowly to a beaker carrying ice-cold water with stirring for quenching. On quenching no precipitate was obtained and extraction was performed with the mixture of DCM:IPA (8.5:1.5) to yield 4-nitro-N-(piperidin-4-yl)benzamide, intermediate-2 (S5I2) after evaporation of mixture of organic solvents. Upon TLC monitoring, it was observed that some fraction of the compound (S5I2) was present in the aqueous phase and was kept for further use.

Appearance: Solid yellow white crystalline powder, mp 205-207℃, Yield 1.07 gm (85%), molecular formula: C₁₂H₁₅N₃O₃

IR (KBr, in cm-1): 3352.97 (Amide N-H) (str.), 3076.05 (Ar. C-H), 2955.43 (Ali. C-H), 1634.46 (Amide C=O) (str.), 1599.61 (N=O) (str.); ¹H NMR (400 MHz, DMSO) δ 8.58 (d, J = 7.8 Hz, 1H), 8.27 (d, J = 8.5 Hz, 2H), 8.04 (d, J = 8.4 Hz, 2H), 3.80 (tdt, J = 11.8, 8.2, 4.3 Hz, 1H), 2.94 (s, 1H), 2.93 – 2.84 (m, 1H), 2.52 – 2.42 (m, 3H), 1.76 – 1.67 (m, 2H), 1.39 (qd, J = 12.0, 4.1 Hz, 2H); ¹³C NMR (101 MHz, DMSO) δ 163.80, 148.90, 140.50, 128.84, 123.42, 47.89, 45.29, 40.15, 39.94, 39.73, 39.52, 39.31, 39.10, 38.90, 32.84; LC/MS (ESI) m/z: 250.18.

Synthesis of 4-nitro-N-(piperidin-4-yl)benzamide derivatives (S5F1-S5F7)

N-(1-acetylpiperidin-4-yl)-4-nitrobenzamide (S5F1)

Acetyl chloride (0.06 mL, 0.96 mmol) was introduced gradually with stirring into the aqueous phase of the compound (S5I2) (200 mg, 0.80 mmol). The final reaction blend was continuously stirred at ambient temperature for 12 hours. Upon the reaction's accomplishment, the final derivative was extracted using a 10 ml mixture of DCM: IPA (8.5:1.5), 03 times. The organic friction was treated with anhydrous sodium sulfate, filtered, and concentrated under a vacuum to give S5F1.

Appearance: Solid white powder, mp 198-200℃, Yield 101 mg (43%), molecular formula: C₁₄H₁₇N₃O₄

IR (KBr, in cm-1): 3353.06 (Amide N-H) (str.), 3075.96 (Ar. C-H), 2955.28 (Ali. C-H), 1634.26 (Amide C=O) (str.), 1599.30 (N=O) (str.); ¹H NMR (400 MHz, DMSO) δ 8.60 (d, J = 7.8 Hz, 1H), 8.28 (d, J = 8.4 Hz, 2H), 8.05 (d, J = 8.4 Hz, 2H), 3.81 (ddt, J = 16.1, 11.6, 5.9 Hz, 1H), 3.48 (s, 2H), 2.94 (d, J = 12.3 Hz, 2H), 2.50 (d, J = 11.1 Hz, 2H), 1.84 – 1.76 (m, 1H), 1.76 – 1.68 (m, 2H), 1.40 (qd, J = 12.0, 4.1 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 163.81, 148.90, 140.49, 128.85, 123.42, 47.86, 45.27, 40.15, 39.94, 39.88, 39.73, 39.52, 39.31, 39.10, 38.90, 32.80; LC/MS (ESI) m/z: 292.18.

N-(1-benzoylpiperidin-4-yl)-4-nitrobenzamide (S5F2)

Benzoyl chloride (0.11 mL, 0.96 mmol) was introduced gradually with stirring into the aqueous phase of the compound (S5I2) (200 mg, 0.80 mmol). The final reaction blend was stirred continuously at atmospheric temperature for 8 hours, leading to the formation of precipitates. After the reaction, the final compound was filtered, washed, and desiccated to yield S5F2.

Appearance: Solid white yellow powder, mp 190-192℃, Yield 126 mg (44%), molecular formula: C₁₉H₁₉N₃O₄

IR (KBr, in cm-1): 3344.73 (Amide N-H) (str.), 3109.22 (Ar. C-H), 2950.22 (Ali. C-H), 1665.64 (Amide C=O) (str.), 1614.97 (N=O) (str.); ¹H NMR (400 MHz, DMSO) δ 8.64 (d, J = 7.7 Hz, 1H), 8.28 (d, J = 8.4 Hz, 2H), 8.04 (d, J = 8.5 Hz, 2H), 7.51 – 7.40 (m, 3H), 7.39 – 7.31 (m, 2H), 4.42 (s, 1H), 4.07 (tdt, J = 11.5, 8.2, 4.2 Hz, 1H), 3.57 (s, 1H), 3.15 (s, 1H), 2.94 (s, 1H), 1.89 (s, 1H), 1.80 (s, 1H), 1.49 (s, 2H); ¹³C NMR (101 MHz, DMSO) δ 169.11, 164.04, 149.00, 140.20, 136.22, 129.49, 128.83, 128.59, 128.52, 126.61, 123.50, 46.85, 40.15, 39.94, 39.73, 39.52, 39.31, 39.10, 38.89; LC/MS (ESI) m/z: 354.19.

4-nitro-N-(1-(4-nitrobenzoyl)piperidin-4-yl)benzamide (S5F3)

4-Nitrobenzoic acid (134 mg, 0.80 mmol) and intermediate-2 (S5I2) (200 mg, 0.80 mmol) were solubilized in dimethylformamide (DMF) (2 mL). Then incorporation of HATU (609 mg, 1.60 mmol), and triethylamine (TEA) (0.44 mL, 3.20 mmol) was done with stirring at a cool temperature. Stirring was sustained at atmospheric temperature for 12 hours. Once the reaction reached completion, the blend was poured into ice-cold water to terminate the reaction. That leads to the precipitation. The residue was treated with ammonium chloride for purification. The pure residue was filtered using Whatman filter paper, rinsed with water, dried, and desiccated to give S5F3.

Appearance: Solid yellow white powder, mp 118-120℃, Yield 210 mg (65%), molecular formula: C₁₉H₁₈N₄O₆

IR (KBr, in cm-1): 3442.45 (Amide N-H) (str.), 3075.65 (Ar. C-H), 2959.95 (Ali. C-H), 1647.16 (Amide C=O) (str.), 1599.14 (N=O) (str.); ¹H NMR (400 MHz, DMSO) δ 8.66 (d, J = 7.6 Hz, 1H), 8.32 – 8.24 (m, 4H), 8.04 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 4.42 (d, J = 13.1 Hz, 1H), 4.09 (t, J = 9.5 Hz, 1H), 3.05 – 2.94 (m, 1H), 1.92 (s, 2H), 1.79 (d, J = 13.0 Hz, 1H), 1.56 (d, J = 12.2 Hz, 2H), 1.44 (d, J = 11.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO) δ 167.16, 164.12, 149.02, 147.80, 142.51, 140.19, 128.85, 127.99, 123.92, 123.50, 46.75, 40.15, 39.94, 39.73, 39.52, 39.31, 39.10, 38.90; LC/MS (ESI) m/z: 399.14.

4-nitro-N-(1-(4-(trifluoromethyl)benzoyl)piperidin-4-yl)benzamide (S5F4)

4-Trifluoromethylbenzoic acid (76 mg, 0.40 mmol) and intermediate -2 (S5I2) (100 mg, 0.40 mmol) were solubilized in dimethylformamide (DMF) (2 mL). An incorporation of HATU (305 mg, 0.80 mmol), and triethylamine (TEA) (0.22 mL, 1.60 mmol) was done with stirring at cool temperature. Stirring was sustained at atmospheric temperature for 8 hours. After the reaction was complete, the blend was introduced into ice-cold water to halt the reaction. That leads to the precipitation. The precipitate was filtered using Whatman filter paper, rinsed with water, and desiccated to obtain S5F4.

Appearance: Solid white powder, mp 210-212℃, Yield 100 mg (59%), molecular formula: C₂₀H₁₈F₃N₃O₄

IR (KBr, in cm-1): 3339.35 (Amide N-H) (str.), 3107.17 (Ar. C-H), 2956.19 (Ali. C-H), 1661.76 (Amide C=O) (str.), 1618.14 (N=O) (str.); ¹H NMR (400 MHz, DMSO) δ 8.65 (d, J = 7.6 Hz, 1H), 8.28 (d, J = 8.4 Hz, 2H), 8.04 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H), 4.42 (d, J = 13.1 Hz, 1H), 4.12 – 4.04 (m, 1H), 3.50 (s, 1H), 3.47 (s, 1H), 3.18 (s, 1H), 1.92 (s, 1H), 1.79 (d, J = 11.5 Hz, 1H), 1.56 (d, J = 11.4 Hz, 1H), 1.45 (s, 1H); ¹³C NMR (101 MHz, DMSO) δ 167.70, 164.09, 149.01, 140.20, 128.84, 127.46, 125.63, 123.50, 46.77, 40.14, 39.94, 39.73, 39.52, 39.31, 39.10, 38.89; LC/MS (ESI) m/z: 422.17.

N-(1-(4-fluorobenzoyl)piperidin-4-yl)-4-nitrobenzamide (S5F5)

4-Fluorobenzoic acid (140 mg, 1.00 mmol) and intermediate-2 (S5I2) (250 mg, 1.00 mmol) were solubilized in dimethylformamide (DMF) (3 mL). With continuous stirring, the incorporation of HATU (762 mg, 2.00 mmol) was done to activate the carboxylic acid, enhance coupling efficiency, and suppress side reactions. Triethylamine (TEA) (0.55 mL, 4.01 mmol) was introduced as a base to neutralize acidic byproducts. Stirring was first carried out at a low temperature and then at atmospheric temperature for 12 hours. Once the reaction was finalized, the resulting blend was introduced into ice-cold water to stop the reaction. That leads to the precipitation. The precipitate was filtered using Whatman filter paper, rinsed with water, dried, and desiccated to give S5F5.

Appearance: Solid yellowish white powder, mp 148-150℃, Yield 244 mg (65%), molecular formula: C₁₉H₁₈FN₃O₄

IR (KBr, in cm-1): 3426.10 (Amide N-H) (str.), 3089.42 (Ar. C-H), 2925.72 (Ali. C-H), 1649.12 (Amide C=O) (str.), 1607.96 (N=O) (str.); ¹H NMR (400 MHz, DMSO) δ 8.64 (d, J = 7.7 Hz, 1H), 8.28 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.5 Hz, 2H), 7.42 (dd, J = 8.4, 5.5 Hz, 2H), 7.25 (t, J = 8.7 Hz, 2H), 4.39 (s, 1H), 4.13 – 4.00 (m, 1H), 3.58 (s, 1H), 1.85 (s, 3H), 1.48 (s, 3H); ¹³C NMR (101 MHz, DMSO) δ 168.24, 164.07, 149.01, 140.21, 132.58, 129.31, 129.23, 128.84, 123.50, 115.60, 115.39, 46.85, 40.14, 39.94, 39.73, 39.52, 39.31, 39.10, 38.89; LC/MS (ESI) m/z: 372.19.

N-(1-nicotinoylpiperidin-4-yl)-4-nitrobenzamide (S5F6)

Nicotinic acid (123 mg, 1.00 mmol) and compound (S5I2) (250 mg, 1.00 mmol) were solubilized in dimethylformamide (DMF) (3 mL). Subsequently, the incorporation of HATU (762 mg, 2.00 mmol), and triethylamine (TEA) (0.55 mL, 4.01 mmol) was done. Stirring continued initially at a cool temperature and then at an environmental temperature for 08 hours. On the accomplishment of the reaction, the reaction blend was transferred into ice-cold water for quenching. As no precipitate was observed, extraction was done with dichloromethane (DCM) and isopropyl alcohol (IPA) mixture (8.5:1.5). After separation, the organic fraction was treated with brine to eliminate any residual salts. The organic friction was desiccated using anhydrous sodium sulfate, subsequently strained, and concentrated under a vacuum to get S5F6.

Appearance: Brownish yellow sticky solid, mp 146-148℃, Yield 250 mg (70%), molecular formula: C₁₈H₁₈N₄O₄

IR (KBr, in cm-1): 3427.57 (Amide N-H) (str.), 3074.76 (Ar. C-H), 2928.12 (Ali. C-H), 1659.20 (Amide C=O) (str.), 1599.07 (N=O) (str.); ¹H NMR (400 MHz, DMSO) δ 8.64 (d, J = 5.6 Hz, 3H), 8.28 (t, J = 7.5 Hz, 2H), 8.03 (d, J = 8.6 Hz, 2H), 7.78 (d, J = 7.9 Hz, 1H), 7.51 – 7.21 (m, 1H), 4.40 (d, J = 13.0 Hz, 1H), 4.06 (dq, J = 11.3, 6.1 Hz, 1H), 3.04 (t, J = 7.3 Hz, 1H), 3.02 – 2.90 (m, 1H), 2.84 (d, J = 3.3 Hz, 1H), 1.93 (dd, J = 17.5, 12.1 Hz, 1H), 1.78 (d, J = 12.8 Hz, 1H), 1.60 – 1.36 (m, 2H); ¹³C NMR (101 MHz, DMSO) δ 166.81, 164.12, 150.16, 149.01, 143.70, 140.20, 128.85, 123.54, 123.50, 120.98, 46.74, 45.81, 40.15, 39.94, 39.73, 39.52, 39.31, 39.10, 38.89; LC/MS (ESI) m/z: 355.18.

N-(1-isonicotinoylpiperidin-4-yl)-4-nitrobenzamide (S5F7)

Isonicotinic acid (158 mg, 1.28 mmol) and intermediate-2, S5I2 (320 mg, 1.28 mmol) were solubilized in dimethylformamide (DMF) (4 mL). Then HATU (976 mg, 2.56 mmol), and triethylamine (TEA) (0.71 mL, 5.13 mmol), were introduced. Stirring was first carried out at room temperature, followed by ambient temperature for 12 hours. Subsequently, the reaction was complete, the blend was added to ice-cold water to terminate the reaction. Extraction was performed using ethyl acetate. After phase separation, the organic fraction was treated with brine to eliminate any remaining salts. The blend was subsequently desiccated using anhydrous sodium sulfate, then strained and dried up under reduced pressure to obtain S5F7.

Appearance: Brownish yellow sticky solid, mp 180-182℃, Yield 405 mg (89%), molecular formula: C₁₈H₁₈N₄O₄

IR (KBr, in cm-1): 3421.75 (Amide N-H) (str.), 3076.42 (Ar. C-H), 2990.77 (Ali. C-H), 1658.01 (Amide C=O) (str.), 1607.69 (N=O) (str.); ¹H NMR (400 MHz, DMSO) δ 8.61 (dt, J = 12.2, 7.3 Hz, 2H), 8.55 (s, 1H), 8.26 (d, J = 8.2 Hz, 2H), 8.02 (d, J = 8.3 Hz, 2H), 7.77 (d, J = 7.9 Hz, 1H), 7.43 (ddd, J = 14.1, 7.9, 4.9 Hz, 1H), 4.06 (ddp, J = 11.6, 8.4, 4.3 Hz, 1H), 3.04 (q, J = 7.2 Hz, 1H), 2.94 (d, J = 16.9 Hz, 1H), 2.84 (d, J = 3.2 Hz, 1H), 2.64 (s, 1H), 1.99 – 1.86 (m, 1H), 1.83 – 1.75 (m, 1H), 1.53 (d, J = 12.1 Hz, 1H), 1.44 (d, J = 16.1 Hz, 1H); ¹³C NMR (101 MHz, DMSO) δ 166.89, 164.18, 162.10, 150.49, 150.07, 149.04, 147.34, 146.80, 140.24, 134.62, 134.03, 132.04, 128.88, 123.73, 123.57, 123.53, 46.83, 46.23, 45.86, 40.70, 40.14, 39.93, 39.72, 39.51, 39.30, 39.10, 38.89, 38.30; LC/MS (ESI) m/z: 355.14.

In-Silico studies

Docking studies were performed using the freely available PyRx (AutoDock Vina Version 4.0) software to design and predict the binding affinity of 4-nitro-N-(piperidin-4-yl)benzamide derivatives (S5F1-S5F7) as GPR119 agonists. The compounds were docked to the GPR119 receptor protein, which was modeled through homology using the MODELLER software. Pharmacokinetic evaluations using SwissADME software, provide predicted biological screening to assess their efficacy in stimulating insulin secretion and glucose homeostasis. While ligand-protein interactions were analyzed and visualized with Discovery Studio Visualizer.

RESULTS AND DISCUSSION:

Chemistry

The synthesis of 4-nitro-N-(piperidin-4-yl)benzamide derivatives (S5F1-S5F7), total 07 numbers of compounds, involves amidation of 4-nitrobenzoic acid with a protected piperidine intermediate, followed by deprotection using TFA. Further modifications were achieved via coupling reactions with substituted benzoic or nicotinic acids/chlorides using peptide coupling reagents, enabling pharmacological optimization. A comprehensive characterization of these derivatives was done to confirm their chemical identity, purity, and structural integrity. Spectroscopic techniques, including NMR, IR, and mass spectrometry, provided detailed insights into functional group presence, molecular connectivity, and molecular weight, ensuring the accuracy of the synthesized structures.

By integrating computational modeling, targeted synthesis, and rigorous analytical validation, this approach provided a rational framework for developing novel GPR119 agonists, which hold potential for diabetes treatment and metabolic disorder management.

The IR (KBr, in cm-1) spectrum of compound (S5I1) exhibited a strong IR peak due to (Amide N-H str.) at 3352.53, (Ar C-H str.) at 3076.05, (C=O str.) at 1644.89, and (N=O str.) at 1601.06. The ¹H NMR spectrum indicated the presence of an N-H proton at δ (ppm) 8.59, an aromatic proton between δ (ppm) 8.28-8.03, and an amide/aliphatic proton between δ (ppm) 4.02-1.32. The ¹³C NMR spectrum also complied with the target structure.

Compound (S5I2) was characterized by its IR (KBr, in cm-1) spectrum that exhibited a characteristic IR peak due to (Amide N-H str.) at 3303.99, (Ar C-H str.) at 3076.05, (C=O str.) at 1634.46, and (N=O str.) at 1599.61. The ¹H NMR spectrum indicated the presence of an N-H proton at δ (ppm) 8.58, an aromatic proton between δ (ppm) 8.27-8.04, and an amide/aliphatic proton between δ (ppm) 3.80-1.39. The ¹³C NMR spectrum also complied with the target structure.

The appearance of characteristic IR (KBr, in cm-1) spectra that exhibited a strong IR peak in the range of: due to (Amide N-H str.) 3442.45-3339.35, (Ar C-H) at 3109.22-3074.76, (C=O str.) at 1665.64-1634.26 and (N=O str.) at 1618.14-1599.07; ¹H NMR spectra: N-H proton of the amide between δ (ppm) 8.40-7.21, aromatic C-H proton between δ (ppm) 8.40-7.21 and C-H aliphatic proton of piperidine between δ (ppm) 4.42-1.36 indicates the synthesis of the desired products (S5F1-S5F7).

In-silico studies

Investigation of molecular docking aimed to evaluate the interactions between the designed compounds and the GPR119 receptor. The results showed that all designed and synthesized compounds displayed notable binding affinity to the GPR119 receptor protein [Table 1]. Among these, S5F4, S5F2, and S5F3 exhibited the highest binding affinities of -12.8, -10.7, and -10.5, respectively, compared to the reference molecule AR231453, which had a binding affinity of -11. These compounds emerged as the most potent ligands. The presence of fluorine atoms, as seen in S5F4, enhances receptor binding affinity due to their electronegativity and small size, and also improves pharmacokinetic properties. For example, incorporating a fluoro group into certain GPR119 agonists has been linked to reduced lipophilicity, which in turn improves glucose tolerance. The incorporation of electron-withdrawing groups, like the nitro group (-NO₂) group, is critical in influencing structure-activity relationships by strengthening receptor binding affinity through enhanced electrostatic interactions with key active site residues.

4-nitro-N-(1-(4-(trifluoromethyl)benzoyl)piperidin-4-yl)benzamide (S5F4)

Compound (S5F4) showed interaction with amino acids of receptor proteins such as Pi-Pi stacking, alkyl, and pi-alkyl interactions. Amino acid PHE A:157, TRP A:265, and TRP A:238 shows Pi-Pi stacked interactions with benzine moiety, ALA A:90, VAL A:93, ILE A:136, LEU A:94, LEU A:242, shows Alkyl and Pi-Alkyl interaction with benzine and piperidine moiety of the nucleus. [Figure 1]

N-(1-benzoylpiperidin-4-yl)-4-nitrobenzamide (S5F2)

Compound (S5F2) showed interaction with amino acids of receptor proteins such as Carbon-Hydrogen bond, Pi-Sigma, Pi-Pi stacking, alkyl, and pi-alkyl interactions. Amino acid THR A:86, GLU A:261 shows a Carbon Hydrogen bond with the oxygen of nitro and benzamide nucleus. TRP A:238 shows Pi-Sigma interaction with the benzene moiety of the nucleus. PHE A:174, PHE A:157, TRP A:265 shows Pi-Pi Stacked interaction with benzine moiety of the nucleus, LEU A:242, VAL A:93, VAL A: 166, PHE A:165, LEU A:169 shows Alkyl and Pi-Alkyl interaction with Benzine and piperidine moiety of the nucleus. [Figure 2]

CONCLUSION:

The synthesized 4-nitro-N-(piperidine-4-yl)benzamide derivatives of substituted benzoic and nicotinic acids/chlorides (S5F1-S5F7) were thoroughly characterized using spectroscopic techniques, confirming their chemical structures. Molecular docking studies revealed strong binding affinities to the GPR119 active site, through interactions like hydrogen bonding and π-π stacking, predicting the probability of enhanced efficacy in in-vitro diabetes studies. Notably, fluoro-substituted derivatives like S5F4 exhibited significant receptor affinity. These compounds adhere to Lipinski's Rule of Five and demonstrate favorable ADME properties, indicating good oral bioavailability and safety. The efficient synthetic approach allows structural modifications using established reaction conditions and key intermediates. By activating the GPR119 receptor, these derivatives may stimulate insulin and GLP-1 secretion, reinforcing their probable therapeutic potential for diabetes treatment. This study highlights the importance of heterocyclic scaffolds in receptor-targeted drug design and contributes to the development of novel GPR119 agonists for metabolic disorders.

ACKNOWLEDGEMENT:

The authors express their gratitude to the Department of Pharmacy, Shri Govindram Seksariya Institute of Technology and Science, Indore, Madhya Pradesh, India, for offering the necessary research facilities, mentorship, and assistance, which contributed to the successful completion of this study.

CONFLICT OF INTERESTS:

The authors declare that they have no conflicts of interest concerning the publication or content of this article.

REFERENCES:

1. Manaithiya A, et al. GPR119 agonists: Novel therapeutic agents for type 2 diabetes mellitus. Bioorganic Chemistry. 2021; 113: 104998.doi: 10.1016/j.bioorg.2021.104998

2. Liu P, et al. Design of Potent and Orally Active GPR119 Agonists for the Treatment of Type II Diabetes. ACS Medicinal Chemistry Letters. 2015; 6 (8): 936-41.doi: 10.1021/acsmedchemlett.5b00207

3. Neelamkavil SF, et al. Discovery of MK-8282 as a Potent G-Protein-Coupled Receptor 119 Agonist for the Treatment of Type 2 Diabetes. ACS Medicinal Chemistry Letters. 2018; 9 (5): 457-61.doi: 10.1021/acsmedchemlett.8b00073

4. Zhu X, et al. The first pharmacophore model for potent G protein-coupled receptor 119 agonist. European Journal of Medicinal Chemistry. 2011; 46 (7): 2901-7.doi: 10.1016/j.ejmech.2011.04.014

5. Li R, et al. Structure of human GPR119-G(s) complex binding APD597 and characterization of GPR119 binding agonists. Frontiers in Pharmacology. 2024; 15: 1310231.doi: 10.3389/fphar.2024.1310231

6. Qian Y, et al. Activation and signaling mechanism revealed by GPR119-Gs complex structures. Nature Communications. 2022; 13 (1): 7033.doi: 10.1038/s41467-022-34696-6

7. Shah U, Kowalski TJ. GPR119 agonists for the potential treatment of type 2 diabetes and related metabolic disorders. Vitamins and Hormones. 2010; 84: 415-48.doi: 10.1016/b978-0-12-381517-0.00016-3

8. Wacker DA, et al. Discovery of 5-chloro-4-((1-(5-chloropyrimidin-2-yl)piperidin-4-yl)oxy)-1-(2-fluoro-4-(methylsulfonyl)phenyl)pyridin-2(1H)-one (BMS-903452), an antidiabetic clinical candidate targeting GPR119. Journal of Medicinal Chemistry. 2014; 57 (18): 7499-508.doi: 10.1021/jm501175v

9. Jang YK, et al. Design, synthesis, and biological evaluation of aryl N-methoxyamide derivatives as GPR119 agonists. Bioorganic & Medicinal Chemistry Letters. 2017; 27 (16): 3909-14.doi: 10.1016/j.bmcl.2017.06.032

10. Shiri F, Teymoori M. In silico approaches to explore structure of new GPR 119 agonists for treatment of type 2 diabetes mellitus. Medicinal Chemistry Research. 2017; 26 (5): 947-61.doi: 10.1007/s00044-017-1808-y

11. Matsuda D, et al. Design, synthesis and biological evaluation of novel 7-azaspiro[3.5]nonane derivatives as GPR119 agonists. Bioorganic & Medicinal Chemistry. 2018; 26 (8): 1832-47.doi: 10.1016/j.bmc.2018.02.032

12. Kubo O, et al. Discovery of a novel series of GPR119 agonists: Design, synthesis, and biological evaluation of N-(Piperidin-4-yl)-N-(trifluoromethyl)pyrimidin-4-amine derivatives. Bioorganic & Medicinal Chemistry. 2021; 41: 116208.doi: 10.1016/j.bmc.2021.116208

13. Shiue CY, et al. N-(N-benzylpiperidin-4-yl)-2-[18F]fluorobenzamide: a potential ligand for PET imaging of sigma receptors. Nuclear Medicine and Biology. 1997; 24 (7): 671-6.doi: 10.1016/s0969-8051(97)00097-8

14. Rafi Q-u-AS, et al. Benzophenone Semicarbazones as Potential alpha-glucosidase and Prolyl Endopeptidase Inhibitor: In-vitro free radical scavenging, enzyme inhibition, mechanistic, and molecular docking studies. 2023.doi:

15. Huang Z-N, et al. Synthesis and structure–activity relationship of N-(piperidin-4-yl)benzamide derivatives as activators of hypoxia-inducible factor 1 pathways. Arch Pharm Res. 2018; 41 (12): 1149-61.doi: 10.1007/s12272-018-1050-2

16. Ngo VTH, et al. Structure-activity relationship investigation of Phe-Arg mimetic region of human glutaminyl cyclase inhibitors. Bioorganic and Medicinal Chemistry. 2018; 26 (12): 3133-44.doi: 10.1016/j.bmc.2018.04.040

Received on 22.03.2025 Revised on 29.05.2025

Accepted on 24.08.2025 Published on 01.10.2025

Available online from October 04, 2025

Research J. Pharmacy and Technology. 2025;18(10):4981-4989.

DOI: 10.52711/0974-360X.2025.00720

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

S.NO	Compound Code	Binding Affinity	Molecular weight	#H-bond acceptors	#H-bond donors	Consensus Log P
1.	S5F1	-10.1	291.3	4	1	0.78
2.	S5F2	-10.7	353.37	4	1	1.94
3.	S5F3	-10.5	398.37	6	1	1.2
4.	S5F4	-12.8	421.37	7	1	2.98
5.	S5F5	-9.9	371.36	5	1	2.24
6.	S5F6	-10.5	354.36	5	1	1.22
7.	S5F7	-10	354.36	5	1	1.21
8.	AR231453 (Reference molecule)	-10.9	505.52	10	1	2.73

Code	S5F1	S5F2	S5F3	S5F4	S5F5	S5F6	S5F7
R