N′-(5-Bromofuran-2-carbonyl)isonicotinohydrazide

Abstract

N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1) was obtained in the form of a colorless solid from the 2-methyl-6-nitrobenzoic anhydride (MNBA)/4-dimethylaminopyridine (DMAP)-catalyzed reaction of 5-bromofuran-2-carboxylic acid and isoniazid in dichloromethane at room temperature with a yield of 83%. The structure of N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1) was elucidated using ¹H NMR, ¹³C NMR, FTIR, and high-resolution mass spectrometry. Molecular docking screening of the title compound (1) on cyclooxygenase-2 (COX-2) protein (PDB ID: 5IKR) indicated that compound (1) has a good binding affinity, suggesting that further structure optimization and in-depth research can be carried out on compound (1) as a potential COX-2 inhibitor.

1. Introduction

Furan carboxamides are furan amides with various pharmacological activities, including antitumor [1], antifungal [2], antimicrobial [3], anticancer [4], and antidiabetic [5]. Thionyl chloride is commonly used as a coupling agent in the synthesis of amides [6]. However, thionyl chloride has been listed in the Chemical Weapons Convention (CWC) and the Law of the Republic of Indonesia Number 9 of 2008 concerning the use of chemicals and prohibitions on the use of chemicals as chemical weapons [7,8]. Furan carboxamides have been successfully synthesized using hydroxybenzotriazole as a coupling reagent [4], but this material is explosive so its use should be avoided [9]. More environmentally friendly synthesis of furan carboxamides can be carried out using 1,1′-carbonyldiimidazole (CDI) as a coupling agent [10]. However, CDI has low reactivity, especially when compared to similar materials [11]. Alternatively, the synthesis of carboxamides can be carried out by utilizing 2-methyl-6-nitrobenzoic anhydride (MNBA) and 4-dimethylaminopyridine (DMAP) as coupling agent. This has been shown to be advantageous since the reaction can be carried out in one pot at room temperature and the resulting compounds possess high purity and percentage yield [12]. Therefore, this study aims to utilize MNBA/DMAP for the synthesis of furan carboxamide from furoic acid, namely N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1). Molecular docking was carried out to understand the molecular behavior of N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1) against cyclooxygenase-2 (COX-2) protein.

2. Results and Discussion

2.1. Chemistry

The synthesis of N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1) was carried out by utilizing the reaction of 5-bromofuran-2-carboxylic acid and isoniazid in the presence of MNBA and DMAP in dichloromethane at room temperature (Scheme 1). The crude product was purified by means of dry-column flash chromatography to obtain pure N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1) in the form of a colorless solid with a yield of 83%. The ¹H NMR spectrum of the product clearly showed two doublet signals at δ 6.84 and 7.32 ppm indicating two furanyl protons, two doublet signals at δ 7.80 and 8.79 ppm indicating four pyridinyl protons, and two broad singlet signals at δ 10.62 and 10.81 ppm indicating proton signals of the NH groups. The presence of the NH group was confirmed by the IR spectrum, in which a single absorption at a wavenumber of 3168.9 cm⁻¹ can be observed, indicating the presence of a secondary NH group. It was also supported by the ¹³C NMR spectrum, which exhibited nine signals corresponding to the nine carbon types in the structure of N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1). The carbon of the two carbonyl groups gave signals at δ 156.71 and 164.85 ppm; and aromatic carbons signals at δ 114.69, 117.87, 121.84, 126.21, 139.80, 148.41, and 151.05 ppm. The presence of these carbonyl groups was confirmed by the IR spectrum which showed absorption at wavenumbers of 1644.2 and 1682.4 cm⁻¹. The high-resolution mass spectrum further confirmed the reaction product as N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1) based on the molecular ion peak at m/z 310.9904 [M + 2H]⁺ in a positive ionization mode.

2.2. Molecular Docking Study

Molecular docking was carried out to investigate the molecular behavior of N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1) at the COX-2 protein binding site. Human cyclooxygenase-2 complexed with mefenamic acid (PDB ID: 5IKR) was chosen as a molecular target to examine the interaction of compound (1) with COX-2 protein. Redocking of mefenamic acid, as the native ligand, at the COX-2 binding site resulted in a binding energy of −7.61 kcal/mol and an RMSD value of 0.55 Å (Figure 1).

The docking results showed that the title compound (1) had a binding energy of −7.89 kcal/mol, which was not significantly different from mefenamic acid. Compound (1) exhibited hydrogen bonding, electrostatic interaction, and hydrophobic interaction (Figure 2). The pyridine core of compound (1) generated hydrophobic interactions with Val349 and Leu352 via π–alkyl interactions. One of the carbonyl groups and two NH groups formed hydrogen bonds with Gly526, Met522, and Ala527, respectively. The furan core formed hydrophobic interactions with Val523, Leu352, and Ala527 in the form of π–sigma and π–alkyl interactions. Furthermore, electrostatic interaction was established through π–cation interaction between the furan core and Arg120. Meanwhile, the bromo atom on the furan moiety formed hydrophobic interactions with Tyr385 and Ala527 in the form of π–alkyl and alkyl interactions, respectively.

3. Materials and Methods

The starting materials and reagents used in this study were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Merck (Rahway, NJ, USA) which were used without further purification. Thin layer chromatography was carried out using Merck 0.20 mm precoated silica gel aluminum plates (Kieselgel 60, F₂₅₄) and was visualized using a 254 nm UV lamp. Dry-column flash chromatography was carried out using Merck silica gel 60H. NMR spectra were obtained in DMSO-d₆, using a Jeol JNM-ECS400 spectrometer (400 MHz) (JEOL Ltd., Akishima, Japan). The high-resolution mass spectrum was recorded using the Thermo Scientific TSQ Vantage Triple State Quadrupole spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), while the infrared spectrum was obtained using the Thermo Scientific Nicolet iS10 FTIR spectrometer.

3.1. Synthesis of N′-(5-Bromofuran-2-carbonyl)isonicotinohydrazide (1)

The solution of 5-bromofuran-2-carboxylic acid (0.30 g; 1.62 mmol), MNBA (0.39 g; 1.16 mmol), and DMAP (0.26 g; 2.12 mmol) in dichloromethane (10 mL) was stirred at room temperature for 60 min. The solution was added with isoniazid (0.13 g; 0.96 mmol) and was further stirred for 5 days. The reaction was monitored by means of thin layer chromatography with ethyl acetate:methanol (5:1) as the eluent. The reaction product was evaporated under reduced pressure, and the residue was purified by means of dry-column flash chromatography with ethyl acetate: n-hexane (3:1) as the eluent to obtain N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1) in the form of a colorless solid (0.25 g, 83% yield); mp: 203 °C; FTIR (KBr, cm⁻¹): 3168.9 (N-H), 1682.4 (C=O amide), 1644.2 (C=O amide); ¹H NMR (DMSO-d₆, 400 MHz): δ 6.84 (1H, d, J = 3.6 Hz, ArH), 7.32 (1H, d, J = 3.6 Hz, ArH), 7.80 (2H, dd, J = 4.4, 1.6 Hz, ArH), 8.79 (2H, dd, J = 4.4, 1.6 Hz, ArH), 10.62 (1H, bs, NH), 10.81 (1H, bs, NH); ¹³C NMR (DMSO-d₆, 100 MHz): δ 114.69, 117.87, 121.84, 126.21, 139.80, 148.41, 151.05, 156.71, 164.85; HRESIMS m/z (pos): calcd. for C₁₁H₁₀BrN₃O₃ [M + 2H]⁺, 310.9906 (⁷⁹Br) and 312.9885 (⁸¹Br); found 310.9904 (⁷⁹Br) and 312.9886 (⁸¹Br) (Supplementary Materials).

3.2. Molecular Docking Study

The crystal structure of the COX-2 protein was obtained from the Protein Data Bank (PDB ID: 5IKR) and prepared using MGLTools 1.5.6 [13]. The 3D structure of N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1) was generated using the MarvinSketch tool [14]. The location of the receptor binding site was set using a grid box placed at the position of the native ligand (mefenamic acid) with a centering of x: 38.042; y: 2.131; z: 61.280, xyz dimensions of 30 × 30 × 30, and a spacing of 0.375 Å. The 200 iterations of the Lamarckian genetic algorithm were performed on the Autodock4.2 program in order to obtain the best binding pose of N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1) at the binding site of COX-2 protein [15]. The complex formed between compound (1) and COX-2 was analyzed using Biovia Discovery Studio 2020 [16].

4. Conclusions

N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1) was successfully synthesized from the reaction of 5-bromofuran-2-carboxylic acid and isoniazid in the presence of MNBA and DMAP with a yield of 83%. Molecular docking showed that N′-(5-bromofuran-2-carbonyl)isonicotinohydrazide (1) interacted with residues of the COX-2 protein through several interactions, namely hydrogen bonding, electrostatic interaction, and hydrophobic interaction. The resulting compound had a good binding affinity with a value of −7.89 kcal/mol. Isonicotinohydrazide has the potential to serve as a lead structure in the development of a COX-2 inhibitor.