Greener Synthesis, In-Silico and Theoretical Analysis of Hydrazides as Potential Antituberculosis Agents (Part 1) ^†

Abstract

Since several decades, our healthcare burden has been increased due to tremendous cases of multidrug-resistant (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) infections, especially in tropical countries. In the present study, we have synthesized ten hydrazides with the use of the greener Chitosan-derived catalyst. This catalyst accomplished the said condensation reaction within 15–30 min at room temperature conditions. All our synthesized compounds showed stronger affinities against Mycobacterium tb and bacterial targets, especially towards 1d7u, than the standard drug ciprofloxacin. One of our compounds retained a lower toxicity (electrophilicity index (ω) 3.1304), low chemical hardness (η: 2.1740), and high softness (S: 0.4600). In the realm of the development of more potent, effective, and safe antituberculosis agents with an effective greener synthesis, our current study would provide more insights on potent analogues containing hydrazine motifs in them.

1. Introduction

Antimicrobial resistance is a severe global healthcare threat, which is hampering the quality of human life [1,2,3,4]. Searching for potent, safe, and effective agents is still a difficult task for medicinal chemists all over the world. Tuberculosis (TB) remains a major global healthcare threat, as reported in WHO in 2019 [4]. Hydrazide–hydrazones motifs were reported for their wider pharmacological potentials, such as being anticonvulsant, anticancer, antiviral, etc. [4]. Recently, computational methodologies are also gaining tremendous attention by medicinal chemists [4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]. Recent applications of the Chitosan catalyst have been elaborated thoroughly, and the relevant literatures are cited in references [25,26,27,28,29].

Considering the stronger antimicrobial potentials of Hydrazide–hydrazones having the azomethine group (–NH–N=CH–), we decided to synthesize newer hydrazides using a greener catalyst, Chitosan hydrochloride, and theoretically tested (3a–3j) for their antimicrobial potentials using several computational approaches [5]. These attempts would also enlighten us on the probable anti-TB mechanisms of previously (in vitro) tested hydrazides [6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24] (Figure 1). Moreover, recently, our group has also reported anti-TB potentials of a variety of potent Hydrazide–hydrazone derivatives.

2. Materials and Methods

All the necessary chemicals required for Figure 2 were procured from Sigma-Aldrich and Merck (Mumbai, India). Raw chitosan (MW = 50,000–190,000 Dalton) was purchased from Sigma-Aldrich. All compounds were synthesized according to the previous literature and characterized once again using various spectroscopic techniques, like proton magnetic resonance (1H-NMR), FT-IR (Infrared spectroscopy), etc. Exhaustive details on catalyst characterization and methods are provided in the supporting information, and the data is coherent with the previous literature.

2.1. Preparation of Chitosan-HCl Catalyst

We synthesized the new chitosan-HCl catalyst by taking raw chitosan (1 g) and allowing it to dissolve in 75 cm³ of 1% HCl. The stirring rate was maintained at 800 rpm, along with frequent heating at 40 °C. Furthermore, the mixture was allowed to pass through cotton to filter undissolved mass. Finally, filtrate was collected and dried to obtain the catalyst [5].

2.2. Synthesis of Derivatives of Hydrazones (3a–3j)

For the synthesis of hydrazides (3a–3j), we took equal amounts of phenyl acetohydrazine (2) (Scheme 1 of Figure 2) or benzohydrazine (0.1 mmol, 3, Scheme 2 of Figure 2) and various substituted aldehydes (1a–1j) in a round-bottom flask containing a catalytic amount of chitosan hydrochloride (20% w/w)/ethanol (Figure 2) [6,7]. This reaction mixture was stirred at room temperature until completion. The crude solid products obtained were then washed with cold alcohol and characterized. All reactions were completed within 15–20 min. The spectral data for synthesized compounds is available in the supporting information.

2.3. Molecular Docking and Theoretical Analysis

The structures of all compounds were drawn using ‘ChemDraw V. 12.1′ and converted into 3D formats. The optimized structures were then docked using the ‘Glide’ module from Schrodinger, LLC, (New York, NY, USA) NY suite, 2021 [8]. All 3D crystal structures for docking were downloaded from the protein database bank (PDB database, www.rcsb.org, accessed on 10 October 2021) [9]. Docking was carried out using known protocols (

Table 1. Glide docking score for the best docked molecule, 3e, along with interacted amino acid residues against various antimicrobial targets.

Sr. No.	Target (PDB Id)	Residues with Contribution Energy (kcal/mol)
1	1ai9 (Candida albicans dihydrofolate reductase)	LYS 57, ALA 115, THR 58, ARG 56 (−7.2)
2	1d7u (2,2-dialkylglycine decarboxylase)	ARG406, LYS 272, ASN 394, SER 271, TRP 138 (−9.746)
3	2x22 (enoyl acyl carrier enzyme)	ALA191, PRO 193, THR 196, MET 199, ILE 202, TRP 222 (−8.32)
4	2xcs (S. aureus Gyrase complex)	DG E:10, DC E:11, DG F:10, DC F:11 (−5.47)
5	3ivx (mycobacterial pantothenate synthase)	GLN 72, TYR 82, LYS 160, HIS 47, THR 186, VAL 184, VAL 187, ALA 49 (−9.23)

) [1,2]. The gas-phase structures of the synthesized compounds (shown in Figure 3) were optimized by means of density functional theory (DFT). The DFT calculation was performed by the hybrid B3LYP method, which is based on the idea of Becke and considers a mixture of the exact (HF) and DFT exchange utilizing the B3 functional, together with the LYP correlation functional. The B3LYP calculations were performed in conjunction with the basis set 6-311++G** (

Table 2. Calculated quantum chemical descriptors.

Comp. Id	EHOMO (eV)	ELUMO (eV)	Gap, D (Debye)	μ (eV)	η (eV)	S (eV-1)	ω (eV)
3a	−6.0906	−1.6796	4.7314	3.8851	2.2055	0.4534	3.4219
3b	−6.2155	−1.5537	5.8692	3.8846	2.3309	0.4290	3.2369
3c	−5.8994	−1.5409	5.1159	3.7201	2.1793	0.4589	3.1753
3d	−6.1532	−1.6587	1.7175	3.9059	2.2473	0.4450	3.3944
3e	−5.8632	−1.5153	5.3443	3.6893	2.1740	0.4600	3.1304
3f	−6.5476	−1.8888	3.7701	4.2182	2.3294	0.4293	3.8192
3g	−6.6409	−2.0006	5.4182	4.3207	2.3202	0.4310	4.0231
3h	−6.3164	−1.6927	6.3137	4.0045	2.3119	0.4326	3.4682
3i	−6.2147	−1.6233	5.2590	3.9190	2.29568	0.4356	3.3451
3j	−6.2198	−1.6279	6.1410	3.9239	2.2960	0.4355	3.3530

) [1].

2.4. The 96-Well Microplate Alamar Blue Assay (MABA) Study and Plausible Mechanism for Anti-TB Activity

The 96-well Microplate Alamar Blue Assay (MABA) allows for the quantitative determination of drug susceptibility against any strain of replicating Mycobacterium tuberculosis to be completed within a week at minimal cost. The observed blue color indicated mycobacterial growth inhibition at a particular concentration of the particular wells. All our synthesized compounds were found to possess moderate anti-TB activity (a minimum of 100 μg/mL of minimum inhibitory concentration (MIC) values) when tested using the microplate Alamar Blue assay (MABA) (Repetitions, n = 3).

3. Results and Discussion

3.1. Docking Studies

Based on the microbial activity of hydrazides derivatives, it seemed reasonable to conduct a molecular docking study with the enzyme dialkylglycine decarboxylate (DGD). The DGD is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that can catalyze both decarboxylation and transamination in its normal catalytic cycle. There are several evidences derived from molecular modeling studies and experimental data demonstrating how these inhibitors bind to the active site of 2,2-dialkylglycine decarboxylase (DGD) [18]. Pantothenate biosynthesis is essential for the virulence of Mycobacterium tuberculosis, and this pathway thus presents potential drug targets against tuberculosis. We determined the crystal structure of pantothenate synthetase (PS) from M. tuberculosis, and its complexes with AMPCPP, pantoate, and a reaction intermediate, pantoyl adenylate, with resolutions from 1.6 to 2 Å. PS catalyzes the ATP-dependent condensation of pantoate and β-alanine to form pantothenate. Its structure reveals a dimer, and each subunit has two domains with a tight association between domains. All our synthesized compounds, 3a–3j, were allowed to dock into the binding pockets of various antimicrobial targets such as the common antibacterial target 2,2-dialkylglycine decarboxylase (pdb id: 1d7u), Candida albicans dihydrofolate reductase (pdb id: 1ai9), M. tuberculosis InhA (pdb id: 2x22), the crystal structure of pantothenate synthetase (pdb id: 3ivx), etc. (Figure 4) [1]. The docking protocol was validated by redocking all in-bound ligands into active binding pockets of the respective crystal structures. The average RMSD value was retained to be around 2 Ǻ. Our molecular docking analysis suggested that compound 3e had the highest XP Gscore of −9.746 kcal/mol for the common antibacterial target (pdb id: 1d7u), indicating stronger antibacterial potentials. Compound 3e interacted with ASN394, ARG 406, LYS 272, SER 54, SER 215 amino acid residues from target 1d7u. The detailed binding scores (kcal/mol) and interacting residues for the best docked molecule 3e are provided in Table 1.

3.2. Theoretical Analysis

The B3LYP-converged geometries of the studied compounds are summarized in Figure 3, Figure 4 and Figure 5. The energy of the highest occupied molecular orbital (E_HOMO), the energy of the lowest unoccupied molecular orbital (E_LUMO), dipole moment (D), and the quantum chemical descriptors including the chemical potential (μ), chemical hardness (η), softness (S), and electrophilicity index (ω) are calculated by known equations (Supporting Material) [1], where I and A are the ionization energy and electron affinity of a species, respectively. Furthermore, the ionization energy (I) and the electron affinity (A) of a species could be calculated by applying the Koopmans’ theory (I = −E_HOMO and A = −E_LUMO), and the quantum chemical descriptors were calculated and summarized in Table 2. The stability and reactivity of molecules are significantly related to their chemical hardness and global softness [1]. The smaller (greater) the value of hardness (softness) that a molecule has, the more reactive it should be. From Table 2, it was observed that 3e exhibited the lowest value of chemical hardness and highest value of global softness among the studied compounds; therefore, it is chemically more reactive and less stable than all other compounds. According to the previous relevant studies [10,11], the toxicity of a species seems to be correlated with its electrophilicity index (ω). From Table 2, it was observed that 3e showed the lowest value of the electrophilicity index (ω) among the tested compounds, which indicates that it should have the lowest toxicity among all the studied compounds [11]. Figure 4 depicts the HOMO and LUMO diagrams for the best docked molecule 3e.

4. Conclusions

In the current study, we have successfully synthesized various hydrazide derivatives (3a–3j) using a greener catalyst (chitosan HCl). The reaction was accompanied with minimal use of solvents and fewer workups. Considering the abundancy of raw unmodified chitosan, chitosan-HCl-mediated reactions may strengthen newer aspects of greener reactions. Hydrazide-hydrazones are typically reported on for their potent antimicrobial potentials. Currently synthesized analogues (3a–3j) showed higher binding scores against common bacterial targets. Moreover, our DFT calculations depicted that compound 3e had better theoretical properties. Combining in silico docking and DFT results, compound 3e may serve as the future hit/lead molecule for the development of potent antimicrobial agents.