A Chitosan Hydrochloride Mediated, Simple and Efficient Approach for the Synthesis of Hydrazones, their in vitro Antimycobacterial Evaluations, and Molecular Modeling Studies (Part III)

A simple, eco-friendly and straightforward synthesis of hy- drazones has been devised that is conducted in the presence of chitosan Hydrochloride (chitosan·HCl) as catalyst in aqueous-ethanol me- dium at room temperature. The current protocol offers metal-free synthesis, adaptability to large-scaleup, good yields, and quicker reac- tion time. All ten synthesized hydrazones also showed good antimycobacterial activity, with minimum inhibitory concentrations (MICs) rang- ing from 3.12 to 6.25  g/mL. One of the products presented strong binding affinity against M. tuberculosis pantothenate synthetase (pdb id: 3IVX) with a Glide docking score of –8.803 kcal/mol. Molecular dynamics simulation analysis of its complex with 3IVX retained good sta- bility over the simulation period of 20 ns.

Hydrazide-hydrazones are chemical moieties that contain an azomethine group (-NH-N=CH-) connected with a (-C=O-) carbonyl group. [1][2][3] They attract continuing interest due to their wide spectrum of pharmacological activities. Compounds bearing such moieties were reported to have a number of bioactivities including anti-inflammatory, analgesic, anticancer, anticonvulsant, EGFR inhibitory, antiprotozoal, and antiviral action. [1][2][3] However, this class of compound is most commonly reported as antimicrobial agents. 2 It is also impressive to note that the hydrazide-hydrazone moiety is also present in the chemical structure of drugs with antimicrobial activity, such as nitrofurazone, furazolidone, and nitrofurantoin.  A typical synthesis of hydrazone involves a condensation reaction between a carbonyl compound and a hydrazide, requiring a dehydrating agent. 29 The use of various acid catalysts such as polystyrene sulfonic acid, glacial acetic acid, choline chloride-oxalic acid, or meglumine, have been reported. [29][30][31][32][33][34][35][36][37][38] Although these protocols are effective in many cases for the synthesis of hydrazone derivatives, some of them have one or more drawbacks, such as the use of volatile organic solvents, unsatisfactory yields, over-oxidization of aldehydes to carboxylic acids, long reaction times, high temperatures, difficulties in product isolation, lack of generality, or the need for special apparatus. 39 Many protecting groups, in particular, are rapidly deprotected under acidic environments. As a result, the development of a more efficient process for synthesizing hydrazone derivatives under environmentally friendly conditions remains extremely desirable. 39 In our previous report, we described the use of chitosan hydrochloride to synthesize a new set of hydrazones. 40 Moreover, from our previous medicinal chemistry knowledge [41][42][43][44][45][46][47][48][49] on hydrazones as antitubercular agents (anti-TB) agents, we noticed that these moieties presented particularly strong anti-TB activities, with typical minimum inhibitory concentrations (MICs) ranging from 1.25 to 100 g/mL. In a continuation of this study, we further extended our work to synthesize a set of hydrazones with chitosan hydrochloride as a catalyst. 40 All synthesized hydrazones were also assessed for their probable binding modes against Mycobacterium tuberculosis pantothenate synthetase using molecular docking analysis. 41,43 Further, the best docked S. N. Mali et al.

Paper SynOpen
compound was also subjected to molecular dynamics analysis to establish the binding stability against the selected target (pdb id: 3IVX). Finally, we calculated in silico ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties to gain a better understanding of the probable pharmacokinetics and toxicities.
To understand the reaction optimizations and their relevant parameters, we used the model reaction 1 [benzaldehyde (1 mmol) and phenylhydrazine (1 mmol); Table 1, entry 1]. For catalyst screening, we used previously reported reaction solvent, 39 aqueous ethanol (water/ethanol, 1:1, v/v). The reaction was carried out at room temperature (27°C ). We had noted that the model reaction with no catalyst yielded 48% product, (E)-1-benzylidene-2-phenylhydrazine after 60 min reaction time. 39 As presented in Table 1, for various catalysts (entries 2-7), yields ranging from 48 to 90% were achieved after 60 min reaction time. 39 In addition, we observed that the reaction time could be reduced with the use of chitosan HCl as a catalyst (27 °C, 60 min, 93% yield; Table 1). Thus, we extended the use of chitosan HCl to the reaction shown in Scheme 1. The catalyst was prepared as per the reported procedure. 40 Surprisingly, the reaction proceeded smoothly in water/ethanol (1:1, v/v) at room temperature with higher yields >93%. 39 Formation of the imine (Schiff base or hydrazone) took place reversibly at slightly acidic pH (5.5-6.5). The reaction did not reach completion when it was carried out without catalyst, so the yield obtained in the absence of catalyst was about 48%. The use of mannitol and meglumine as catalysts was previously reported for imine bond formation in aqueous ethanol. Both these compounds are weakly alkali in aqueous ethanol, which leads to irreversible dehydration of the tetrahedral intermediate. Chitosan hydrochloride maintains acidic pH in aqueous ethanol (liberate free HCl in aqueous ethanol) and favors the formation of hydrazone, which is not possible in pure organic solvent such as dichloromethane, methanol, or absolute ethanol. Chitosan is a polymeric material so it is not soluble in pure dichloromethane, methanol, or pure ethanol, but its hydrochloride (15 wt%) liberates trace amounts of HCl in aqueous ethanol, which was sufficient for hydrazone formation.
The variation of yield may be explained by the polarity of the solvent, which is determined by the amount of free HCl liberated from chitosan·HCl. We found that an increase in temperature for our newly optimized reaction had no significant effect on the product yield. However, a reduction in time required for reaction completion from 15 to 10 min-utes was observed. Taking account of environmental considerations, this reaction can also be carried out using water/ethanol (1:1, v/v) as solvent at room temperature. From Table 2, we can also see the effects of various solvents and catalytic loading of chitosan HCl for model reaction 1 (shown in Table 1). The reaction using EtOH and water gave the corresponding product (E)-1-benzylidene-2-phenylhydrazine in higher yields than with glycerin ( Table 2, entries 3 and 4), while the results with other solvents such as CH 2 -Cl 2 , and polyethylene glycol (PEG) 400 were not satisfactory. 39 Our further analysis demonstrated that the best choice of solvent system was aqueous ethanol (water/ethanol, 1:1, v/v) (entries 6 and 7), and that the yield of product was higher with a catalytic load of 15 wt% than with 10 wt%. When the catalytic amount of chitosan·HCl was increased to 20 wt% a yield of final product of 93% was achieved after 15 minutes reaction. Finally, the catalytic load of chi-tosan·HCl 20 wt% was kept constant for optimization of the reaction shown in Scheme 1, with a solvent system of aqueous ethanol (water/ethanol, 1:1, v/v) at room temperature.  S. N. Mali et al. Table 2 Initial Optimization of Reaction Conditions with Various Solvents a As shown in Figure 1, a variety of substituted aromatic aldehydes, irrespective of the presence of electron-donating or electron-withdrawing functional groups attached to the benzene ring, reacted with phenylhydrazine to give the desired products in high to excellent yields. 39 To elaborate the reaction scope of the model reaction 1 (Table 1), we additionally varied the aldehydes with the same catalyst system (see the Supporting Information). Encouraged by these results, we next applied this protocol to the reaction shown in Scheme 1 with a range of aromatic aldehydes (1a-j). Without any further optimization, we were pleased to see good yields of final products 3a-j ( Figure 1). The underlying reaction mechanism for this reaction catalyzed by chi-tosan·HCl is depicted in Figure 2. S. N. Mali et al.

Figure 2 Reaction mechanism
In vitro Antimycobacterial Activity In vitro antimycobacterial activity was assessed by using a microplate Alamar Blue assay (MABA) 43 against Mycobacteria tuberculosis (Vaccine strain, H37RV strain): ATCC No. 27294 ( Figure 3). We recorded minimum inhibitory concentrations (MICs; preventing the color change from blue to pink) with reference to three anti-TB drugs, namely, pyrazinamide, ciprofloxacin and streptomycin, as standard. The results suggested that compounds 3c-e had lower MICs than other compounds, i.e., 3.12 g/mL each. The other compounds exhibited MIC values of 6.25 g/mL. It is important to note that the synthesized hydrazones had MICs that were comparable to those of in vitro anti-TB standard drugs such as pyrazinamide (3.12 g/mL), ciprofloxacin (3.12 g/mL) and streptomycin (6.25 g/mL). 39

Molecular Dynamics (MD) Analysis
We performed MD simulation analysis to establish the stability of the docked molecule 3b towards the chosen target 3IVX. 55 The total duration of simulation was kept at 20 ns, and the model was comprised of 288 residues of chain A, 4299 atoms, and charge of -4. Ensemble class was kept at NTP mode ( Figure 5). In order to see structural changes, trajectory analyses were made for various parameters such as root mean square deviation (RMSD) and root mean square fluctuation (RMSF).

RMSD and RMSF
The backbone changes (N, C, C) of the protein were analyzed via RMSD and RMSF evaluations. 55 The protein-ligand RMSD graph shows that the complex has fluctuations over the simulation period of 5 ns and was stabilized after 7.5 ns of simulation time. The protein RMSD value was retained below 2.4 Å, which is generally acceptable and is considered as a good indicator of stability. The RMSF was used to investigate the fluctuation of the complexes as a function of time. 55 The protein RMSF value was also retained below 4.5 Å. The N-terminal had higher fluctuation compared with the C-terminal.

In Silico ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) Analysis
Pharmacokinetics plays an important role in the development of safer drugs. Popular Lipinski's Ro5 (Ro5: molecular weight > 500, CLogP >5.0, sum of nitrogen and oxygen (N, O) atoms > 10 and hydrogen bond donors > 5) was developed to set 'drugability' guidelines from an oral bioavailability perspective for small molecules. Considering this rule, we observed no violations of Ro5 for compounds 3a-j. Furthermore, water-solubility values (LogS) were also determined to be within the range of -3.027 to -3.982 for the hydrazones 3a-j. For the best docked compound, 3b, the Acute Oral Toxicity value of 2.031 kg/mol was calculated from the 'admetSAR' server. 56 Compounds, 3b-e demonstrated higher human oral bioavailability compared to the other compounds. For all compounds, eye corrosion and eye irritations were found to be on the 'negative' side. However, all compounds would likely have 'category III, Acute Oral Toxicity' (Category III has LD 50 values <500 mg/kg but less than 5000 mg/kg). Other important parameters such as cytochrome 450 enzymes substrate or inhibitions are repre-  (Tables S1 and S2). As per QikProp calculations, compound 3b had a QPPCaco permeability of 2654.171 (representing good Caco-2 cell permeability value). Other parameters such as QPlogBB, QPPM-DCK, and QPlogHERG, were calculated to be within acceptable limits suggested by QikProp, Schrodinger, LLC, NY, 2022. 57 To summarize, we have synthesized a set of 10 hydrazones using chitosan·HCl as a catalyst. All compounds shown good anti-TB activity when tested against Mycobacteria tuberculosis (Vaccine strain, H37RV strain): ATCC No. 27294. Moreover, compound 3b had a higher docking score (-8.803 kcal/mol) than other standard drugs such as ciprofloxacin, streptomycin or pyrazinamide. MD simulation analysis of complex 3b:3ivx demonstrated good stability, as depicted by lower values of RMSD and RMSF. Our in silico analysis also indicates that compounds 3a-j are likely to exhibit Grade III, Acute Oral Toxicity with no carcinogenicity as calculated from 'admetSAR' server.
The melting point of hydrazones 3a-j were measured with a digital Optimelt melting-point apparatus and are uncorrected. Fouriertransform infrared (FTIR) spectra were recorded with a Perkin Elmer Tensor-II model, and a Perkin Elmer Lambda-25 double beam spectrophotometer was employed to record absorption data. A Bruker Avance III 500 NMR instrument was used to record the 1 H NMR spectra of the final products in CDCl 3 using tetramethylsilane (TMS) as an internal reference. Mass spectral data were recorded with a Thermo Scientific, USA (Model: ultimate 3000, LTQ XL) instrument with an electrospray ionization (ESI) source.
Raw chitosan (MW = 50,000-190,000 Dalton) was purchased from Sigma-Aldrich. All compounds were synthesized according to the reported procedures and characterized using spectroscopic techniques ( 1 H NMR, FTIR, etc). Exhaustive details on catalyst characterization and methods are provided in the Supporting Information, and the data is consistent with the reported data. 40 Chitosan hydrochloride (20 wt%) was added to a round-bottom flask containing carbonyl compound (1 mmol) and corresponding hydrazine (1 mmol) in water-ethanol (1:1, 4 mL) as solvent. The reaction, (Scheme 1 and Table  1, model reaction 1) was carried out at room temperature, maintaining a stirring rate of 250-300 rpm. The progress of reaction was monitored by TLC (EtOAc/hexane, 9:1). All products were extracted using EtOAc followed by addition of water (5 mL) and EtOAc (5 mL). The organic phase was then dried with sodium sulfate with subsequent removal of organic solvent using a rotary evaporator under vacuum. Crude products were recrystallized (petroleum ether/EtOAc, 1:1) and their melting points, yields and other physical properties were measured.

UV Spectral Analysis and Photoluminescence Study
A detailed description of this analysis is included in the Supporting Information.