Pyridine- and Thiazole-Based Hydrazides with Promising Anti-inflammatory and Antimicrobial Activities along with Their In Silico Studies

Abstract

A new class of compounds formed by the linkage of −C(O)–NH– with pyridine and thiazole moieties was designed, synthesized, and characterized by various spectral approaches. The newly characterized compounds were evaluated for their antimicrobial as well as anti-inflammatory properties. The in vitro anti-inflammatory activity of these compounds was evaluated by denaturation of the bovine serum albumin method and showed inhibition in the range of IC₅₀ values—46.29–100.60 μg/mL. Among all the tested compounds, compound 5l has the highest IC₅₀ value and compound 5g has the least IC₅₀ value. On the other hand, antimicrobial results revealed that compound 5j showed the lowest MIC values and compound 5a has the highest MIC values. Furthermore, molecular docking of the active compounds demonstrated a better docking score and interacted well with the target protein. Physicochemical parameters of the titled compounds were found suitable in the reference range only. The in silico molecular docking study revealed their COX-inhibitory action. Compound 5j emerged as a significant bioactive molecule among the synthesized analogues.

Introduction

Inflammation is provoked by injuring any tissue in the body or by infection through pathogens, such as bacteria and viruses, allergens, irritants, toxic compounds, and so forth, either exogenously or endogenously.¹ This may also include injury, surgery, autoimmune disorder, adult respiratory distress syndrome, long-term exposure to industrial chemicals, and reoxygenation injuries. The inflammation process is catalyzed by easily accessible molecules in the body such as histamine, prostaglandins, leukotrienes, oxygen- and nitrogen-derived free radicals, serotonin, bradykinin, and interleukins. Extravagant inflammation may lead to stroke or heart diseases, lupus, cancer, tissue injury, physiological decompensation, organ damage, and death.² The risks associated with the inflammation process pose a challenge to the medicinal chemists to search for more effective anti-inflammatory agents. A majority of the existing anti-inflammatory compounds, especially those with proven clinical efficiency, are acidic in nature, such as aspirin, indomethacin, flufenamic acid, ibuprofen, and so forth. Nonsteroidal anti-inflammatory drugs (NSAIDs) that act on the affected tissues^3,4 by inhibiting the cyclooxygenase (COX) involved in the synthesis of prostaglandins are one of the major class of drugs used to treat inflammation.⁵⁻⁹

Various natural as well as synthetic compounds containing the thiazole core moiety have been explored for their antitumor,^10,11 antimicrobial, anti-HIV,¹² anticoagulant,¹³ anti-inflammatory,¹⁴ and antioxidant¹⁵ properties since many decades. The available literature also supports the bioactivities of thiazole scaffolds (Figure 1).

Figure 1.

Structure of thiazole-based molecules as anti-inflammatory agents.

Compound A demonstrated its selective cannabinoid CB2 agonists with anti-inflammatory activity.¹⁶ Compound B is a thiazole derivative exhibiting anti-inflammatory property,¹⁷ and compound C is a 1,3,4-oxadiazole-benzothiazole-pyridine conjugate which showed promising COX inhibition.¹⁸

In recent decades, microbial infections are widespread and one of the life-threatening problems. To overcome these situations, researchers are trying hard to discover more efficient and effective antimicrobial candidates against both drug-sensitive as well as drug-resistant strains. Mahmoud et al.,¹⁹ Adole et al.,²⁰ Wang et al.,²¹ and Eryılmaz et al.(22) reported various thiazole containing antimicrobial candidates. Some of the known reported bioactive compounds containing pyridine and thiazole having amide linkages are shown in Figure 2. Based on the available literature, the thiazole nucleus has been considered as one of the promising bioactive scaffolds in the wide range of biological systems.

Figure 2.

Structure of pyridine-, thiazole-, and amide-containing bioactive molecules.

Various pyridine containing anti-inflammatory and antimicrobial agents are also reported in the literature. Researchers like Fan et al.,²³ Bhila et al.,²⁴ Desai et al.,²⁵ Rani and Reddy,²⁶ and Rashdan et al.(27) reported some antimicrobial agents. Laddha and Bhatnagar,²⁸ Kumar et al.,²⁹ and Thirumurugan et al.(30) reported some pyridine-based anti-inflammatory agents. In view of these observations,³¹ we also aimed to synthesize some hydrazides linked to pyridine-containing thiazole as potent bioactive scaffolds. The strategy used in this study is presented in Figure 3.

Figure 3.

Strategy used in designing the titled molecules.

Results and Discussion

Chemistry

The detailed synthetic strategy for the target compounds is illustrated in Scheme 1. The structures and the yield of the synthesized compounds are represented in Figure 4. Initially, 3-cyanopyridine (1) was converted to pyridine-3-carbothiamide (2) by treating with P₄S₁₀. Compound 2 upon refluxing with ethyl-2-chloroacetoacetate resulted in ethyl-5-methyl-2-(pyridine-3-yl)thiazole-4-carboxylate (3). The resulting ester was treated with hydrazine hydrate to yield 5-methyl-2-(pyridine-3-yl)thiazole-4-carbohydrazide (4). Further, 5-methyl-2-(pyridine-3-yl)thiazole-4-carbohydrazide was condensed with an aromatic aldehyde to afford the target compounds (5a–l). The formation of 5a–l was confirmed by the absence of IR bands around 3330 cm^–1 and scissoring at 1589 cm^–1 due to the NH₂ group and the presence of CH stretching at 2860 cm^–1. A singlet at δ 8 ppm confirmed the formation of −CH=N.

Scheme 1. Synthetic Pathway of the Thiazole-Based Hydrazides (5a–l).

Reagents: (i): P₄S₁₀, ethanol, reflux, 70 °C, 4 h; (ii): ethyl 2-chloroacetoacetate, ethanol, reflux, 70 °C, 8 h; (iii): hydrazine hydrate, ethanol, reflux, 70 °C, 4 h; (iv): Ar-CHO, ethanol, reflux, 70 °C, 12 h.

Figure 4.

Structure of the synthesized compounds.

Biological Activity

In Vitro Anti-inflammatory Activity (Denaturation of the Bovine Serum Albumin Method)

Inflammation is the biological response that takes place when the healthy tissues get damaged or injured by harmful pathogens or by the alteration of neighboring protein tissues. Because of the lack of groundwork, animal risk management, and ethical disputes, in vivo studies have potential complications in order to carry out the animal studies. The leading cause of inflammation is the alteration of the native state of the tissue proteins. In the in vivo studies, the denaturation of proteins takes place because of the synthesis of autoantigens, and the compounds which inhibit denaturation are potentially useful for the discovery of novel anti-inflammatory drugs. The inhibitory effect on denaturation of proteins can be accessed by using a heat-induced protein denaturation process using diclofenac sodium as the standard drug. For the protein denaturation process to take place, the main important property to be maintained in several NSAIDs evaluation is the physiological pH (6.2–6.5) range of the reaction medium. All the synthesized thiazole-based hydrazide derivatives were tested for their in vitro anti-inflammatory activity by inhibition of the protein (bovine albumin) denaturation method using diclofenac sodium as a standard drug. Percentage inhibition of the synthesized compounds was determined using varying concentrations that is, 20–100 μg/mL. The IC₅₀ values were determined, and the results and graphical representations are tabulated in Table 1 and Figure 5, respectively. From the results, it is clear that some of the synthesized compounds showed better activity (compounds 5j, 5k, and 5l). IC₅₀ values of the synthesized compounds were in the range of 46.29–100.60 μg/mL because of the structural variations of the substrate. By varying the substitutions, the potency of the compounds changed. Compound 5l has the highest inhibition among the synthesized compounds which have 4-hydroxy-3-methoxyphenyl, and it has more inhibition than 5b (which has 3,4-dimethoxy substitution) and 5i (which has a hydroxyl group at 4th position). The lowest inhibition was observed for 5g, which has the 4-benzyloxyphenyl group. The compounds containing heterocyclic rings, 5k, and 5e showed better inhibition when compared with the compounds having aromatic substituents such as 5a, 5c, and 5g. Compounds 5b, 5d, 5f, 5h, and 5i (3,4-dimethoxy, 4-methyl, 3,4-dimethyl, 4-N,N-dimethyl, and 4-hydroxy) showed moderate inhibition. It can be concluded finally that the presence of the hydroxyl group on the phenyl ring and/or the presence of heterocyclic moieties exhibit good inhibition.

Table 1. Percentage Inhibition of Egg Albumin Protein Denaturation by the Synthesized Compounds^a.

	inhibition of protein denaturation (%)
con (μg/mL)	std*	5a*	5b*	5c*	5d*	5e*	5f*	5g*	5h*	5i*	5j*	5k*	5l*
20	42.98 ± 1.28	11.66 ± 0.95	22.66 ± 1.05	12.76 ± 0.99	24.46 ± 1.19	28.63 ± 0.68	17.65 ± 1.34	9.94 ± 0.53	19.18 ± 0.71	25.49 ± 0.85	30.66 ± 1.04	31.42 ± 0.95	32.63 ± 1.07
40	57.08 ± 1.6	20.51 ± 0.82	30.51 ± 2.22	22.51 ± 2.33	35.06 ± 0.56	39.23 ± 1.37	27.27 ± 1.52	16.82 ± 1.74	27.92 ± 1.59	31.18 ± 1.28	40.01 ± 1.33	41.00 ± 1.24	43.20 ± 1.87
60	75.54 ± 1.26	28.25 ± 1.47	42.86 ± 2.43	28.91 ± 1.44	48.94 ± 1.53	51.18 ± 1.26	35.26 ± 1.42	26.00 ± 1.00	37.33 ± 1.43	49.24 ± 1.60	53.98 ± 1.78	56.72 ± 2.00	59.51 ± 2.46
80	83.56 ± 0.96	39.55 ± 0.92	52.54 ± 0.90	39.65 ± 1.08	61.43 ± 1.24	65.57 ± 1.33	49.58 ± 0.98	37.61 ± 1.99	49.89 ± 1.45	58.12 ± 0.56	68.39 ± 1.00	71.47 ± 0.90	78.24 ± 1.37
100	94.60 ± 0.90	52.56 ± 0.80	69.65 ± 1.21	58.28 ± 1.29	73.82 ± 1.45	77.37 ± 0.94	62.06 ± 1.57	49.70 ± 0.73	65.78 ± 1.64	71.07 ± 0.34	80.34 ± 0.95	83.46 ± 2.21	87.97 ± 1.37
IC50 μg/mL	35.03	95.11	76.12	85.79	61.29	58.62	80.68	100.60	80.18	62.74	55.58	52.89	46.29

^aValues are expressed as mean ± SD, n = 3, and * Correlation is significant at the 0.01 level.

Figure 5.

Graphical representation of in vitro protein denaturation results of the compounds.

The structure activity relationship study of the active compound 5l was deduced based on the IC₅₀ value; it clearly reveals that the presence of hydroxyl and methoxy groups is responsible for the enhanced inhibition. The presence of the hydroxyl substituent on the 4th place and the methoxy substituent on the 3rd position enhanced the anti-inflammatory activity drastically.

In Vitro Antimicrobial Activity

Minimal inhibitory concentration of the synthesized compounds showed the highest to least values because of the structural modifications of the substrate. Minimal inhibitory concentration values along with mean ± SD and graphical representations of antibacterial and antifungal activities are tabulated in Tables 2 and 3 and in Figures S11 and S12, respectively.

Table 2. MIC Values (μg/mL) of Antibacterial Evaluation of the Titled Compounds^a.

compound	S. aureus	B. subtilis	E. coli	P. aeruginosa
5a	127.66 ± 1.52	127.33 ± 1.15	128.33 ± 0.57	127.33 ± 1.15
5b	127.00 ± 1.73	65.00 ± 1.73	128.33 ± 0.57	127.00 ± 1.73
5c	64.00 ± 1.00	32.66 ± 1.15	64.33 ± 0.57	64.00 ± 1.00
5d	63.00 ± 1.00	31.66 ± 0.57	65.00 ± 1.00	64.33 ± 1.52
5e	15.66 ± 0.57	8.33 ± 0.57	17.33 ± 1.52	16.00 ± 1.00
5f	33.00 ± 1.73	15.33 ± 1.15	31.66 ± 1.52	31.33 ± 1.15
5g	63.66 ± 0.57	33.00 ± 1.73	64.66 ± 1.15	64.00 ± 1.00
5h	31.66 ± 0.57	17.00 ± 1.73	32.66 ± 1.15	32.00 ± 2.00
5i	15.33 ± 1.15	7.00 ± 1.00	17.00 ± 1.00	16.33 ± 0.57
5j	1.66 ± 0.57	1.32 ± 0.57	2.66 ± 1.52	2.33 ± 0.57
5k	3.66 ± 1.52	2.00 ± 1.00	4.66 ± 1.15	4.00 ± 1.00
5l	4.00 ± 01.73	4.33 ± 0.57	7.00 ± 1.00	7.00 ± 1.00
tetracycline	0.68 ± 0.54	0.66 ± 0.28	1.66 ± 1.15	1.66 ± 1.15
streptomycin	1.00 ± 00	1.00 ± 00	1.66 ± 1.15	1.00 ± 0.86

^aValues are expressed as mean ± SD, n = 3, and * correlation is significant at the 0.01 level.

Table 3. MIC Values (μg/mL) of Antifungal Evaluation of the Titled Compounds^a.

compound	A. flavus	T. atroviridae	P. citranum	C. albicans
5a	126.33 ± 2.08	126.00 ± 1.73	124.33 ± 2.30	128.33 ± 0.57
5b	128.33 ± 2.08	129.00 ± 2.00	124.00 ± 1.00	126.00 ± 2.00
5c	129.00 ± 1.73	127.33 ± 1.15	126.66 ± 2.30	62.33 ± 1.52
5d	64.00 ± 1.00	65.66 ± 1.52	67.00 ± 2.64	32.00 ± 1.00
5e	16.66 ± 1.15	15.66 ± 2.51	16.33 ± 1.52	8.33 ± 1.52
5f	32.66 ± 3.05	33.33 ± 1.52	32.00 ± 1.00	14.33 ± 1.52
5g	63.33 ± 1.15	62.66 ± 1.52	65.00 ± 1.73	130.33 ± 2.08
5h	64.6 ± 3.05	65.00 ± 1.73	65.33 ± 1.52	33.66 ± 2.08
5i	14.66 ± 2.30	15.00 ± 1.73	15.33 ± 1.15	7.66 ± 1.52
5j	2.66 ± 1.15	2.33 ± 0.57	1.66 ± 0.57	1.66 ± 1.15
5k	3.00 ± 1.00	5.33 ± 1.52	4.66 ± 1.15	3.33 ± 1.52
5l	3.00 ± 1.00	5.33 ± 1.52	3.66 ± 1.52	4.00 ± 2.00
fluconazole	2.00 ± 0.00	2.00 ± 0.00	2.00 ± 0.00	1.00 ± 0.00
nystatin	3.33 ± 1.52	1.66 ± 1.15	2.00 ± 1.73	3.66 ± 2.08

^aValues are expressed as mean ± SD, n = 3, and * correlation is significant at the 0.01 level.

The variation of the position of the substituents and the introduction of the heterocyclic ring were explored to study the structure–activity relationship (SAR) of the synthesized compounds. Results obtained from the antimicrobial studies indicated that the compounds 5j, 5k, 5l, 5e, and 5i showed significant minimal inhibitory concentration (MIC) values, of which the compound 5j showed MIC values as that of the standard drugs against the tested bacterial and fungal strains. The enhanced antimicrobial activity is due to either the presence of a hydroxyl group on the aromatic ring and or the presence of the heterocyclic moiety in the synthesized derivatives when compared with other compounds having the aromatic moiety. The least activity was shown by compounds 5a, 5b, and 5c with 4-chloro, 3,4-dimethoxyphenyl, and 3-nitrophenyl moieties, respectively. The moderate antimicrobial property was observed in the case of compounds 5f, 5d, 5g, and 5h, which contain 3,4-dimethyl, 4-methyl, and 4-benzyloxyphenyl groups.

The structural activity relationship study deduced that the alterations in the aryl ring play the significant role for enhancing the activity. Compound 5j with the combinations of bromo substitution at the 5th position and hydroxyl substitution at the 2nd position produces effective and enhanced antimicrobial properties.

In silico prediction of physicochemical and ADME parameters: in the field of medicinal chemistry, Lipinski’s rule of five and Veber’s rule are the guidelines for medicinal chemists searching drug-like chemical compounds with effective oral potential. It is understood that fine-tuning the physicochemical properties (PCPs) of a lead molecule could alter the ADME property. Hence, close inspection was placed on the PCPs, by calculating the guiding parameters such as molecular weight (MW), number of hydrogen bond acceptors (HBAs), number of hydrogen bond donors (HBDs) calculated log P (clog P), number of rotatable bonds (NRBs), and polar surface area (PSA). Compounds with optimal PCPs (MW < 500, HBA < 10, HBD < 5, log P < 5, NRB < 10, and PSA < 140 Å) have higher probability for oral absorption. The predicted parameters of the titled compounds (Table 4) satisfied the Lipinski’s rule of five. Caco-2 permeability of all the predicted compounds lies within the range except compound 5c, which showed a less permeability of 91.567 nm/s. 50% of the compounds revealed the predicted results of 100% oral human absorption, and the rest of the compounds exhibited the values within the range of 77–96%. Overall, the predicted parameters of the compounds were satisfied with the physicochemical parameters, which are exhibited by most of the clinically approved drugs. Based on the prediction studies, these compounds may not face any problems in the mere future.

Table 4. In silico Predicted Physicochemical Parameters of the Titled Compounds.

code	MWa	SASAb	donorHBc	accptHBd	log P o/we	PCacof	log Sg	#rotorh	% human oral absorptioni
5a	356.829	649.025	1	5.5	3.973	1059.54	–5.876	4	100
5b	382.436	705.878	1	7	3.667	839.026	–5.698	6	100
5c	367.381	671.77	1	6.5	2.701	91.567	–5.446	5	77.874
5d	336.411	663.689	1	5.5	3.875	951.014	–5.852	4	100
5e	312.345	571.87	1	6	2.687	976.977	–4.332	4	96.192
5f	350.437	689	1	5.5	4.093	982.991	–7.296	4	100
5g	428.508	788.776	1	6.25	5.283	1154.27	–4.073	7	100
5h	365.452	700.173	1	6.5	3.836	958.45	–6.293	5	100
5i	338.383	600.548	2	6.25	2.442	247.973	–5.946	5	84.098
5j	417.279	619.16	2	6.25	3.392	927.915	–6.187	5	100
5k	361.42	686.978	2	5.5	3.752	529.314	–5.307	4	100
5l	368.409	682.421	2	7	2.926	313.646	–4.982	6	88.762

^aMolecular weight, in Da (range for 95% of drugs: 130–725 Da).

^bTotal solvent-accessible volume in cubic angstroms using a probe with a 1.4 Å radius. (500–2000).

^cNo. of hydrogen bonds donated by the molecule (range for 95% of drugs: 0–6).

^dNo. of hydrogen bonds accepted by the molecule (range for 95% of drugs: 2–20).

^ePredicted octanol/water partition coefficient log P (acceptable range: −2.0 to 6.5).

^fPredicted aqueous solubility; S in mol/L (acceptable range: −6.5 to 0.5).

^gApparent Caco-2 permeability (nm/s) (<25 poor, >500 great).

^hNumber of nontrivial (not CX3) and nonhindered (not alkene, amide, small ring) rotatable bonds. (0–15).

ⁱ% human oral absorption (>80 high and <25 poor).

Molecular Docking Studies

To find out the putative binding mode of the significantly active compound against the targeted protein COX-2, docking studies were performed. Before the docking studies of the test compound, root mean square deviation (RMSD) of the X-ray pose and redocked pose (Figure 6) of the co-crystallized ligand in the target protein was checked and found to be 0.20 Å, suggesting that the docking protocol could be relied on for the docking studies.

Figure 6.

Superimposed view of the X-ray native pose of the ligand (Celecoxib) with its docking pose in the active site of the target (PDB-3LN1) [color interpretation: white—X-ray native pose of the ligand, pink—docked pose of the ligand].

In-depth scrutiny of the 3D and 2D poses of the co-crystal ligand (Celecoxib) was performed (Figures 7 and 8), and celecoxib unveiled four hydrogen bond interactions within the target with a highest docking score of −12.4 kcal/mol and energy of −62.50 kcal/mol (5). The amino acid residues such as GLN-178, SER-339, ARG-499, and PHE-504 were actively involved in the hydrogen bond formation with celecoxib. Apart from the hydrogen bond interaction, celecoxib also revealed two aromatic bond interactions with the amino acid residues HIS-75 and TYR-341; ARG-106 was involved in π–π-cationic interaction with the pyrazole moiety of celecoxib.

Figure 7.

Networking of the co-crystal ligand exhibited various interactions in the active site of the protein (3LN1) [color interpretation: black—hydrogen bond, blue—aromatic bond, green—π–π cation interactions].

Figure 8.

2D representation of the co-crystal ligand [color interpretation: magenta—hydrogen bond, red—cation interactions.

Table 5. Docking Analysis of Celecoxib and Significantly Active Compounds 5j, 5k, and 5l.

compound code	Glide score (kcal/mol)
5j	–9.80
5k	–7.80
5l	–7.10

code (PDB-3LN1)	H-bond	aromatic bond	π–π interactions	Glide score (kcal/mol)	Glide energy (kcal/mol)
co-crystal ligand (Celecoxib)	GLN-178	HIS-75		–12.40	–62.50
	SER-339	TYR-341
	ARG-499
	PHE-504
5j		SER-516		–9.80	–46.80

Similarly, significantly active compound 5j (Figures 9 and 10) also exhibited only one aromatic bond interaction with the amino acid residue SER-516 through the pyridine moiety. The compound exhibited a docking score of −9.80 kcal/mol and docking energy of −46.80 kcal/mol, which are comparatively less than those of the standard drug celecoxib.

Figure 9.

Networking of the significantly active compound 5j exhibited various interactions in the active site of the protein (3LN1) [color interpretation: blue—aromatic bond].

Figure 10.

2D representation of the significantly active compound 5j.

Conclusions

In the present study, we developed thiazole-based hydrazides as anti-inflammatory and antimicrobial agents. The in vitro anti-inflammatory study of the target compounds revealed that compounds 5j, 5k, and 5l are significant inhibitors among the series. The in vitro antimicrobial assay showed that compounds 5j, 5k, and 5l are effective against all the tested strains. The phenolic group, along with methoxy or bromo substituents, showed better activity when compared with other substituents. Compounds 5j and 5l displayed better activity in the series. Therefore, compounds 5j and 5l can be used for further studies. Pharmacokinetic studies also underlined that the active compound has better absorption, distribution, metabolism, and elimination profiles. More interestingly, compounds 5j, 5k, and 5l showed significant biological activity with safe pharmacokinetic properties. The molecular docking study also supports the effective interactions with target molecules. Based on the present study, we can conclude that this is the potential route for the development of new effective antimicrobial as well as anti-inflammatory agents.

Experimental Section

Materials and Methods

All the chemicals and solvents used for the present study were purchased from commercial suppliers of Sigma-Aldrich, India, and Spectrochem Pvt. Ltd., India, and used without purification. Reactions were carried out using standard techniques. Melting points of the newly synthesized compounds were determined in an open capillary tube and were uncorrected. To monitor the progress of the reaction and purity of the product, thin layer chromatography (Merck silica gel 60 F254-coated aluminum plates) was used. The mixture of ethyl acetate and hexane solvents was used as the mobile phase and visualized under UV light at 254 nm. Shimadzu-FTIR was used to record FTIR spectra (γ_max in cm^–1). The Bruker AVANCE II 400 spectrometer (with 5 mm PABBO BB-1H tubes) was used to record NMR spectra, using DMSO-d₆ as a solvent and TMS as an internal standard (the chemical shift in δ ppm and coupling constants (J) were expressed in parts per million (ppm) and hertz, respectively). VARIOEL-III (Elementar analysensysteme GmBH) was used for the elemental analysis.

Procedure for the Synthesis of Pyridine-3-carbothiamide (2)

A solution of P₄S₁₀ (2 equiv) and 3-cyanopyridine 1 (1 equiv) in ethanol was taken in a round-bottomed flask and stirred for 30 min at ambient temperature. The reaction mixture was then refluxed for 4 h. After the completion of the reaction [monitored by TLC (EtOAc/hexane 3:7)], the reaction mixture was cooled to room temperature, diluted with cold water, and extracted with chloroform (3 × 200 mL). The combined organic extract was dried over CaCl₂ and concentrated to dryness. Further, the crude residue was recrystallized to afford a pure compound.³²

Procedure for the Synthesis of Ethyl-5-methyl-2-(pyridine-3-yl)thiazole-4-carboxylate (3)

A mixture of ethyl-2-chloro acetoacetate (1.2 equiv) and compound 2 (1 equiv) dissolved in ethanol (50 mL) was refluxed for 8 h. After completion of the reaction [monitored by TLC (EtOAc/hexane 3:7)], the reaction mixture was cooled to ambient temperature. The reaction mixture was quenched with ice cold water. The solids precipitated out were collected by filtration, washed with ice cold water, and dried under reduced pressure to afford intermediate 3.³³

Procedure for the Synthesis of 5-Methyl-2-(pyridine-3-yl)thiazole-4-carbohydrazide (4)

To the stirred solution of intermediate compound 3 (1.5 equiv) in ethanol (50 mL), hydrazine hydrate (2.5 equiv) was added at ambient temperature and heated to reflux for 4 h. The reaction progress was monitored by TLC (EtOAc/hexane 3:7). After completion of the reaction, the reaction mass was poured onto crushed ice and stirred for half an hour. The solid was filtered and dried under vacuum to get intermediate compound 4.³⁴

General Procedure for the Synthesis of N′-Arylmethylidene-5-methyl-2-(pyridin-3-yl)thiazole-4-carbohydrazides (5)

A mixture of intermediate compound 4 (1 equiv) and aromatic aldehyde (1.2 equiv) in ethanol (50 mL) was stirred under reflux for about 12 h. The reaction mixture was cooled to 0–10 °C, filtered, and dried for 6 h. It was further recrystallized by the DMF–DMSO mixture to afford the pure titled target compounds.³⁵ Purity of the synthesized compounds was in the range of 95–99%.

N′-(4-Chlorobenzylidene)-5-methyl-2-(pyridin-3-yl)thiazole-4-carbohydrazide (5a)

Yellow solid; Yield: 82%; mp > 300 °C; FT-IR (KBr cm^–1): 3270 (N–H), 3041 (Ar–H), 2964 (C–H), 1665 (NH–CO), 1610 (C=N), 653 (C–S–C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.48 (s, 3H, thz-CH₃), 7.38–7.33 (d, 2H, Ar–H, J = 7.8 Hz), 7.60–7.57 (m, 1H, Pyr-H), 7.85 (d, 2H, Ar–H, J = 7.8 Hz), 8.14 (s, 1H, =CH), 8.39 (d, 1H, Pyr-H, J = 7.2 Hz), 8.73 (d, 1H, Pyr-H, J = 4 Hz), 9.21 (s, 1H, Pyr-H), 11.95 (s, 1H, N–H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 15.2, 115.4, 115.6, 127.5, 130.3, 130.4, 130.7, 131.1, 134.5, 134.9, 149.3, 151.4, 155.7, 157.2, 160.2, 162.3, 168.5; LCMS (m/z): 357.05 [M + H]⁺, 359.32 [(M + 2) + H]⁺; Elemental analysis of C₁₇H₁₃N₄ClOS Calcd: C, 57.22; H, 3.67; N, 15.70. Found: C, 57.20; H, 3.65; N, 15.72.

N′-(3,4-Dimethoxybenzylidene)-5-methyl-2-(pyridin-3-yl)thiazole-4-carbohydrazide (5b)

Pale yellow solid; Yield: 80%; mp 230–232 °C; FT-IR (KBr cm^–1): 3447 (N–H), 3014 (Ar–H), 2839 (C–H), 1648 (−NHCO), 1597 (C=N), 651 (C–S–C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.79 (s, 3H, thz-CH₃), 3.81 (s, 3H,–OCH₃), 3.85 (s, 3H, −OCH₃), 7.04 (d, 1H, Ar–H, J = 8.4 Hz), 7.21–7.23 (m, 1H, Ar–H), 7.45 (s, 1H, Ar–H), 7.56–7.59 (m, 1H, Pyr-H), 8.03 (s, 1H, =CH), 8.32 (d, 1H, Pyr-H, J = 8 Hz), 8.71 (d, 1H, Pyr-H, J = 4 Hz), 9.14 (s, 1H, Pyr-H), 11.80 (s, 1H, −NH); ¹³C NMR (DMSO-d₆, 100 MHz): δ 15.2, 56.8, 110.1, 114.1, 122.1, 127.5, 129.4, 131.1, 134.5, 134.9, 148.6, 149.3, 149.8, 150.5, 151.4, 155.7, 157.2, 168.5, 181.7; LCMS (m/z): 383.10 [M + H]⁺; Elemental analysis of C₁₉H₁₈N₄O₃S Calcd: C, 59.67; H, 4.74; N, 14.65. Found: C, 59.65; H, 4.75; N, 14.63.

5-Methyl-N′-(3-nitrobenzylidene)-2-(pyridin-3-yl)thiazole-4-carbohydrazide (5c)

Yellow solid; Yield: 75%; mp 266–268 °C; FT-IR (KBr cm^–1): 3440 (N–H), 3041 (Ar–H), 2868 (C–H), 1651 (−NHCO), 1609 (C=N), 650 (C–S–C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.69 (s, 3H, thz-CH₃), 7.56–7.59 (m, 1H, Pyr-H), 7.78 (d, 1H, Ar–H, J = 7.2 Hz), 8.04 (d, 1H, Ar–H, J = 7.6 Hz), 8.23 (d, 1H, Ar–H, J = 7.6 Hz), 8.35 (s, 1H, =CH), 8.4 (s, 1H, Ar–H), 8.42 (d, 1H, Pyr-H, J = 8 Hz), 8.75 (d, 1H, Pyr-H, J = 4 Hz), 9.24 (s, 1H, Pyr-H), 11.75 (s, 1H, −NH); ¹³C NMR (DMSO-d₆, 100 MHz): δ 53.6, 107.1, 110.6, 120.1, 123.2, 125.1, 135.7, 144.7, 149.0, 150.5, 160.1, 160.8, 172.5; LCMS (m/z): 368.23 [M + H]⁺; Elemental analysis for C₁₇H₁₃N₅O₃S Calcd: C, 55.58; H, 3.57; N, 19.06. Found: C, 55.60; H, 3.60; N, 19.05.

5-Methyl-N′-(4-methylbenzylidene)-2-(pyridin-3-yl)thiazole-4-carbohydrazide (5d)

Yellow solid; Yield: 85%; mp 216–218 °C; FT-IR (KBr cm^–1): 3444 (N–H), 3049 (Ar–H), 2861 (C–H), 1655 (−NHCO), 1605 (C=N), 652 (C–S–C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.43 (s, 3H, CH₃), 2.61 (s, 3H, thz-CH₃), 7.32 (d, 2H, Ar–H, J = 7.8 Hz), 7.52–7.55 (m, 1H, Pyr-H), 7.76 (d, 2H, Ar–H, J = 7.8 Hz), 8.01 (s, 1H, =CH), 8.1 (d, 1H, Pyr-H, J = 7.8 Hz), 8.70 (d, 1H, Pyr-H, J = 4.6 Hz), 9.24 (s, 1H, Pyr-H), 11.72 (s, 1H, N–H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 28.1, 54.2, 108.3, 110.5, 123.1, 124.2, 126.5, 134.2, 146.8, 148.9, 152.6, 161.6, 174.2; LCMS (m/z): 337.42 [M + H]⁺; Elemental analysis for C₁₈H₁₆N₄OS Calcd: C, 64.27; H, 4.79; N, 16.65. Found: C, 64.25; H, 4.80; N, 16.63.

N′-[(furan-2-ylmethylidene)-5-methyl]-2-(pyridin-3-yl)thiazole-4-carbohydrazide (5e)

Yellow solid; Yield: 68%; mp 120–122 °C; FT-IR (KBr cm^–1): 3434 (N–H), 3040 (Ar–H), 2860 (C–H), 1635 (−NHCO), 1609 (C=N), 658 (C–S–C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.62 (s, 3H, thz-CH₃), 6.53 (t, 1H, furan-H, J = 3.6 Hz), 7.29 (d, 1H, furan-H, J = 3.6 Hz), 7.44–7.47 (m, 1H, Pyr-H), 7.65 (d, 1H, furan-H, of J = 1.8 Hz), 7.85 (s, 1H, =CH), 8.22 (d, 1H, Pyr-H, J = 7.4 Hz), 8.68 (d, 1H, Pyr-H, J = 4.7 Hz), 9.18 (s, 1H, Pyr-H), 11.12 (s, 1H, N–H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 55.5, 108.0, 111.8, 120.9, 124.3, 126.4, 134.3, 147.3, 149.1, 151.6, 160.1, 175.3; LCMS (m/z): 313.35 [M + H]⁺; Elemental analysis for C₁₅H₁₂N₄O₂S Calcd: C, 57.68; H, 3.87; N, 17.94. Found: C, 57.69; H, 3.88; N, 17.95.

N′-(3,4-Dimethylbenzylidene)-5-methyl-2-(pyridin-3-yl)thiazole-4-carbohydrazide (5f)

Pale yellow solid; Yield: 82%; mp 210–212 °C; FT-IR (KBr cm^–1): 3445 (N–H), 3024 (Ar–H), 2865 (C–H), 1621 (−NHCO), 1618 (C=N), 659 (C–S–C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.28 (s, 3H, −CH₃), 2.31 (s, 3H, −CH₃), 2.56 (s, 3H, thz-CH₃), 7.01 (d, 1H, Ar–H, J = 8.4 Hz), 7.18–7.21 (m, 1H, Ar–H), 7.38 (s, 1H, Ar–H), 7.56–7.58 (m, 1H, Pyr-H), 8.01 (s, 1H, =CH), 8.30 (d, 1H, Pyr-H, J = 8 Hz), 8.68 (d, 1H, Pyr-H, J = 4 Hz), 9.13 (s, 1H, Pyr-H), 11.78 (s, 1H, −NH); ¹³C NMR (DMSO-d₆, 100 MHz): δ 28.1, 54.5, 107.5, 110.8, 121.0, 123.4, 126.7, 134.5, 146.8, 148.1, 151.4, 161.5, 175.2; LCMS (m/z): 351.28 [M]⁺; Elemental analysis for C₁₉H₁₈N₄OS Calcd: C, 65.12; H, 5.18; N, 15.99. Found: C, 65.10; H, 5.16; N, 15.97.

N′-(4-(Benzyloxy)benzylidene)-5-methyl-2-(pyridin-3-yl)thiazole-4-carbohydrazide (5g)

Yellow solid; Yield: 70%; mp 208–210 °C; FT-IR (KBr cm^–1): 3432 (N–H), 3073 (Ar–H), 2866 (C–H), 1653 (−NHCO), 1622 (C=N), 648 (C–S–C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.63 (s, 3H, thz-CH₃), 5.16 (s, 2H, CH₂), 7.18 (d, 2H, Ar–H, J = 7.8 Hz), 7.36 (m, 1H, Ar–H), 7.38 (d, 2H, Ar–H, J = 7.7 Hz), 7.47 (d, 2H, Ar–H, J = 7.7 Hz), 7.53–7.56 (m, 1H, Pyr-H), 7.87 (d, 2H, Ar–H, J = 7.8 Hz), 8.02 (s, 1H, =CH), 8.11 (d, 1H, Pyr-H, J = 7.6 Hz), 8.71 (d, 1H, Pyr-H, J = 4.6 Hz), 9.25 (s, 1H, Pyr-H), 11.73 (s, 1H, N–H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 55.6, 70.9, 108.3, 111.5, 114.8, 120.1, 124.4, 126.6, 127.1, 127.6, 128.9, 132.3, 133.7, 136.7, 146.8, 149.0, 151.5, 161.8, 164.8, 176.6; LCMS (m/z): 429.51 [M + H]⁺; Elemental analysis for C₂₄H₂₀N₄O₂S Calcd: C, 67.27; H, 4.70; N, 13.08. Found: C, 67.25; H, 4.72; N, 13.09.

N′-(4-Dimethylaminobenzylidene)-5-methyl-2-(pyridine-3-yl)thiazole-4-carbohydrazide (5h)

Yellow solid; Yield: 81%; mp 212–214 °C; FT-IR (KBr cm^–1): 3435 (N–H), 3043 (Ar–H), 2849 (C–H), 1654 (−NHCO), 1610 (C=N), 653 (C–S–C); ¹HNMR (DMSO-d₆, 400 MHz): δ 2.79 (s, 3H, thz-CH₃), 2.99 (s, 6H, N–Me), 6.81 (d, 1H, Ar–H, J = 8.8 Hz), 7.6 (d, 4H, Ar–H, J = 8.4 Hz), 8.0 (s, 1H, =CH), 8.39 (d, 1H, Pyr-H, J = 8.4 Hz), 8.73 (d, 1H, Pyr-H, J = 4.4 Hz), 9.19 (s, 1H, Pyr-H), 11.66 (s, 1H, N–H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 19.3, 52.1, 112.5, 120.4, 121.6, 124.9, 129.2, 134.3, 145.4, 147.5, 151.9, 161.6, 161.8, 167.5; LCMS (m/z): 366.46 [M + H]⁺; Elemental analysis for C₁₉H₁₉N₅OS Calcd: C, 62.45; H, 5.24; N, 19.16. Found: C, 62.45; H, 5.25; N, 19.15.

N′-(4-Hydroxybenzylidene)-5-methyl-2-(pyridin-3-yl)thiazole-4-carbohydrazide (5i)

Yellow solid; Yield: 83%; mp above 300 °C; FT-IR (KBr cm^–1): 3434 (N–H), 2980 (Ar–H), 2832 (C–H), 1664 (−NHCO), 1615 (C=N), 641 (C–S–C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.65 (s, 3H, thz-CH₃), 7.21 (d, 2H, Ar–H, J = 8.2 Hz), 7.76–7.78 (m, 1H, Pyr-H), 7.82 (d, 2H, Ar–H, J = 8.2 Hz), 8.10 (s, 1H, =CH), 8.42 (d, 1H, Pyr-H, J = 7.6 Hz), 8.72 (d, 1H, Pyr-H, J = 4.7 Hz), 9.15 (s, 1H, Pyr-H), 9.93 (s, 1H, O–H), 11.72 (s, 1H, N–H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 49.1, 112.3, 120.1, 121.3, 124.3, 129.1, 131.2, 133.1, 136.1, 146.7, 148.2, 151.3, 161.3, 171.6; LCMS (m/z): 339.39 [M + H]⁺; Elemental analysis for C₁₇H₁₄N₄O₂S Calcd: C, 60.34; H, 4.17; N, 16.56. Found: C, 60.32; H, 4.15; N, 16.57.

N′-(5-Bromo-2-hydroxybenzylidene)-5-methyl-2-(pyridin-3-yl)thiazole-4-carbohydrazide (5j)

Yellow solid; Yield: 78%; mp 258–260 °C; FT-IR (KBr cm^–1): 3432 (N–H), 2966 (Ar–H), 2866 (C–H), 1672 (−NHCO), 1612 (C=N), 651 (C–S–C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.69 (s, 3H, thz-CH₃), 6.99 (d, 1H, Ar–H, J = 8.4 Hz), 7.48 (d, 1H, Ar–H, J = 8.4 Hz), 7.78–7.80 (m, 1H, Pyr-H), 7.82 (s, 1H, Ar–H), 8.02 (s, 1H, =CH), 8.19 (d, 1H, Pyr-H, J = 7.6 Hz), 8.68 (d, 1H, Pyr-H, J = 4.7 Hz), 9.20 (s, 1H, Pyr-H), 10.52 (s, 1H, O–H), 11.70 (s, 1H, N–H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 51.2, 112.8, 120.0, 120.6, 124.5, 129.5, 131.8, 133.3, 135.8, 146.6, 147.3, 152.3, 161.1, 170.6; LCMS (m/z): 418.28 [M + H]⁺, 420.21 [(M + 2) + H]⁺; Elemental analysis for C₁₇H₁₃N₄BrO₂S Calcd: C, 48.93; H, 3.14; N, 13.43. Found: C, 48.91; H, 3.15; N, 13.44.

N′-[(1H-indol-3-yl)methylidene]-5-methyl-2-(pyridin-3-yl)thiazole-4-carbohydrazide (5k)

Yellow solid; Yield: 84%; mp above 300 °C; FT-IR (KBr cm^–1): 3438 (N–H), 2981 (Ar–H), 2866 (C–H), 1651 (−NHCO), 1614 (C=N), 658 (C–S–C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.62 (s, 3H, thz-CH₃), 7.04–7.14 (m, 2H, indolyl-H), 7.65 (m, 3H, indolyl-H), 7.79–7.81 (m, 1H, Pyr-H), 8.05 (s, 1H, =CH), 8.22 (d, 1H, Pyr-H, J = 7.4 Hz), 8.68 (d, 1H, Pyr-H, J = 4.7 Hz), 9.18 (s, 1H, Pyr-H), 10.23 (s, 1H, indole N–H), 11.12 (s, 1H, N–H); ¹³C NMR (DMSO-d₆, 100 MHz): δ 54.5, 108.0, 111.8, 116.3, 118.4, 120.2, 123.9, 124.4, 126.2, 128.1, 134.1, 147.7, 148.8, 152.2, 161.2, 175.5; LCMS (m/z): 362.42 [M + H]⁺; Elemental analysis for C₁₉H₁₅N₅OS Calcd: C, 63.14; H, 4.18; N, 19.38. Found: C, 61.90; H, 4.30; N, 20.03.

N′-(4-Hydroxy-3-methoxybenzylidene)-5-methyl-2-(pyridin-3-yl)thiazole-4-carbohydrazide (5l)

Yellow solid; Yield: 85%; mp 262–264 °C; FT-IR (KBr cm^–1): 3441 (N–H), 2975 (Ar–H), 2865 (C–H), 1672 (−NHCO), 1619 (C=N), 655 (C–S–C); ¹H NMR (DMSO-d₆, 400 MHz): δ 2.76 (s, 3H, thz-CH₃), 3.83 (s, 3H, −OCH₃), 7.01 (m, 1H, Ar–H), 7.31–7.33 (m, 1H, Ar–H), 7.40 (s, 1H, Ar–H), 7.58–7.56 (m, 1H, Pyr-H), 8.08 (s, 1H, =CH), 8.34 (d, 1H, Pyr-H, J = 8 Hz), 8.69 (d, 1H, Pyr-H, J = 4 Hz), 9.15 (s, 1H, Pyr-H), 9.65 (s, 1H, −OH), 11.82 (s, 1H, −NH); ¹³C NMR (DMSO-d₆, 100 MHz): δ 50.9, 114.4, 120.2, 121.2, 124.9, 126.0, 129.9, 131.1, 133.1, 135.2, 146.5, 147.2, 151.4, 161.9, 171.7; LCMS (m/z): [M + H]⁺; Elemental analysis for C₁₈H₁₆N₄O₃S Calcd: C, 58.68; H, 4.38; N, 15.21. Found: C, 58.69; H, 4.40; N, 15.22.

Biological Activity

In Vitro Anti-inflammatory Activity (Denaturation of the Bovine Serum Albumin Method)

The anti-inflammatory activity of the target compounds was determined by denaturation of the bovine serum albumin technique as per the Mizushima and Kobayashi³⁶ and Sakat et al.(37) reported method. The test sample contains the test compound and 1% aqueous solution of the bovine albumin fraction, and the pH of the reaction mixture was adjusted to pH 7.4 using suitable stripping solutions. Further, test samples were incubated at 37 °C for 20 min, and then, they was heated to 51 °C for 20 min. Later, it was cooled to ambient temperature, and the obtained turbidity of the sample was measured at 660 nm using a UV–visible spectrophotometer. The experiment was performed in triplicates using diclofenac sodium as the standard drug.

The anti-inflammatory activity of the titled compounds was estimated based on the percentage of inhibition of albumin denaturation using the following equation

Antimicrobial Activity by the Macrodilution Broth Method

The antimicrobial susceptibility of the target compounds was evaluated by the NCCLS macrodilution broth method. The MIC of the target compounds was evaluated according to the Clinical and Laboratory Standards Institute (CLSI)³⁸ protocol. As the definition of the Clinical and Laboratory Standards Institute document³⁹ states, “the MIC is the lowest concentration of the antimicrobial agent that completely inhibits growth of the organism in the tubes or microdilution wells as detected by the unaided eye”. Hence, the standard procedure was followed. Antimicrobial evaluation was carried out for the synthesized novel thiazole-based hydrazides against fungal strains such as Aspergillus flavus (A. flavus) (MTCC 1316), Trichoderma atroviridae (T. atroviridae) (MTCC 28036), Penicillium citranum (P. citranum) (MTCC 9849), and Candida albicans (C. albicans) (MTCC 461) and bacterial strains such as Staphylococcus aureus (S. aureus) (MTCC 9660), Bacillus subtilis (B. subtilis) (MTCC 441), Escherichia coli (E. coli) (MTCC 443), and Pseudomonas aeruginosa (P. aeruginosa) (MTCC 424) at varying concentrations (0.125–128 μg/mL). Nystatin and tetracycline were used as a standard drug for antifungal and antibacterial evaluation, respectively. Sterile test tubes containing the target compounds were placed in sterile Mueller Hinton broth (MHB) medium for bacteria and sterile Saboraud dextrose broth (SDB) for the fungi for which the test microorganisms were added. The MHB and SDB tubes with and without target compounds were used as the control. The MIC was detected as the lowest concentration of the target compound containing the tube showing no visible growth of the test microorganism. The experiment was performed in triplicates, and the data were analyzed by SPSS 20.0 software.

Statistical Analysis

Statistical analysis was carried out using SPSS software, version 20.0. The experiments were carried out in triplicates, and the data were expressed as mean ± standard deviation by one-way ANOVA. Turkey’s multiple comparison test was used to determine significant differences between the standard and synthesized compounds. Correlation analysis was carried out using Pearson’s correlation analysis using p = 0.01.

In Silico Prediction of Physicochemical and ADME Parameters

Physicochemical parameters of the designed compounds were predicted in silico using the QikProp module of Schrödinger. Various parameters predicted were molecular weight (M.Wt.), total solvent-accessible volume (TSAV), number of hydrogen bond donors (HBDs), number of hydrogen bond acceptors (HBAs), van der Waals polar surface area (PSA) of nitrogen and oxygen atoms, octanol/water partition coefficient (log P), aqueous solubility (log S), predicted Caco-2 cell permeability in nm/s (PCaco), apparent Madin Darby canine kidney (MDCK) permeability, and percentage of human oral absorption.³⁹⁻⁴¹ The details of the predicted values are presented in 5.

Molecular Docking Studies

Molecular docking of the significantly active compound was performed using the Glide module of Schrödinger software⁴² installed on the Intel Xenon W3565 processor and Ubuntu enterprise version 14.04 as an operating system. The selected target protein structure was retrieved from the RCSB protein data bank.⁴³ The titled ligand was drawn using Chemdraw 18.0 PerkinElmer software.

Ligand Preparation

The ligands used as an input for the docking study was sketched by ChemDraw software, and the structure was cleaned up for bond alignment. Then, ligands were incorporated into the workstation, and the energy was minimized using the OPLS3e (Optimized Potentials for Liquid Simulations) force field in LigPrep⁴³ (version 2019-1, Schrödinger). This minimization helps to assign bond orders, addition of hydrogens to the ligands, and conversion of 2D to the 3D structure for the docking studies. The generated output file (best conformations of the ligands) was further used for docking studies.

Protein Preparation

The protein preparation wizard⁴⁴ (version 2019–1, Schrödinger) was the main tool in Schrödinger to prepare and minimize the protein. The hydrogen atom was added to the protein, and charges were assigned. Het states were generated using Epik at pH 7.0 ± 2.0. The protein was preprocessed and refined, and the protein was modified by analyzing the workspace water molecules and others. The critical water molecules remained the same, and the rest of the molecules apart from heteroatoms was deleted. Finally, the protein was minimized using the OPLS3 force field. A grid was created by considering the co-crystal ligand, which was included in the active site of the protein of the selected target (PDB-3LN1).⁴⁵ After the final step of docking with the co-crystal ligand in the XP mode, RMSD was checked to validate the protein, and the RMSD value was found to be 0.20 Å.

Receptor Grid Generation

A receptor grid was generated around the protein (PDB: 3LN1) by choosing the inhibitory ligand (X-ray pose of the ligand in the protein). The centroid of the ligand was selected to create a grid box around it, and the van der Waal radius of the receptor atoms was scaled to 1.00 Å with a partial atomic charge of 0.25.

Docking Validation

The most straightforward way of validating the accuracy of specified parameters for docking studies is to redock the co-crystallized ligand back into the binding site of the protein and calculate the RMSD value between the crystallographic orientation and the docking pose. The calculation of RMSD is a convenient method to study how much a structure has diverged from its initial geometry. Lower RMSD value between its docked pose and crystallographic orientation is an indication of the suitability of the docking protocol. Therefore, before the screening of all ligands, the co-crystal structure of 3LN1 was chosen, and their inhibitor was redocked back to their active site. The RMSD value of the crystallographic orientation and the best docked pose was found to be 0.20 Å.^46,47

Docking Analysis

Docking and analysis of molecular docking were performed using the above prepared ligand and protein as the input. The results of the docking study were analyzed with the help of the XP visualizer (Version 2019-1, Schrödinger). All docking calculations were performed using the extra precision (XP) mode. A scaling factor of 0.8 and a partial atomic charge of less than 0.15 were applied to the atoms of the protein. The glide docking score was used to determine the best docked confirmation from the output. The interactions of these docked conformations were investigated further using the XP visualizer, and the results are depicted in the tables (Tables 1 and 5) and pictures (Figures 6–10).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c03386.

• FTIR, NMR (¹H and ¹³C), and LCMS spectra of the specific compounds and graphical representations of results of the antibacterial and antifungal evaluation of the compounds (PDF)

The authors declare no competing financial interest.