4-Chlorophenyl-N-furfuryl-1,2,4-triazole Methylacetamides as Significant 15-Lipoxygenase Inhibitors: an Efficient Approach for Finding Lead Anti-inflammatory Compounds.

Lipoxygenases (LOXs) are a class of enzymes that catalyze the production of pro-inflammatory mediators, such as leukotrienes and lipoxins, via an arachidonic acid cascade as soon as they are released from the membrane phospholipids after tissue injury. In continuation of our efforts in search for new LOX inhibitors, a series of chlorophenyl-furfuryl-based 1,2,4-triazole derivatives were prepared and evaluated for their 15-LOX inhibitory activities. A simple precursor, 4-chlorobenzoic acid (a), was consecutively transformed into benzoate (1), hydrazide (2), semicarbazide (3), and N-furfuryl 5-(4-chlorobenzyl)-4H-1,2,4-triazole (4), which when further merged with electrophiles (6a-o) resulted in end products (7a-o). The structural elucidations of the newly synthesized compounds (7a-o) were carried out by Fourier transform infrared, 1H-, 13C NMR spectroscopy, EI-MS, and HR-EI-MS spectrometry. The inhibitive capability of compounds (7a-o) on soybean 15-LOX was performed in vitro using the chemiluminescence method. The compounds 7k, 7o, 7m, 7b, and 7i demonstrated potent activities (IC50 17.43 ± 0.38, 19.35 ± 0.71, 23.59 ± 0.68, 26.35 ± 0.62, and 27.53 ± 0.82 μM, respectively). These compounds revealed 79.5 to 98.8% cellular viability as measured by the MTT assay at 0.25 mM concentration. The structure-activity relationship (SAR) studies showed that the positions and the nature of substituents bonded to the phenyl ring are important in the determination of 15-LOX inhibitory activities. ADME, in silico, and density functional theory studies supported the evidence as yet another class of triazoles with potential lead properties in search for anti-LOX compounds with a safe gastrointestinal safety profile for various inflammatory diseases. Further work is in progress on the synthesis of more derivatives in search for anti-inflammatory agents.

Introduction

Lipoxygenases (LOXs) are dioxygenase enzymes that incorporate oxygen into polyunsaturated fatty acids with (cis,cis)-1,4-pentadiene, such as arachidonic acid and linoleic acid, to produce their respective derivatives.[1−3] LOXs are found in a wide variety of organisms, including plants, fungi, and animals.[4] First, the LOX enzyme was discovered in soybean about 70 years ago, sparking substantial research into the enzyme’s role in plants.[5] In 1974, Hamberg and Samuelsson discovered that human platelets create 12S-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) when they are incubated with arachidonic acid.[6,7] This led to the hypothesis that LOXs could be found in animal tissues too. LOXs are classified as non-heme iron dioxygenases[8] and are named after the carbon chain position in arachidonic acid at which the enzyme catalyzes lipid peroxidation (e.g., 12-LOX oxygenates arachidonic acid at the 12-carbon).[9] 5-, 8-, 12-, and 15-LOXs are distinguished by the substrates of oxygenated positions in the arachidonic acid they utilize. 15-LOX is composed of two isoenzymes: 15-LOX-1 (also known as 12/15-LOX) and 15-LOX-2 (Figure ).[10,11]

Figure 1

(A) Depiction of different types of LOX enzymes acting on various positions of arachidonic acid and (B) illustration of the LOX reaction mechanism.[15]

The human 15-LOX-1 (h-15-LOX-1) is involved in the biosynthesis of anti-inflammatory and pro-inflammatory mediators such as lipoxins and eoxins and is found in both humans and animals.[12,13] This enzyme is found in high concentrations in the monocytes, bronchoalveolar epithelial cells, eosinophils, and macrophages of patients with asthma. Despite the fact that h-15-LOX-1 has been identified as a critical target in the drug discovery process for a variety of inflammatory diseases, the discovery of highly potent h-15-LOX-1 inhibitors and their potential application as a novel therapeutic strategy are still in the early stages of development.[14]

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely used and effective drugs on the market to get relief from inflammation. NSAIDs protect inflammatory responses by inhibiting the cyclooxygenase pathway of arachidonic acid metabolism. The long-term use of these medications can result in a variety of gastrointestinal, renal, and hepatic problems.[16] Few drugs are available on the market based on the LOX inhibition mechanism as anti-inflammatory agents, although there have been several reports of 15-LOX-1 inhibitors having heterocyclic cores ranging from 11-dihydro-5-thia-11-aza-benzo[a]-fluorene,[17] tryptamine-sulfonamide,[18] indolizine,[19] MLO94,[20] pyrazole-based sulfonamide,[21] and imidazole-based sulfonamide (Figure ).[22] They may be mildly effective, but because of their unfavorable physicochemical features and poor ligand efficiency values, these inhibitors are unlikely to be used therapeutically in clinical studies.[18,22]

Figure 2

Rationale of the present research plan and reported LOX inhibitors.[17−22]

Among the different types of heterocyclic compounds, 1,2,4-triazole derivatives are one of the most intriguing molecules because of their strong binding affinity for biological targets and their low toxicity in animal experiments, which make them ideal for the treatment of inflammatory diseases.[23−25] Recently, we have reported that 1,2,4-triazole derivatives possess azinane and phenylcarbamoyl moieties as 15-LOX inhibitors with excellent to good IC50 values.[26] In addition to the triazole moiety, several furan-based scaffolds have been identified with remarkable biological activity including nematocidal, insecticidal, antibacterial, antifungal, antiviral, antioxidant, anti-inflammatory, antimicrobial, anthelmintic, and anti-nociceptive properties.[27,28] A methoxyphenyl group has been shown to have anticancer effects in these compounds.[29]

Keeping in view the importance of both scaffolds, it was thought that some unique compounds having these groups in their structures would be desirable in order to generate more potent bioactive molecules with improved activities. The compounds can be synthesized using the molecular hybridization concept, led by different pharmaceutical industries, as an effective way to create bioactive molecules with improved inhibitory effects and lower toxicity by merging multiple bioactive pharmacophores together in a single compound.[30]

In this study, we synthesized novel 1,2,4-triazoles as potent 15-LOX inhibitors with fewer side effects by incorporating N-methylacetamide and furfural ring into the molecules. All the derivatives were successfully synthesized with good yield and were characterized by modern techniques. In vitro biological evaluation was carried out, and compounds were identified as potent to excellent 15-LOX inhibitors associated with low cellular toxicity. ADME, docking, and density functional theory (DFT) studies supported the in vitro findings in search for “lead” compounds as anti-inflammatory agents.

Results and Discussion

Chemistry

In the current work, the preparation of the new triazoles (7a–o) was carried out, and the details are described in Scheme . At first, ethyl 4-chlorobenzoate (1) was synthesized via the reaction of 4-chlorobenzoic acid with ethanol, which produced 4-chlorobenzohydrazide (2) by reacting with hydrazine hydrate. The 1H NMR spectrum of 2 presented two singlets at δ 3.91 (1H, −NH) and 8.01 (2H, −NH2), which deduced the −NHNH2 moiety. The hydrazide (2) then reacted with furfuryl isothiocyanate to afford N-furfuryl-5-(4-chlorophenyl)thiosemicarbazide (3), which was refluxed in basic media (10% NaOH) to synthesize an intramolecular cyclized compound, 5-(4-chlorophenyl)-4-furfuryl-4H-1,2,4-triazole-3-thiol (4). Meanwhile, the N-alkyl/aralkyl/aryl of 2-bromopropanamides (6a–o), the other reactant of target compounds, was synthesized by vigorously shaking alkyl/aralkyl/aryl amines (5a–o; Table ) separately with 2-bromopropyl bromide in an alkaline solution (pH 9–10). Compounds (7a–o) were attained by the subsequent reaction of compound 4 separately with the second reactants (6a–o) to obtain desired compounds, 7a–o, respectively. This work was executed via a protocol, as illustrated in the experimental part.

Scheme 1

Protocol for the Synthesis of Triazole Amides (7a–o) from 4-Chloroabenzoic Acid

Table 1

Alkyl/Aralkyl/Aryl-Substituted Amines (5a–o)

The structures of title compounds (7a–o) were confirmed by different spectroscopic techniques such as 1D-NMR (1H- and 13C NMR), 2D-NMR (HMQC, HMBC, and COSY), and mass spectrometry (EI-MS and HR-EI-MS). In the IR spectrum of compound 7g, the N–H stretching band associated with the amino group was observed at 3331 cm–1 as predicted. The amide C=O stretching bands emerging at 1641 cm–1 verified the fabrication of the designed compound. Likewise, the aromatic and aliphatic C–H stretching vibrations were subjected to the bands at 3021 and 2919 cm–1, respectively. The C=C and C=N stretching bands appeared at 1612 and 1538 cm–1, and lastly, C–N stretching was observed at 1220 cm–1. In the 1H NMR spectrum of compound 7k, the annotation of the distinctive a quadrat peak at δ 4.60 for the S–CH proton is a clear confirmation of the formation of the thioacetamides of the end product. In addition, two methyl singlets attached to the phenyl ring at the amide end appeared at δ 2.13 and 2.25, and the third singlet belonging to aromatic methine of the same phenyl was detected at δ 7.21 (1H, br s, H-6‴), whereas a doublet of methyl appeared at δ 1.70 (3H, d, J = 7.0 Hz, H-3″), and two doublets belonging to the methine protons of the furfuryl moiety became apparent at δ 6.34 and 7.40. Moreover, two doublet peaks at δ 5.19 (1H, d, J = 16.0 Hz, CH2α) and 5.27 (1H, q, J = 16.0 Hz, CH2β) assigned to methylene of the furfuryl and a quadrat at δ 4.62 (1H, q, J = 8.0 Hz, H-2″) were allocated to the sulfur-linked methine proton. A multiplet displayed at δ 6.91 (1H, d, J = 7.5 Hz, H-4‴) and 7.05 (1H, d, J = 7.5 Hz, H-3‴) earmarked the methine protons of the phenyl group. The 4-chlorophenyl moiety is recognized by two doublet peaks at δ 7.55 (2H, d, J = 8.6 Hz, H-3′,5′) and 7.64 (2H, d, J = 8.6 Hz, H-2′,6′). The 13C NMR spectrum (both BB and DEPT) of 7k disclosed a total of 22 carbon signals for 24 carbons, which supported the presence of three methyl, one methylene, 11 methine, and nine quaternary carbon atoms. The SCH2 and C=O carbons at δ 18.4, 47.5, and 171.3, respectively, also corroborated the formation of the thiopropanamide bonds of the end product. The two methyl carbons of the phenyl group became visible at 17.6 and 21.0, respectively. Besides, the methylene sulfur-attached carbon appeared at δ 47.5, and the signals at δ 42.9, 111.3, 111.4, 144.5, and 147.5 were raised by the furfuryl nucleus attached to the triazole ring. The signals at δ 124.3, 130.3, 131.3, and 138.5 marked the presence of the 4-chlorophenyl ring. The nitrogen attached to the phenyl ring displayed peaks at δ 126.1, 127.8, 129.8, 131.1, 135.6, and 136.7. The two quaternary carbons of the triazole ring presented their peaks at δ 152.9 and 155.9. The molecular formula of C24H23N4O2ClS was deduced using the data of HR-EI-MS, which exhibited the molecular ion [M]+ peak at m/z of 466.1250. The spectral data for the remaining derivatives of the series are presented in the experimental part.

LOX Inhibitory Activities and SAR Studies

This in vitro study demonstrates the inhibitory profiles of 1,2,4-triazoles (7a–o) against soybean 15-LOX using quercetin (IC50 4.86 ± 0.14 μM) and baicalein (IC50 2.24 ± 0.13 μM) as reference molecules (Table ). All compounds exhibit appreciable, from potent to excellent, inhibitions (IC50 17.43 ± 0.38 to 82.34 ± 0.58 μM) against the said enzyme. Admitting that the activity is a cumulative response of the gross molecule, a minimal SAR has been established by examining the effects of different alkyl/aralkyl/aryl groups (being a single erratic part of all molecules) on the inhibitive profiles of these molecules. The compounds 7k and 7o exhibit strong anti-LOX activities with IC50 values of 17.43 ± 0.38 and 19.35 ± 0.71 μM, respectively. Compound 7k is the most potent compound of all, and its activity is attributed to the electron-donating mesomeric effect of two methyl groups present at 2 and 5 positions. While comparing the inhibitory potential of 7k (IC50 17.43 ± 0.38 μM) with other compounds having the same substituents, it may be inferred that the electron-donating effect of dimethyl groups fluctuates significantly when attached to different positions at the phenyl group so as to exhibit diversified inhibitory activities ranging from IC50 17.43 ± 0.38 to 72.62 ± 0.49 μM. When the activity of the second potent inhibitor 7o (IC50 19.35 ± 0.71 μM) was compared with that of 7k, it was deduced that the absence of methyl substituents on the phenyl ring slightly diminished the activity. Furthermore, 7m and 7b demonstrated potent inhibition profiles (IC50 23.59 ± 0.68 and IC50 26.35 ± 0.62 μM, respectively), which can be explained by the fact that the presence of two methyl groups at adjacent carbons in 7m and the saturated n-propyl group at nitrogen in 7b increases the lipophilicity responsible for its potent activity. Excellent activity (IC50 32.14 ± 0.57 μM) was displayed by 7c that may be imputed to the saturated ring having a greater electronic charge, which can provide an enviable site for hydrophobic interactions. Significant equipotent activities were manifested by analogues 7a, 7d, 7f, 7g, and 7l (IC50 41.83 ± 0.35, 42.58 ± 0.76, 45.42 ± 0.72, 42.15 ± 0.67, and 41.27 ± 0.75 μM, respectively) and advocated that the other substituents of the molecules were not playing their role in maintaining enzyme inhibitory profiles. By collating the activity of 7g (IC50 42.15 ± 0.67 μM) with that of 7h (IC50 82.34 ± 0.58 μM), it is concluded that the OCH3 group present in the latter vetoes the activity, which may be due to the electron-withdrawing inductive effect of oxygen. Eventually, upon scrutinizing the SAR amongst the synthesized compounds, it may be alleged that the positions along with the nature of substituents bonded to the phenyl ring pose noteworthy fallout on 15-LOX inhibitory activity.

Table 2

15-LOX Inhibitory and Cytotoxicity Profiles of Compounds 7a–o (Mean ± SEM, n = 3)

	soybean 15-LOX assay		MTT assay
comp	inhibition (%) at 0.25 mM	IC50 (μM)	cell viability (%) at 0.25 mM
7a	81.41 ± 1.47	41.83 ± 0.35	93.23 ± 1.2
7b	89.27 ± 1.55	26.35 ± 0.62	90.12 ± 1.1
7c	87.34 ± 1.75	32.14 ± 0.57	89.23 ± 1.2
7d	81.65 ± 1.93	42.58 ± 0.76	85.12 ± 1.3
7e	68.32 ± 1.53	82.32 ± 0.48	86.21 ± 1.1
7f	82.13 ± 1.85	45.42 ± 0.72	90.21 ± 1.2
7g	83.53 ± 1.73	42.15 ± 0.67	92.12 ± 1.1
7h	68.25 ± 1.86	82.34 ± 0.58	67.53 ± 1.3
7i	89.45 ± 1.62	27.53 ± 0.82	91.12 ± 1.2
7j	71.53 ± 1.38	72.62 ± 0.49	95.51 ± 1.1
7k	91.23 ± 1.43	17.43 ± 0.38	94.43 ± 1.2
7l	82.34 ± 1.89	41.27 ± 0.75	92.21 ± 1.3
7m	89.61 ± 1.76	23.59 ± 0.68	82.21 ± 1.2
7n	81.35 ± 1.58	53.25 ± 0.76	85.21 ± 1.2
7o	92.67 ± 1.85	19.35 ± 0.71	89.21 ± 1.1
quercetin	92.63 ± 1.24	4.86 ± 0.14
baicalein	93.79 ± 1.27	2.24 ± 0.13
cyclophosphamide			56.50 ± 1.9
cisplatin			51.70 ± 1.8
curcumin			73.90 ± 1.5

Cellular Viability Assay

The cytotoxic properties of compounds (7a–o) toward blood mononuclear cells (MNCs) were determined at higher concentrations of 0.25 mM by the MTT assay, and data are given in Table . Results show that all these compounds, especially the most active ones, were the least toxic toward MNCs under the given experimental conditions. If the assay is carried out at their respective IC50 values, these compounds will show viability closer to 100%, which will exhibit further intactness at these values. Collectively, the data show that all compounds are the least toxic to the studied cells under the given assay conditions.

ADME Studies

A close relation between the polar surface area, rotatable bonds, and oral bioavailability of the drug is also established, and molecules with a polar surface area <140 Å2 and rotatable bonds <10 possess good oral bioavailability.[31] The molecular properties of compounds (7a–o) are given in Table . The data show that all compounds obeyed Lipinski’s rule. Log P (values between 2 and 4) or log D values (between 1 and 3) are good indicators of drug lipophilicity.[32,33] The drug-like properties of all compounds especially the most active 7k, 7m, and 7o are highly encouraged. Human intestinal absorption (HIA) is an important biopharmaceutical parameter for designing a drug molecule. To achieve high levels of bioavailability, the drug molecule needs to be highly dissolvable and stable in the gastrointestinal lumen and should also be significantly absorbed in the small and large intestines. In the current studies, all our compounds show high HIA values.[32]

Table 3

ADME Properties of Compounds 7a–o

comp	M log P	S + log P	S + log D	m. wt.	M_NO	T_PSA	HBDH	HIA
7a	3.332	4.609	4.609	482.992	7	82.18	1	0.005
7b	2.698	3.464	3.464	404.921	6	72.95	1	0.008
7c	3.565	4.809	4.809	459.013	6	72.95	1	0.006
7d	3.348	3.986	3.986	452.965	6	72.95	1	0.008
7e	3.616	4.359	4.359	452.965	6	72.95	1	0.005
7f	3.823	4.694	4.694	466.992	6	72.95	1	0.006
7g	3.636	4.398	4.398	468.965	7	82.18	1	0.005
7h	3.636	4.398	4.398	468.965	7	82.18	1	0.005
7i	3.823	4.663	4.663	466.992	6	72.95	1	0.005
7j	3.823	4.752	4.752	466.992	6	72.95	1	0.005
7k	3.823	4.696	4.696	466.992	6	72.95	1	0.005
7l	3.823	4.623	4.622	466.992	6	72.95	1	0.005
7m	3.823	4.786	4.786	466.992	6	72.95	1	0.006
7n	3.823	4.766	4.765	466.992	6	72.95	1	0.005
7o	3.405	4.109	4.109	438.938	6	72.95	1	0.014

Molecular Docking Studies

The major contributing amino acids of the active pocket involved in binding interactions were Trp772, Phe144, Asp768, Thr529 Arg533, Glu244, Lys526, Arg182, Val126, Ala145, and His147. In protein–ligand interactions, hydrogen bonding is of great importance as it helps to find the potent inhibitor. Compound 7k has shown the binding energy of −35.44 kJ/mol. As shown in Figure , in compound 7k, hydrogen bonding was found between Trp772 and the hydrogen attached with the nitrogen of N-methylcetamide. Arg533 formed π-cation with the furan ring of compound 7k. Val126 and Val520 made π-alkyl interactions with the p-xylene substitution of compound 7k. Phe144, Ala145, Hiss147, Phe143, Asn146, and Asn128 amino acid residues were involved in van der Waals interactions. Compound 7m has shown the binding energy of −37.4 kJ/mol. In compound 7m, Trp772 was the amino acid residue involved in one hydrogen bond with the hydrogen of the furan ring. Two π-alkyl interactions were found. One π-alkyl interaction was seen by Val126 with the furan ring and triazole of this compound. Another π-alkyl interaction was found between Val520 and chlorobenzene of 7m, and Lys526 formed π-cation interaction with the furan ring. The amino acids Phe144, Ala145, Tyr525, and Arg182 made van der Waals interactions with 7m. Compound 7o has shown the binding energy of −34.32 kJ/mol. In compound 7o, His515 was found to make one hydrogen bond with the oxygen of isobutyramide, and one π-alkyl interaction was formed between Val126 and the chlorobenzene part of this compound. One more interaction of π-anion was made by Asp243 with toluene of 7o, and Ala145 made a halogen bond with chlorine attached to it. Figure also shows that the amide π-stacked interaction was also established between Val520 and chlorobenzene. It also reveals that Thr529, Cys127, Asn146, Trp772, Phe144, Asn128, and Arg182 amino acid residues were involved in van der Waals interactions.

Figure 3

2D and 3D binding interactions of the most effective inhibitors (7k, 7m, and 7o) within the active pocket of LOX.

Density Functional Theory

The geometry optimizations were carried out by DFT/B3LYP using the basis set STO-3G. Optimized structures are shown in Figure while the geometric parameters are elaborated in Table .

Figure 4

Optimized structures of compounds 7a–o.

Table 4

Optimized Geometric Parameters of the Compounds 7a–o

	gas phase
comp	optimization energy (hartree)	polarizability (α) (a.u.)	dipole moment (Debye)
7a	–2204.7	224.5	5.67
7b	–1933.3	146.3	5.80
7c	–2047.9	162.2	6.44
7d	–2082.9	170.0	5.62
7e	–2082.9	171.9	5.75
7f	–2082.9	173.4	5.40
7g	–2121.5	177.7	5.70
7k	–2156.8	175.9	5.79
7i	–2121.5	178.3	5.68
7j	–2121.5	179.1	5.88
7k	–2121.5	178.6	5.99
7l	–2121.5	177.9	5.96
7m	–2121.5	180.6	5.53
7n	–2121.5	180.1	5.64
7o	–2044.4	166.8	5.51
Quercetin	–1083.7	115.0	3.89
Baicalein	–936.0	108.2	4.67

The frontier molecular orbital structures include HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) with specified energy values and energy gaps. On the basis of these energies, the behavior, reactivity, and kinetic stabilities of compounds are predicted. The smaller the orbital energy gap, the more the compound is polarized, has high chemical reactivity and low kinetic stability, and is termed as a soft molecule. The higher HOMO–LUMO (Figure ) energy gap denotes high kinetic stability and low chemical reactivity because it is energetically unfavorable to add electrons to a high-lying LUMO. HOMO is the outermost orbital containing electrons and tends to donate these electrons, while LUMO is the innermost orbital containing free places to accept electrons. In the present studies, compound 7m showed the lowest energy gap at 0.027 eV, which indicated it to be the most highly reactive among all compounds (Table ). Compound 7a showed a large energy gap at 0.079 eV, exhibiting high kinetic stability with low chemical reactivity. It had the highest HOMO energy at −0.117 eV. Thus, it would be the best electron donor, while the compound with the lowest LUMO energy value was 7a at 0.037, which would be the best electron acceptor. On the basis of high chemical reactivity, compounds 7h and 7n were the second-best compounds with the same value of the energy gap of 0.032 eV. Compound 7f is at the third rank of chemical reactivity with the value of the energy gap of 0.035 eV. The energetic parameters along with hardness and softness have been illustrated in Table .

Figure 5

HOMO–LUMO structures of compounds 7a–o.

Table 5

Energetic Parameters of Compounds 7a–o

comps	EHOMO (eV)	ELUMO (eV)	ΔEgap (eV)	hardness (η)	softness (S)
7a	–0.117	0.037	0.079	0.077	6.50
7b	–0.239	0.204	0.035	0.221	2.26
7c	–0.242	0.202	0.040	0.222	2.25
7d	–0.240	0.203	0.036	0.222	2.26
7e	–0.244	0.201	0.043	0.223	2.25
7f	–0.236	0.202	0.035	0.219	2.28
7g	–0.244	0.201	0.043	0.223	2.25
7h	–0.235	0.203	0.032	0.219	2.29
7i	–0.244	0.202	0.042	0.223	2.25
7j	–0.243	0.202	0.042	0.222	2.25
7k	–0.244	0.201	0.042	0.223	2.25
7l	–0.244	0.201	0.043	0.223	2.25
7m	–0.229	0.202	0.027	0.216	2.32
7n	–0.234	0.202	0.032	0.218	2.30
7o	–0.239	0.202	0.037	0.220	2.27
quercetin	–0.223	0.203	0.426	0.213	2.35
baicalein	–0.224	0.192	0.416	0.208	2.40

Conclusions

In continuation of our efforts to find a “lead” as anti-LOX, a new series of 4-chlorophenyl-N-furfuryl-1,2,4-triazole methylacetamide derivatives were synthesized, characterized, and evaluated against 15-LOX. Compounds 7k, 7o, 7m, 7b, and 7i were found as potent inhibitors with excellent cellular viability and low toxicity as measured by the MTT assay. The SAR studies of compounds implied that the type and position of substituents had notable footprints on the activities. ADME, DFT, and molecular docking studies further supported these molecules as potential “leads” against 15-LOX and promising candidates as anti-inflammatory agents. Our struggle in search for new “leads” remains continued.

Experimental Section

Materials and Methods

The requisite material and solvents of analytical grade were obtained from Sigma Aldrich and Alfa Aesar. The 1H- and 13C NMR spectra were obtained from a Bruker device working at 400 and 100 MHz, respectively, using tetramethylsilane as the internal standard. The IR spectra were achieved on a Shimadzu 460 FTIR spectrometer using a KBr disk. The mass spectra were commenced to be conducted on a JMSA 500 mass spectrometer and a JMS H × 110 spectrometer with a data system. The Gallen Kemp electro-thermal apparatus was used to find out the melting points of synthesized compounds.

Synthetic Procedures

Synthesis of Ethyl 4-Chlorobenzoate (1)

4-Chlorobenzoic acid (a, 10 g) was taken in a 500 mL round-bottom flask and dissolved in 300 mL of ethanol; then, 6 mL of conc. H2SO4 was added. The mixture was refluxed for about 6–7 h at 65–75 °C. The reaction progress was observed by TLC, and at the accomplishment of reaction, a redundant amount of cold water was added. A colorless oily product of ethyl 4-chlorobenzoate (1) was obtained at pH 8–9, maintained by Na2CO3, and separated by solvent extraction using dichloromethane. The physical and spectroscopic data of 1 are as follows:

Ethyl 4-Chlorobenzoate (1)

Colorless oil; yield: 86%; bp: 237–239 °C; IR (KBr disk, νmax cm–1): 3027 (Ar–H), 2952 (C–H), 1753 (C=O), 1611–1522 cm–1 (Ar–C=C); 1H NMR (400 MHz, CDCl3, ppm): δ 1.24 (3H, t, J = 6.8 Hz, CH3–CH2–O), 4.43 (2H, q, J = 6.8 Hz, CH3–CH2–O), 7.44 (2H, d, J = 8.5 Hz, H-3,5), 7.90 (2H, d, J = 8.4 Hz, H-2,6); 13C NMR (100 MHz, CDCl3, ppm): δ 14.1 (CH3–CH2–O), 60.2 (CH3–CH2–O), 126.4 (C-2,6), 130.5 (C-3,5), 130.2 (C-1), 136.2 (C-4), 170.2 (C==O); HR-EI-MS (m/z): 184.0296 [M]+ calcd for C9H9ClO2, 184.0282.

Synthesis of 4-Chlorobenzohydrazide (2)

In a 250 mL round-bottom flask, 9.8 mL of ethyl 4-chlorobenzoate (1; 0.05 mol) was taken, and 35 mL of hydrazine hydrate (80%) was added slowly while refluxing. The reaction mixture was refluxed for 5–6 h. The solvent was evaporated under vacuum, and yellow precipitates were formed, which were filtered, washed with cold water, and desiccated. The physical and spectroscopic data of 2 are as follows:

4-Chlorobenzohydrazide (2)

Yellow amorphous powder; yield: 96%; mp: 163–165 °C; IR (KBr disk, νmax cm–1): 3391, 3352 (N–H), 3032 (Ar–H), 2955 (C–H), 1675 (C=O), 1611–1515 cm–1 (Ar–C=C); 1H NMR (400 MHz, CDCl3, ppm): δ 7.53 (2H, d, J = 8.4 Hz, H-3,5), 7.80 (2H, d, J = 8.4 Hz, H-2,6); 13C NMR (100 MHz, CD3OD): δ 126.4 (C-3,5), 129.2 (C-2,6), 130.2 (C-1), 135.9 (C-4), 169.2 (C=O); HR-EI-MS (m/z): 170.0247 [M]+ calcd for C7H7ClN2O, 170.0238.

Synthesis of N-Furfuryl-1-(4-chlorobenzyl)thiosemicarbazide (3)

Compound 2 (8.9 g, 0.05 mol) was taken in a 50 mL round-bottom flask. An equimolar amount of furfuryl isothiocyanate (5.98 mL, 0.05 mol) was added drop-wise with stirring, followed by the addition of 30 mL of methanol as a solvent. The reaction mixture was refluxed and stirred for 8–10 h. Precipitates were formed on cooling and were filtered, washed, and dried. The physical state and spectroscopic data of 3 are given as follows

N-Furfuryl-1-(4-chlorobenzyl)thiosemicarbazide (3)

White powder; yield: 97%; mp: 153–156 °C; IR (KBr disk, νmax cm–1): 3033 (Ar–H), 2932 (C–H), 1631–1550 cm–1 (Ar–C=C, C=N); 1H NMR (400 MHz, CDCl3, ppm): δ N-fur [6.33 (1H, d, J = 1.8 Hz, H-2), 6.34 (1H, m, H-3), 7.41 (1H, d, J = 1.8 Hz, H-4)], 7.46 (2H, d, J = 8.4 Hz, H-3,5), 7.86 (2H, d, J = 8.4 Hz, H-2,6), 7.26 (5H, br s, N-Ph); 13C NMR (100 MHz, CDCl3, ppm): δ 126.1 (C-3,5), 130.9 (C-2,6), 131.0 (C-1), 137.1 (C-4), N-fur [111.2 (C-2), 110.8 (C-3), 143.7 (C-4), 149.0 (C-1)], 168.1 (C=O), 179.6 (C=S); HR-EI-MS (m/z): 309.0354 [M]+ calcd for C13H12N3O2ClS, 309.0339.

Synthesis of 5-(4-chlorobenzyl)-N-furfuryl-4H-1,2,4-triazole-3-thiol (4)

The semicarbazide 3 (0.05 mol, 15.5 g) was dissolved in 10% aqueous NaOH (30 mL) in a 100 mL round-bottom flask and refluxed for 6–8 h. After observing a single spot on TLC, the reaction mixture was transferred into ice-cold water and acidified to pH 5–6 by using dilute HCl. The white precipitates that appeared were filtered, washed, and desiccated. The physical and spectroscopic data of 4 are as follows:

5-(4-Chlorobenzyl)-N-furfuryl-4H-1,2,4-triazole-3-thiol (4)

White powder; yield: 94%; mp: 196–200 °C; IR (KBr disk, νmax cm–1): 3034 (Ar–H), 2936 (C–H), 1632–1551 cm–1 (Ar–C=C, C=N); 1H NMR (400 MHz, CDCl3, ppm): δ N-fur [5.14 (2H, s, CH2), 6.30 (1H, d, J = 1.7 Hz, H-2), 6.31 (1H, m, H-3), 7.47 (1H, m, H-3)], 7.51 (2H, d, J = 8.5 Hz, H-3′,5′), 7.61 (2H, d, J = 8.5 Hz, H-2′,6′); 13C NMR (100 MHz, CD3OD): δ N-fur [42.3 (CH2), 110.5 (C-2), 111.5 (C-3), 144.0 (C-4), 148.3 (C-1)], 129.1 (C-1‴), 129.6 (C-3′,5′), 130.1 (C-2′,6′), 131.5 (C-1′), 137.2 (C-4′), 152.6 (C-3), 156.1 (C-5), 168.7 (C-1″); HR-EI-MS (m/z): 291.0247. [M]+ calcd for C13H10N3OClS, 291.0233.

General Procedure for the Synthesis of Compounds 6a–o

Quantified amounts of alkyl/aralkyl/aryl amines (5a–o, 0.01 mol) were added separately in a quick-fit Erlenmeyer flask already having 20% aqueous Na2CO3 solution to keep the pH 9–10. The 2-bromopropionyl bromide (0.01 mol) was added drop by drop over 2–5 min with vigorous wobbling till the appearance of precipitates. Stirring was continued further for 1–2 h until cognate precipitates were formed, which were filtered, washed with cold water, and dried to get the respective electrophile(s), N-alkyl/aralkyl/aryl-substituted-2-bromopropionamides (6a–o).

General Method for the Synthesis of Compounds 7a–o

In a 50 mL round-bottom flask, 0.2 g (0.0006 mol) of compound 4 was taken and dissolved in ethanol. Furthermore, the KOH (0.6 mmol, 0.036 g) was added, and the mixture was stirred for 30 min at room temperature. The electrophiles (6a–o) were added at a slow pace separately to the mixture and additionally stirred for 4–5 h. An excess of cold water was added, and the precipitates of the desired products (7a–o) were formed separately, which were filtered, washed, and dried.

Spectral Characterization of Compounds 7a–n

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-(4-ethoxyphenyl)methylacetamide (7a)

Colorless amorphous powder; yield: 93%; mp: 176–180 °C; IR (KBr disk, νmax cm–1): 3361 (N–H), 3032 (Ar–H), 2928 (C–H), 1671 (C=O), 1613–1555 (Ar–C=C, C=N), 1248 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.29 (3H, t, J = 7.0 Hz, CH3–CH2–O), 1.60 (3H, d, J = 7.1 Hz, H-3″), 3.91 (2H, q, J = 7.0 Hz, CH3–CH2–O), 4.38 (1H, q, J = 7.1 Hz, H-2″), N-fur [5.10 (1H, d, J = 16.0 Hz, CH2α), 5.20 (1H, q, J = 16.0 Hz, CH2β), 6.24–6.26 (2H, m, H-3,4), 7.32 (1H, d, J = 1.8 Hz, H-5)], 6.74 (2H, d, J = 8.0 Hz, H-2‴,6‴), 7.35 (2H, d, J = 8.0 Hz, H-3‴,5‴), 7.53 (2H, d, J = 8.7 Hz, H-2′,6′), 7.47 (2H, d, J = 8.7 Hz, H-3′,5′); 13C NMR (100 MHz, CD3OD): δ 14.2 (C-3″), 17.6 (CH3–CH2–O), 47.5 (C-2″), N-fur [42.1 (CH2), 110.4 (C-3), 110.5 (C-4), 143.6 (C-5), 146.5 (C-2)], 63.5 (CH3–CH2–O), 114.5 (C-2‴,6‴), 121.5 (C-3‴,5‴), 123.0 (C-4′), 129.4 (C-3′,5′), 130.4 (C-2′,6′), 137.8 (C-1‴), 151.9 (C-4‴), 154.9 (C-3), 155.9 (C-5), 169.4 (C-1″); HR-EI-MS (m/z): 482.1199 [M]+ calcd for C24H23N4O3ClS, 482.1179.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-n-propylmethylacetamide (7b)

White amorphous powder; yield: 91%; mp: 137–141 °C; IR (KBr disk, νmax cm–1): 3354 (N–H), 3031 (Ar–H), 2933 (C–H), 1671 (C=O), 1616–1556 (Ar–C=C, C=N), 1246 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 0.77 (3H, t, J = 7.2 Hz, H-3‴), 1.37 (2H, hexet, J = 7.2 Hz, H-2‴), 1.46 (3H, d, J = 7.2 Hz, H-3″), 3.02 (2H, t, J = 7.2 Hz, H-1‴), 4.15 (2H, q, J = 7.2 Hz, H-2″), N-fur [5.16 (1H, d, J = 16.0 Hz, CH2α), 5.22 (1H, q, J = 16.0 Hz, CH2β), 6.17 (1H, d, J = 2.9 Hz, H-3), 6.25 (1H, t, J = 2.9 Hz, H-4), 7.35 (1H, d, J = 2.9 Hz, H-5)], 7.49 (2H, d, J = 8.5 Hz, H-2′,6′), 7.57 (2H, d, J = 8.5 Hz, H-3′,5′); 13C NMR (100 MHz, CD3OD): δ 10.2 (C-3‴), 17.4 (C-1″), 22.1 (C-2‴), 41.1 (C-1‴), 46.9 (C-2″), N-fur [41.5 (CH2), 109.4 (C-3), 110.2 (C-4), 143.3 (C-5), 147.6 (C-2)], 129.0 (C-2′,6′), 130.3 (C-3′,5′), 124.9 (C-1′), 136.7 (C-4′), 150.9 (C-3), 155.2 (C-5), 171.8 (C-1″); HR-EI-MS (m/z): 404.1093 [M]+ calcd for C19H21N4O2ClS, 404.1073.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-cyclohexylmethylacetamide (7c)

White amorphous powder; yield: 91%; mp: 157–161 °C; IR (KBr disk, νmax cm–1): 3349 (N–H), 3026 (Ar–H), 2929 (C–H), 1663 (C=O), 1611–1557 (Ar–C=C, C=N), 1242 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.15–1.31 (6H, m, H-3‴,4‴,5‴), 1.61–1.84 (4H, m, H-2‴,6‴), 1.57 (3H, d, J = 6.5 Hz, H-3″), 3.57 (1H, m, H-1‴), 4.28 (1H, q, J = 6.5 Hz, H-2″), N-fur [5.21 (1H, d, J = 16.0 Hz, CH2α), 5.29 (1H, q, J = 16.0 Hz, CH2β), 6.32–6.36 (2H, m, H-3,4), 7.42 (1H, d, J = 2.1 Hz, H-5)], 7.56 (2H, d, J = 7.5 Hz, H-3′,5′), 7.59 (2H, d, J = 7.5 Hz, H-2′,6′); 13C NMR (100 MHz, CD3OD): δ 18.6 (C-3″), 25.5 (C-2‴), 26.1 (C-6‴), 33.1 (C-3‴-5‴), 47.7 (C-2″), 48.1 (C-1‴), N-fur [42.9 (CH2), 111.3 (C-3), 111.5 (C-4), 144.5 (C-5), 147.7 (C-2)], 124.3 (C-1′), 130.3 (C-3′,5′), 131.3 (C-2′,6′), 138.6 (C-4′), 152.7 (C-3), 155.7 (C-5), 171.5 (C-1″); HR-EI-MS (m/z): 444.1406 [M]+ calcd for C22H25N4O2ClS, 444.1386.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio-N-benzylmethylacetamide (7d)

White amorphous powder; yield: 94%; mp: 140–142 °C; IR (KBr disk, νmax cm–1): 3363 (N–H), 3034 (Ar–H), 2930 (C–H), 1673 (C=O), 1611–1552 (Ar–C=C, C=N), 1250 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.59 (3H, d, J = 6.5 Hz, H-3″), 4.29 (2H, s, H-7‴), 4.41 (1H, q, J = 6.5 Hz, H-2″), N-fur [5.12 (1H, d, J = 16.0 Hz, CH2α), 5.19 (1H, q, J = 16.0 Hz, CH2β), 6.23 (1H, d, J = 2.1 Hz, H-3), 6.38 (1H, m, H-4), 7.43 (1H, d, J = 2.1 Hz, H-4)], 7.19–7.27 (5H, m, H-2‴-6‴), 7.58 (2H, d, J = 8.3 Hz, H-3′,5′), 7.63 (2H, d, J = 8.3 Hz, H-2′,6′); 13C NMR (100 MHz, CD3OD): δ 18.6 (C-3″), 48.2 (C-2″), 44.4 (C-7‴), N-fur [42.8 (CH2), 110.8 (C-3), 111.6 (C-4), 144.7 (C-5), 149.7 (C-2)], 126.3 (C-1′), 128.3 (C-4‴), 128.6 (C-3‴,5‴), 129.6(C-2‴,6‴), 130.4 (C-3′,5′), 131.6 (C-2′,6′), 138.1 (C-1‴), 139.5 (C-4′), 152.1 (C-3), 156.1 (C-5), 173.3 (C-1″); HR-EI-MS (m/z): 452.1093 [M]+ calcd for C23H21N4O2ClS, 452.1073.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-(o-tolyl)methylacetamide (7e)

White amorphous powder; yield: 91%; mp: 139–143 °C; IR (KBr disk, νmax cm–1): 3357 (N–H), 3034 (Ar–H), 2936 (C–H), 1672 (C=O), 1619–1558 (Ar–C=C, C=N), 1247 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.70 (3H, d, J = 8.0 Hz, H-3″), 2.19 (3H, s, CH3), 4.60 (1H, q, J = 8.0 Hz, H-2″), N-fur [5.23 (1H, d, J = 16.0 Hz, CH2α), 5.30 (1H, q, J = 16.0 Hz, CH2β), 6.32 (1H, d, J = 2.1 Hz, H-3), 6.35 (1H, m, H-4), 7.42 (1H, d, J = 2.1 Hz, H-5)], 7.11–7.20 (3H, m, H-3‴-5‴), 7.35 (1H, d, J = 7.3 Hz, H-6‴), 7.58 (2H, d, J = 8.3 Hz, H-3′,5′), 7.64 (2H, d, J = 8.3 Hz, H-2′,6′); 13C NMR (100 MHz, CD3OD): δ 18.1 (CH3), 18.6 (C-3″), 47.1 (C-2″), N-fur [42.9 (CH2), 111.2 (C-3), 111.5 (C-4), 144.7 (C-5), 148.2 (C-2)], 125.0 (C-2‴), 126.0 (C-4‴), 127.2 (C-6‴), 127.4 (C-3‴,5‴), 130.4 (C-3′,5′), 131.5 (C-2′,6′), 132.8 (C-1′),136.2 (C-1‴), 137.5 (C-4′), 152.9 (C-3), 156.3 (C-5), 171.8 (C-1″); HR-EI-MS (m/z): 452.1093 [M]+ calcd for C23H21N4O2ClS, 452.1073.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-(m-tolyl) Methylacetamide (7f)

White amorphous powder; yield: 91%; mp: 128–130 °C; IR (KBr disk, νmax cm–1): 3350 (N–H), 3027 (Ar–H), 2930 (C–H), 1664 (C=O), 1612–1558 (Ar–C=C, C=N), 1243 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.66 (3H, d, J = 6.0 Hz, H-3″), 2.29 (3H, s, CH3), 4.44 (1H, q, J = 8.0 Hz, H-2″), N-fur [5.19 (1H, d, J = 16.0 Hz, CH2α), 5.29 (1H, q, J = 16.0 Hz, CH2β), 6.28 (1H, d, J = 1.8 Hz, H-3), 6.32 (1H, m, H-4), 7.30 (1H, d, J = 1.8 Hz, H-5)], 6.92 (1H, d, J = 7.9 Hz, H-6‴), 7.17 (1H, t, J = 7.9 Hz, H-5‴), 7.31 (1H, d, J = 7.9 Hz, H-4‴), 7.33 (1H, s, H-2‴), 7.54 (2H, d, J = 7.0 Hz, H-3′,5′), 7.57 (2H, d, J = 7.0 Hz, H-2′,6′); 13C NMR (100 MHz, CD3OD): δ 18.4 (C-3″), 21.5 (CH3), 48.3 (C-2″), N-fur [42.9 (CH2), 111.1 (C-3), 111.5 (C-4), 144.6 (C-5), 148.1 (C-2)], 118.1 (C-2‴), 121.5 (C-6‴), 124.8 (C-1′), 126.2 (C-4‴), 129.6 (C-5‴), 130.3 (C-3′,5′), 131.5 (C-2′,6′), 138.4 (C-1‴), 138.9 (C-3‴), 139.7 (C-4′), 152.5 (C-3), 156.2 (C-5), 170.9 (C-1″); HR-EI-MS (m/z): 452.1093 [M]+ calcd for C23H21N4O2ClS, 452.1073.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-(2-ethylphenyl)methylacetamide (7g)

White amorphous powder; yield: 90%; mp: 108–110 °C; IR (KBr disk, νmax cm–1): 3355 (N–H), 3030 (Ar–H), 2932 (C–H), 1665 (C=O), 1616–1552 (Ar–C=C, C=N), 1245 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.09 (3H, t, J = 7.4 Hz, CH3–CH2), 1.69 (3H, d, J = 6.4 Hz, H-3″), 2.54 (2H, q, J = 7.4 Hz, CH3–CH2), 4.60 (1H, q, J = 6.4 Hz, H-2″), N-fur [5.25 (1H, d, J = 16.0 Hz, CH2α), 5.32 (1H, q, J = 16.0 Hz, CH2β), 6.29 (1H, d, J = 1.8 Hz, H-3), 6.35 (1H, m, H-4), 7.43 (1H, d, J = 1.8 Hz, H-5)], 7.17 (1H, d, J = 7.6 Hz, H-6‴), 7.20 (1H, d, J = 7.6 Hz, H-3‴), 7.24 (1H, t, J = 7.6 Hz, H-4‴), 7.31 (1H, t, J = 7.6 Hz, H-5‴), 7.59 (2H, d, J = 8.1 Hz, H-3′,5′), 7.66 (2H, d, J = 8.1 Hz, H-2′,6′); 13C NMR (100 MHz, CD3OD): δ 14.9 (CH3–CH2), 18.5 (C-3″), 25.3 (CH3–CH2), 47.9 (C-2″), N-fur [42.9 (CH2), 111.0 (C-3), 111.7 (C-4), 144.8 (C-5), 148.9 (C-2)], 126.3 (C-1′), 127.3 (C-6‴), 127.4 (C-3‴), 128.0 (C-4‴), 130.0 (C-5‴), 130.4 (C-3′,5′), 131.7 (C-2′,6′), 135.7 (C-1‴), 138.1(C-2‴), 140.2 (C-4′), 152.7 (C-3), 156.7 (C-5), 172.5 (C-1″); HR-EI-MS (m/z): 466.1250 [M]+ calcd for C24H23N4O2ClS, 466.1230.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-(2-methoxyphenyl)methylacetamide (7h)

White amorphous powder; yield: 89%; mp: 104–106 °C; IR (KBr disk, νmax cm–1): 3348 (N–H), 3025 (Ar–H), 2930 (C–H), 1658 (C=O), 1618–1553 (Ar–C=C, C=N), 1238 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.67 (3H, d, J = 6.2 Hz, H-3″), 3.82 (3H, s, CH3O), 4.60 (1H, q, J = 6.2 Hz, H-2″), N-fur [5.22 (1H, d, J = 16.0 Hz, CH2α), 5.27 (1H, q, J = 16.0 Hz, CH2β), 6.27 (1H, d, J = 1.8 Hz, H-3), 6.32 (1H, m, H-4), 7.39 (1H, d, J = 1.8 Hz, H-4)], 6.92 (1H, t, J = 7.5 Hz, H-4‴), 6.97 (1H, d, J = 7.5 Hz, H-6‴), 7.10 (1H, t, J = 7.5 Hz, H-5‴), 7.56 (2H, d, J = 8.0 Hz, H-3′,5′), 7.61 (2H, d, J = 8.0 Hz, H-2′,6′), 8.00 (1H, d, J = 7.5 Hz, H-3‴); 13C NMR (100 MHz, CD3OD): δ 18.2 (C-3″), 47.8 (C-2″), 56.3 (CH3O), N-fur [42.9 (CH2), 111.1 (C-3), 111.6 (C-4), 144.7 (C-5), 148.8 (C-2)], 111.7 (C-3‴), 121.9 (C-6‴), 122.5 (C-5‴), 125.1 (C-1′), 126.2 (C-4‴), 127.7 (C-1‴), 129.4 (C-3′,5′), 131.6 (C-2′,6′), 138.5 (C-4′), 151.0 (C-2‴), 155.6 (C-3), 156.4 (C-5), 171.2 (C-1″); HR-EI-MS (m/z): 468.1042 [M]+ calcd for C23H21N4O3ClS, 468.1022.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-(2,3-dimethylphenyl)methylacetamide (7i)

Colorless amorphous powder; yield: 94%; mp: 126–128 °C; IR (KBr disk, νmax cm–1): 3339 (N–H), 3021 (Ar–H), 2921 (C–H), 1649 (C=O), 1620–1544 (Ar–C=C, C=N), 1230 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.72 (3H, d, J = 6.5 Hz, H-3″), 2.07 (3H, s, CH3), 2.27 (3H, s, CH3), 4.61 (1H, q, J = 6.5 Hz, H-2″), N-fur [5.25 (1H, d, J = 16.0 Hz, CH2α), 5.32 (1H, q, J = 16.0 Hz, CH2β), 6.33–6.36 (2H, m, H-3,4), 7.42 (1H, d, J = 2.2 Hz, H-5)], 7.04–7.11 (3H, m, H-4‴-6‴), 7.59 (2H, d, J = 8.6 Hz, H-3′,5′), 7.67 (2H, d, J = 8.6 Hz, H-2′,6′); 13C NMR (100 MHz, CD3OD): δ 14.2 (CH3), 18.7 (C-3″), 20.6 (CH3), 47.8 (C-2″), N-fur [43.0 (CH2), 111.3 (C-3), 111.5 (C-4), 144.7 (C-5), 147.9 (C-2)], 124.3 (C-6‴), 124.7 (C-1′), 126.5 (C-5‴), 129.2 (C-4‴), 130.5 (C-2′,6′), 131.6 (C-3′,5′), 132.7 (C-2‴), 135.7 (C-3‴), 138.6 (C-4′), 138.7 (C-1‴), 153.9 (C-3), 156.1 (C-5), 171.8 (C-1″); HR-EI-MS (m/z): 466.1230 [M]+ calcd for C24H23N4O2ClS, 466.1230.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-(2,4-dimethylphenyl)methylacetamide (7j)

White amorphous powder; yield: 91%; mp: 90–92 °C; IR (KBr disk, νmax cm–1): 3339 (N–H), 3028 (Ar–H), 2930 (C–H), 1651 (C=O), 1618–1545 (Ar–C=C, C=N), 1232 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.68 (3H, d, J = 6.1 Hz, H-3″), 2.12 (3H, s, CH3), 2.27 (3H, s, CH3), 4.55 (1H, q, J = 6.1 Hz, H-2″), N-fur [5.24 (1H, d, J = 16.0 Hz, CH2α), 5.31 (1H, q, J = 16.0 Hz, CH2β), 6.27 (1H, d, J = 1.5 Hz, H-3), 6.34 (1H, m, H-4), 7.42 (1H, d, J = 1.5 Hz, H-5)], 6.98 (1H, d, J = 7.6 Hz, H-5‴), 7.02 (1H, s, H-3‴), 7.19 (1H, d, J = 7.6 Hz, H-6‴), 7.57 (2H, d, J = 8.5 Hz, H-3′,5′), 7.64 (2H, d, J = 8.5 Hz, H-2′,6′); 13C NMR (100 MHz, CD3OD): δ 18.0 (CH3), 18.6 (C-3″), 20.9 (CH3), 48.2 (C-2″), N-fur [43.0 (CH2), 110.9 (C-3), 111.6 (C-4), 144.8 (C-5), 148.9 (C-2)], 126.3 (C-1′), 126.4 (C-6‴), 128.0 (C-5‴), 130.4 (C-3′,5′), 131.7 (C-2′,6′), 132.2 (C-3‴), 133.8 (C-4‴), 133.9 (C-2‴), 137.5 (C-1‴), 138.2 (C-4′), 152.6 (C-3), 156.7 (C-5), 172.2 (C-1″); HR-EI-MS (m/z): 466.1230 [M]+ calcd for C24H23N4O2ClS, 466.1230.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-(2,5-dimethylphenyl)methylacetamide (7k)

White amorphous powder; yield: 94%; mp: 157–161 °C; IR (KBr disk, νmax cm–1): 3331 (N–H), 3021 (Ar–H), 2919 (C–H), 1641 (C=O), 1612–1538 (Ar–C=C, C=N), 1220 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.70 (3H, d, J = 7.0 Hz, H-3″), 2.13 (3H, s, CH3), 2.25 (3H, s, CH3), 4.60 (1H, q, J = 7.0 Hz, H-2″), N-fur [5.19 (1H, d, J = 16.0 Hz, CH2α), 5.27 (1H, q, J = 16.0 Hz, CH2β), 6.34 (2H, m, H-3,4), 7.40 (1H, br s, H-5)], 6.91 (1H, d, J = 7.5 Hz, H-4‴), 7.05 (1H, d, J = 7.5 Hz, H-3‴), 7.21 (1H, br s, H-6‴), 7.55 (2H, d, J = 8.6 Hz, H-3′,5′), 7.64 (2H, d, J = 8.6 Hz, H-2′,6′); 13C NMR (100 MHz, CD3OD): δ 17.6 (CH3), 18.4 (C-3″), 21.1 (CH3), 47.5 (C-2″), N-fur [42.9 (CH2), 111.3 (C-3), 111.4 (C-5), 144.5 (C-5), 147.6 (C-2)], 124.3 (C-1′), 126.1 (C-4‴), 127.8 (C-6‴), 129.8 (C-2‴), 130.3 (C-3′,5′), 131.1 (C-3‴), 131.3 (C-2′,6′), 135.6 (C-5‴), 136.7 (C-1‴), 138.5 (C-4′), 152.9 (C-3), 155.9 (C-5), 171.3 (C-1″); HR-EI-MS (m/z): 466.1250 [M]+ calcd for C24H23N4O2ClS, 466.1230.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-(2,6-dimethylphenyl)methylacetamide (7l)

White amorphous powder; yield: 95%; mp: 116–118 °C; IR (KBr disk, νmax cm–1): 3342 (N–H), 3032 (Ar–H), 2915 (C–H), 1632 (C=O), 1617–1545 (Ar–C=C, C=N), 1231 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.72 (3H, d, J = 6.5 Hz, H-3″), 2.11 (6H, s, 2 × CH3), 4.66 (1H, q, J = 6.5 Hz, H-2″), N-fur [5.25 (1H, d, J = 16.0 Hz, CH2α), 5.35 (1H, q, J = 16.0 Hz, CH2β), 6.31 (1H, d, J = 2.8 Hz, H-3), 6.36 (1H, m, H-4), 7.45 (1H, d, J = 2.8 Hz, H-5)], 7.04–7.09 (3H, m, H-3‴-5‴), 7.59 (2H, d, J = 8.3 Hz, H-3′,5′), 7.68 (2H, d, J = 8.3 Hz, H-2′,6′); 13C NMR (100 MHz, CD3OD): δ 18.3 (2 × CH3), 19.0 (C-3″), 49.6 (C-2″), N-fur [42.9 (CH2), 110.9 (C-3), 111.7 (C-4), 144.8 (C-5), 149.1 (C-2)], 126.3 (C-1′), 128.6 (C-4‴), 129.1 (C-3‴,5‴), 130.5 (C-2′,6′), 131.7 (C-3′,5′), 134.9 (C-1‴), 136.8 (C-2‴,6‴), 138.2 (C-4′), 152.6 (C-3), 156.6 (C-5), 172.2 (C-1″); HR-EI-MS (m/z): 466.1250 [M]+ calcd for C24H23N4O2ClS, 466.1230.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-(3,4-dimethylphenyl)methylacetamide (7m)

White amorphous powder; yield: 90%; mp: 138–140 °C; IR (KBr disk, νmax cm–1): 3335 (N–H), 3026 (Ar–H), 2919 (C–H), 1643 (C=O), 1618–1543 (Ar–C=C, C=N), 1226 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.64 (3H, d, J = 6.4 Hz, H-3″), 2.20 (6H, s, 2 × CH3), 4.40 (1H, q, J = 6.4 Hz, H-2″), N-fur [5.18 (1H, d, J = 16.0 Hz, CH2α), 5.29 (1H, q, J = 16.0 Hz, CH2β), 6.25 (1H, d, J = 2.1 Hz, H-3), 6.31 (1H, m, H-4), 7.39 (1H, d, J = 2.1 Hz, H-5)], 7.04 (1H, d, J = 8.5 Hz, H-6‴), 7.26 (1H, m, H-2‴,5‴), 7.53 (4H, br s, H-2′,3′,5′,6′); 13C NMR (100 MHz, CD3OD): δ 18.4 (C-3″), 19.3 (CH3), 19.9 (CH3), 48.4 (C-2″), N-fur [42.9 (CH2), 111.0 (C-3), 111.6 (C-4), 144.7 (C-4), 148.6 (C-1)], 118.7 (C-6‴), 122.3 (C-2‴), 125.7 (C-1′), 130.3 (C-2′,6′), 130.8 (C-5‴), 131.5 (C-3′,5′), 134.0 (C-4‴), 136.9 (C-3‴), 138.0 (C-1‴), 138.2 (C-4′), 152.3 (C-3), 156.6 (C-5), 170.9 (C-1″); HR-EI-MS (m/z): 466.1250 [M]+ calcd for C24H23N4O2ClS, 466.1230.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-(3,5-dimethylphenyl)methylacetamide (7n)

White amorphous powder; yield: 92%; mp: 148–151 °C; IR (KBr disk, νmax cm–1): 3326 (N–H), 3017 (Ar–H), 2912 (C–H), 1636 (C=O), 1612–1534 (Ar–C=C, C=N), 1217 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.67 (3H, d, J = 6.4 Hz, H-3″), 2.24 (6H, s, 2 × CH3), 4.49 (1H, q, J = 6.4 Hz, H-2″), N-fur [5.15 (1H, d, J = 16.0 Hz, CH2α), 5.26 (1H, q, J = 16.0 Hz, CH2β), 6.32 (2H, br s, H-3,4), 7.38 (1H, d, J = 1.3 Hz, H-5)], 6.74 (1H, s, H-4‴), 7.15 (2H, s, H-2‴,6‴), 7.52 (2H, d, J = 8.7 Hz, H-2′,6′), 7.60 (2H, d, J = 8.7 Hz, H-3′,5′); 13C NMR (100 MHz, CD3OD): δ 18.3 (C-3″), 21.5 (2 × CH3), 48.2 (C-2″), N-fur [42.9 (CH2), 111.4 (C-3,4), 144.3 (C-5), 147.1 (C-2)], 118.4 (C-2‴,6‴), 123.6 (C-1′), 126.9 (C-4‴), 130.2 (C-2′,6′), 131.2 (C-3′,5′), 138.4 (C-1‴), 138.6 (C-4′), 139.2 (C-3‴,5‴), 152.8 (C-3), 155.6 (C-5), 170.3 (C-1″); HR-EI-MS (m/z): 466.1250 [M]+ calcd for C24H23N4O2ClS, 466.1230.

2-((5-(4-Chlorophenyl)-4-(furan-2-ylmethyl)-4H-1,2,4-triazol-3-yl)thio)-N-phenylmethylacetamide (7o)

White amorphous powder; yield: 93%; mp: 154–160 °C; IR (KBr disk, νmax cm–1): 3330 (N–H), 3018 (Ar–H), 2915 (C–H), 1625 (C=O), 1611–1530 (Ar–C=C, C=N), 1213 cm–1 (C–N); 1H NMR (400 MHz, CD3OD): δ 1.66 (3H, d, J = 6.4 Hz, H-3″), 4.45 (1H, q, J = 6.4 Hz, H-2″), N-fur [5.15 (1H, d, J = 16.0 Hz, CH2α), 5.26 (1H, q, J = 16.0 Hz, CH2β), 6.28 (1H, d, J = 2.1 Hz, H-3), 6.31 (1H, m, H-4), 7.38 (1H, d, J = 2.1 Hz, H-5)], 7.09 (1H, t, J = 7.6 Hz, H-4‴), 7.30 (2H, t, J = 7.6 Hz, H-3‴,5‴), 7.51 (2H, d, J = 7.6 Hz, H-2‴,6‴), 7.53 (2H, d, J = 8.5 Hz, H-2′,6′), 7.55 (2H, d, J = 8.5 Hz, H-3′,5′); 13C NMR (100 MHz, CD3OD): δ 18.4 (C-3″), 48.2 (C-2″), N-fur [42.8 (CH2), 111.0 (C-3), 111.4 (C-4), 144.5 (C-5), 147.8 (C-2)], 120.7 (C-2‴,6‴), 124.6 (C-1′), 125.3 (C-4‴), 129.6 (C-3‴,5‴), 130.2 (C-3′,5′), 131.3 (C-2′,6′), 138.3 (C-1‴), 138.8 (C-4′), 152.4 (C-3), 156.0 (C-5), 170.7 (C-1″); HR-EI-MS (m/z): 438.0937 [M]+ calcd for C22H19N4O2ClS, 438.0917.

15-LOX Inhibition Assay

The soybean 15-LOX assay was essentially carried out by a chemiluminescence method as described earlier using quercetin and baicalein as standards.[34] The reaction mixture contained borate buffer, test compounds, and 15-LOX, followed by the addition of luminol and cytochrome C. Reaction was started by the addition of linoleic acid as the substrate, and relative counts were measured in 100–300 s in a 96-well plate reader. EZ-Fit enzyme kinetics software was used for the calculations of IC50 values.

Cell Viability Assay

The cell viability assay was carried out as reported earlier.[26,34] Mononuclear cells were separated by loading fresh blood taken from a healthy volunteer onto lymphocyte separation medium (density 1.077 g/mL) and used in the MTT assay as described.

Computational Approach

ADME Studies

The ADME (absorption, distribution, metabolism, and excretion) properties of compounds were determined using MedChem Designer software ver. 3.0, and data are given in Table . According to Lipinski’s rule of five, good oral bioavailability is observed if the molecular weight of the compound is <500, H-bond donors are <5, H-bond acceptors are <10, and logP value is < 5.[32,35] These compounds obeyed the rule. Moreover, the gastrointestinal safety profile was performed using the online web server ADMET lab 2.0.[35] The list of SMILES was uploaded on these online web servers, and it generated the required properties of the desired compounds.

Molecular Docking

Molecular docking is an essential tool for attaining possible binding poses for ligands. For this purpose, the molecular operating environment (MOE) 2015.10[36] was utilized to find the active site where ligands would bind and give the best binding poses. We took the protein from the RCSB PDB id: 3pzw.[37] The protein was prepared by opening it in the MOE window. Hetatoms and water molecules were deleted, and only polar hydrogen atoms were added. 3D protonated charges were added in the structures. MMFF94x force-field was selected. A separate database for adding ligands was made, and dummies of the active pocket in a targeted receptor were created. The receptor was selected before docking was started. Visualization was accomplished by opening the output file of results in the MOE window. The best pose with a minimum binding energy value was selected.

DFT Calculations

DFT calculations were performed using the Gaussian 09W program[38] using the DFT/B3LYP calculation setup with the basic set STO-3G. After performing, the Gaussian checkpoint file was visualized in GaussView 6.0.[39] It enabled us to determine the optimized structures and HOMO–LUMO orbital structures, which further assisted in determining the deep molecular properties of the selected compounds.