Convergent Synthesis, Kinetics and Computational Comprehensions of Indole- (Phenyl)triazole Bi-heterocycles Amalgamated with Propanamides as Elastase Inhibitors

doi:10.21203/rs.3.rs-4361622/v1

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Convergent Synthesis, Kinetics and Computational Comprehensions of Indole- (Phenyl)triazole Bi-heterocycles Amalgamated with Propanamides as Elastase Inhibitors

https://doi.org/10.21203/rs.3.rs-4361622/v1

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In the study presented here, 4-(1H-indol-3-yl)butanoic acid was sequentially converted into ethyl 4-(1H-indol-3-yl)butanoate, 4-(1H-indol-3-yl)butanohydrazide and 5-[3-(1H-indol-3-yl)propyl]-1,2,4-triazole-2-thiol as a nucleophile. In a parallel series of reactions, various electrophiles were synthesized by reacting aryl amines with 3-bromopropanoyl chloride to afford N-(aryl)-3-bromopropanamides. Then the nucleophilic substitution reaction of 5 was carried out with different electrophiles, to achieve final bi-heterocyclic derivative. The structural confirmation of all the synthesized compounds was done by IR, ¹H-NMR, ¹³C-NMR and CHN analysis data. The inhibitory effects of these bi-heterocyclic propanamides were evaluated against elastase, and all these molecules were identified as potent inhibitors relative to the standard used. The kinetics mechanism was ascribed by evaluating the Lineweaver–Burk plots, which revealed that compound 9d inhibited elastase competitively to form an enzyme–inhibitor complex. The inhibition constant K_i calculated from Dixon plots for this compound was 0.51 µM. These molecules also exhibited mild cytotoxicity toward red blood cell membranes, when analyzed through hemolysis. So, these molecules might be deliberated as nontoxic medicinal scaffolds for dealing with the elastase related ailments such as lungs diseases, cyclic neutropenia, pruritic skin disease and liver infection.

Indole

triazoles

propanamides

anilines

Elastase

hemolysis

Indole is derived from two words ‘indigo’ and ‘oleum’, as it was first separated from the reaction of indigo dye and oleum. Since then, the study of indigo dye developed indole chemistry. Indole is structurally a benzopyrrole ring (benzene and pyrrole rings fused at -2 and − 3 positions of pyrrole ring). Indole moiety is most commonly found in naturally occurring fungal metabolites, marine natural compounds, and indole alkaloids. Derivatives of indole are well known for their anticancer, anti-ulcer, anti-malarial, anti-convulsant, anti-inflammatory, antibiotics, analgesic, and numerous biological activities [1].

Heterocyclic triazoles are the vital building blocks in the formation of drugs. 1,2,4-Triazoles with large heterocyclic ring systems are integrated with a wide range of other therapeutically potent compounds to enhance their medicinal values. 1,2,4-Triazole derivatives are known to exhibit a wide range of biological activities [2], such as antidepressant [3], antibacterial [4–7] anticonvulsant [8] antifungal [9], anti-inflammatory [10], antiviral [11], analgesic [12], antitubercular [13], anticancer [14] and central nervous system (CNS) stimulants [15]. The enzyme inhibition potential of triazole against different enzymes is also found in the literature [16, 17].

Elastases are powerful serine proteases that belong to the chymotrypsin like elastase family. Probably it is the most destructive enzyme known for inflammation of connective tissue, which hydrolytically breakdown extracellular matrix like elastin (elastic fiber that supports connective tissues with collagen), fibronectin, collagen, and proteoglycans [18]. Pancreatic elastase is produced by the acinar cells of the pancreas. In the beginning, it is produced in an inactive form called zymogen and later on, it is activated in the duodenum by an enzyme trypsin. Elastase is reported for uncontrolled proteolytic damage in several pathological conditions [19–21]. That is why elastase is a striking therapeutic objective for pulmonary diseases, inhibition activity of elastase enzyme is a useful method to protect human skin against aging [22].

Nowadays microorganisms have developed immunity against already present drugs; besides toxicity and narrow spectrum activity are the reasons for the development of new drug compounds. Keeping given pharmacological importance of indole and triazoles moieties as elastase enzyme inhibitors [23–28] shown in Fig. 1, it was found worthwhile to synthesize new potent drugs including propanamides in their nucleus as shown in Scheme 1. We aim in our present work to create new leads of indole-triazole derivatives which will show better cytotoxic activities. Different interactions of our synthetic derivatives and enzyme active sites will be analyzed by molecular docking studies.

2.1. Chemistry

Various heterocyclic derivatives having propanamide group in them were synthesized in fine yields via multi-step convergent synthesis, according to Scheme-1. 4-(1H-Indol-3-yl)butanoic acid (1) was converted into ethyl 4-(1H-indol-3-yl)butanoate (2) by esterification using concentrated H₂SO₄and EtOH. As we know esterification is a reversible reaction, to avoid this fear, EtOH was used as reactant as well as solvent. Weak base was added to neutralize extra H₂SO₄and left over 4-(1H-indol-3-yl)butanoic acid 1 with excess of water followed by solvent extraction to get the product 2. During solvent extraction, ester got transferred to organic layer while salts moved to aqueous layer resulting in separation of ester 2 as brownish liquid, from other compounds. Second step was the conversion of ester 2 to respective 4-(1H-indol-3-yl)butanohydrazide (3) by adding calculated amount of N₂H₄.H₂O with organic solvent like methanol and refluxing it for 14 hours. The resulting carbohydrazide 3 was a light brown coloured solid, which was further reacted with phenyl isothiocyanate (4) resulting in the formation of heterocyclic ring of 5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazole-3-thiol (5), having mercapto substituent at its C-3 position. Different electrophiles N-(aryl)-3-bromopropanamides (8a-k) were synthesized in a parallel series of reaction by using various aryl amines, 6a-k, with 3-bromopropanoyl chloride (7). In the last step, two independently synthesized compounds 5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazole-3-thiol (5) and N-(aryl)-3-bromopropanamides (8a-k) were coupled together in the presence of LiH using aprotic polar medium to get required derivatives, 9a-k.

The structural analysis of one of the compounds is discussed here in detail for the articulacy of the reader. The molecule 9h was obtained as a brown amorphous solid. Its molecular formula, C₃₀H₃₁N₅SO, was established by CHN analysis data and its molecular ion peak in its EI-MS spectrum m/z 509, Figure 2. The number of proton and carbon resonances in its ¹H-NMR and ¹³C-NMR spectra were also in agreement with its deduced molecular formula. Different functionalities in this molecule were depicted by absorption bands in its IR spectrum (KBr, cm^-1) appearing at υ 3219 (N-H str.), 3092 (C-H str. of aromatic ring), 2931 (C-H str. of aliphatic), 1581 (C=C aromatic str.), 1155 (C-N-C bond str.), 670 (C-S str.) cm^-1. With the help of ¹H-NMR spectrum of this molecule, the indole heterocyclic core was identified clearly by the characteristic signals at δ 10.72 (s, 1H, NH-1), 7.37 (br.d, J = 7.5 Hz, 3H, H-7, H-2''' & H-6'''), 7.31 (br.d, J = 8.1 Hz, 1H, H-4), 7.05-7.02 (m, 2H, H-6 & H-3'''''), 6.97 (dist.d, J = 2.0 Hz, 1H, H-2), 6.94 (br.t, J = 7.7 Hz, 1H, H-5). The phenyl ring attached at 1-postion of 1,2,4-triazole was characterized by two resonances at δ 7.54-7.50 (m, 3H, H-3''', H-4''' & H-5''') and 7.37 (br.d, J = 7.5 Hz, 3H, H-7, H-2''' & H-6'''). Similarly, the resonances at δ 7.15 (br.s, 1H, H-6'''''), 7.05-7.02 (m, 2H, H-6 & H-3'''''), 6.88 (br.d, J = 7.5 Hz, 1H, H-4'''''), 2.22 (s, 3H, CH₃-5''''') and 2.09 (s, 3H, CH₃-2''''') ppm, were typical for a 2,5-dimethylphenyl group substituted at nitrogen atom in the molecule. The C and N-substituted propanamido group was recognized by two signals at δ 9.30 (s, 1H, CONH), 3.12 (t, J = 7.1 Hz, 2H, CH₂-4'''') and 2.42 (t, J = 7.2 Hz, 2H, CH₂-2''''). In the up-field region of spectrum, the signals of three intervening methylene groups at δ 2.66 (t, J = 7.4 Hz, 2H, CH₂-3'), 2.59 (t, J = 7.5 Hz, 2H, CH₂-1') and 1.89 (q, J = 7.4 Hz, 2H, CH₂-2'), were helpful to ascertain the connectivity of indole moiety from its 3-position to the 5-position of 1,2,4-triazole scaffold. The ¹H-NMR spectrum of this compound has been shown in Figure 3A while Figure 3B displayed the expanded aromatic region. The expanded aliphatic part of this spectrum has been shown in Figure 3C. The significant connectivity’s carbon with hydrogen further confirms the structure of compound 9h by its HMBC spectrum and is shown in Figure 4.

The carbon skeleton of this molecule was also fully supported by its ¹³C-NMR spectrum, shown in the Figure 5. The ¹³C-NMR spectrum depicted the signals of all the sixteen carbons, where the most downfield quaternary carbons signals at δ 155.52 (C-5'') and 149.56 (C-2'') belonged to the cyclized 1,2,4-triazole ring, thus confirming the formation of this ring. The other three quaternary carbons appearing at δ 136.24 (C-8), 127.01 (C-9) and 113.52 (C-3) ppm, were attribute of the indole core. The methine carbon resonances appearing at δ 122.19 (C-2), 120.77 (C-6), 118.19 (C-7), 118.05 (C-5), 111.26 (C-4) were also coherent with the indole moiety. The phenyl group substituted at nitrogen atom of 1,2,4-triazole was ascertained by one quaternary carbon signal at δ 133.13 (C-1'''), and three signals of five methine carbons resonances at δ 129.08 (C-4'''), 129.78 (C-3''' & C-5''') and 127.19 (C-2''' & C-6'''). The C and N-substituted propanamido group was rational by four discrete resonances, one for a carbonyl group at δ 168.69 (C-1'''') and two methylene group at δ 35.74 (C-2'''') and 28.00 (C-3''''). The 2,5-dimethylphenyl group substituted at the nitrogen atom was demonstrated by eight signals at δ 135.92 (C-1'''''), 134.79 (C-5'''''), 129.98 (C-3'''''), 128.53 (C-2'''''), 125.79 (C-4'''''), 125.58 (C-6'''''), 20.51 (CH₃-5'''''), 17.41 (CH₃-2''''') while three methylene signals appearing in up-field region at δ 26.96 (C-2'), 24.35 (C-1') and 23.96 (C-3'), were assignable to those methylene groups, which were connecting the indole and triazole moieties together.

In its mass spectrum, the molecular ion peak of parent molecule was observed at m/z 509, which was further cleaved in following ways (Figure 6):

So, on the basis of aforesaid cumulative evidences, the structure of 9h was confirmed and it was named as N-(2,5-dimethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9h). A similar protocol was exercised for the structural characterization (Figure S1-S20) of other derivatives in the synthesized series.

2.2. Elastase inhibition activity

Synthesized compounds 9a-k was subjected to elastase enzyme inhibition to test their inhibitory potential. It is clear from the resulting IC₅₀ (µM) values given in Table 1 that all the synthesized compounds 9a-k showed significant inhibitory potential against elastase enzyme relative to the standard Oleanolic acid with IC₅₀value 13.453 ± 0.015 µM. Considering the inhibitory potential of synthesized compounds 9a-k with different substituted R (alkyl) groups on them, a captive SAR (structure activity relationship) was rationalized. The general structural units of the examined compounds are labeled in Figure 7.

2.3. Structure activity relationship (SAR)

Compound 9a, 9b and 9c were having a single substitution of methyl group at ortho, meta- and para-positions in aryl part, respectively (Figure 8). The IC₅₀values of these compounds, 9a (IC₅₀ = 0.324 ± 0.039 µM), 9b (IC₅₀ = 1.231 ± 0.077 µM) and 9c (IC₅₀ = 3.538 ± 0.741µM) showed significant enzyme inhibition as compared to the standard oleanolic acid (IC₅₀ = 13.453 ± 0.015 µM). In the mutual comparison of these compounds, the molecule having ortho-substitution exhibited more inhibitory potential followed by meta and then para substitution, probably due to the reason that the aryl part in former molecule was more compact in size.

There was a noteworthy increase in the activity of compound 9d (IC₅₀ = 0.142 ± 0.014 µM) when it was substituted with a little bulky ethyl group at para position, instead of single methyl group in 9c. But in contrast, activity was comparatively reduced when a more polar ethoxy group was substituted at same para position in compound 9e (IC₅₀ = 38.338 ± 0.993µM), probably due to the electronic factors (Figure 9).

In dimethylated compounds with substitution of methyl groups at 2,3 and 2,4-positions (9f & 9g), very comparable results were obtained. The molecule with 2,5-subsitution (9h; IC₅₀ = 13.548 ± 0.516 µM) was less active while the compound with 2,6-dimethyl groups (9i; IC₅₀ = 0.819 ± 0.094 µM) was more active among these. Hereby, both ortho-positions were occupied by the small methyl groups, the molecule became more suited for the inhibition of this enzyme probably, due to the more compactness in the size of aryl part (Figure 10).

Similarly, the comparison of 3,4 and 3,5-dimethyl derivatives (Figure 11) exposed that 9k (IC₅₀ = 0.199 ± 0.027µM) behaved as a better inhibitor than 9j (IC₅₀ = 1.371 ± 0.881 µM), probably due to the presence of methyl groups symmetrically on meta-positions in former molecule, which presumably made it more suited for the inhibition of studied enzyme.

2.4. Kinetic mechanism

To accredit the inhibitory mode of these bi-heterocycles against elastase, the inhibition kinetic studies were assayed. Based on the obtained IC₅₀ results, the most potent compound 9d was chosen to determine their inhibition type and inhibition constant. The kinetic results of the enzyme by the Lineweaver-Burk plot of 1/V versus substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide 1/[S] in the presence of different inhibitor concentrations gave a series of straight lines. The result of the Lineweaver-Burk plot of 9d showed that V_max remained the same without significantly affecting the slopes. K_m increased with increasing concentration, while V_max remained the same with insignificant differences. This behavior specified that 9d inhibited the enzyme in a competitive manner (Figure 12A). The second plot (Figure 12B) of slope against a concentration of 9d exposed EI dissociation constant. K_i was calculated from the inhibitor concentration of 9d versus the slope and K_i was found to be 0.51 µM.

2.5. Computational Comprehensions

2.5.1. Optimized Molecular Structure Analysis

The DFT method was applied to analyze the optimized (the lowest energy conformation) molecular structures of the ligands. All the ligands from 9a to 9k were fully optimized by using B3LYP method with 6-31G* basis set to obtain the minimized energy states of the ligands. The optimized molecular geometry parameters of compound 9a are presented as an example from among the members of the series. These parameters included bond lengths, bond angles and dihedral angles. The optimized geometry of 9a is displayed in Figure 13. The carbonyl group (C=O) showed bond length of 1.223Å which is almost same as to average carbonyl bond length 1.23Å. The N10-N11 bond length of triazole ring showed the value of 1.387Å. This shorter bond length is due to double bonds in triazole ring. The C1-C2, C8-N10, N45-C37 and C38-C1 were displayed at 1.54Å, 1.309Å, 1.383Å and 1.502Å, respectively. The bond between C-54-C62 is found to be 1.512Å. The three bond angles that we mentioned are present between C8-N12-C9, C9-S26-C27 and C48-N50-C51 and their angles has been found to be 104.1°, 98.6° and 129.4° and the torsion angles between C33-C32-C38-C1 were formed dihedral to be 1.73°, which indicated a planer arrangement of atoms that is completely shown in Figure S21 of supporting information.

2.5.2. The frontier molecular orbitals (FMOs)

The study of FMOs are necessary for the investigation of molecular structure and electrochemical behavior of our designed ligands (Mumit, et al., 2020).The FMOs usually consists of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO). The HOMO and LUMO plots of designed compounds were shown in Figure 2 while complete set of these orbitals for all compounds were given in Figure S22 of supporting information. The ionization potential is closely associated with the HOMO, which is the outermost orbital occupied by electrons. And LUMO left unoccupied by an electron is thought to be the first innermost orbital that is empty and associated with binding affinity. Energy levels of FMOs in molecules provide information on the probability of charge transfer and distribution in HOMO and LUMO orbitals. A thorough examination of HOMOs and LUMOs of designed compounds uncovers that HOMOs are mainly delocalized on terminal side of 4-phenyl-4H-1,2,4-triazole which is behaving as electron donor for all designed molecules. While on the other hands, the LUMOs are delocalized on central moiety of 1H-indole which is acting as electron acceptor moiety in all compounds. Overall, an intramolecular charge transfer is obvious from the study of FMOs of all compounds.

2.5.3. Global reactivity descriptors (GRD)

The GRDs based on DFT calculations are crucial to get insights into the molecular reactivity and electronic properties of the molecules. The GRDs usually consists of the ionization energy (I), electron affinity (A), chemical potential, global hardness, and electrophilicity. By using energies of HOMO and LUMO orbitals as an input, theses parameters were calculated. The methodological details with working equations were given in our previous works [29, 30]. The ionization energies of all the compounds under study were somewhat similar, yet molecules 9a and 9j showcased lower ionization energies than the rest, which indicateed that they may lose electron more quickly to ionize themselves. The molecule 9a, 9g and 9j showed relatively larger chemical hardness indicating their resistance to electronic cloud polarization while for others were collected in Figure S23 of supporting information. The GRDs might be helpful for structure-activity relationships where such descriptors are used quantitatively to build molds for new predictions.

Table 2. Global reactivity parameters of 9a-9k compounds including ionization energy (I), electron affinity (A), chemical hardness (η), chemical potential (μ), electronegativity index (χ), electrophilicity index (ω) and softness (S).

Global Reactivity Parameters		9a	9b	9c	9d	9e	9f	9g	9h	9i	9j	9k
Ionization Energy (I)	5.22		5.24	5.24	5.24	5.24	5.24	5.23	5.24	5.25	5.23	5.24
Electron Affinity (A)	0.73		0.75	0.75	0.75	0.76	0.75	0.74	0.75	0.77	0.74	0.75
Chemical Hardness (η)	2.25		2.24	2.24	2.24	2.24	2.24	2.25	2.24	2.24	2.25	2.24
Chemical potential (μ)	-2.97		-3.00	-2.99	-2.99	-3.00	-2.99	-2.99	-2.99	-3.01	-2.99	-2.99
Electronegativity index (χ)	2.97		3.00	2.99	2.99	3.00	2.99	2.99	2.99	3.01	2.99	2.99
Electrophilicity index (ω)	5.66		5.64	5.65	5.65	5.61	5.65	5.66	5.65	5.60	5.67	5.66
Softness (S)	0.45		0.45	0.45	0.45	0.45	0.45	0.45	0.45	0.45	0.45	0.45

2.5.4. Protein and Ligands Interactions

In structure-based drug designing molecular docking provides pivotal role for evaluating for potential ligand and target (protein) interactions [31]. As a result of the protein being docked against various ligands, various ligand poses and rankings are obtained. Afterwards ligand with higher value of binding affinity and best pose with zero RMSD was selected. Then molecular interactions of selected ligands are visualized by utilizing discovery studio visualizer. The results of molecular docking in the form of binding affinity are tabulated in Table 3 and Intermolecular interactions between ligand and protein are illustrated in Figure 15.

Table 3. Binding energies kcal/mol of all designed ligand docked with protein PDB ID 7est.

Compounds	Protein	Binding Energy (kcal/mol)
9a		-7.3
9b		-8.1
9c		-7.6
9d	7est (Elastase)	-7.5
9e		-6.8
9f		-6.1
9g		-7.5
9h		-7.2
9i		-7.0
9j		-8.0
9k		-6.6

According to the binding affinities of different ligands the ligands 9b, 9j and 9c have higher negative value of binding affinities but 9b is highest binding affinity value from all. It is crucial to note that highest binding affinity means the best pose of ligand has most favorable interaction with protein. Additionally, our calculated binding energies showed a respectable correlation with the corresponding experimental inhibition constants that were determined using experimental methods. The ligand 9b has -8.1 kcal/mol highest binding energy and favorable pose of ligand formed two hydrogen bonds with GLU-70 residue of proteins and TYR-82 formed pi-pi stacked bond. LEU-151, LEU-73 and ALA-39 formed alky and Pi-alkyl interaction with ligand.ARG-65A formed unfavorable interaction due to two bonds formation of hydrogen atom. And ligand formed Pi donor hydrogen bond with LEU-171 amino acid pf protein. Ligand 9c that showed binding energy -7.6 formed two hydrogen bonds with two residues of GLN-206 and GLN-321 of protein.LEU-123 formed Pi donor hydrogen bond with ligands. THR-207 formed Pi sigma bond with ligand.PRO-28 and VLA-122 formed Alkyl and Pi Alkyl bonds. The binding energy of designed compound 9j is -7.0 and interacted with ligand through three conventional hydrogen bonds with VAL-216, HIS-57, SER-195 residues of protein.HIS-57 also formed Pi-cation bond and TRP-57 Pi -Pi T Shaped with ligand.VLA99 and ALA99A interacted with ligand through Pi-Alkyl bonds.

2.5.5. Molecular electrostatic potentials (MEPs)

Electrostatic potential maps of studied compounds are obtained at same level of theory and are displayed in Figure 16. MEPs plots are characteristic tool for highlighting the reactivity, nucleophilic, and electrophilic sites in a molecule [32]. They determine the most favorable position for nucleophilic or electrophilic attack with assistance of different color codes [30]. In MEPs maps, maximum electronic density is indicated by red color while region of lowest electronic density is shown by blue color. The decreasing trend for electronic density in MEPs follows the pattern of red > orange > yellow > green > blue. In our designated compounds, maximum negative potential is indicated by red color over the oxygen atom due to its higher electronegativity which can be favorable site for electrophilic attack. The presence of yellow color in surroundings of sulfur atom speaks for relatively lower negative potential due to lower electronegative character of sulfur as compared to oxygen. Slight negative potential is also found over the terminal phenyl rings as indicated by yellow color. In all the designated compounds, maximum positive potential is concentrated over the triazole ring which predicts the possibility of favorable nucleophilic attack over this position. Therefore, overall presence of different colors leads to predict nucleophilic and electrophilic sites of attack in a molecule and to explore chemical and electronic properties completely explained in Figure S24 –S25 of supporting information.

2.5.6. Density of States (DOS)

The density of states is an important technique to apprehend the importance of different moieties in a molecular compound. Total and partial DOS were calculated by projecting them on the individual fragments of the molecules. For DOS calculations, the molecule 9a was chosen because it is best representative of the overall series of the compounds. The three fragments including 4-phenyl-4H-1,2,4-triazole, 1H-indole and 3-(methylthio)-N-(o-tolyl)propanamide were divided as Fragment A, and B and C, respectively as shown in Figure 17. The DOS around the orbital gap showed a significant contribution of electronic states from Fragment A (red) while one the other hands, significant states can be seen in LUMO region from Fragment B (blue). The contribution of fragment C is mainly deep in valence and conduction regions around -9.0 to -11.0 eV and from 3.5 to 5.8 eV, respectively. Overall dominancy was from electronic states of 3-(methylthio)-N-(o-tolyl)propanamide moiety as seen by the blue line of DOS plots.

From the presented study, it was concluded that the newly synthesized indole-(phenyl)triazole bi-heterocycles amalgamated with a propanamide entity were acquired through a convergent approach in excellent yields and their structures were articulated by IR, ¹H-NMR, ¹³C-NMR, HMBC, EI-MS and CHN analysis data. All the synthesized derivatives depicted effective potential against elastase enzyme and showed lower IC₅₀ values as compared to standard used. Moreover, all the compounds were attributed with mild hemolysis. The kinetics mechanism was exposed by lineweaver-burk plots which revealed that, 9d, inhibited elastase competitively with an inhibition constants (K_i) of 0.51 µM. All these results, attributed these bi-heterocyclic propanamides as useful and praiseworthy addition to the list of potential therapeutic agents suitable for the treatment of elastase related ailments.

4.1. General information

Chemicals were purchased from Sigma Aldrich & Alfa Aesar (Germany) and solvents of Analytical grades were supplied by local suppliers. By using open capillary tube method, melting points were taken on Griffin and George apparatus and were uncorrected. By using thin layer chromatography (with ethyl acetate and n-hexane (30:70) as mobile phase), initial purity of compounds was detected at 254 nm. IR peaks were recorded on a Jasco-320-A spectrometer by using KBr pellet method. ¹H-NMR signals were recorded at 600 MHz and ¹³C-NMR at 150 MHz in DMSO-d₆ using Bruker spectrometers.

4.2. Synthesis of ethyl 4-(1H-indol-3-yl)butanoate (2)

4-(1H-Indol-3-yl)butanoic acid (0.2 mol.; 1) dissolved in absolute ethanol (70 mL) and catalytic amount of concentrated sulfuric acid (20 mL) was taken in a 500 mL round bottom (RB) flask and refluxed for 8 hours until the maximum completion of reaction, supervised through TLC. At the end, reaction mixture was neutralized with 10% aqueous sodium carbonate (40 mL). The product was isolated by solvent extraction by chloroform (50 mL × 3). The solvent was distilled off and the required ester, 2, was obtained as reddish brown [33, 34].

4.2.1. Synthesis of 4-(1H-indol-3-yl)butanohydrazide (3)

4-(1H-Indol-3-yl)butanoate (0.15 mol.; 2) in 60 mL methanol and hydrazine monohydrate (80%; 25 mL) was taken in a 500 mL round bottom flask. The reaction mixture was stirred for 14 hours at room temperature (RT). After absolute conversion, the acid hydrazide was obtained by distilling methanol off from the reaction mixture. The precipitates were filtered, washed with cold n-hexane and air-dried to get pure 4-(1H-indol-3-yl)butanohydrazide (3) [33, 34].

4.2.2. Synthesis of 5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazole-3-thiol (5)

4-(1H-Indol-3-yl)butanohydrazide (0.13 mol.; 3) in absolute ethanol (30 mL) and KOH (0.13 mol) were taken in a round bottom flask. Phenyl isothiocyanate (4) was added subsequently. Mixture was refluxed for 16 hours. On completion of the reaction, excess chilled distilled water and dil. HCl was added to adjust pH 5–6. The precipitates were filtered, washed and dried to get desired cyclized product, 5 [35].

4.2.3. Preparation of N-(Aryl)-3-bromopropanamides (8a-k)

Preparation of various N-(aryl)-3-bromopropanamides (8a-k) was carried out by reaction of various aryl amines (6a-k) with 3-bromopropanoyl chloride (7) in equimolar quantities (0.001 moles) and shaking manually in 10% aqueous Na₂CO₃ solution. Solid precipitates were formed after 20–30 min, which were filtered and washed with cold distilled water to obtain the desired electrophiles, 8a-k.

4.2.4. Synthesis of N-(aryl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamides (9a-k)

5-[3-(1H-Indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazole-3-thiol (0.2 g, 5) was added in DMF (5 mL) contained in a 250 mL round bottom flask at room temperature, added one pinch of LiH and stirred for 30 min. Then, different N-(aryl)-3-bromopropanamides (8a-k) were added in equimolar amounts, one by one separately, in each respective reaction and stirred for 60–70 hours. The completion of the reaction was monitored by TLC and after its completion; the reaction mixture was quenched with ice cold water (100 mL). The respective derivatives, 9a-k, were collected through filtration or solvent extraction according to nature of the product.

4.3. Spectral characterization of synthesized compounds, 9a-k

4.3.1. 3-({5-[3-(1H-Indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(2-methylphenyl)propanamide (9a)

Light brown sticky liquid; Mol. formula C₂₉H₂₉N₅SO; Mol. mass 495 gmol^− 1; yield: 74%; IR (KBr, cm^− 1): υ 3217 (N-H str.), 3055 (C-H str. of aromatic ring), 2878 (C-H str. of aliphatic), 1583 (C = C aromatic str.), 1156 (C-N-C bond str.), 680 (C-S str.) cm^− 1; ¹H-NMR (600 MHz, DMSO-d₆, δ/ppm): δ 10.72 (s, 1H, NH-1), 9.37 (s, 1H, CONH), 7.55–7.51 (m, 3H, H-3''', H-4''' & H-5'''), 7.40–7.36 (m, 3H, H-7, H-2''' & H-6'''), 7.31 (br.d, J = 8.2 Hz, 2H, H-4 & H-5'''''), 7.19 (br.d, J = 7.5 Hz, 1H, H-3'''''), 7.14 (br.t, J = 7.5 Hz, 1H, H-4'''''), 7.08 (br.t, J = 7.6 Hz, 1H, H-5'''''), 7.04 (br.t, J = 7.2 Hz, 1H, H-6), 6.97 (dist.d, J = 1.9 Hz, 1H, H-2), 6.93 (br.t, J = 7.1 Hz, 1H, H-5), 3.33 (t, J = 6.7 Hz, 2H, CH₂-3''''), 2.81 (t, J = 6.7 Hz, 2H, CH₂-2''''), 2.68–2.64 (m, 2H, CH₂-3'), 2.59 (t, J = 7.6 Hz, 2H, CH₂-1'), 2.14 (s, 3H, CH₃-2'''''), 1.89 (q, J = 7.3 Hz, 2H, CH₂-2'); ¹³C-NMR (150 MHz, DMSO-d₆, δ/ppm): δ 168.96 (C-1''''), 155.53 (C-5''), 149.57 (C-3''), 136.21 (C-8), 136.10 (C-1'''''), 133.09 (C-1'''), 131.71 (C-2'''''), 130.19 (C-3'''''), 129.82 (C-4'''), 129.80 (C-3''' & C-5'''), 127.18 (C-2''' & C-6'''), 126.99 (C-9), 125.81 (C-6'''''), 125.15 (C-4'''''), 125.05 (C-5'''''), 122.18 (C-2), 120.78 (C-6), 118.18 (C-7), 118.05 (C-5), 113.51 (C-3), 111.26 (C-4), 35.49 (C-2''''), 27.96 (C-3''''), 26.94 (C-2'), 24.32 (C-1'), 23.93 (C-3'), 17.83 (CH₃-2'''''); Anal. Calc. for C₂₉H₂₉N₅SO (495.21): C, 70.28; H, 5.90; N, 14.13. Found: C, 70.20; H, 5.81; N, 14.09. EI-MS (m/z): 495 (C₂₉H₂₉N₅SO) ̇⁺ (M)⁺, 352 (C₁₉H₂₀N₄SO)⁺, 334 (C₁₉H₁₈N₄S) ̇⁺, 245 (C₁₃H₁₅N₃S)⁺, 191 (C₉H₉N₃S)⁺, 158 (C₁₁H₁₂N)⁺, 143 (C₁₀H₉N)⁺, 130 (C₉H₈N)⁺, 106 (C₇H₈N)⁺, 91 (C₆H₅N)⁺, 78 (C₅H₄N)⁺.

4.3.2. 3-({5-[3-(1H-Indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(3-methylphenyl)propanamide (9b)

Light grey amorphous solid; Mol. formula C₂₉H₂₉N₅SO; Mol. mass 495 gmol^− 1; yield: 74%; melting point 140–141 ^oC; IR (KBr, cm^− 1): υ 3217 (N-H str.), 3057 (C-H str. of aromatic ring), 2874 (C-H str. of aliphatic), 1583 (C = C aromatic str.), 1157 (C-N-C bond str.), 682 (C-S str.) cm^− 1; ¹H-NMR (600 MHz, DMSO-d₆, δ/ppm): δ 10.69 (s, 1H, NH-1), 10.00 (s, 1H, CONH), 7.51 (br.s, 3H, H-3''', H-4''' & H-5'''), 7.43 (br.s, 1H, H-2'''''), 7.35 (br.s, 3H, H-7, H-2''' & H-6'''), 7.33 (br.s, 1H, H-6'''''), 7.31 (br.d, J = 8.1 Hz, 1H, H-4), 7.15 (br.t, J = 7.6 Hz, 1H, H-5'''''), 7.03 (br.t, J = 7.5 Hz, 1H, H-6), 6.93 (br.s, 1H, H-2), 6.91 (br.t, J = 7.8 Hz, 1H, H-6), 6.85 (br.d, J = 7.3 Hz, 1H, H-4'''''), 4.42 (t, J = 7.2 Hz, 2H, CH₂-3''''), 2.90 (t, J = 7.2 Hz, 2H, CH₂-2''''), 2.61 (t, J = 7.2 Hz, 2H, CH₂-3'), 2.48 (t, J = 7.3 Hz, 2H, CH₂-1'), 2.24 (s, 3H, CH₃-3'''''), 1.81 (q, J = 7.2 Hz, 2H, CH₂-2'); ¹³C-NMR (150 MHz, DMSO-d₆, δ/ppm): δ 168.10 (C-1''''), 166.45 (C-5''), 150.86 (C-3''), 138.91 (C-1'''''), 137.77 (C-3'''''), 136.22 (C-8), 134.05 (C-1'''), 129.49 (C-4'''), 129.37 (C-3''' & C-5'''), 128.43 (C-5'''''), 128.05 (C-2''' & C-6'''), 126.93 (C-9), 123.86 (C-4'''''), 122.22 (C-2), 120.78 (C-6), 119.72 (C-2'''''), 118.14 (C-7), 118.06 (C-5), 116.37 (C-6'''''), 113.25 (C-3), 111.24 (C-4), 34.54 (C-2''''), 30.59 (C-3''''), 25.91 (C-2'), 24.70 (C-1'), 23.64 (C-3'), 21.08 (CH₃-2'''''); Anal. Calc. for C₂₉H₂₉N₅SO (495.21): C, 70.28; H, 5.90; N, 14.13. Found: C, 70.22; H, 5.80; N, 14.09. EI-MS (m/z): 495 (C₂₉H₂₉N₅SO) ̇⁺ (M)⁺, 352 (C₁₉H₂₀N₄SO)⁺, 334 (C₁₉H₁₈N₄S) ̇⁺, 245 (C₁₃H₁₅N₃S)⁺, 191 (C₉H₉N₃S)⁺, 158 (C₁₁H₁₂N)⁺, 143 (C₁₀H₉N)⁺, 130 (C₉H₈N)⁺, 106 (C₇H₈N)⁺, 91 (C₆H₅N)⁺, 78 (C₅H₄N)⁺.

4.3.3. 3-({5-[3-(1H-Indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(4-methylphenyl)propanamide (9c)

Dark brown sticky liquid; Mol. formula C₂₉H₂₉N₅SO; Mol. mass 495 gmol^− 1; yield: 81%; IR (KBr, cm^− 1): υ 3219 (N-H str.), 3055 (C-H str. of aromatic ring), 2874 (C-H str. of aliphatic), 1585 (C = C aromatic str.), 1156 (C-N-C bond str.), 681 (C-S str.) cm^− 1; ¹H-NMR (600 MHz, DMSO-d₆, δ/ppm): δ 10.71 (s, 1H, NH-1), 9.89 (s, 1H, CONH), 7.55–7.54 (m, 3H, H-3''', H-4''' & H-5'''), 7.44 (br.d, J = 7.0 Hz, 2H, H-2''''' & H-6'''''), 7.36 (br.s, 3H, H-7, H-2''' & H-6'''), 7.31 (br.d, J = 7.7 Hz, 1H, H-4), 7.08 (br.d, J = 6.8 Hz, 2H, H-3''''' & H-5'''''), 7.04 (br.t, J = 7.2 Hz, 1H, H-6), 6.97 (br.s, 1H, H-2), 6.93 (br.t, J = 6.5 Hz, 1H, H-5), 3.31 (t, J = 5.7 Hz, 2H, CH₂-3''''), 2.76 (t, J = 5.7 Hz, 2H, CH₂-2''''), 2.66 (t, J = 7.2 Hz, 2H, CH₂-3'), 2.58 (t, J = 7.2 Hz, 2H, CH₂-1'), 2.23 (s, 3H, CH₃-4'''''), 1.88 (q, J = 6.9 Hz, 2H, CH₂-2'); ¹³C-NMR (150 MHz, DMSO-d₆, δ/ppm): δ 168.68 (C-1''''), 155.53 (C-5''), 149.54 (C-3''), 136.46 (C-1'''''), 136.23 (C-8), 133.10 (C-1'''), 132.00 (C-4'''''), 129.76 (C-4'''), 129.69 (C-3''' & C-5'''), 128.99 (C-3''''' & C-5'''''), 127.20 (C-2''' & C-6'''), 127.01 (C-9), 122.18 (C-2), 120.77 (C-6), 119.03 (C-2''''' & C-6'''''), 118.18 (C-7), 118.05 (C-5), 113.52 (C-3), 111.26 (C-4), 36.01 (C-2''''), 27.79 (C-3''''), 26.96 (C-2'), 24.33 (C-1'), 23.95 (C-3'), 20.33 (CH₃-4'''''); Anal. Calc. for C₂₉H₂₉N₅SO (495.21): C, 70.28; H, 5.90; N, 14.13. Found: C, 70.21; H, 5.83; N, 14.09. EI-MS (m/z): 495 (C₂₉H₂₉N₅SO) ̇⁺ (M)⁺, 352 (C₁₉H₂₀N₄SO)⁺, 334 (C₁₉H₁₈N₄S) ̇⁺, 245 (C₁₃H₁₅N₃S)⁺, 191 (C₉H₉N₃S)⁺, 158 (C₁₁H₁₂N)⁺, 143 (C₁₀H₉N)⁺, 130 (C₉H₈N)⁺, 106 (C₇H₈N)⁺, 91 (C₆H₅N)⁺, 78 (C₅H₄N)⁺.

4.3.4. N-(4-Ethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9d)

Dark brown sticky liquid; Mol. formula C₃₀H₃₁N₅SO; Mol. mass 509 gmol^− 1; yield: 89%; IR (KBr, cm^− 1): υ 3185 (N-H str.), 3080 (C-H str. of aromatic ring), 2928 (C-H str. of aliphatic), 1587 (C = C aromatic str.), 1153 (C-N-C bond str.), 680 (C-S str.) cm^− 1; ¹H-NMR (600 MHz, DMSO-d₆, δ/ppm): δ 10.74 (s, 1H, NH-1), 9.87 (s, 1H, CONH), 7.51–7.50 (m, 3H, H-3''', H-4''' & H-5'''), 7.46–7.43 (m, 2H, H-2''''' & H-6'''''), 7.37–7.36 (m, 3H, H-7, H-2''' & H-6'''), 7.31 (br.d, J = 8.0 Hz, 1H, H-4), 7.10 (br.d, J = 7.9 Hz, 2H, H-3''''' & H-5'''''), 7.05 (br.t, J = 7.6 Hz, 1H, H-6), 7.01 (br.s, 1H, H-2), 6.93 (br.t, J = 7.1 Hz, 1H, H-5), 4.39 (t, J = 6.1 Hz, 2H, CH₂-3''''), 2.76–2.73 (m, 2H, CH₂-2''''), 2.66 (t, J = 7.3 Hz, 2H, CH₂-3'), 2.62 (t, J = 7.1 Hz, 2H, CH₂-1'), 2.53–2.52 (m, 2H, CH₃CH₂-4'''''), 1.95–1.88 (m, 2H, CH₂-2'), 1.12 (t, J = 7.4 Hz, 3H, CH₃CH₂-4'''''); ¹³C-NMR (150 MHz, DMSO-d₆, δ/ppm): δ 168.66 (C-1''''), 153.06 (C-5''), 149.52 (C-3''), 138.58 (C-4'''''), 136.55 (C-1'''''), 136.23 (C-8), 133.90 (C-1'''), 129.70 (C-4'''), 129.69 (C-3''' & C-5'''), 127.79 (C-2''' & C-6'''), 127.00 (C-9), 125.50 (C-3''''' & C-5'''''), 122.21 (C-2), 120.78 (C-6), 118.18 (C-7), 118.06 (C-5), 119.25 (C-2''''' & C-6'''''), 113.57 (C-3), 111.27 (C-4), 37.03 (C-2''''), 27.52 (CH₃CH₂-4'''''), 27.29 (C-3''''), 27.22 (C-2'), 23.99 (C-1'), 23.76 (C-3'), 15.61 (CH₃CH₂-4'''''); Anal. Calc. for C₃₀H₃₁N₅SO (509.22): C, 70.70; H, 6.13; N, 13.74. Found: C, 70.66; H, 6.09; N, 13.71. EI-MS (m/z): 509 (C₃₀H₃₁N₅SO) ̇⁺ (M)⁺, 366 (C₂₀H₂₂N₄SO)⁺, 334 (C₁₉H₁₈N₄S) ̇⁺, 245 (C₁₃H₁₅N₃S)⁺, 191 (C₉H₉N₃S)⁺, 158 (C₁₁H₁₂N)⁺, 143 (C₁₀H₉N)⁺, 130 (C₉H₈N)⁺, 120 (C₈H₁₀N)⁺, 91 (C₆H₅N)⁺, 78 (C₅H₄N)⁺.

4.3.5. N-(4-Ethoxyphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9e)

Dark brown sticky liquid; Mol. formula C₃₀H₃₁N₅SO₂; Mol. mass 525 gmol^− 1; yield: 89%; IR (KBr, cm^− 1): υ 3187 (N-H str.), 3083 (C-H str. of aromatic ring), 2928 (C-H str. of aliphatic), 1587 (C = C aromatic str.), 1153 (C-N-C bond str.), 679 (C-S str.) cm^− 1; ¹H-NMR (600 MHz, DMSO-d₆, δ/ppm): δ 10.73 (s, 1H, NH-1), 9.76 (s, 1H, CONH), 7.53–7.51 (m, 3H, H-3''', H-4''' & H-5'''), 7.44–7.39 (m, 3H, H-7, H-2''''' & H-6'''''), 7.36 (br.d, J = 7.8 Hz, 2H, H-2''' & H-6'''), 7.32 (br.d, J = 8.0 Hz, 1H, H-4), 7.04 (br.dt, J = 0.9 & 7.2 Hz, 1H, H-6), 7.00 (dist.d, J = 2.1 Hz, 1H, H-5), 6.93–6.92 (m, 1H, H-2), 6.83–6.80 (m, 2H, H-3''''' & H-5'''''), 3.99–3.93 (m, 2H, CH₃CH₂O-4'''''), 3.53 (t, J = 8.7 Hz, 2H, CH₂-3''''), 3.03 (t, J = 8.7 Hz, 2H, CH₂-2''''), 2.73 (t, J = 7.5 Hz, 2H, CH₂-3'), 2.66 (t, J = 7.4 Hz, 2H, CH₂-1'), 1.93 (q, J = 7.4 Hz, 2H, CH₂-2'), 1.31–1.28 (m, 3H, CH₃CH₂O-4'''''); ¹³C-NMR (150 MHz, DMSO-d₆, δ/ppm): δ 168.34 (C-1''''), 155.51 (C-5''), 153.09 (C-4'''''), 153.04 (C-3''), 136.23 (C-8), 133.95 (C-1'''), 131.94 (C-1'''''), 129.70 (C-3''', 4''' & C-5'''), 129.01 (C-2''' & C-6'''), 127.01 (C-9), 125.51 (C-2''''' & C-6'''''), 122.21 (C-2), 120.78 (C-3''''' & C-5'''''), 120.71 (C-6), 118.18 (C-7), 118.06 (C-5), 113.57 (C-3), 111.27 (C-4), 63.17 (CH₃CH₂O-4'''''), 35.74 (C-2''''), 27.30 (C-2' & C-3''''), 23.99 (C-1'), 23.73 (C-3'), 14.63 (CH₃CH₂O-4'''''); Anal. Calc. for C₃₀H₃₁N₅SO₂ (522.21): C, 68.55; H, 5.94; N, 13.32. Found: C, 68.49; H, 5.91; N, 13.26. EI-MS (m/z): 525 (C₃₀H₃₁N₅SO₂) ̇⁺ (M)⁺, 334 (C₁₉H₁₈N₄S) ̇⁺, 245 (C₁₃H₁₅N₃S)⁺, 191 (C₉H₉N₃S)⁺, 158 (C₁₁H₁₂N)⁺, 143 (C₁₀H₉N)⁺, 130 (C₉H₈N)⁺, 91 (C₆H₅N)⁺, 78 (C₅H₄N)⁺.

4.3.6. N-(2,3-Dimethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9f)

Light grey amorphous solid; C₃₀H₃₁N₅SO; Mol. Mass: 509 gmol^− 1; yield: 78%; melting point 149^oC; IR (KBr, cm^− 1): υ 3184 (N-H str.), 3080 (C-H str. of aromatic ring), 2926 (C-H str. of aliphatic), 1589 (C = C aromatic str.), 1151 (C-N-C bond str.), 684 (C-S str.) cm^− 1; ¹H-NMR (600 MHz, DMSO-d₆, δ/ppm): δ 10.73 (s, 1H, NH-1), 9.51 (s, 1H, CONH), 7.52 (br.s, 2H, H-3''' & H-5'''), 7.49 (br.s, 1H, H-4'''), 7.36 (dist.d, J = 6.9 Hz, 3H, H-7, H-2''' & H-6'''), 7.30 (br.d, J = 7.9 Hz, 1H, H-4), 7.12 (s, 1H, H-6'''''), 7.03 (br.t, J = 7.6 Hz, 1H, H-6), 6.99 (br.s, 2H, H-4''''' & H-5'''''), 6.96 (br.s, 1H, H-2), 6.93 (dist.t, J = 6.3 Hz, 1H, H-5), 4.42 (t, J = 6.1 Hz, 2H, CH₂-3''''), 2.91 (t, J = 6.6 Hz, 2H, CH₂-2''''), 2.64 (dist.t, J = 7.6 Hz, 2H, CH₂-3'), 2.60–2.58 (m, 2H, CH₂-1'), 2.22 (s, 3H, CH₃-3'''''), 2.04 (s, 3H, CH₃-2'''''), 1.84 (dist.q, J = 7.1 Hz, 2H, CH₂-1'); ¹³C-NMR (150 MHz, DMSO-d₆, δ/ppm): δ 168.18 (C-1''''), 153.06 (C-5''), 150.82 (C-3''), 136.80 (C-3'''''), 136.22 (C-8), 135.93 (C-1'''''), 134.07 (C-1'''), 131.18 (C-2'''''), 129.56 (C-4'''), 129.51 (C-3''' & C-5'''), 128.07 (C-2''' & C-6'''), 127.01 (C-9), 125.53 (C-4'''''), 125.03 (C-5'''''), 123.61 (C-6'''''), 122.21 (C-2), 120.77 (C-6), 118.17 (C-7), 118.04 (C-5), 113.26 (C-3), 111.26 (C-4), 33.89 (C-2''''), 27.31 (C-3''''), 26.03 (C-2'), 24.83 (C-1'), 23.78 (C-3'), 20.08 (CH₃-3'''''), 13.95 (CH₃-2'''''); Anal. Calc. for C₃₀H₃₁N₅SO (509.22): C, 70.70; H, 6.13; N, 13.74. Found: C, 70.64; H, 6.09; N, 13.72. EI-MS (m/z): 509 (C₃₀H₃₁N₅SO) ̇⁺ (M)⁺, 366 (C₂₀H₂₂N₄SO)⁺, 334 (C₁₉H₁₈N₄S) ̇⁺, 245 (C₁₃H₁₅N₃S)⁺, 191 (C₉H₉N₃S)⁺, 158 (C₁₁H₁₂N)⁺, 143 (C₁₀H₉N)⁺, 130 (C₉H₈N)⁺, 120 (C₈H₁₀N)⁺, 91 (C₆H₅N)⁺, 78 (C₅H₄N)⁺.

4.3.7. N-(2,4-Dimethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9g)

Light grey amorphous solid; Mol. formula: C₃₀H₃₁N₅SO; Mol. Mass: 509 gmol^− 1; yield: 71%; melting point 105–106 ^oC; IR (KBr, cm^− 1): υ 3181 (N-H str.), 3078 (C-H str. of aromatic ring), 2926 (C-H str. of aliphatic), 1586 (C = C aromatic str.), 1150 (C-N-C bond str.), 684 (C-S str.) cm^− 1; ¹H-NMR (600 MHz, DMSO-d₆, δ/ppm): δ 10.72 (s, 1H, NH-1), 9.37 (s, 1H, CONH), 7.54–7.51 (m, 2H, H-3''' & H-5'''), 7.50–7.49 (m, 1H, H-4'''), 7.37–7.35 (m, 3H, H-7, H-2''' & H-6'''), 7.31 (br.d, J = 8.0 Hz, 1H, H-4), 7.22 (br.d, J = 7.9 Hz, 1H, H-6'''''), 7.04 (br.t, J = 7.9 Hz, 1H, H-6), 7.00 (br.s, 1H, H-2'''''), 6.97 (dist.d, J = 1.8 Hz, 1H, H-2), 6.93–6.89 (m, 2H, H-5 & H-5'''''), 4.42 (t, J = 7.2 Hz, 2H, CH₂-3''''), 2.90 (t, J = 7.2 Hz, 2H, CH₂-2''''), 2.65 (t, J = 7.4 Hz, 2H, CH₂-3'), 2.61 (t, J = 7.3 Hz, 2H, CH₂-1'), 2.23 (s, 3H, CH₃-4'''''), 2.12 (s, 3H, CH₃-2'''''), 1.83 (q, J = 7.3 Hz, 2H, CH₂-2'); ¹³C-NMR (150 MHz, DMSO-d₆, δ/ppm): δ 168.09 (C-1''''), 153.06 (C-5''), 150.84 (C-3''), 136.22 (C-8), 134.20 (C-1'''''), 134.09 (C-1'''), 133.54 (C-2'''''), 131.74 (C-4'''''), 130.65 (C-3'''''), 129.51 (C-4'''), 129.41 (C-3''' & C-5'''), 128.07 (C-2''' & C-6'''), 126.94 (C-9), 126.29 (C-5'''''), 125.21 (C-6'''''), 122.20 (C-2), 120.77 (C-6), 118.14 (C-7), 118.05 (C-5), 113.27 (C-3), 111.26 (C-4), 39.93 (C-2''''), 27.31 (C-3''''), 26.02 (C-2'), 24.81 (C-1'), 23.76 (C-3'), 20.42 (CH₃-4'''''), 17.70 (CH₃-2'''''); Anal. Calc. for C₃₀H₃₁N₅SO (509.22): C, 70.70; H, 6.13; N, 13.74. Found: C, 70.67; H, 6.06; N, 13.70. EI-MS (m/z): 509 (C₃₀H₃₁N₅SO) ̇⁺ (M)⁺, 366 (C₂₀H₂₂N₄SO)⁺, 334 (C₁₉H₁₈N₄S) ̇⁺, 245 (C₁₃H₁₅N₃S)⁺, 191 (C₉H₉N₃S)⁺, 158 (C₁₁H₁₂N)⁺, 143 (C₁₀H₉N)⁺, 130 (C₉H₈N)⁺, 120 (C₈H₁₀N)⁺, 91 (C₆H₅N)⁺, 78 (C₅H₄N)⁺.

4.3.8. N-(2,5-Dimethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9h)

Brown amorphous solid; Mol. formula: C₃₀H₃₁N₅SO; Mol. Mass: 509 gmol^− 1; yield: 92%; melting point 133 − 34 ^oC; IR (KBr, cm^− 1): υ 3219 (N-H str.), 3092 (C-H str. of aromatic ring), 2931 (C-H str. of aliphatic), 1581 (C = C aromatic str.), 1155 (C-N-C bond str.), 670 (C-S str.) cm^− 1; ¹H-NMR (600 MHz, DMSO-d₆, δ/ppm): δ 10.72 (s, 1H, NH-1), 9.30 (s, 1H, CONH), 7.54–7.50 (m, 3H, H-3''', H-4''' & H-5'''), 7.37 (br.d, J = 7.5 Hz, 3H, H-7, H-2''' & H-6'''), 7.31 (br.d, J = 8.1 Hz, 1H, H-4), 7.15 (br.s, 1H, H-6'''''), 7.05–7.02 (m, 2H, H-6 & H-3'''''), 6.97 (dist.d, J = 2.0 Hz, 1H, H-2), 6.94 (br.t, J = 7.7 Hz, 1H, H-5), 6.88 (br.d, J = 7.5 Hz, 1H, H-4'''''), 3.33 (t, J = 6.7 Hz, 2H, CH₂-3''''), 2.81 (t, J = 6.7 Hz, 2H, CH₂-2''''), 2.66 (t, J = 7.4 Hz, 2H, CH₂-3'), 2.59 (t, J = 7.5 Hz, 2H, CH₂-1'), 2.22 (s, 3H, CH₃-5'''''), 2.09 (s, 3H, CH₃-2'''''), 1.89 (q, J = 7.4 Hz, 2H, CH₂-2'); ¹³C-NMR (150 MHz, DMSO-d₆, δ/ppm): δ 168.69 (C-1''''), 155.52 (C-5''), 149.56 (C-3''), 136.24 (C-8), 135.92 (C-1'''''), 134.79 (C-5'''''), 133.13 (C-1'''), 129.98 (C-3'''''), 129.78 (C-3''' & C-5'''), 129.08 (C-4'''), 128.53 (C-2'''''), 127.19 (C-2''' & C-6'''), 127.01 (C-9), 125.79 (C-4'''''), 125.58 (C-6'''''), 122.19 (C-2), 120.77 (C-6), 118.19 (C-7), 118.05 (C-5), 113.52 (C-3), 111.26 (C-4), 35.74 (C-2''''), 28.00 (C-3''''), 26.96 (C-2'), 24.35 (C-1'), 23.96 (C-3'), 20.51 (CH₃-5'''''), 17.41 (CH₃-2'''''); Anal. Calc. for C₃₀H₃₁N₅SO (509.22): C, 70.70; H, 6.13; N, 13.74. Found: C, 70.65; H, 6.10; N, 13.69. EI-MS (m/z): 509 (C₃₀H₃₁N₅SO) ̇⁺ (M)⁺, 366 (C₂₀H₂₂N₄SO)⁺, 334 (C₁₉H₁₈N₄S) ̇⁺, 245 (C₁₃H₁₅N₃S)⁺, 191 (C₉H₉N₃S)⁺, 158 (C₁₁H₁₂N)⁺, 143 (C₁₀H₉N)⁺, 130 (C₉H₈N)⁺, 120 (C₈H₁₀N)⁺, 91 (C₆H₅N)⁺, 78 (C₅H₄N)⁺.

4.3.9. N-(2,6-Dimethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9i)

Pink amorphous solid; Mol. formula: C₃₀H₃₁N₅SO; Mol. Mass: 509 gmol^− 1; yield: 78%; melting point 113–114 ^oC; IR (KBr, cm^− 1): υ 3224 (N-H str.), 3088 (C-H str. of aromatic ring), 2928 (C-H str. of aliphatic), 1585 (C = C aromatic str.), 1153 (C-N-C bond str.), 667 (C-S str.) cm^− 1; ¹H-NMR (600 MHz, DMSO-d₆, δ/ppm): δ 10.72 (s, 1H, NH-1), 9.40 (s, 1H, CONH), 7.54–7.49 (m, 3H, H-3''', H-4''' & H-5'''), 7.37–7.35 (m, 3H, H-7, H-2''' & H-6'''), 7.31 (br.d, J = 8.1 Hz, 1H, H-4), 7.07–7.02 (m, 4H, H-6, H-3''''', H-4''''' & H-5'''''), 6.96 (dist.d, J = 1.7 Hz, 1H, H-2), 6.92 (br.t, J = 7.2 Hz, 1H, H-5), 4.42 (t, J = 7.1 Hz, 2H, CH₂-3''''), 2.92 (t, J = 7.1 Hz, 2H, CH₂-2''''), 2.64 (t, J = 7.2 Hz, 2H, CH₂-3'), 2.59–2.57 (m, 2H, CH₂-1'), 2.11 (s, 6H, CH₃-2''''' & CH₃-6'''''), 1.86–1.83 (m, 2H, CH₂-2'); ¹³C-NMR (150 MHz, DMSO-d₆, δ/ppm): δ 167.72 (C-1''''), 166.50 (C-5''), 150.84 (C-3''), 136.25 (C-8), 135.13 (C-2''''' & C-6'''''), 134.99 (C-1'''''), 134.08 (C-1'''), 129.53 (C-4'''), 129.44 (C-3''' & C-5'''), 128.06 (C-3''''' & C-5'''''), 127.55 (C-2''' & C-6'''), 126.95 (C-9), 126.34 (C-4'''''), 122.21 (C-2), 120.81 (C-6), 118.19 (C-7), 118.08 (C-5), 113.27 (C-3), 111.28 (C-4), 34.29 (C-2''''), 33.36 (C-3''''), 26.11 (C-2'), 24.85 (C-1'), 23.84 (C-3'), 18.03 (CH₃-2''''' & CH₃-6'''''); Anal. Calc. for C₃₀H₃₁N₅SO (509.22): C, 70.70; H, 6.13; N, 13.74. Found: C, 70.62; H, 6.09; N, 13.71. EI-MS (m/z): 509 (C₃₀H₃₁N₅SO) ̇⁺ (M)⁺, 366 (C₂₀H₂₂N₄SO)⁺, 334 (C₁₉H₁₈N₄S) ̇⁺, 245 (C₁₃H₁₅N₃S)⁺, 191 (C₉H₉N₃S)⁺, 158 (C₁₁H₁₂N)⁺, 143 (C₁₀H₉N)⁺, 130 (C₉H₈N)⁺, 120 (C₈H₁₀N)⁺, 91 (C₆H₅N)⁺, 78 (C₅H₄N)⁺.

4.3.10. N-(3,4-Dimethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9j)

Pink amorphous solid; Mol. formula C₃₀H₃₁N₅SO; Mol. mass 509 gmol^− 1; yield: 91%; melting point 156–157 ^oC; IR (KBr, cm^− 1): υ 3226 (N-H str.), 3089 (C-H str. of aromatic ring), 2928 (C-H str. of aliphatic), 1585 (C = C aromatic str.), 1150 (C-N-C bond str.), 667 (C-S str.) cm^− 1; ¹H-NMR (600 MHz, DMSO-d₆, δ/ppm): δ 10.71 (s, 1H, NH-1), 9.91 (s, 1H, CONH), 7.51 (br.s, 3H, H-3''', H-4''' & H-5'''), 7.36 (br.s, 3H, H-7, H-2''' & H-6'''), 7.34 (br.d, J = 8.0 Hz, 1H, H-6'''''), 7.31–7.29 (br.s, 2H, H-4 & H-2'''''), 7.05–7.01 (m, 2H, H-6 & H-5'''''), 6.93 (br.s, 1H, H-2), 6.91 (br.t, J = 7.8 Hz, 1H, H-5), 4.41 (t, J = 7.2 Hz, 2H, CH₂-3''''), 2.88 (t, J = 7.3 Hz, 2H, CH₂-2''''), 2.67 (t, J = 7.2 Hz, 2H, CH₂-3'), 2.61 (t, J = 7.1 Hz, 2H, CH₂-1'), 2.15 (s, 3H, CH₃-4'''''), 2.14 (s, 3H, CH₃-3'''''), 1.81 (q, J = 7.2 Hz, 2H, CH₂-2'); ¹³C-NMR (150 MHz, DMSO-d₆, δ/ppm): δ 167.82 (C-1''''), 153.06 (C-5''), 150.85 (C-3''), 136.75 (C-3'''''), 136.22 (C-8), 136.14 (C-1'''''), 134.07 (C-1'''), 130.84 (C-4'''''), 129.56 (C-4'''), 129.49 (C-3''' & C-5'''), 128.08 (C-2''' & C-6'''), 127.02 (C-9), 126.94 (C-5'''''), 122.25 (C-2), 120.78 (C-6), 120.44 (C-2'''''), 118.18 (C-7), 118.06 (C-5), 116.74 (C-6'''''), 113.25 (C-3), 111.26 (C-4), 34.50 (C-2''''), 27.31 (C-3''''), 25.91 (C-2'), 24.74 (C-1'), 23.67 (C-3'), 19.55 (CH₃-3'''''), 18.71 (CH₃-4'''''); Anal. Calc. for C₃₀H₃₁N₅SO (509.22): C, 70.70; H, 6.13; N, 13.74. Found: C, 70.67; H, 6.09; N, 13.69. EI-MS (m/z): 509 (C₃₀H₃₁N₅SO) ̇⁺ (M)⁺, 366 (C₂₀H₂₂N₄SO)⁺, 334 (C₁₉H₁₈N₄S) ̇⁺, 245 (C₁₃H₁₅N₃S)⁺, 191 (C₉H₉N₃S)⁺, 158 (C₁₁H₁₂N)⁺, 143 (C₁₀H₉N)⁺, 130 (C₉H₈N)⁺, 120 (C₈H₁₀N)⁺, 91 (C₆H₅N)⁺, 78 (C₅H₄N)⁺.

4.3.11. N-(3,5-Dimethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9k)

Light yellow colored amorphous powder; Mol. Formula: C₃₀H₃₁N₅SO; Mol. mass: 509 g/mol; yield: 84%; melting point 142–143°C; IR (KBr, cm^− 1): υ: 3282 (N-H str.), 2945 (C-H aromatic str.), 1664 (C = O str.), 1599 (C = C aromatic str.), 1535, 1473, 1456(Str. for oxadiazole), 1155 (C-O-C str.), 1116 (C = N str.), 689 (C-S str.); ¹H-NMR (600 MHz, DMSO-d₆, δ/ppm): δ 10.72 (s, 1H, NH-1), 9.76 (s, 1H, CONH), 7.53–7.52 (m, 3H, H-3''', H-4''' & H-5'''), 7.38–7.35 (m, 3H, H-7, H-2''' & H-6'''), 7.31 (br.d, J = 7.9 Hz, 1H, H-4), 7.20 (br.s, 2H, H-2''''' & H-6'''''), 7.04 (br.t, J = 7.4 Hz, 1H, H-6), 7.00 (br.s, 1H, H-2), 6.93 (br.t, J = 7.2 Hz, 1H, H-5), 6.66 (br.s, 1H, H-4'''''), 3.10 (t, J = 6.9 Hz, 2H, CH₂-3''''), 2.65 (t, J = 7.1 Hz, 2H, CH₂-3'), 2.58 (t, J = 7.2 Hz, 2H, CH₂-1'), 2.38 (t, J = 7.0 Hz, 2H, CH₂-2''''), 2.20 (s, 6H, CH₃-3''''' & CH₃-5'''''), 1.88 (quintet, J = 7.2 Hz, 2H, CH₂-2'). ¹³C-NMR (150 MHz, DMSO-d₆, δ/ppm): δ 170.13 (C-1''''), 155.48 (C-5''), 149.46 (C-3''), 139.03 (C-1'''''), 137.50 (C-3''''' & C-5'''''), 136.23 (C-8), 133.99 (C-1'''), 129.77 (C-4'''), 129.69 (C-3''' & C-5'''), 127.23 (C-2''' & C-6'''), 127.02 (C-9), 125.52 (C-4'''''), 122.21 (C-2), 120.77 (C-6), 118.19 (C-7), 118.04 (C-5), 116.84 (C-2''''' & C-6'''''), 113.52 (C-3), 111.25 (C-4), 34.92 (C-2''''), 31.69 (C-3''''), 26.93 (C-2'), 24.36 (C-1'), 23.95 (C-3'), 21.10 (CH₃-3''''' & CH₃-5'''''); Anal. Calc. for C₃₀H₃₁N₅SO (509.22): C, 70.70; H, 6.13; N, 13.74. Found: C, 70.65; H, 6.09; N, 13.70. EI-MS (m/z): 509 (C₃₀H₃₁N₅SO) ̇⁺ (M)⁺, 366 (C₂₀H₂₂N₄SO)⁺, 334 (C₁₉H₁₈N₄S) ̇⁺, 245 (C₁₃H₁₅N₃S)⁺, 191 (C₉H₉N₃S)⁺, 158 (C₁₁H₁₂N)⁺, 143 (C₁₀H₉N)⁺, 130 (C₉H₈N)⁺, 120 (C₈H₁₀N)⁺, 91 (C₆H₅N)⁺, 78 (C₅H₄N)⁺.

4.4. In vitro elastase inhibition assay

The elastase (Elastase from porcine pancreas) inhibition activity was performed following already reported method [36–38] with few modifications. In order to perform the inhibition of elastase activity, the amount of released p-nitroaniline, which was hydrolyzed from the substrate (N-succinyl-Ala-Ala-Ala-p-nitroanilide) by elastase, was determined by measuring the absorbance at 410 nm. In detail, 0.8 mM solution of N-succinyl-Ala-Ala-Ala-p-nitroanilide was prepared in a 0.2 M Tris-HCl buffer (pH 8.0) and this buffer (130 µL) was added to the test sample (10 µL) in a 96 well microplate. The microplate was pre-incubated for 10 min at 25ºC before an elastase (0.0375 Unit/mL) stock solution (10 µL) was added. After enzyme addition, the microplate was kept at 25ºC for 30 min, and the absorbance was measured at 410 nm using microplate reader (SpectraMax ABS, USA). IC₅₀ values were calculated by nonlinear regression using Graph Pad Prism 5.0 (Graph Pad, San Diego, CA USA). All experiments were carried out in triplicate. The elastase inhibition activities were calculated according to the following formula:

Elastase inhibition activity (%) = (OD_control – OD_sample × 100) / OD_control

Where OD_control and OD_sample represents the optical densities in the absence and presence of sample, respectively. Oleanolic acid was used as the standard inhibitor for elastase.

4.5. Kinetics assay

Kinetic analysis was carried out to determine the mode of inhibition by the following our reported method [24]. The compound 9d was selected on the basis of most potent IC₅₀ values. Kinetics was carried out by varying the concentration of N-succinyl-Ala-Ala-Ala-p-nitroanilide the presence of different concentrations of compound 9d (0.00, 0.071, 0.142 and 0.284 µM). Briefly the N-succinyl-Ala-Ala-Ala-p-nitroanilide concentrations was changed from 2, 1, 0.5, 0.25, 0.125 and 0.0625 mM for N-succinyl-Ala-Ala-Ala-p-nitroanilide kinetics studies and remaining procedure was same for all kinetic studies as describes in elastase inhibition assay protocol. Maximal initial velocities were determined from initial linear portion of absorbance up to 10 minutes after addition of enzyme at per minute interval. The inhibition type on the enzyme was assayed by Lineweaver-Burk plot of inverse of velocities (1/V) versus inverse of substrate concentration 1/ [S] mM^− 1. The EI dissociation constant K_i was determined by secondary plot of 1/V versus inhibitor concentration. The results were processed by using SoftMaxPro.

4.6. Computational Methodology Details

In the present research, all the designed molecules were optimized by using gaussian 16 [39] program. GaussView 6 [40] was utilized as a visualizer software to display molecular orbitals and maps of electrostatic potential. The density functional theory (DFT) approach was used with 6-31G* basis set and B3LYP functional for achieving the minimized state of the energies and other properties of the molecules. Quantum chemical geometry optimization have been performed for all the molecules. Optimized molecular geometries were used for studying molecular docking interactions. The three-dimensional structure of the protein porcine pancreatic elastase was fetched from protein data bank (https://www.rcsb.org) having PDB ID 7est for molecular docking investigations. The resolution of protein in x-ray crystal structure is 1.8 Å. The elastase protein consists of one chain with 240 amino acids. Then protein was purified in discovery studio visualizer by removing co-crystallized ligands and water molecules. Missing residues of protein were confirmed from visualizer afterward Kollman charges and polar hydrogens were added through autodock tools. Autodock vina (Huey, 2012) was utilized to check the binding affinity of protein-ligand complex by doing molecular docking. For molecular docking with protein, the optimized ligands served as input structures. For porcine pancreatic elastase, the grid dimensions and active locations were selected based on prior research [41] while all default settings were chosen for docking analysis. Discovery studio visualizer [42] was used to determine the 2-D and 3-D interactions.

4.7. Statistical Analysis

All the measurements were carried out in triplicate and statistical); Analysis was performed by Microsoft Excel 2010. The results are presented as mean ± SEM with 96% CL.

Conflict of Interest

The authors have no conflicts of interest to declare that are relevant to the content of this article.

Author Contribution

"M.A.A., A-U-R. and S.Z.S.: conceptualization, data curation, Writing-review & editing. S., M.N. and S.M.: methodology, writing-original draft, H.R. and M.S.: formal analysis, software, S.A.A.S.: investigation, validation, A.R.C.: funding acquisition, S.J.K.: investigation, Writing-review & editing. All authors reviewed the manuscript."

Acknowledgement

The authors from Pakistan acknowledge the funding from the ORIC office of the Government College University Lahore vide Notification No. 21/ORIC/24 dated 04-01-2024. The authors from Saudi Arabia extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding the work through group Research Project under grant number RGP2/276/45. The authors extend their appreciation to the Deanship of Graduate Studies and Scientific Research at the University of Bisha for supporting this work through the Fast-Track Research Support Program.

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Convergent Synthesis, Kinetics and Computational Comprehensions of Indole- (Phenyl)triazole Bi-heterocycles Amalgamated with Propanamides as Elastase Inhibitors

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Abstract

Figures

1. Introduction

2. Results and discussion

2.1. Chemistry

3. Conclusion

4. Experimental

4.1. General information

4.2. Synthesis of ethyl 4-(1H-indol-3-yl)butanoate (2)

4.2.1. Synthesis of 4-(1H-indol-3-yl)butanohydrazide (3)

4.2.2. Synthesis of 5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazole-3-thiol (5)

4.2.3. Preparation of N-(Aryl)-3-bromopropanamides (8a-k)

4.2.4. Synthesis of N-(aryl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamides (9a-k)

4.3. Spectral characterization of synthesized compounds, 9a-k

4.3.1. 3-({5-[3-(1H-Indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(2-methylphenyl)propanamide (9a)

4.3.2. 3-({5-[3-(1H-Indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(3-methylphenyl)propanamide (9b)

4.3.3. 3-({5-[3-(1H-Indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)-N-(4-methylphenyl)propanamide (9c)

4.3.4. N-(4-Ethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9d)

4.3.5. N-(4-Ethoxyphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9e)

4.3.6. N-(2,3-Dimethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9f)

4.3.7. N-(2,4-Dimethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9g)

4.3.8. N-(2,5-Dimethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9h)

4.3.9. N-(2,6-Dimethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9i)

4.3.10. N-(3,4-Dimethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9j)

4.3.11. N-(3,5-Dimethylphenyl)-3-({5-[3-(1H-indol-3-yl)propyl]-4-phenyl-4H-1,2,4-triazol-3-yl}sulfanyl)propanamide (9k)

4.4. In vitro elastase inhibition assay

4.5. Kinetics assay

4.6. Computational Methodology Details

4.7. Statistical Analysis

Declarations

Conflict of Interest

Author Contribution

Acknowledgement

References

Additional Declarations

Supplementary Files

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