Next Article in Journal
Pharmacological Treatments and Natural Biocompounds in Weight Management
Next Article in Special Issue
Nitrogen Containing Heterocycles as Anticancer Agents: A Medicinal Chemistry Perspective
Previous Article in Journal
The Use of Triphenyl Phosphonium Cation Enhances the Mitochondrial Antiplatelet Effect of the Compound Magnolol
Previous Article in Special Issue
Pyrrolyl and Indolyl α-γ-Diketo Acid Derivatives Acting as Selective Inhibitors of Human Carbonic Anhydrases IX and XII
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Pharmaceuticals
Volume 16
Issue 2
10.3390/ph16020211
pharmaceuticals-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Bio-Oriented Synthesis and Molecular Docking Studies of 1,2,4-Triazole Based Derivatives as Potential Anti-Cancer Agents against HepG2 Cell Line

by
Naheed Akhter
1,
Sidra Batool
2,
Samreen Gul Khan
2,*,
Nasir Rasool
2,
Fozia Anjum
2,
Azhar Rasul
3,
Şevki Adem
4,
Sadaf Mahmood
2,
Aziz ur Rehman
5,
Mehr un Nisa
6,
Zainib Razzaq
2,
Jørn B. Christensen
7,
Mohammed A. S. Abourehab
8,
Syed Adnan Ali Shah
9,10,* and
Syahrul Imran
10,11
1
Department of Biochemistry, Faculty of Life Science, Government College University Faisalabad, Faisalabad 38000, Pakistan
2
Department of Chemistry, Drug Design and Medicinal Chemistry Laboratory, Faculty of Physical Science, Government College University, Faisalabad 38000, Pakistan
3
Department of Zoology, Faculty of Life Sciences, Government College University Faisalabad, Faisalabad 38000, Pakistan
4
Department of Chemistry, Faculty of Sciences, Çankırı Karatekin University, 18100 Çankırı, Turkey
5
Department of Chemistry, Government College University, Lahore 54000, Pakistan
6
Department of Chemistry, University of Lahore, Lahore 40100, Pakistan
7
Department of Chemistry, Faculty of Science, University of Copenhagen, 2100 Copenhagen, Denmark
8
Department of Pharmaceutics College of Pharmacy, Umm Al-Qura University, Makkah 21955, Saudi Arabia
9
Faculty of Pharmacy, Universiti Teknologi MARA Cawangan Selangor Kampus Puncak Alam, Bandar Puncak Alam 42300, Selangor D. E., Malaysia
10
Atta-ur-Rahman Institute for Natural Product Discovery (AuRIns), Universiti Teknologi MARA Cawangan Selangor Kampus Puncak Alam, Bandar Puncak Alam 42300, Selangor D. E., Malaysia
11
Faculty of Applied Sciences, Universiti Teknologi MARA Shah Alam, Shah Alam 40450, Selangor D.E., Malaysia
 
add Show full affiliation list
 
remove Hide full affiliation list
*
Authors to whom correspondence should be addressed.
Pharmaceuticals 2023, 16(2), 211; https://doi.org/10.3390/ph16020211
Submission received: 5 December 2022 / Revised: 20 January 2023 / Accepted: 23 January 2023 / Published: 30 January 2023
(This article belongs to the Special Issue Nitrogen Containing Scaffolds in Medicinal Chemistry)
Browse Figures
Review Reports Versions Notes

Abstract

:
Triazole-based acetamides serve as important scaffolds for various pharmacologically active drugs. In the present work, structural hybrids of 1,2,4-triazole and acetamides were furnished by chemically modifying 2-(4-isobutylphenyl) propanoic acid (1). Target compounds 7a–f were produced in considerable yields (70–76%) by coupling the triazole of compound 1 with different electrophiles under different reaction conditions. These triazole-coupled acetamide derivatives were verified by physiochemical and spectroscopic (HRMS, FTIR, 13CNMR, and 1HNMR,) methods. The anti-liver carcinoma effects of all of the derivatives against a HepG2 cell line were investigated. Compound 7f, with two methyl moieties at the ortho-position, exhibited the highest anti-proliferative activity among all of the compounds with an IC50 value of 16.782 µg/mL. 7f, the most effective anti-cancer molecule, also had a very low toxicity of 1.190.02%. Molecular docking demonstrates that all of the compounds, especially 7f, have exhibited excellent binding affinities of −176.749 kcal/mol and −170.066 kcal/mol to c-kit tyrosine kinase and protein kinase B, respectively. Compound 7f is recognized as the most suitable drug pharmacophore for the treatment of hepatocellular carcinoma.

1. Introduction

In the 21st century, cancer and other infectious diseases are the most prevalent causes of death globally [1]. According to the World Health Organization (WHO), 11.5 million deaths are expected by 2030 due to cancer [2]. Among all types of cancer, hepatocellular carcinoma is among the leading causes of death, accounting for approximately 92% mortality rates worldwide [3,4]. Thus, the development of new anti-cancer drugs remains a huge clinical need for improving therapeutic efficacy and controlling cancer [5]. The development of multi-target anti-cancer agents is the major focus of researchers globally due to the different drawbacks associated with already-used chemotherapeutics such as undesirable side effects, a lack of selectivity, systemic toxicity, and the emergence of multidrug resistance [6,7,8,9]. Intensive efforts must be made to discover and develop new, effective, tailored –anti-cancer agents with better safety profiles and drug-like properties.
Structurally modified nitrogen-containing heterocyclic moieties have a broad spectrum of applications for the development of novel therapeutic drugs as shown in Figure 1 [10,11]. Approximately 75% of Food and Drug Administration-approved drugs are nitrogen-based moieties [12]. Nitrogen-containing heterocyclic compounds have been synthesised in large numbers in recent times. They exhibit anti-tubercular [13], anti-cancer [14], anti-fungal [15], anti-microbial [16], anti-viral [17], and other biological properties such as genotoxicity and lipid peroxidation [18], and anti-inflammatory properties [19].
Molecular hybridization is an easy and efficient method to combine various important drug pharmacophores. Our ongoing research focuses on the design and synthesis of pharmacologically active, diverse polyvalent scaffolds as anti-cancer agents. The versatile nature of 1,2,4-triazole has been reported to be of great importance in medicinal chemistry, such as for its anti-cancer [20], anti-fungal [21], anti-bacterial [22], anti-microbial, and anti-tumor properties [23], as well as pyrophosphatases and phosphodiesterase [24]. Acetamide has been identified as the most significant pharmacophore of anti-cancer drugs [25].
On this basis, we have created a hybrid of acetamide and 1,2,4-triazole pharmacophore by chemical derivatization of 2-(4-isobutylphenyl)propanoic acid in an attempt to avoid tumor progression. On each side of triazole and acetamide, we developed a molecular framework with hydrophobic aryl rings. It improves the solubility of the drug and its candidacy as a good drug molecule. Earlier, we reported the synthesis of various structural hybrids of oxadiazole-based acetamide [26,27,28,29], and it has been proven from the literature that heterocycle-based compounds possess good anti-cancer activity [30]. Thus, in an extension of our earlier research on heterocycles, –COOH group of 2-(4-isobutylphenyl)propanoic acid was cyclized into a 1,2,4-triazole ring 4.

2. Results

2.1. Chemistry

In the current study, 2-(4-isobutylphenyl)propanoic acid has been chemically modified with improved clinical utility. Different N-arylated 5-aryl-1,2,4-triazole-coupled acetamides (7a–f) have been synthesized in good yields by replacement of the H group of SH with various electrophiles.
Scheme 1 depicts the synthetic route of final compounds 7a–f. The Fischer esterification method was used to create compound 2 by refluxing compound 1 with absolute CH3OH at 76 °C for 3–4 h [31,32]. Compound 2 was slowly refluxed at 76 °C for 3–4 h with hydrazine hydrate [32] in CH3OH and yielded 2-(4-isobutylphenyl)propane hydrazide (3). Molecule 3 was converted into its respective 5-(1-(4-isobutylphenyl) ethyl)-1,2,4-triazole-2-thiol (4) by slowly heating it at 225 °C for 3–6 h with methyl isothiocyanate in 10% NaOH and absolute CH3OH. Upon completion, the reaction was acidified to pH 4–5 with conc. HCl. Upon acidification, precipitates appeared that had been separated as compound 4 by the process of filtration. At room temperature, compound 4 was treated with various N-arylated aralkyl/alkyl/aryl 2-bromoacetamides (6a–f) along with DCM and NaH as catalysts. The structures of triazole-coupled acetamide scaffolds were verified by physiochemical and spectroscopic (HRMS, FTIR, 13CNMR, and 1HNMR) methods.
In the 1HNMR spectrum, –NH protons of the acetamide were the most deshielded and their chemical shift value was observed around 10.27–9.64 ppm. In propanoic acid, the protons of the aliphatic region were among the most shielded, with values ranging from 0.86–0.84 ppm. The presence of acetamide was confirmed by the appearance of signals around 4.04 ppm for the CH2 group and in the range of 10.27–9.64 ppm for the NH group. In 13CNMR, the appearance of C=O signals in the range of 164.15–168 ppm confirmed the synthesis of acetamide. The presence of a triazole ring in the final derivatives was also confirmed by the appearance of signals in the range of 158.15 ppm and at 29.58 ppm for N-CH3. In the 1HNMR spectrum, signals for N-CH3 were observed around 3.28 ppm. A distinctive peak around 22.0 ppm corresponded to the protons of two -CH3 carbon nuclei. Signals for the CH2 groups were observed between 44.18 to 20.19 ppm. By introducing some electron-withdrawing substitutions and comparing them to electron-donating group substitutions, we reported structure–activity relationships for the phenyl group. 3,5-disubstituted triazole nuclei have a versatile nature and are important in the pharmaceutical industry. On the basis of their medicinal importance, the anti-cancer activities of all of the compounds were checked.

2.2. Anti-proliferative Potential

The anti-hepatocellular activity of afforded N-arylated 1,2,4-triazole coupled acetamides (7a–f) was evaluated via MTT assay and these structural hybrids were screened against a liver cancer HepG2 cell line [33]. All of the compounds demonstrated mild to outstanding anti-cancer activity, as shown in Table 1. Among all of the compounds, 7f, with two methyl groups at positions 2 and 6 of the phenyl ring, displayed the best anti-cancer potential with IC50 = 16.782 µg/mL. Compound 7a, which contains a methyl at position 2 of the phenyl ring, also displayed good anti-cancer activity with an IC50 value of 20.667 µg/mL but less than 7f. Compounds 7b, 7c, and 7e also exhibited a significant anti-cancer effect but less than 7f and 7a. Compound 7d, with an electron-withdrawing Cl substituent, displayed the lowest anti-hepatocellular activity with a 39.667 µg/mL IC50 value.
The cell viability of all of the compounds was further evaluated using various concentrations (3.125–200 µg) to test the dose response and % inhibition relationship as shown in Table 2.
Figure 2 shows that compounds 7f and 7a produced the best results at a 25 µg/mL dose among all of the compounds.
The dose response and % inhibition of the most potent compound, 7f, was checked at various concentrations (Figure 3).

2.3. Hemolytic Activity Potential

Hemolytic activity was investigated by the reported method [34] using Triton-X-100 as a standard. All of the triazole-based acetamide derivatives showed very low cytotoxicity, as shown in Table 1. Molecule 7f also presented very low toxicity at 1.19 ± 0.02 relative to reference Triton-X-100. All of the other compounds had moderately good hemolytic activity. Compounds 7a (2.46%), 7b (4.43%), 7c (4.32%), 7e (4.19%), and 7d (7.33%) also exhibited low cytotoxicity.

2.4. Structure–Activity Relationship of 7a–f

The anti-hepatocellular potential of all of the derivatives, 7a–f, was evaluated against a HepG2 cancer cell line at various concentrations via MTT assay. The most potent derivative was 7f, which contains two methyl groups at ortho-positions of the phenyl ring (IC50 value of 16.782 µM). Compound 7d, which contained an electron-withdrawing Cl-group in an orthogonal position, showed the lowest anti-cancer potency with an IC50 value of 39.667 µM. The anti-proliferative activity of all of the derivatives was decreased in the following order: 7f > 7a > 7b > 7c > 7e > 7d. This proves that the attachment of an electron-donating CH3 group at the ortho-position increases the anti-cancer activity of the compounds. Based on the results of the SAR of N-arylated 5-aryl-1,2,4-triazole-coupled acetamide scaffolds 7a–f, it was determined that the -CH3 motif at the ortho-position of the phenyl ring improved the anti-proliferative potential of the compounds.

2.5. Molecular Docking

Molecular docking screenings were carried out to theoretically predict the most promising protein targets of compounds by molecular docking to some cancer targets. Five major targets in the treatment of cancer have been identified: human Aurora B kinase, phosphatidylinositol 3-kinase alpha (PI3Kalpha), the signal transducer and activator of transcription 3 (STAT3), protein kinase B (Akt), and c-kit tyrosine kinase (c-Kit). The web page http://www.swisstargetprediction.ch/ (Accessed on 7 November 2022) was used to study the potential anti-cancer effect of new synthesized molecules [35]. The results suggest that molecules may be effective against kinase targets.
Some important kinases and important targets in cancer treatment were determined by the literature review. c-kit tyrosine kinase endorses cellular proliferation by activating signal transduction mechanisms in response to stem cell factor adhesion [36]. Akt plays a vital role in internal cell signalling by accelerating cellular survival and proliferation. Its path becomes irregular during cancer [37]. Aurora kinase B regulates the cell cycle and is ubiquitously expressed in cancerous cells. [38]. The insulin-like growth factor-1 receptor (IGF-1R) has a vital role in cells in conjunction with PI3K–AKT and Ras–Raf–MEK signalling cascades, which control proliferation and apoptosis within cells. It is considered an important therapeutic target because of its deregulation of solid tumor types [39]. Phosphatidylinositol 3-kinase alpha is an intracellular lipid kinase that regulates cell survival, development, proliferation, and metabolism. It has been linked to a number of human cancers [40]. STAT3 is a secret transcription factor; it is regarded as an appealing target of anti-cancer therapeutics [41].
Table 3 shows the selected targets, grid box coordinates, their protein data bank codes, and Moldock scores. The docking findings demonstrate that molecules have the ability to affect a variety of targets. c-kit and Akt specifically are anticipated to have a high binding potential with cancer therapeutic targets, as demonstrated in Table 3.
Figure 4 demonstrates 2D and 3D diagrammatic representations displaying the molecular bindings between the reference ligand SIT and the active pocket of c-Kit tyrosine kinase.
The docking findings demonstrate that all of the compounds have good binding affinity to kinase protein. The protein c-kit tyrosine kinase is expected to interact strongly with cancer-targeted therapeutics. Table 4 shows the docked complexes’ categories, modes of interactions, binding affinities, and by-products. Each ligand’s hydrophobic contacts and hydrogen bonding interactions were evaluated within the receptor protein’s binding pocket.
Compound 7f bonded to c-kit tyrosine kinase with the most suitable binding pose and a low binding energy of −176.749 kcal/mol. It formed an H-bond with Asp810 and Glu640. Hydrophobic interactions occurred such as the pi-sigma bond with Thr670, His790, and Val643. Other hydrophobic interactions involved alkyl interactions with Val603, Val668, Leu644, Leu783, and Ile571, pi-sulfur interactions with Cys788 and Lys623, and amide-pi-stacked interactions with Cys809 residue. It also interacts with Val654, Ile789, Leu647, Ile808, Ile653, and Phe811 residues through van der Waals interaction. The 2D and 3D diagrammatic expressions of the binding interactions of 7f and c-kit tyrosine kinase are shown in Figure 5.
Compound 7a combines with c-kit tyrosine kinase with the most suitable binding poses with a low binding energy of −173.411 kcal/mol. Hydrophobic interactions of 7a involved a pi-sigma bond with Leu595 and Leu644 and alkyl interactions with Cys673, Cys809, Ala621, Val654, Tyr672, and Phe811. It also interacts with Gly676, Leu799, Asp810, Leu647, Leu783, His790, Ile808, Glu640, Thr670, Val603, and Asp677 residues by van der Waals interactions. The 2D and 3D diagrammatic expressions of the binding interactions of 7a and c-kit tyrosine kinase are shown in Figure 6.
The docked conformations of reference ligand X39 and protein kinase B were investigated in order to calculate qualitative estimates and the molecular basis of the analyzed bioactive molecules. Figure 7 shows 2D and 3D diagrammatic representations of the molecular bindings between the reference ligand X39 and kinase B protein’s active pocket.
According to the docking findings, all triazole-coupled acetamide scaffolds have a significant ability to influence the protein kinase B moiety. Akt is anticipated to have a high affinity for carcinoma therapeutic targets. Table 3 shows the docked complexes’ binding interactions, classifications, kinds of interactions, and interacting residues. Each ligand’s hydrophobic contacts and hydrogen bonding interactions were assessed within the receptor protein’s binding site. Table 5 describes the ligand conformations that demonstrated the greatest biological activity, as well as their suitable interactions in the receptors.
Compound 7a had the most suitable binding poses with a binding energy of −166.843 kcal/mol to protein kinase B. Compound 7a had the most suitable binding poses with a low energy of −166.843 kcal/mol to the catalytic site of protein kinase B. 7a bonded to protein kinase B via H-bonding with Gly164, Phe163, and Thr162, a carbon–hydrogen bond with SerC9, pi-sigma interaction with Gly161, and pi–sulfer interaction with Asp293. It bonded with residues Leu183, val166, and Lys181 via hydrophobic alkyl interactions. Asn280, Lys277, Glu279, ArgC6, Lys160, Glu236, Gly159, Lys165, and ThrC8 residues involve van der Waals interactions among protein kinase B and compound 7a (Figure 8).
7f and the protein kinase B complex is stabilized by H-bonding and van der Waals interactions. 7f combines with the active site of the protein via H-bonding with Gly161, ArgC6, and Asp293. Hydrophobic bonds are involved with alkyl interactions with Lys181 and Leu183 and pi-sigma interactions with Val166 residues. Figure 9 demonstrates 2D and 3D diagrams displaying the most suitable binding interactions between 7f and the active pocket of the kinase B protein.

3. Materials and Methods

3.1. General

In the present research, all of the starting materials were of analytical grade and were purchased from Alfa Aesar or Sigma Aldrich. 2-(4-isobutylphenyl) propanoic acid was used as a starting material. A Stuart SMP10 melting point apparatus was used for determining the melting point of all of the derivatives. The structures of all of the triazole-based scaffolds were confirmed by spectroscopy and physiochemical methods. An FT-IR spectrophotometer (4000–400 cm−1) by BRUKER was used at the Hi-Tech Lab, GC University, Faisalabad. 1HNMR spectra were recorded on an Bruker Advance 500 MHz spectrophotometer using DMSO-d6, on 5 mm diameter tubes at the University of Copenhagen, Denmark. An Bruker Advance NMR spectrophotometer was used to record 13C NMR spectra at 75 MHz by using DMSO-d6, on 5 mm diameter tubes. The reactions were supervised by thin layer chromatography.

3.2. General Procedure for the Synthesis of Synthesized N-Arylated 5-Aryl-1,2,4-Triazole-Coupled Acetamide Scaffolds 7a–f

3.2.1. Synthesis of Methyl 2-(4-Isobutylphenyl)Propanoate (2)

Compound 2 was prepared by the reported method [42]. The compound (2) was obtained as a pale yellow, oily liquid. Yield: (90%); b.p. 263–265 °C; IR (KBr) cm−1: 1736.32, 1203.36, 1162.83; 1H NMR (400 MHz, CDCl3) δ 7.15 (d, 2H, J = 8.0 Hz), 7.05 (d, 2H, J = 8.0 Hz), 3.67 (q, 1H), 3.60 (s, 3H), 2.40 (d, 2H, J = 8.0 Hz), 1.82 (m,1H), 1.44 (d, 3H, J = 8.0 Hz), 0.85 (d, 6H, J = 8.0 Hz). 13C NMR (101 MHz, CDCl3) δ 175.22 (C=O), 140.49 (C-1), 137.63 (C-4), 129.04 (C-2 & C-6), 127.31 (C-3 & C-5), 52.09 (OCH3), 44.99 (CH2-9), 40.11 (CH-7), 30.25 (CH-10), 22.21 (CH3-11 & CH3-12), 18.50 (CH3-8). HRMS (ESI+): m/z calculated for [(C14H20O2)+H]+: 220.1463; found: 220.1460. Element analysis: C, 76.30; H, 9.16% (Figures S1 and S6).

3.2.2. Synthesis of 2-(4-Isobutylphenyl)Propanehydrazide (3)

Compound 3 was prepared by the reported method [43]. 2-(4-isobutylphenyl) propane hydrazide was separated as a white, crystalline solid. Yield: (88%); m.p. 77–78 °C; IR (KBr) cm−1: 3272.76, 2963.13, 1640.12, 1604.83, 1466.29, 1366.62, 906.66, 686.83. 1H NMR (400 MHz, CDCl3) δ 9.50 (s, 1H), 7.13 (d, 2H, J = 8.0 Hz), 7.06 (d, 2H, J = 8.0 Hz), 3.48 (d, 2H), 3.46 (q, 1H), 2.40 (d, 2H, J = 8.0 Hz), 1.81 (m,1H), 1.48 (d, 3H, J = 8.0 Hz), 0.84 (d, 6H, J = 8.0 Hz). 13C NMR (101 MHz, CDCl3) δ 175.24 (C=O), 140.49 (C-1), 137.59 (C-4), 129.57 (C-2 & C-6), 127.30 (C-3 & C-5), 44.96 (CH2-9), 40.11 (CH-7), 30.25 (CH-10), 22.19 (CH3-11 & CH3-12), 18.23 (CH3-8). HRMS (ESI+): m/z calculated for [(C13H20N2O)+H]+. 220.1576; found: 220.1574. Element analysis: C, 70.85; H, 9.16; N, 12.72% (Figures S2 and S7).

3.2.3. Synthesis of 5-(1-(4- Isobutylphenyl)Ethyl)-1,2,4-Triazole -2-Thiol (4)

In the current study, methyl isothiocyanate and 2-(4-isobutylphenyl) propane hydrazide (0.02 mol) were dissolved in 10% KOH soln. in an equimolar amount. For 10–11 h, the mixture was set on refluxing at 95 °C. Thin-layer chromatography was used for monitoring the reaction. Upon completion, cold water was added to afford the precipitates of product. Water was used to filter and wash the precipitates. The precipitates were further purified with an ethanolic recrystallization process. The 5-(1-(4-isobutylphenyl)ethyl)-1,2,4- triazole-2-thiol scaffold was crystalized as an off-white solid. 1HNMR (400 MHz, CDCl3) δ 11.77 (s, 1H, SH), 7.05 (d, 2H, J = 8.0 Hz), 6.99 (d, 2H, J = 8.0 Hz), 3.96 (q, 1H), 3.18 (s, 3H), 2.39 (d, 2H, J = 8.0 Hz), 1.80 (m,1H), 1.62 (d, 3H, J = 8.0 Hz), 0.83 (d, 6H, J = 8.0 Hz). 13C NMR (101 MHz, CDCl3) 155.16 (C-1 & C-4), 141.43 (C-1), 137.11 (C-4), 129.94 (C-2 & C-6), 126.87 (C-3 & C-5), 45.03 (CH2-9), 37.60 (CH-7), 30.67 (CH-10), 30.15 (CH3-N), 22.22 (CH3-11 & CH3-12), 20.20 (CH3-8). HRMS (ESI+): m/z calculated for [(C15H21N3S)+H]+: 275.1456; found: 275.1454. Element analysis: C, 65.41; H, 7.69; N, 15.27; S, 11.65% (Figures S3–S5 and S8).

3.2.4. Synthesis of N–Aryl/Alkyl 2-Bromoroacetamides 6a–f

Compounds 6a–f were synthesized using the reported method [27]. In an RBF, 12.0 moles N-substituted alkyl/aryl amines (5a–f) were dissolved in 10.0 mL of 5% Na2CO3 solution. Bromoacetyl bromide (12.0 mmoles) was gradually added to the reaction mixture described above. Upon reaction completion, n-hexane was added to afford arylated derivatives as precipitates which were further purified with an ethanolic recrystallization process or column chromatography technique using ethyl acetate–petroleum ether (1:9).

3.2.5. Synthesis of N-Arylated 5-(1-(4-Isobutylphenyl)Ethyl)-1,2,4-Triazole-2-yl- 2-Sulfanyl Coupled Acetamide Derivatives 7a–f

Various N-arylated 5-(1-(4-isobutylphenyl)ethyl)-1,2,4-triazole-2-yl-2-sulfanyl-coupled acetamide compounds were prepared in good yield by thoroughly mixing 4 (0.02 mol) with an equimolar amount of N-alkyl/aryl 2-bromoacetamides 6a–f using DMF and NaH (0.01 mol). Thin-layer chromatography was used for monitoring the reaction. Upon reaction completion, n-hexane was added to afford arylated derivatives as precipitates which were further purified with an ethanolic recrystallization process or column chromatography technique using ethyl acetate–petroleum ether (1:9).

3.2.6. N-(2-Methylphenyl)-2-((5-(1-(4-isobutylphenyl)ethyl)-4-methyl-4H-1,2,4-triazol-3-yl)thio)Acetamide (7a)

White, amorphous solid. Yield 73%, m.p 122–124 °C. IR: ν (cm−1): 3270, 1696, 1524, 1488, 1306, 1082, 756. 1HNMR (500 MHz, DMSO) δ 9.64 (s, 1H), 7.40 (d, J = 5.0 Hz), 7.20 (d, J = 5.0 Hz), 7.15 (t, 1H), 7.09–7.06 (m, 5H), 4.33–4.29 (q, 1H), 4.04 (s, 2H), 3.28 (s, 3H), 2.40 (d, 2H, J = 10.0 Hz), 2.15 (s, 3H), 1.83–1.78 (m, 1H) 1.60 (d, J = 5.0 Hz), 0.85 (d, J= 10.0 Hz). 13C NMR (126 MHz, DMSO) δ 165.93, 158.08, 149.15, 139.59, 139.36, 135.90, 131.21, 130.27, 129.27, 126.81, 125.92, 125.18, 124.32, 44.15, 37.11, 35.47, 29.99, 29.53, 22.14, 20.93, 17.67 (Figures S9–S11). HRMS (ESI+): m/z calculated for [(C24H30N4OS)+H]+: 423.2119; found: 423.2214 (Figure S27). Analysis calculated for C24H30N4OS, C, 68.21; H, 7.16; N, 13.26; S, 7.59%.

3.2.7. N-(4-Bromo-2-Mthylphenyl)-2-((5-(1-(4-Isobutylphenyl)Ethyl)-4-Methyl-4H-1,2,4-Triazol-3-yl)Thio)Acetamide (7b)

Off-white, amorphous solid. m.p 122–124 °C. Yield 71%. IR: ν (cm−1): 3370, 1670, 1528, 1470, 1306, 659.93. 1HNMR (500 MHz, DMSO) δ 9.68 (s, 1H), 7.43 (s, 1H), 7.40 (d, J = 5.0 Hz, 1H), 7.34 (dd, J = 10.0 Hz, 1H), 7.08–7.05 (m, 4H), 4.33–4.2 (q, 1H), 4.04 (s, 2H), 3.27 (s, 3H), 2.40 (d, 2H, J = 10.0 Hz), 2.15 (s, 3H), 1.80–1.75 (m, 1H), 1.60 (d, J = 10.0 Hz, 3H), 0.85 (d, J = 10.0 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 166.17, 158.09, 149.05, 139.53, 139.30, 135.34, 133.77, 132.67, 129.25, 128.71, 126.78, 125.93, 44.15, 37.10, 35.47, 30.00, 29.53, 22.14, 20.92, 17.40 (Figures S12–S14). HRMS (ESI+): m/z calculated for [(C24H29BrN4OS)+H]+: 501.1324; found: 503.1313 (Figure S28). Analysis calculated for C24H29BrN4OS. Elemental Analysis: C, 57.48; H, 5.83; N, 11.17; S, 6.39.

3.2.8. N-(4-Ethylphenyl)-2-((5-(1-(4-Isobutylphenyl)Ethyl)-4-Methyl-4H-1,2,4-Triazol-3-yl)Thio)Acetamide (7c)

Off-white, amorphous solid. Yield 75%, m.p 100–102 °C. IR: ν (cm−1): 3235, 1682, 1517, 1468, 1320, 695. 1HNMR (500 MHz, DMSO) δ 10.18 (s, 1H), 7.43–7.42 (d, 2H, J = 5.0 Hz), 7.14–7.13 (d, 2H, J = 5.0 Hz), 7.05–7.01, (m, 4H,) 4.32–4.29 (q, 1H), 3.98 (s, 2H), 3.26 (s, 3H), 2.39(d, 2H, J = 5.0 Hz), 1.81–1.76 (m, 1H), 1.59 (d, J = 12, 3H), 1.17–1.14 (t, 3H) 0.84 (d, J = 5.0 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 165.44, 157.97, 149.05, 139.46, 139.35, 138.87, 136.40, 129.25, 127.91, 126.68, 119.14, 44.15, 37.84, 35.49, 29.97, 29.52, 27.54, 22.14, 20.89, 15.60 (Figures S15–S17). HRMS (ESI+): m/z calculated for [(C25H32N4OS)+H]+: 437.2375; found: 437.2366 (Figure S29). Analysis calculated for C25H32N4OS, C, 68.77; H, 7.39; N, 12.83; S, 7.34.

3.2.9. N-(2-Chlorophenyl)-2-((5-(1-(4-Isobutylphenyl)Ethyl)-4-Methyl-4H-1,2,4-Triazol-3-yl)Thio)Acetamide (7d)

White, amorphous solid. Yield 70%, m.p 123–125 °C. IR: ν (cm−1): 3330, 1691, 1515, 1452, 1315, 1081, 757. 1HNMR (500 MHz, DMSO) δ 9.90 (s, 1H), 7.75 (d, 1H, J = 10.0 Hz), 7.50 (d, 1H, J =10.0 Hz), 7.33–7.31 (t, 1H, J = 5.0 & 10.0 Hz), 7.21–7.18 (t, 1H, J = 5.0 & 10.0 Hz), 7.10–7.06 (m, 4H), 4.33–4.29 (q, 1H), 4.12 (s, 2H), 3.28 (s, 3H), 2.40(d, 2H, J = 5.0 Hz), 1.79–1.76 (m, 1H) 1.60 (d, J = 5.0 Hz, 3H), 0.84 (d, J = 10.0 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 166.56, 158.12, 148.93, 139.65, 139.28, 134.53, 129.50, 129.27, 127.43, 126.81, 126.14, 125.14, 44.07, 37.07, 35.48, 30.04, 29.53, 22.13, 20.97 (Figures S18–S20). HRMS (ESI+): m/z calculated for [(C23H27ClN4OS)+H]+: 444.1672; found: 444.1697 (Figure S30). Analysis calculated for C23H27ClN4OS, C, 62.36; H, 6.14; N, 12.65; S, 7.24.

3.2.10. N-(Phenyl)-2-((5-(1-(4-Isobutylphenyl)Ethyl)-4-Methyl-4H-1,2,4-Triazol-3-yl)Thio)Acetamide (7e)

White, amorphous solid. Yield 74%, m.p 88–90 °C. IR: ν (cm−1): 3250, 1686, 1525, 1436, 1303, 1082, 757. 1HNMR (500 MHz, DMSO) δ 10.27 (s, 1H), 7.54 (d, 2H, J = 10.0 Hz), 7.32–7.29 (t, 2H, J = 5.0 & 10.0 Hz), 7.10–7.02 (m, 5H), 4.31–4.29 (q, 1H), 4.01 (s, 2H), 3.29 (s, 3H), 2.39(d, 2H, J = 5.0 Hz), 1.79–1.76 (m, 1H), 1.59 (d, J = 5.0 Hz, 3H), 0.85 (d, J = 10.0 Hz, 6H). 13CNMR (126 MHz, DMSO), δ 165.75, 158.10, 149.03, 139.60, 139.33, 138.68, 129.25, 128.74, 126.77, 123.42, 119.04, 44.10, 37.79, 35.41, 29.88, 29.49, 22.14, 20.89 (Figures S21–S23). HRMS (ESI+): m/z calculated for [(C23H28N4OS)+H]+: 409.2044; found: 409.2054 (Figure S31). Analysis calculated for C23H28N4OS, C, 67.62; H, 6.91; N, 13.71; S, 7.85.

3.2.11. N-(2,6-Dimethylphenyl)-2-((5-(1-(4-Isobutylphenyl)Ethyl)-4-Methyl-4H-1,2,4-Triazol-3-yl)Thio)Acetamide (7f)

Off-white, amorphous solid. Yield 76%, m.p 150–152 °C. IR: ν (cm−1): 3272, 1640, 1516, 1445, 1388, 1081, 694. 1HNMR (500 MHz, DMSO) δ 9.68 (s, 1H), 7.12–7.02 (m, 7H), 4.40–4.23 (q, 1H), 4.06 (s, 2H), 3.29 (s, 3H), 2.29 (d, 2H, J = 10.0 Hz), 2.13 (s, 6H), 1.81–1.78 (m, 1H), 1.61 (d, J = 5.0 Hz, 3H), 0.85 (d, J= 10.0 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 165.46, 157.98, 149.10, 139.60, 139.38, 135.06, 134.57, 129.26, 127.57, 126.83, 126.49, 44.15, 36.59, 35.46, 29.97, 29.54 (Figures S24–S26). HRMS (ESI+): m/z calculated for [(C25H32N4OS)+H]+: 437.2375; found: 437.2366 (Figure S32). Analysis calculated for C25H32BN4OS, C, 68.77; H, 7.39; N, 12.83; S, 7.34.

3.3. Experimental Procedures for Biological Activities

3.3.1. Cell Culture and Treatment

Human HepG2 liver cancer cell lines were cultured by Dulbecco’s modified Eagle’s medium. It is composed of 100 μg/mL streptomycin, 100 units/mL penicillin, and 10% FBS. A humidified atmosphere was provided for incubation at 37 °C with 5% CO2. The anti-hepatocellular therapeutic potential of triazole-based scaffolds was evaluated by dissolving its different concentrations in 0.05% DMSO.

3.3.2. Evaluation of Cell Viability

An MTT assay was applied for evaluation of cell viability against the HepG2 cell line [44]. In short, different concentrations of new triazole-based scaffolds were incubated with HepG2 cell lines for 48 h. After incubation, 5 mg/mL of 10 µL MTT solution was added in each plate and they were further incubated at 37 °C for 4 h. The percentage of cell viability was calculated at 490 nm after the addition of 150 μL DMSO into a microplate reader (Thermo Scientific, Waltham, MA, USA).

3.3.3. Hemolytic Activity Potential

Hemolytic activity was investigated by the reported method [45,46] using Triton-X-100 as standard.

3.4. Molecular Docking of Triazole-Coupled Acetamides

Docking experiments for all of the scaffolds were carried out in order to comprehend the potential interaction process of the synthesized anti-cancer compounds on the HepG2 cancer cell line. The website https://www.rcsb.org was used for drawing the structures of PI3Kalpha, Akt, c-kit tyrosine kinase, human Aurora B kinase, and STAT3 from the RCSB Protein Data Bank under the PDB IDs of 4FA6, 2X39, 1T46, 4AF3, and 6NJS, respectively [36,37,38,39,47]. ChemDraw 20.1.1 was used to create and reduce the 3D SDF structures of all of the compounds, which were then transferred to MarvinSketch. Prior to docking, the target proteins’ frameworks were evaluated, and errors in amino acid structures were rectified using Molegro Virtual Docker software [48]. The grid boxs’ centers were chosen to be the co-crystallized ligands of proteins. They re-docked in order to validate the in silico process. Molegro Virtual Docker was applied to dock active chemicals 10 times to the target proteins’ receptors. The sequences with the lowest interaction affinity and excellent connections with the targets were separated for further detailed analysis. The molecular bindings between the target and new derivatives were visualized in 2D using Discovery Studio Visualizer Software 2021.

4. Conclusions

A series of new anti-cancer compounds (7a–f) were synthesized in moderate to good yield (73–76%) by combining compound 4 with various electrophiles under different reaction conditions (Table 1, Scheme 1). Because of its low bioavailability of 38–49%, Sorafenib necessitates a significant daily dose in cancer therapy. Sorafenib is a very costly medicine with many side effects. We have incorporated various electron-donating and electron-withdrawing groups into electrophiles to test structure–activity relationships at various concentrations. All of the molecules demonstrated medium to outstanding anti-cancer activity, comparable to sorafenib, which diversified according to aryl ring substitution, as shown in Table 1. These triazole-based acetamide derivatives also exhibited low cytotoxicity, with values ranging from 7.33% to 1.19% in comparison to the 100% cytotoxicity exhibited by the reference standard Triton X100. Compounds 7f and 7a showed the highest anti-cancer potential, with IC50 values of 16.782 µg/mL and 20.667 µg/mL, respectively. On the other hand, the triazole derivative containing an electron-withdrawing chloro moiety demonstrated the least anti-proliferative activity with an IC50 value of 39.667 µg/mL. The sequence of anti-cancer potential was found to be 7f > 7a > 7b > 7c > 7e > 7d. The anti-cancer potential of all of the compounds was further investigated by molecular docking studies and the results were in accordance with in-vitro studies. In silico studies have shown that the molecules have strong affinity for kinase targets. Molecules 7f and 7a have shown their anti-cancer effects, especially by affecting Akt and c-lit molecular targets. According to in silico modelling studies, 7f has an outstanding docking score with the lowest binding energy of −170.066 kcal/mol, which is lower than the reference ligand X39 for protein kinase B (−130.624 kcal/mol). We concluded that compound 7f contained electron-donating methyl groups at the 2 and 6 position of the aryl ring and showed good anti-cancer activity, low cytotoxicity, and good thrombolytic activity. Thus, compound 7f might be utilized to synthesize new anti-cancer drugs in the near future.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph16020211/s1, Figure S1: 1HNMR spectrum of compound 2, Figure S2: 1HNMR spectrum of compound 3, Figure S3: 1HNMR spectrum of compound 4, Figure S4: 1HNMR spectrum of compound 4 (aromatic region), Figure S5: 1HNMR spectrum of compound 4 (aliphatic region), Figure S6: 13C NMR spectrum of compound 2, Figure S7: 13C NMR spectrum of compound 3, Figure S8: 13C NMR spectrum of compound 4, Figure S9: 1H NMR spectrum of 7a, Figure S10: COSYH NMR spectrum of 7a, Figure S11: 13C NMR spectrum of 7a, Figure S12: 1H NMR spectrum of 7b, Figure S13: COSYH NMR spectrum of 7b, Figure S14: 13C NMR spectrum of 7b, Figure S15: 1H NMR spectrum of 7c, Figure S16: COSYH NMR spectrum of 7c, Figure S17: 13C NMR spectrum of 7c, Figure S18: 1H NMR spectrum of 7d, Figure S19: COSYH NMR spectrum of 7d, Figure S20: 13C NMR spectrum of 7d, Figure S21: 1H NMR spectrum of 7e, Figure S22: COSYH NMR spectrum of 7e, Figure S23: 13C NMR spectrum of 7e, Figure S24: 1H NMR spectrum of 7f, and Figure S25: COSYH NMR spectrum of 7f. Figure S26: 13C NMR spectrum of 7f. Figure S27: HRMS spectrum of compound 7a, Figure S28: HRMS spectrum of compound 7b, Figure S29: HRMS spectrum of compound 7c, Figure S30: HRMS spectrum of compound 7d, Figure S31: HRMS spectrum of compound 7e, and Figure S32: HRMS spectrum of compound 7f.

Author Contributions

Conceptualization and experimental design, S.G.K. and S.A.A.S.; methodology, N.A., S.I., S.G.K. and M.A.S.A.; experimental work, S.B., S.G.K., Ş.A., A.u.R., F.A. and S.M.; software, J.B.C. and Ş.A.; writing—original draft preparation, N.A., S.B., A.R., Z.R., S.G.K. and M.A.S.A.; writing—review and editing, Ş.A., Z.R., N.A., A.u.R., S.B., S.G.K., M.u.N., Z.R., J.B.C., A.R., S.A.A.S., F.A. and M.A.S.A.; data curation, J.B.C., S.I., S.M., S.A.A.S., F.A., N.R., Z.R., M.u.N. and S.G.K.; visualization, S.G.K., A.u.R., Ş.A., M.u.N. and J.B.C.; supervision, S.G.K.; project administration, S.G.K., N.A. and M.A.S.A.; funding acquisition, S.A.A.S. All authors have read and agreed to the published version of the manuscript.

Funding

The authors would like to acknowledge the Ministry of Higher Education (MOHE) Malaysia for financial support under the Fundamental Research Grant Scheme (FRGS) with sponsorship reference number FRGS/1/2019/STG05/UITM/02/9. The authors would also like to acknowledge the Universiti Teknologi MARA for the financial support under the reference number 600-IRMI/FRGS 5/3 (424/2019).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and Supplementary Material.

Acknowledgments

The authors would like to acknowledge Research Management Centre (RMC), Office of Deputy Vice-Chancellor (Research & Innovation), Universiti Teknologi MARA 40450 Shah Alam Selangor Darul Ehsan Malaysia for financial support.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of all of the compounds are available from the authors.

References

  1. Kamal, A.; Bharathi, E.V.; Reddy, J.S.; Ramaiah, M.J.; Dastagiri, D.; Reddy, M.K.; Viswanath, A.; Reddy, T.L.; Shaik, T.B.; Pushpavalli, S.; et al. Synthesis and biological evaluation of 3,5-diaryl isoxazoline/isoxazole linked 2,3-dihydroquinazolinone hybrids as anticancer agents. Eur. J. Med. Chem. 2011, 46, 691–703. [Google Scholar] [CrossRef] [PubMed]
  2. Tahiliani, H.T.; Purohit, A.P.; Desai, S.C.; Jarwani, P.B. Retrospective analysis of histopathological spectrum of premalignant and malignant colorectal lesions. Cancer Res. Stat. Treat. 2021, 4, 472–478. [Google Scholar] [CrossRef]
  3. Jang, J.-W.; Song, Y.; Kim, K.M.; Kim, J.-S.; Choi, E.K.; Kim, J.; Seo, H. Hepatocellular carcinoma-targeted drug discovery through image-based phenotypic screening in co-cultures of HCC cells with hepatocytes. BMC Cancer 2016, 16, 810. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Rumgay, H.; Arnold, M.; Ferlay, J.; Lesi, O.; Cabasag, C.J.; Vignat, J.; Laversanne, M.; McGlynn, K.A.; Soerjomataram, I. Global burden of primary liver cancer in 2020 and predictions to 2040. J. Hepatol. 2022, 77, 1598–1606. [Google Scholar] [CrossRef]
  5. Zheng, Z.; Liu, Q.; Kim, W.; Tharmalingam, N.; Fuchs, B.B.; Mylonakis, E. Antimicrobial activity of 1,3,4-oxadiazole derivatives against planktonic cells and biofilm of Staphylococcus aureus. Futur. Med. Chem. 2018, 10, 283–296. [Google Scholar] [CrossRef] [PubMed]
  6. Hanahan, D.; Weinberg, R.A. Hallmarks of cancer: The next generation. Cell 2011, 144, 646–674. [Google Scholar] [CrossRef] [Green Version]
  7. Chen, J.-S.; Chou, C.-H.; Wu, Y.-H.; Yang, M.-H.; Chu, S.-H.; Chao, Y.-S.; Chen, C.-N. CC-01 (chidamide plus celecoxib) modifies the tumor immune microenvironment and reduces tumor progression combined with immune checkpoint inhibitor. Sci. Rep. 2022, 12, 1100. [Google Scholar] [CrossRef]
  8. Liu, Q.; Zhang, B.; Wang, Y.; Wang, X.; Gou, S. Discovery of phthalazino[1,2-b]-quinazolinone derivatives as multi-target HDAC inhibitors for the treatment of hepatocellular carcinoma via activating the p53 signal pathway. Eur. J. Med. Chem. 2021, 229, 114058. [Google Scholar] [CrossRef]
  9. Chen, C.; Li, X.; Zhao, H.; Liu, M.; Du, J.; Zhang, J.; Yang, X.; Hou, X.; Fang, H. Discovery of DNA-Targeting HDAC Inhibitors with Potent Antitumor Efficacy In Vivo That Trigger Antitumor Immunity. J. Med. Chem. 2022, 65, 3667–3683. [Google Scholar] [CrossRef]
  10. Khan, I.; Ibrar, A.; Abbas, N.; Saeed, A. Recent advances in the structural library of functionalized quinazoline and quinazolinone scaffolds: Synthetic approaches and multifarious applications. Eur. J. Med. Chem. 2014, 76, 193–244. [Google Scholar] [CrossRef]
  11. Khwaza, V.; Mlala, S.; Oyedeji, O.; Aderibigbe, B. Pentacyclic Triterpenoids with Nitrogen-Containing Heterocyclic Moiety, Privileged Hybrids in Anticancer Drug Discovery. Molecules 2021, 26, 2401. [Google Scholar] [CrossRef] [PubMed]
  12. Gomha, S.M.; Abdel-Aziz, H.M.; El-Reedy, A.A.M. Facile Synthesis of Pyrazolo[3,4-c]pyrazoles Bearing Coumarine Ring as Anticancer Agents. J. Heterocycl. Chem. 2018, 55, 1960–1965. [Google Scholar] [CrossRef]
  13. Kaproń, B.; Czarnomysy, R.; Wysokiński, M.; Andrys, R.; Musilek, K.; Angeli, A.; Supuran, C.T.; Plech, T. 1,2,4-Triazole-based anticonvulsant agents with additional ROS scavenging activity are effective in a model of pharmacoresistant epilepsy. J. Enzym. Inhib. Med. Chem. 2020, 35, 993–1002. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Li, Y.-S.; Tian, H.; Zhao, D.-S.; Hu, D.-K.; Liu, X.-Y.; Jin, H.-W.; Song, G.-P.; Cui, Z.-N. Synthesis and bioactivity of pyrazole and triazole derivatives as potential PDE4 inhibitors. Bioorganic Med. Chem. Lett. 2016, 26, 3632–3635. [Google Scholar] [CrossRef]
  15. de Andrade, M.A.R.; Lima, B.J.S.; de Jesus, A.A.; Teixeira, L.D.A.C.; de Souza, C.R.S.; Fontes, R.B.; Mendonça, N.P.V.; Gonçalves, H.S.; Cesar, A.S.; Aragão, M.T. Burnout Syndrome in COVID-19: An analysis of physicians from the public and private health system in the state of Sergipe. Res. Soc. Dev. 2022, 11, e3911729602. [Google Scholar] [CrossRef]
  16. Kumar, S.; Khokra, S.L.; Yadav, A. Triazole analogues as potential pharmacological agents: A brief review. Futur. J. Pharm. Sci. 2021, 7, 106. [Google Scholar] [CrossRef]
  17. Han, S.; Zhang, F.-F.; Xie, X.; Chen, J.-Z. Design, synthesis, biological evaluation, and comparative docking study of 1,2,4-triazolones as CB1 receptor selective antagonists. Eur. J. Med. Chem. 2014, 74, 73–84. [Google Scholar] [CrossRef]
  18. Zhang, W.; Yuan, J. Poly(1-Vinyl-1,2,4-triazolium) Poly(Ionic Liquid)s: Synthesis and the Unique Behavior in Loading Metal Ions. Macromol. Rapid Commun. 2016, 37, 1124–1129. [Google Scholar] [CrossRef] [Green Version]
  19. Cao, X.; Wang, W.; Wang, S.; Bao, L. Asymmetric synthesis of novel triazole derivatives and their in vitro antiviral activity and mechanism of action. Eur. J. Med. Chem. 2017, 139, 718–725. [Google Scholar] [CrossRef]
  20. Hu, G.; Wang, C.; Xin, X.; Li, S.; Li, Z.; Zhao, Y.; Gong, P. Design, synthesis and biological evaluation of novel 2,4-diaminopyrimidine derivatives as potent antitumor agents. New J. Chem. 2019, 43, 10190–10202. [Google Scholar] [CrossRef]
  21. Ni, T.; Ding, Z.; Xie, F.; Hao, Y.; Bao, J.; Zhang, J.; Yu, S.; Jiang, Y.; Zhang, D. Design, Synthesis, and In Vitro and In Vivo Antifungal Activity of Novel Triazoles Containing Phenylethynyl Pyrazole Side Chains. Molecules 2022, 27, 3370. [Google Scholar] [CrossRef] [PubMed]
  22. Zhang, G.-F.; Liu, X.; Zhang, S.; Pan, B.; Liu, M.-L. Ciprofloxacin derivatives and their antibacterial activities. Eur. J. Med. Chem. 2018, 146, 599–612. [Google Scholar] [CrossRef] [PubMed]
  23. Li, Z.; Xu, L.; Zhu, L.; Zhao, Y.; Hu, T.; Yin, B.; Liu, Y.; Hou, Y. Design, synthesis and biological evaluation of novel pteridinone derivatives possessing a hydrazone moiety as potent PLK1 inhibitors. Bioorganic Med. Chem. Lett. 2020, 30, 127329. [Google Scholar] [CrossRef] [PubMed]
  24. Akhtar, T.; Hameed, S.; Al-Masoudi, N.P.; Khan, K.M. Synthesis and anti-HIV activity of new chiral 1,2,4-triazoles and 1,3,4-thiadiazoles. Heteroat. Chem. 2007, 18, 316–322. [Google Scholar] [CrossRef]
  25. Irfan, A.; Faiz, S.; Rasul, A.; Zafar, R.; Zahoor, A.F.; Kotwica-Mojzych, K.; Mojzych, M. Exploring the Synergistic Anticancer Potential of Benzofuran–Oxadiazoles and Triazoles: Improved Ultrasound- and Microwave-Assisted Synthesis, Molecular Docking, Hemolytic, Thrombolytic and Anticancer Evaluation of Furan-Based Molecules. Molecules 2022, 27, 1023. [Google Scholar] [CrossRef]
  26. Gul, S.; Rehman, A.U.; Abbasi, M.A.; Khan, K.M.; Nafeesa, K.; Siddiqa, A.; Akhtar, M.N.; Shahid, M.; Subhani, Z. Synthesis, antimicrobial evaluation and hemolytic activity of 2-[[5-alkyl/aralkyl substituted-1,3,4-oxadiazol-2-yl]thio]-N-[4-(4-morpholinyl)phenyl]acetamide derivatives. J. Saudi Chem. Soc. 2014, 21, S425–S433. [Google Scholar] [CrossRef]
  27. Khan, S.G.; Bokhari, T.H.; Anjum, F.; Akhter, N.; Rasool, S.; Shah, S.A.A.; Shahid, M.; Arshad, A. Synthesis, characterization, antibacterial, hemolytic and thrombolytic activity evaluation of 5-(3-chlorophenyl)-2-((N-(substituted)-2-acetamoyl) sulfanyl)-1, 3, 4-oxadiazole derivatives. Pak. J. Pharm. Sci. 2020, 33, 871–876. [Google Scholar]
  28. Mahmood, S.; Khan, S.G.; Rasul, A.; Christensen, J.B.; Abourehab, M.A.S. Ultrasound Assisted Synthesis and In Silico Modelling of 1,2,4-Triazole Coupled Acetamide Derivatives of 2-(4-Isobutyl phenyl)propanoic acid as Potential Anticancer Agents. Molecules 2022, 27, 7984. [Google Scholar] [CrossRef]
  29. Aziz-ur-Rehman; Khan, S.G.; Naqvi, S.A.R.; Ahmad, M.; Akhtar, N.; Bokhari, T.H.; Irfan, M.; Usman, A.; Batool, S.; Rasool, S. Synthesis, spectral analysis and biological evaluation of 2-{[(morpholin-4-yl) ethyl] thio}-5-phenyl/aryl-1, 3, 4-oxadiazole derivatives. Pak. J. Pharm. Sci. 2021, 34, 441–446. [Google Scholar] [CrossRef]
  30. Amewu, R.K.; Sakyi, P.O.; Osei-Safo, D.; Addae-Mensah, I. Synthetic and Naturally Occurring Heterocyclic Anticancer Compounds with Multiple Biological Targets. Molecules 2021, 26, 7134. [Google Scholar] [CrossRef]
  31. Zabiulla; Gulnaz, A.; Mohammed, Y.H.E.; Khanum, S.A. Design, synthesis and molecular docking of benzophenone conjugated with oxadiazole sulphur bridge pyrazole pharmacophores as anti-inflammatory and analgesic agents. Bioorganic Chem. 2019, 92, 103220. [Google Scholar] [CrossRef] [PubMed]
  32. Iqbal, K.; Jamal, Q.; Iqbal, J.; Afreen, M.S.; Sandhu, M.Z.A.; Dar, E.; Farooq, U.; Mushtaq, M.F.; Arshad, N.; Iqbal, M.M. Synthesis of N-substituted acetamide derivatives of azinane-bearing 1,3,4-oxadiazole nucleus and screening for antibacterial activity. Trop. J. Pharm. Res. 2017, 16, 429. [Google Scholar] [CrossRef] [Green Version]
  33. Thongnest, S.; Chawengrum, P.; Keeratichamroen, S.; Lirdprapamongkol, K.; Eurtivong, C.; Boonsombat, J.; Kittakoop, P.; Svasti, J.; Ruchirawat, S. Vernodalidimer L, a sesquiterpene lactone dimer from Vernonia extensa and anti-tumor effects of vernodalin, vernolepin, and vernolide on HepG2 liver cancer cells. Bioorganic Chem. 2019, 92, 103197. [Google Scholar] [CrossRef] [PubMed]
  34. Shahzadi, I.; Parveen, B.; Ahmad, S.; Zahoor, A.F.; Rasul, A.; Zahid, F.M. In-vitro cytotoxic evaluation, hemolytic and thrombolytic potential of newly designed acefylline based hydrazones as potent anti-cancer agents against human lung cancer cell line (A549). Pak. J. Pharm. Sci. 2022, 35, 885–889. [Google Scholar] [PubMed]
  35. Daina, A.; Michielin, O.; Zoete, V. SwissTargetPrediction: Updated data and new features for efficient prediction of protein targets of small molecules. Nucleic Acids Res. 2019, 47, W357–W364. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Mol, C.D.; Dougan, D.R.; Schneider, T.R.; Skene, R.J.; Kraus, M.L.; Scheibe, D.N.; Snell, G.P.; Zou, H.; Sang, B.-C.; Wilson, K.P. Structural Basis for the Autoinhibition and STI-571 Inhibition of c-Kit Tyrosine Kinase. J. Biol. Chem. 2004, 279, 31655–31663. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. McHardy, T.; Caldwell, J.J.; Cheung, K.-M.; Hunter, L.J.; Taylor, K.; Rowlands, M.; Ruddle, R.; Henley, A.; Brandon, A.D.H.; Valenti, M.; et al. Discovery of 4-Amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamides As Selective, Orally Active Inhibitors of Protein Kinase B (Akt). J. Med. Chem. 2010, 53, 2239–2249. [Google Scholar] [CrossRef] [PubMed]
  38. Elkins, J.M.; Santaguida, S.; Musacchio, A.; Knapp, S. Crystal Structure of Human Aurora B in Complex with INCENP and VX-680. J. Med. Chem. 2012, 55, 7841–7848. [Google Scholar] [CrossRef]
  39. Degorce, S.L.; Boyd, S.; Curwen, J.O.; Ducray, R.; Halsall, C.T.; Jones, C.D.; Lach, F.; Lenz, E.M.; Pass, M.; Pass, S.; et al. Discovery of a Potent, Selective, Orally Bioavailable, and Efficacious Novel 2-(Pyrazol-4-ylamino)-pyrimidine Inhibitor of the Insulin-like Growth Factor-1 Receptor (IGF-1R). J. Med. Chem. 2016, 59, 4859–4866. [Google Scholar] [CrossRef]
  40. Zhang, Q.; Sang, F.; Qian, J.; Lyu, S.; Wang, W.; Wang, Y.; Li, Q.; Du, L. Identification of novel potential PI3Kα inhibitors for cancer therapy. J. Biomol. Struct. Dyn. 2020, 39, 3721–3732. [Google Scholar] [CrossRef]
  41. Bai, L.; Zhou, H.; Xu, R.; Zhao, Y.; Chinnaswamy, K.; McEachern, D.; Chen, J.; Yang, C.-Y.; Liu, Z.; Wang, M.; et al. A Potent and Selective Small-Molecule Degrader of STAT3 Achieves Complete Tumor Regression In Vivo. Cancer Cell 2019, 36, 498–511.e17. [Google Scholar] [CrossRef] [PubMed]
  42. Sardar, A.; Abid, O.-U.; Daud, S.; Fakhar-E-Alam, M.; Siddique, M.H.; Ashraf, M.; Shahid, W.; Ejaz, S.A.; Atif, M.; Ahmad, S.; et al. Design, synthesis, in vitro and in silico studies of naproxen derivatives as dual lipoxygenase and α-glucosidase inhibitors. J. Saudi Chem. Soc. 2022, 26, 101468. [Google Scholar] [CrossRef]
  43. Alderawy MQ, A.; Alrubaie LA, R.; Sheri, F.H. Synthesis, Characterization of Ibuprofen N-Acyl-1, 3, 4-Oxadiazole Derivatives and Anti-cancer Activity against MCF-7 Cell Line. Syst. Rev. Pharm. 2020, 11, 681–689. [Google Scholar] [CrossRef]
  44. Shaikh, M.H.; Subhedar, D.D.; Nawale, L.; Sarkar, D.; Khan, F.A.K.; Sangshetti, J.N.; Shingate, B.B. 1,2,3-Triazole derivatives as antitubercular agents: Synthesis, biological evaluation and molecular docking study. MedChemComm 2015, 6, 1104–1116. [Google Scholar] [CrossRef]
  45. Abutaha, N.; Al-Mekhlafi, F.A.; Almutairi, B.O.; Wadaan, M.A. S-phase cell cycle arrest, and apoptotic potential of Echium arabicum phenolic fraction in hepatocellular carcinoma HepG2 cells. J. King Saud Univ.—Sci. 2021, 34, 101735. [Google Scholar] [CrossRef]
  46. Win, T.S.; Malik, A.A.; Prachayasittikul, V.S.; Wikberg, J.E.; Nantasenamat, C.; Shoombuatong, W. HemoPred: A web server for predicting the hemolytic activity of peptides. Futur. Med. Chem. 2017, 9, 275–291. [Google Scholar] [CrossRef]
  47. Le, P.T.; Cheng, H.; Ninkovic, S.; Plewe, M.; Huang, X.; Wang, H.; Bagrodia, S.; Sun, S.; Knighton, D.R.; Rogers, C.M.L.; et al. Design and synthesis of a novel pyrrolidinyl pyrido pyrimidinone derivative as a potent inhibitor of PI3Kα and mTOR. Bioorganic Med. Chem. Lett. 2012, 22, 5098–5103. [Google Scholar] [CrossRef]
  48. Thomsen, R.; Christensen, M.H. MolDock: A New Technique for High-Accuracy Molecular Docking. J. Med. Chem. 2006, 49, 3315–3321. [Google Scholar] [CrossRef]
Pharmaceuticals 16 00211 g001 550
Figure 1. Structure of some common 1,2,4-triazole-based drugs.
Figure 1. Structure of some common 1,2,4-triazole-based drugs.
Pharmaceuticals 16 00211 g001
Pharmaceuticals 16 00211 sch001 550
Scheme 1. Synthesis of 1,2,4-triazole-cored acetamides 7a–f.
Scheme 1. Synthesis of 1,2,4-triazole-cored acetamides 7a–f.
Pharmaceuticals 16 00211 sch001
Pharmaceuticals 16 00211 g002 550
Figure 2. Dose response and % cell viability of compounds 7a–f at different concentrations.
Figure 2. Dose response and % cell viability of compounds 7a–f at different concentrations.
Pharmaceuticals 16 00211 g002
Pharmaceuticals 16 00211 g003 550
Figure 3. Dose response and % inhibition relationship of compound 7f at different concentrations.
Figure 3. Dose response and % inhibition relationship of compound 7f at different concentrations.
Pharmaceuticals 16 00211 g003
Pharmaceuticals 16 00211 g004 550
Figure 4. Binding of reference ligand STI in the active site of protein c-kit tyrosine kinase.
Figure 4. Binding of reference ligand STI in the active site of protein c-kit tyrosine kinase.
Pharmaceuticals 16 00211 g004
Pharmaceuticals 16 00211 g005 550
Figure 5. Binding interaction of docked ligand 7f in the active site of c-Kit Tyrosine Kinase.
Figure 5. Binding interaction of docked ligand 7f in the active site of c-Kit Tyrosine Kinase.
Pharmaceuticals 16 00211 g005
Pharmaceuticals 16 00211 g006 550
Figure 6. Binding interaction of 7a in the active site of c-kit tyrosine kinase B.
Figure 6. Binding interaction of 7a in the active site of c-kit tyrosine kinase B.
Pharmaceuticals 16 00211 g006
Pharmaceuticals 16 00211 g007 550
Figure 7. Binding interaction of reference ligand (X39) with the active sites of the protein kinase binding pocket.
Figure 7. Binding interaction of reference ligand (X39) with the active sites of the protein kinase binding pocket.
Pharmaceuticals 16 00211 g007
Pharmaceuticals 16 00211 g008 550
Figure 8. Binding interactions of compound 7a with the protein kinase B binding pocket.
Figure 8. Binding interactions of compound 7a with the protein kinase B binding pocket.
Pharmaceuticals 16 00211 g008
Pharmaceuticals 16 00211 g009 550
Figure 9. Binding interactions of compound 7f with the protein kinase B binding pocket.
Figure 9. Binding interactions of compound 7f with the protein kinase B binding pocket.
Pharmaceuticals 16 00211 g009
Table
Table 1. The anti-hepatocellular carcinoma and hemolytic activities of triazole-coupled acetamides 7a–f.
Table 1. The anti-hepatocellular carcinoma and hemolytic activities of triazole-coupled acetamides 7a–f.
CompoundAlkyl/ArylCell Viability
IC50 Value (µg/mL)		Hemolytic Activity
(Mean% ± S.D)
7a2-methyl phenyl20.6672.46 ± 0.31
7b2-methyl-4-bromo phenyl33.5652.43 ± 0.11
7c4-ethyl phenyl39.0024.32 ± 0.24
7d2-chloro phenyl39.6677.33 ± 0.42
7ephenyl39.1054.19 ± 0.02
7f2,6-dimethyl phenyl16.7821.19 ± 0.02
Sorafenib 05.971
PBS 0.00 ± 0.0
Triton-X-100 100 ± 0.0
Table
Table 2. % inhibition of derivatives 7a–f at different concentrations (µg/mL).
Table 2. % inhibition of derivatives 7a–f at different concentrations (µg/mL).
Concentration
(µg/mL)		7a7b7c7d7e7f
20089.40 ± 0.3489.50 ± 0.0687.51 ± 0.2087.70 ± 0.1888.00 ± 1.4488.36 ± 0.31
10088.17 ± 0.2689.39 ± 0.9178.82 ± 10.7878.33 ± 0.7887.34 ± 2.0987.71 ± 1.34
5075.18 ± 5.9988.64 ± 1.1173.66 ± 15.5476.69 ± 5.6374.24 ± 13.1686.57 ± 0.56
2563.98 ± 1.6731.90 ± 0.3022.82 ± 0.2919.52 ± 1.4424.01 ± 6.1285.90 ± 0.30
12.527.70 ± 6.4625.23 ± 4.6719.14 ± 1.2519.11 ± 17.7120.99 ± 3.1333.96 ± 2.47
6.2521.44 ± 6.2424.75 ± 4.6217.35 ± 1.3217.32 ± 1.4020.52 ± 7.1119.55 ± 4.07
3.12511.57 + 8.8510.33 + 0.9615.40 ± 2.0114.27 ± 4.2718.65 ± 0.508.40 ± 4.54
DMSO (-ve Control)0.00.00.00.00.00.0
Table
Table 3. Docking data of scaffolds 7a–f to cancer proteins with grid box coordinates, codes, and mol. docking scores.
Table 3. Docking data of scaffolds 7a–f to cancer proteins with grid box coordinates, codes, and mol. docking scores.
TargetsProtein Kinase B (Akt)
(PKB)		c-Kit Tyrosine Kinase
(c-Kit)		Human Aurora B Kinase
(AURKB)		Phosphatidylinositol 3-Kinase Alpha (PI3Kalpha)Signal Transducer and Activator of Transcription 3 (STAT3)
PDB ID2X391T464AF34FA66NJS
Center of dockingX:43X:28X:21X:44X:13
CoordinatesY:31Y:26Y:-22Y:14Y:56
Z:111Z:39Z:-10Z:31Z:0.32
Reference LigandX39STIVX60TAKQV
LigandsMol. Dock
Score
(Kcal/mol)		Mol. Dock Score (Kcal/mol)Mol. Dock Score (Kcal/mol)Mol. Dock Score (Kcal/mol)Mol. Dock Score (Kcal/mol)
7a
7b		−166.843
−166.371		−173.411
−167.882		−145.234
−138.33		−139.389
−136.267		−125.105
−120.348
7c−162.234−167.814−137.943−134.596−118.623
7d−154.675−158.747−132.083−124.135−107.246
7e−156.207−161.394−136.421−131.706−113.82
7f−170.066−176.749−149.617−149.36−125.441
Reference
Molecules		−130.624−181.533−144.231−112.819−197.521
Table
Table 4. Docking of scaffolds 7a–f to protein c-kit tyrosine kinase.
Table 4. Docking of scaffolds 7a–f to protein c-kit tyrosine kinase.
Ligand(ACE) (kcal/mol)CategoryTypesInteracting Residues
7a−173.411H-bondAlkylLEU595, LYS623, VAL654, LEU644, LEU595,LEU644, CYS673, CYS809, and VAL668.
HydrophobicPi-alkylTYR672.
7b−167.882H-bondSulfur-XCYS809.
HydrophobicC-alkyl


Pi-alkyl		LEU595, VAL654, LEU644, CYS809, LEU595, ILE808, LEU644, AL654.
TYR672, HIS790, and PHE811.
7c−167.814H-bondConventional GLU640.
HydrophobicPi-AlkylHIS790.
HydrophobicC-alkylVAL643, VAL603, LYS623, VAL668, LEU783, CYS788, LYS623, LEU644, VAL668, ALA621, and CYS788.
7d−158.747H-bondConventional GLU640 and ASP810.
HydrophobicC-alkyl
Pi-alkyl		ILE808.
VAL603, VAL643, LEU783, CYS788, CYS809, LEU595, VAL603, VAL643, and LEU783.
7e−161.394H-bondConventional CYS673,
Hydrophobic
C-alkyl
Pi-alkyl		LEU595, VAL654, LEU644, CYS809, LYS623, LEU644, LEU644, TYR672, and VAL668.
7f
−176.749H-bondConventional H-bond
Pi-sigma		GLU640, ASP810,
And HIS790.
HydrophobicAlkylVAL603, LYS623, VAL643, LEU783, CYS788, LYS623, LEU644, VAL668, CYS809, ILE571, and CYS788.
Reference Ligand H-bondConventional ALA232, GLU236, MET282, ASP293, GLU279,
STI−181.533OtherC-H bondMET229, GLY159,
HydrophobicC-alkylVAL166, LEU158, ALA179, LYS181, and LEU183.
Table
Table 5. Docking of scaffolds 7a–f to protein kinase B.
Table 5. Docking of scaffolds 7a–f to protein kinase B.
Ligand(ACE) (kcal/mol)CategoryTypesInteracting Residues
7a−166.843
H-bondConventional
C-H bond		GLY164.
SER9.
Other
Hydrophobic		Pi-sulfur
C-alkyl		PHE163.
VAL166, LYS181, and EU183.
7b−166.371H-bondConventionalCYS809.
HydrophobicC-alkylLYS181, VAL166, and LEU296.
7c−162.234HydrophobicAlkylLYS181, VAL166, and LEU183.
7d−154.675H-bondConventionalGLY161, LEU158.
HydrophobicC-H bond
C-alkyl
Pi-alkyl		ASP293.
VAL166, LYS181.
PHE239, and PHE439.
7e−156.207H-bondC-H bondCYS673.
HydrophobicAlkylLEU296.
7f
−170.066H-bondConventional
C-H bond		GLY161, ASP293, and ARG6.
ASP293.
HydrophobicC-alkylLYS181 and LEU183.
Reference Ligand H-bondConventional ALAA232, ASPA293, META282, GLUA279, GLUA236
X39−130.624OtherC-H bondMET229, GLY159,
HydrophobicC-alkylVAL166, LEU A183, LEU A158, ALA179, LYS and A181.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Akhter, N.; Batool, S.; Khan, S.G.; Rasool, N.; Anjum, F.; Rasul, A.; Adem, Ş.; Mahmood, S.; Rehman, A.u.; Nisa, M.u.; et al. Bio-Oriented Synthesis and Molecular Docking Studies of 1,2,4-Triazole Based Derivatives as Potential Anti-Cancer Agents against HepG2 Cell Line. Pharmaceuticals 2023, 16, 211. https://doi.org/10.3390/ph16020211

AMA Style

Akhter N, Batool S, Khan SG, Rasool N, Anjum F, Rasul A, Adem Ş, Mahmood S, Rehman Au, Nisa Mu, et al. Bio-Oriented Synthesis and Molecular Docking Studies of 1,2,4-Triazole Based Derivatives as Potential Anti-Cancer Agents against HepG2 Cell Line. Pharmaceuticals. 2023; 16(2):211. https://doi.org/10.3390/ph16020211

Chicago/Turabian Style

Akhter, Naheed, Sidra Batool, Samreen Gul Khan, Nasir Rasool, Fozia Anjum, Azhar Rasul, Şevki Adem, Sadaf Mahmood, Aziz ur Rehman, Mehr un Nisa, and et al. 2023. "Bio-Oriented Synthesis and Molecular Docking Studies of 1,2,4-Triazole Based Derivatives as Potential Anti-Cancer Agents against HepG2 Cell Line" Pharmaceuticals 16, no. 2: 211. https://doi.org/10.3390/ph16020211

APA Style

Akhter, N., Batool, S., Khan, S. G., Rasool, N., Anjum, F., Rasul, A., Adem, Ş., Mahmood, S., Rehman, A. u., Nisa, M. u., Razzaq, Z., Christensen, J. B., Abourehab, M. A. S., Shah, S. A. A., & Imran, S. (2023). Bio-Oriented Synthesis and Molecular Docking Studies of 1,2,4-Triazole Based Derivatives as Potential Anti-Cancer Agents against HepG2 Cell Line. Pharmaceuticals, 16(2), 211. https://doi.org/10.3390/ph16020211

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop