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. 2025 Dec 12;10(51):63276–63292. doi: 10.1021/acsomega.5c09752

Design and Synthesis of Thiadiazole Derivatives as Dual EGFR/COX‑2 Inhibitors with Anticancer and Anti-inflammatory Activities

Arzu HIDIR 1,2, Derya OSMANİYE 1,3, Begüm Nurpelin SAĞLIK ÖZKAN 1,3, Serkan LEVENT 3,4, Zafer Asım KAPLANCIKLI 1,5, Yusuf ÖZKAY 1,3,*
PMCID: PMC12756739  PMID: 41487259

Abstract

In this study, 20 new compounds with thiadiazole structures were synthesized based on Alpelisib. The benzene ring, a bioisostere of the pyridine ring in the lead compound, was used in the synthesized compounds, and derivatives containing different substituents were used to observe modifications that could affect activity. Furthermore, because combined therapies are known to be more effective in cancer therapy, attempts were made to design compounds capable of dual inhibition of EGFR-COX-2 by adding sulfonamide substitutions to some compounds. The structures of the compounds were confirmed by IR, 1H NMR, 13C NMR, and HRMS spectral analyses. A549 and MCF-7 cell lines were used to measure anticancer activity, and in vitro assays were conducted. For compounds 3j and 3o, further activity studies, molecular docking, and molecular dynamics studies were performed. The compounds’ EGFR and COX-2 inhibitory potential was investigated.

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1. Introduction

Cancer, in its most general definition, is a complex disease characterized by unregulated cell division, local tissue invasion, and metastases and comprises many diseases. − While cancer has been a common problem in humans and animals throughout history, the incidence of cancer has been increasing annually, particularly in recent years due to changing lifestyles, nutritional habits, increased life expectancy, and exposure to carcinogenic substances. , In fact, one in four people is likely to develop cancer in their lifetime, and in Western societies, one in three people develops cancer and 20% of those diagnosed die. , Cancer cells experience several unique conditions compared to other cells in the body. These include a state of uncontrolled growth, where the cell cycle becomes uncontrolled. Genetic changes disrupt the rigidly organized structure of healthy cells, with the activation of numerous oncogenes and the suppression of genes responsible for tumor suppression. Cancer cells then undergo numerous cell divisions, leading to exponential growth. Another characteristic of cancer cells is their resistance to programmed cell death by inhibiting apoptosis and cellular senescence. Finally, these cancerous cells form new blood vessels, invade different tissues, and spread to distant parts of the body.

Cancer is a treatable disease if diagnosed early enough. This is because the cancer is usually small enough to allow surgical removal or shrinkage through chemotherapy or radiotherapy. The primary goal of cancer therapy is to eliminate cancerous tissue or cells from the body. However, when this is not possible, the goal is to extend the patient’s lifespan and improve their quality of life. ,

When we look at the main methods of cancer therapy, surgery, radiation therapy, and the use of anticancer agents, namely, chemotherapy, stand out. These methods can be used alone or sequentially or simultaneously for the treatment of a specific type of cancer.

Chemotherapy was first introduced in 1941 when Goodman and Gilman used nitrogen mustard to lymphoma patients. Since then, many different chemotherapy agents have been developed, and chemotherapeutics can be categorized as conventional and new generation. ,

Although there is evidence that conventional therapeutics increase survival, slow progression, and reduce tumor size, the problems they create as a result of their use have increased the need for chemotherapeutics with a broad therapeutic range, low toxicity, and more specific properties. Therefore, targeted and specific small molecules that prevent uncontrolled cell growth by affecting cell signaling and various biological pathways have gained importance in new drug development. , The new generation of drugs gaining importance can be divided into two groups: small inhibitory molecules and monoclonal antibodies. Their distinguishing feature from conventional drugs is that they are cytostatic, not cytotoxic. They exert their effects by interacting with receptors within or outside the cell, or by interacting with effectors in the signaling pathways of these receptors. Ultimately, they inhibit the uncontrolled growth and development of cancer cells by disrupting their signaling pathways.

One of the most important groups of signal transduction mechanisms is protein kinases, which emerged in the early 1980s. Protein kinases are involved in the transfer of phosphate groups. Structural alterations in these enzymes have been implicated in cancer development. Protein kinases play a role in cell differentiation, promote cell survival, and regulate the cell cycle. Malfunctions in their activity due to cancer are also associated with various signal transduction pathways. Examples of these signal transduction pathways include the Ras/Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways, which are of great importance. PI3K/Akt/mTOR, a key signal transduction pathway in cells, plays a particularly important role in cancer cell survival, growth, and the development of drug resistance to chemotherapeutics, making it an attractive tool for the development of new drug molecules. Activation of this pathway is generally mediated by mutation or amplification of PIK3CA, the gene encoding the catalytic subunit (p110α) of Class I PI3K. ,−

In recent years, different targets and molecules have been tested for the development of new anticancer agents. A comprehensive review published by Nemr et al. reported that phosphodiesterase enzyme 5 (PDE5) inhibitors can also be used for antiproliferative purposes. Increased PDE5 expression has been reported in various human cancer cell lines, and sildenafil and vardenafil inhibited tumor growth. A study by Abdalla et al. investigated the efficacy of anti-inflammatory agents, such as salicylic acid/aspirin analogs, sulindac, and indomethacin, as inhibitors of cyclin-dependent kinases (CDKs), namely, CDK2, CDK6, and CDK12. Anthranilic acid showed activity against CDK2, CDK6, and CDK12 at 0.67, 0.48, and 0.82 μM, respectively. In a study by Fadaly et al., various benzenesulfonamide derivatives were synthesized and their activities as inhibitors of carbonic anhydrase IX (CA IX) and carbonic anhydrase XII (CA XII) were investigated. Among the synthesized compounds, 4-(4-(5-(4-methoxyphenyl)-1-(4-(4-methoxyphenyl)­thiazol-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)-5-methyl-1H-1,2,3-triazol-1-yl)­benzenesulfonamide showed activities of 0.025 and 0.031 μM against CA IX and CA XII, respectively. In a study by Ewieda et al., new thiazolopyrimidine derivatives were synthesized and their activities as topoisomerase II inhibitors were investigated. Ethyl 5-(4-bromophenyl)-3-(4-methylphenyl)-7-phenyl-5H-thiazolo­[3,2-a]­pyrimidine-6-carboxylate hydrobromide showed activity against topoisomerase at 0.94 μM. In a study by Mohamed et al., sulfonamide-based chalcone derivatives were synthesized and their histone deacetylase (HDAC), carbonic anhydrase (CA), and antitubulin polymerization activities were investigated. Of these compounds, (E)-2-(4-(3-(4-bromophenyl)-3-oxoprop-1-en-1-yl)­phenoxy)-N-(4-sulfamoylphenyl)­acetamide showed activity with IC50 values of 134.22 nM for HDAC1 and 66.29 nM for HDAC2, inhibition constants of 72.03 ± 2.58 nM for CA IX and 50.76 ± 2.58 nM for CA XII and IC50 value of 2.56 μM in the antitubulin polymerization assay. In a study conducted by Abd El-Mawgoud et al., new thieno­[2,3-d]­pyrimidine analogs were synthesized in order to investigate their VEGFR-2/AKT dual inhibition potential. Among these compounds, N-(5-(2,9-diaminothieno­[3,2-e]­1,3,4-triazolo­[1,5-c]­pyrimidin-8-yl)-1,3,4-thiadiazol-2-yl)­benzamide showed activity against VEGFR-2 and AKT-1 with IC50 values of 0.164 and 0.452 μM, respectively. Novel 3,4-diaminothieno­[2,3-b]­thiophene-2,5-dicarbohydrazide derivatives that may be EGFRWT, EGFRT790M, and tubulin polymerization inhibitors were synthesized by Aboelez et al. Of these derivatives, 3,4-diamino-N′2,N′5-bis­(1-phenylethylidene)­thieno­[2,3-b]­thiophene-2,5-dicarbohydrazide showed activity against EGFRT790M and EGFRWT with IC50 values of 24.6 and 28.1 nM, respectively, and an antitubulin polymerization IC50 value of 5.1 μM. In another study, Fadaly et al. synthesized new pyrazole derivatives that may be inhibitors of EGFRWT, EGFRL858R/T790M, and COX-2, and investigated their activities. Among these compounds, (E)-2-((4-(((3-(4-bromophenyl)-1-(4-(methylsulfonyl)­phenyl)-1H-pyrazol-4-yl)­methylene) amino)­phenyl)­amino)-2-oxoethyl nitrate showed activity against COX-2, EGFRWT, EGFRL858R/T790M with the values of 0.437, 0.452, and 0.031 μM, respectively. −

Epidermal growth factor (EGFR), one of the targets of kinase inhibitors, consists of a family of four receptors: ErbB1, also known as EGFR or HER1; ErbB2, also known as HER2; ErbB3, also known as HER3; and ErbB4, also known as HER4. Autophosphorylation of these receptors activates the PI3K/Akt and MAPK/ERK pathways. When the EGFR is activated, it stimulates PI3K, which converts PIP2 to PIP3. PIP3 binds to the Akt serine/threonine kinase and recruits it to the plasma membrane, where phosphorylation of Akt at T308 and S473 activates transcription factors that contribute to cell growth and survival. ,

Alpelisib (Figure ) is an inhibitor that selectively targets the alpha isoform of PI3K. It is the first PI3K inhibitor approved by the FDA on May 24, 2019, in combination with fulvestrant for the treatment of postmenopausal women and men with PIK3CA mutation, HER2-negative, hormone receptor-positive, advanced or metastatic breast cancer. ,

1.

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Design strategy of target compounds.

In cancer treatments, combination therapies targeting different mechanisms have proven to be more effective through synergistic and/or additive mechanisms. The simultaneous use of two different drugs is challenging due to drug–drug interactions and various dose-limiting toxicities resulting from pharmacokinetic profiles. Therefore, the development of compounds that target multiple mechanisms in a single molecule is of great importance.

Considering previous studies on target molecules that can be dually inhibited by EGFR, cyclooxygenase-2 (COX-2) is an important target. Studies have provided evidence for the anticancer effects of aspirin, suggesting that COX inhibitors could be used in anticancer drug development. Certain mediators, including COX-2, have been found to promote cancer development. Overexpression of COX-2 has been associated with cancer survival, immune evasion, repopulation of cancer stem cells during treatment, and ultimately, the development of treatment resistance in many different cancer types. Furthermore, a review by the National Cancer Institute revealed that nonsteroidal anti-inflammatory drugs (NSAIDs) that selectively inhibit COX-2 may exhibit anticancer activity. Studies with these compounds using cell culture and rodent models have demonstrated the potential to inhibit angiogenesis and restore normal apoptosis. Considering COX-2 inhibitor molecules, celecoxib (Figure ) and valdecoxib (Figure ) can be given as examples, and when looking at the literature, it was seen that the pharmacophore group of the compounds was −SO2NH2 group. ,

With the aim of providing a glimmer of hope for cancer, a disease that has been fought for many years but still lacks a radical cure, the aim was to synthesize a new generation of small inhibitor molecules. Based on the information provided in this thesis, we aimed to develop new dual-action anticancer compounds. Based on the fact that the mechanism of action of alpeslisib extends to EGFR, molecules capable of dual inhibition of EGFR and COX-2 were designed. A thiadiazole ring was used instead of the thiazole ring in the alpesilib molecule, a benzene ring, which is a bioisostere of pyridine, and compounds containing various substituted benzene rings were synthesized to test the effect of the CF3 substituent. Care was taken to ensure that the compounds contained a three-ring system and included an amide structure. For COX-2 inhibition, the sulfonamide structure, which is generally prominent in molecules, was utilized, and this structure was selected as a substituent in some compounds. Following the compound designs, 20 new compounds were synthesized within the scope of this study, and their anticancer effects were investigated.

2. Results and Discussion

2.1. Chemistry

In this study, 20 new compounds containing N-(5-((2-oxo-2-(phenylamino)­ethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide structures were obtained. The studies were carried out in three steps. In the first step, aniline derivatives were converted to 2-chloro-N-phenylacetamide derivatives using chloroacetyl chloride, and then in the second step, four different benzoyl chloride derivatives were added to the 5-amino-1,3,4-thiadiazol-2-thiol compound. In the last step, the acetamide derivatives synthesized in the first step were added to the main structure. After all the synthesis steps were completed, spectral analyses were started to elucidate the structures (Scheme and Table ).

1. General Procedure of the Synthesis of Targeted Compounds.

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1. Chemical Structure of the Synthesized Derivatives (2a–2j).

compound R1 R2 compound R1 R2
3a –H 3,4-Cl 3k –H 4-F
3b –CF3 3,4-Cl 3l –CF3 4-F
3c –F 3,4-Cl 3m –F 4-F
3d –Cl 3,4-Cl 3n –Cl 4-F
3e –SO2NH2 3,4-Cl 3o –SO2NH2 4-F
3f –H 4-Cl 3p –H 4-OCH3
3g –CF3 4-Cl 3r –CF3 4-OCH3
3h –F 4-Cl 3s –F 4-OCH3
3i –Cl 4-Cl 3t –Cl 4-OCH3
3j –SO2NH2 4-Cl 3u –SO2NH2 4-OCH3
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When the structures of the synthesized compounds are examined, the amide (−NHCO) structure stands out as the common structure. , When the spectra are examined, bands belonging to the N–H stretching vibrations are observed in the range of 3300–3000 cm–1. Another band belonging to the amide structure, the CO stretching vibrations, was obtained in the range of 1650–1750 cm–1. These band values are considered consistent when compared with literature.

When the structures of the compounds are examined, the benzene and thiadiazole ring systems and the −CH2 bridge are notable for their commonality. The protons belonging to the −CH2 bridge, present in all structures, were observed as singlets between 4.07 and 4.31 ppm. Peaks belonging to the benzene rings were observed between 7.07 and 8.36 ppm, and multiplet peaks due to F–H splitting were observed, particularly in the peaks belonging to benzene rings containing F in their structure. Peaks belonging to the proton in the amide structure bound to the thiadizole structure were observed as singlets or multiplets in the 13.22–13.30 range in some compounds due to F–H splitting but were not observed in some spectra due to the proton being attached to a heteroatom. The proton in the amide structure bound to the −CH2 bridge was observed as singlets or multiplets in the 10.16–10.76 range and again in the 3.03 range due to F–H splitting. In the 3p–3u compounds, the protons in the methoxy (−OCH3) structure bound to the benzene ring were observed as singlets in the 3.81–4.03 range. In the 3e, 3j, 3o, and 3u compounds, the protons in the −SO2NH2 structure were observed within the protons in the benzene ring. Spectral analysis results were found to be consistent with data in the literature. ,

Generally expected peaks were observed in the 13C spectra of the compounds. Specifically, the CN peaks of the thiadiazole ring and the CO peaks of the amide structures were observed in the range of 150.20–171.70 ppm, which is consistent with the literature. The peaks of the −CH2 bridge were observed in the range of 38.04–38.51 ppm, and the peaks of −OCH3 found in 3p–3u compounds were observed in the range of 55.74–55.90 ppm. Other aromatic carbons in the general structure were observed in the range of 112.14–157.79 ppm. ,

Mass spectra of the compounds were obtained by high-resolution mass analysis using electron spray ionization. Since [M + H]^+ molecules are detected by this method, M+1 peaks are expected to be observed in the spectra, and appropriate peaks were observed in all compounds.

2.2. Cytotoxicity Analysis

When the cytotoxic effects of the compounds on the A549 cell line are examined, unfortunately, no compound is more effective than doxorubicin. The two most effective derivatives against this cell line are 3j at 20.682 ± 0.984 μM and 3g at 21.128 ± 0.996 μM. Doxorubicin, on the other hand, exhibits activity at almost half the dose of the compounds, with an IC50 value of 10.985 ± 0.247 μM. However, 3j and 3g are not cytotoxic against NIH3T3 cells, with IC50 values of 85.604 ± 3.164 and >100 μM, respectively. They exhibit selective cytotoxic effects against the A549 cell line. When considering their cytotoxic effects on the MCF-7 cell line, compounds 3j (2.375 ± 0.108 μM) and 3o (2.884 ± 0.124 μM) stand out. They exhibit activity comparable to doxorubicin, with an IC50 value of 1.940 ± 0.084 μM. Furthermore, their IC50 values of 3j = 85.604 ± 3.164 μM and 3o = 81.561 ± 2.739 μM against NIH3T3 cells demonstrated that they were not cytotoxic and could be potential drug candidates selective for the MCF-7 cell line.

The IC50 values of the compounds against A549, MCF-7, and NIH3T3 cell lines are given (Table ).

2. IC50 (μM) Values of Compounds 3a–3u .

  IC50 (μM)
compound A549 MCF-7 NIH3T3
3a >100 >100 >100
3b 46.548 ± 1.452 >100 67.128 ± 2.369
3c >100 7.940 ± 0.384 43.623 ± 2.041
3d >100 >100 >100
3e >100 10.987 ± 0.402 70.784 ± 2.502
3f >100 >100 >100
3g 21.128 ± 0.996 27.068 ± 1.255 >100
3h >100 >100 >100
3i >100 12.064 ± 0.589 24.896 ± 1.178
3j 20.682 ± 0.984 2.375 ± 0.108 85.604 ± 3.164
3k 69.085 ± 2.802 >100 >100
3l 41.098 ± 1.964 30.469 ± 1.374 46.151 ± 1.825
3m 29.410 ± 1.210 35.702 ± 1.262 60.274 ± 2.140
3n >100 >100 >100
3o 25.150 ± 1.187 2.884 ± 0.124 81.561 ± 2.739
3p >100 >100 >100
3r >100 43.065 ± 2.061 47.608 ± 1.774
3s >100 >100 76.145 ± 2.859
3t >100 >100 >100
3u >100 6.415 ± 0.298 55.762 ± 2.112
doxorubicin 10.985 ± 0.247 1.940 ± 0.084 >100
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2.3. Kinase Inhibition Assay

Kinases are important drug targets, particularly for the development of oncological drugs. Their role in every aspect of cell signaling, such as growth and proliferation, and their ability to block this signaling, which is disrupted in cancer cells due to carcinogenesis, and their ability to be inhibited with small molecules are among the factors that make them attractive targets.

In this study, since Alpelisib was used as the starting point, and the primary signaling mechanism through which this drug acts is its binding to EGFR, a protein kinase, compounds 3j and 3o were subjected to an EGFR kinase inhibition test due to their superior cytotoxic and apoptotic effects compared to other compounds. Erlotinib was used as a positive control in the study. Enzyme inhibition assays were conducted with the assumption that the enzyme inhibition potential of the purified standard compounds from the kit was 0. The results are shown below in Table .

3. IC50 Values (μM) of Compounds against 3j–3o EGFR Enzyme.

compound EGFR IC50 (μM)
3j 0.020 ± 0.0008
3o 0.028 ± 0.0012
erlotinib 0.002 ± 0.0001
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2.4. COX-2 Enzyme Inhibition Assay

Because compounds were intended to exhibit dual activity, some compounds were designed to resemble COX-2 inhibitors. Compounds were evaluated for their inhibitory activity against the COX-2 enzyme in the final step of bioactivity studies. Ibuprofen, which inhibits both COX-1 and COX-2 without any affinity for either, and the selective COX-2 inhibitors nimesulide and celecoxib were used as reference compounds. The inhibitory activities of the compounds against COX-2 are listed in Table .

4. IC50 Values (μM) of Compounds 3a–3u against the COX-2 Enzyme.

compound COX-2 IC50 (μM) compound COX-2 IC50 (μM)
3a 2.395 ± 0.104 3k >1000
3b >1000 3l 0.974 ± 0.045
3c >1000 3m >100
3d 6.723 ± 0.312 3n 0.721 ± 0.030
3e 0.320 ± 0.014 3o 0.208 ± 0.009
3f 0.874 ± 0.039 3p >1000
3g >100 3r >100
3h >1000 3s 8.401 ± 0.370
3i 3.785 ± 0.177 3t >100
3j 0.192 ± 0.008 3u 0.375 ± 0.016
ibuprofen 5.326 ± 0.218 celecoxib 0.132 ± 0.005
  nimesulide 1.684 ± 0.079  
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Compounds 3a 2.395 ± 0.104 μM, 3e 0.320 ± 0.014 μM, 3f 0.874 ± 0.039 μM, 3i 3.785 ± 0.177 μM, 3j 0.192 ± 0.008 μM, 3l 0.974 ± 0.045 μM, 3n 0.721 ± 0.030 μM, 3o 0.208 ± 0.009 μM, and 3u 0.375 ± 0.016 μM showed much better activity with IC50 values compared to the ibuprofen, which has IC50 of 5.326 ± 0.218 μM. However, because COX-2 inhibitors are used in cancer therapy, the COX-2 inhibition potential of compounds is more important. In this regard, the IC50 values of compounds 3e, 3f, 3j, 3l, 3n, 3o, and 3u are significantly lower than those of nimesulide, which has an IC50 of 1.684 ± 0.079 μM, indicating their greater efficacy. Unfortunately, no compounds exhibited activity at concentrations as low as celecoxib’s IC50 of 0.132 ± 0.005 μM, but the most effective serine derivative was 3u, with an IC50 of 0.375 ± 0.016 μM.

2.5. Flow Cytometric Analysis

Apoptosis, a naturally occurring programmed cell death mechanism, is used to eliminate unwanted or dysfunctional cells that develop over time in the body. Furthermore, it plays critical roles in homeostasis and cell development, making it particularly important for long-lived mammals. Anticancer drugs act by inducing apoptosis in most cells, making it a valuable tool for in vitro anticancer efficacy studies.

When a cell undergoes apoptosis, phosphatidylserine, located in the lipid layer of the cytoplasmic portion of the cell membrane, migrates to the outer layer of the cell membrane, and this occurs during the early apoptosis phase. Annexin V is a protein that can bind to phosphatidylserine and, when interacted with fluorescent substances, allows the detection of apoptotic cells by flow cytometry. When Annexin V-FITC and EI are used as fluorescent substances, cells are classified into quadrants (necrosis, early apoptosis, and late apoptosis) based on their apoptosis and necrosis states, based on the complexes they form with the respective dyes, as determined by flow cytometry analysis. In the upper right quadrant, late apoptotic cells bind to both substances; in the lower right quadrant, early apoptotic cells bind only to Annexin V; in the upper left quadrant, necrotic cells bind only to EI; and in the lower left quadrant, healthy cells do not bind to either substance. ,

When the cytotoxic effects of the compounds synthesized as part of this thesis were examined, it was observed that they exhibited better activity in the MCF-7 cell line. Therefore, the relevant method was applied to compounds 3j and 3o, which were found to be most effective in this cell line, and the results (Table ) and quadrants (Figure ) were obtained.

5. Apoptosis Percentages of Compounds 3j and 3o on the MCF-7 Cell Line.

  MCF-7 cell line
compound % necrosis % late apoptosis % early apoptosis
3j 4.08% 15.56% 20.20%
3o 4.32% 20.05% 28.48%
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Flow cytometric analysis diagram of compounds 3j and 3o in the MCF-7 cell line.

When the effects of the compounds on the MCF-7 cell line were examined, it was seen that 3j induced necrosis at 4.08%, late apoptosis at 15.56%, and early apoptosis at 20.20% at the IC50 concentration, while 3o induced necrosis at 4.32%, late apoptosis at 20.05%, and early apoptosis at 28.48%. Since chemotherapeutic agents that exhibit necrotic effects are known to be cytotoxic, it can be said that these agents have very good effects.

2.6. Molecular Docking Studies

To investigate the potential interactions of compounds 3j and 3o, which have the highest potential to inhibit COX-2 and EGFR enzymes, with the active sites of these enzymes, docking studies were performed using the COX-2 enzyme crystal (PDB: 3LN1 ) and the EGFR enzyme crystal (PDB: 1M17. For this purpose, the docking technique, performed using the Glide program, was used, and the most probable poses were obtained using GlideScore SP. Figure shows the most probable docking poses obtained for the COX-2 enzyme, while Figure shows the docking poses for the EGFR enzyme.

3.

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Two-dimensional (A) and three-dimensional (B) views of the interaction of compound 3j with the COX-2 enzyme active site. Two-dimensional (C) and three-dimensional (D) views of the interaction of compound 3o with the COX-2 enzyme active site.

4.

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Two-dimensional (A) and three-dimensional (B) views of the interaction of compound 3j with the EGFR enzyme active site. Two-dimensional (C) and three-dimensional (D) views of the interaction of compound 3o with the EGFR enzyme active site.

As can be seen in Figure from studies on the COX-2 enzyme, the carbonyl group adjacent to the p-chloro phenyl and p-fluoro phenyl rings in compounds 3j and 3o forms an H-bond with the hydroxyl of Tyr341. Unlike the other compounds, this carbonyl group in 3j forms an H-bond with the amino group of Arg106. In compound 3o, the p-fluoro phenyl ring forms a π–π interaction with the phenyl of Tyr341. The sulfonamide group, a common structure in both compounds, is quite important in terms of polar interactions. The amine group in this group is observed to form four H-bonds with the amino acids Hyd75, Gln178, Leu338, and Ser339. It was also determined that the oxygen atoms in the sulfonamide group in the structure interact with the amino acids Arg499 and Phe504 via two H-bonds. The interactions observed on the sulfonamide group indicate that the compounds fit closely into the side pocket of the COX-2 enzyme active site, which is thought to be responsible for selectivity. All the data obtained support the potent, selective inhibitory activity of compounds 3j and 3o against the COX-2 enzyme in vitro.

Figure shows the docking poses of compounds 3j and 3o obtained using the EGFR enzyme. The resulting poses show that these compounds occupy an optimal position within the EGFR enzyme to block the enzyme, similar to the COX-2 enzyme. Examining the resulting docking poses reveals that both compounds provide the same interactions and occupy a similar conformation in the enzyme’s active site. The amine structure in the sulfonamide group in the structure forms an H-bond with the carbonyl of Asn818. Another H-bond was also detected between the nitrogen atom of the thiadiazole ring and the amine group of Met769. These interactions are known to occur via key amino acids in the EGFR enzyme active site. All the data obtained clearly demonstrate that compounds 3j and 3o have the potential to potently inhibit the EGFR enzyme.

2.7. Molecular Dynamics Simulation (MD)

In this study, a 100 ns MD simulation was performed in an open hydration environment to evaluate the stability of the docking complex formed between the promising molecules 3j and 3o and the enzymes COX-2 (PDB code: 3LN1) and EGFR (PDB code: 1M17). The molecular dynamics simulation evaluations performed with the relevant enzymes are presented below. Tables and present the data analyses obtained from the molecular dynamics simulation studies of the complexes formed by 3j and 3o against the respective enzymes. The results obtained from these molecular dynamics simulation studies are presented in Figures , , , and .

6. Molecular Dynamics Simulation Analysis of 3j–COX-2 and 3o–COX-2 Complexes.

complex RMSD amino acids that interact more than 15% interaction fractions
3j–COX-2 2.4 Å Arg106 (H-bond: %48 ve %100) water-mediated H-bonding (blue) His75, Val102, Arg106, Gln178, His337, Leu338, Ser339, Gly340, Tyr341, Phe343, Tyr371, Arg499, Pro500, Asp501, Ile503, Phe504, Gly505, Met508, Val509, Glu510, Ser516
Gln178 (H-bond: %16)
Leu338 (H-bond: %79)
Ser339 (H-bond: %93)
Arg499 (H-bond: %33)
Ile503 (H-bond: %45) H-bond (green) Arg106, Gln178, Leu338, Ser339, Tyr341, Arg499, Ile503, Phe504, Ser516
Phe504 (H-bond: %96)
Ser516 (H-bond: %44) ionic interaction (pink) Arg106
Tyr341 (π–π interaction: %22 ve %32) hydrophobic interaction (purple) Val74, Leu78, Ile98, Tyr101, Val102, Val335, Leu338, Tyr341, Phe343, Arg499, Phe504, Val509, Ala513, Leu517
3o–COX-2 2.7 Å Arg106 (H-bond: %35 ve %100) water-mediated H-bonding (blue) His75, Gln178, Gln336, Leu338, Ser339, Gly340, Tyr341, Phe343, Tyr371, Arg499, Pro500, Met508, Glu510, Ser516
Gln178 (H-bond: %16)
Leu338 (H-bond: %77)
Ser339 (H-bond: %98)
Arg499 (H-bond: %16) H-bond (green) His75, Arg106, Gln178, Leu338, Ser339, Tyr341, Arg499, Ile503, Phe504
Ile503 (H-bond: %12)
Phe504 (H-bond: %95) ionic interaction (pink) Arg106
Tyr341 (π–π interaction: %28 ve %39) hydrophobic interaction (purple) Val74, Leu78, Trp85, Ile98, Tyr101, Val102, Val335, Leu338, Tyr341, Phe343, Phe504, Val509, Ala513, Leu517
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7. Molecular Dynamics Simulation Analysis of 3j–EGFR and 3o–EGFR Complexes.

complex RMSD amino acids that interact more than 15% interaction fractions
3j–EGFR 3 Å Lys704 (H-bond: %24 ve %35) water-mediated H-bonding (blue) Lys692, Val693, Leu694, Phe699, Lys704, Ala719, Lys721, Glu738, Cys751, Arg752, Leu764, Thr766, Gln767, Met769, Pro770, Phe771, Cys773, Asp776, Glu780, Arg817, Asn818, Thr830, Asp831, Phe832
Met769 (H-bond: %44) H-bond (green) Lys704, Lys721, Thr766, Met769, Arg817, Asn818, Thr830, Asp831
Thr830 (H-bond: %40) ionic interaction (pink) Lys692, Lys704
Asp831 (H-bond: %39) hydrophobic interaction (purple) Leu694, Phe699, Val702, Ala719, Lys721, Leu768, Met769, Phe771, Tyr777, Leu820
3o–EGFR 2.7 Å Phe699 (π- π interaction: %11) water-mediated H-bonding (blue) Lys692, Leu694, Ser696, Ala698, Phe699, Lys704, Ala719, Lys721, Glu738, Cys751, Leu764, Thr766, Gln767, Met769, Pro770, Phe771, Cys773, Asp776, Glu780, Arg817, Asn818, Thr830, Asp831, Phe832
Lys704 (H-bond: %14)
Met769 (H-bond: %20)
Lys721 (H-bond: %30)
Arg817 (H-bond: %22) H-bond (green) Lys692, Gly695, Ser696, Lys704, Lys721, Thr766, Met769, Cys773, Arg817, Asn818, Thr830, Asp831
Asn818 (H-bond: %41)
Thr830 (H-bond: %21) ionic interaction (pink) Lys692, Lys704, Pro770, Asp831
Asp831 (H-bond: %64) hydrophobic interaction (purple) Lys692, Leu694, Phe699, Val702, Ala719, Lys721, Pro770, Asp813, Leu820
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5.

5

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MD simulation results performed with compound 3j–COX-2 complex. (A) Compound RMSD values. (B) Amino acids that interact more than 15% in the enzyme active site. (C) Interaction percentages of the compound with amino acids. (D) Interaction fractions by complex during simulation.

6.

6

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MD simulation results performed with compound 3o–COX-2 complex. (A) Compound RMSD values. (B) Amino acids that interact more than 15% in the enzyme active site. (C) Interaction percentages of the compound with amino acids. (D) Interaction fractions by complex during simulation.

7.

7

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MD simulation results performed with the compound 3j–EGFR complex. (B) Amino acids that interact more than 15% in the enzyme active site. (C) Interaction percentages of the compound with amino acids. (D) Interaction fractions by complex during simulation.

8.

8

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MD simulation results performed with the compound 3o–EGFR complex. (B) Amino acids that interact more than 15% in the enzyme active site. (C) Interaction percentages of the compound with amino acids. (D) Interaction fractions by complex during simulation.

RMSD was used to check the stability of the model during the simulations. The RMSD plot as a function of simulation time is presented in Figures –. The RMSD value should be between 1 and 3. Since the graph we obtained remains between these specified values (Max 3 Å), it would be correct to say that stability is maintained for the complexes in question.

In Figures –, graph B displays amino acids that interact more than 15% in the enzyme active site. The amino acid interaction types and percentages obtained from the B graphs of these complexes are given in Tables and . Figures – present interaction number and residue graphs. In Figures –, graph D presents the interaction fractions by complex during the simulation. Here, blue represents water-mediated H-bonding; green represents H-bonding; pink represents ionic interaction; and purple represents hydrophobic interactions. Interactions with amino acids important for the enzyme active site are clearly visible in these graphs. The data obtained from these graphs are presented in Tables and .

When we look at these interactions, the almost continuous interactions of 3j with Val102, Arg106, Ser339, Try341, and Phe 504 and 3o with Val102, Arg106, Leu338, Ser339, Try341, and Phe 504 throughout the simulation are noteworthy.

3. Conclusions

Cancer is a complex disease whose incidence is constantly increasing due to increasing carcinogen exposure due to our changing lifestyles, but which still lacks a radical cure. Alpelisib, a PI3K inhibitor, was used as the starting point in this study. PI3K was observed to primarily interact with and affect EGFR, while studies conducted in combination with EGFR revealed that COX-2 is a key target. Within the scope of this study, 20 compounds were designed and their EGFR and COX-2 inhibitory potential was investigated. Alpelisib was selected to measure the biological activities of the compounds using MCF-7 due to its approval for breast cancer, and the A549 cell line was selected to observe its effects on lung cancer, which is in a similar anatomical region. The most effective compound for the A549 cell line was 3j, at 20.682 ± 0.984 μM. For MCF-7, compounds 3j (2.375 ± 0.108 μM) and 3o (2.884 ± 0.124 μM) stand out. Considering their COX-2 inhibitory effects, 9 of the 20 compounds showed demonstrated activity at lower concentrations compared to the nonselective inhibitor ibuprofen, which is especially promising. Notably, compounds 3e, 3f, 3j, 3l, 3n, 3o, and 3u showed better efficacy compared to selective COX-2 inhibitor nimesulide. The presence of the sulfonamide structure in the structures of compounds 3e, 3j, 3o, and 3u may have an impact on their COX-2 inhibitory potential. When considering the kinase inhibitory potential of the compounds, 3j was found to be a better inhibitor than 3u. The significance of compound 3j in both EGFR and COX-2 inhibition made it a molecule capable of exhibiting the expected dual inhibition during compound design. Compounds 3j and 3o showed promising activity in their studies. Their molecular docking and dynamics studies were used for further testing. Their favorable docking and strong interaction with the active sites of COX-2 and EGFR enzymes suggest that they could be used for cancer therapy. Comparing compounds 3j and 3o, the IC50 values of 3j against A549 and MCF-7 cell lines suggest that 3j is the most effective compound, as it inhibits COX-2 and EGFR enzymes more potently than 3o in in vitro assays.

4. Materials and Methods

4.1. Chemistry

4.1.1. General

All reagents were purchased from commercial suppliers and used without purification. Uncorrected melting points (M.p.) were determined on a Mettler Toledo-MP90 Melting Point System. Analytical studies were performed to determine the structures of the compounds. 1H-NMR and 13C-NMR spectra were obtained within the scope of NMR studies. The instrument was a Bruker brand with a power of 300 MHz. IR spectra were obtained using a Shimadzu brand FTIR spectrometer. Mass spectra of the compounds were determined by LCMS-IT-TOF. High-resolution mass spectra indicate the purities of the compounds to four decimal places.

4.1.2. General Synthesis of 2-Chloro-N-phenylacetamide Derivatives

Enough (1 mol) of the appropriate aniline derivative was taken and dissolved in THF, and TEA (1.2 mol) was added and placed in an ice bath. Chloroacetyl chloride (1.1 mol), diluted with THF, was added dropwise. The reaction was then stirred for an hour. The reaction was then monitored by TLC. The THF was removed in vacuo, and the remaining residue was washed with water, dried, and crystallized from ethanol.

4.1.3. Synthesis of 2-Chloro-N-(4-sulfamoylphenyl)­acetamide

Sulfanilamide (1 mol) was dissolved in DMF and placed in an ice bath. Chloroacetyl chloride (1.1 mol) diluted with DMF was added dropwise. The reaction was then allowed to stir for an hour. The reaction was checked with TLC, and the precipitated residue was filtered off, washed with water, dried, and crystallized from ethanol.

4.1.4. Synthesis of N-(5-Mercapto-1,3,4-thiadiazol-2-yl)­benzamide Derivatives

5-Amino-1,3,4-thiadiazole-2-thiol (0.0375 mol) dissolved in THF.TEA (0.045 mol) was added, and the mixture was placed in an ice bath. The appropriate benzoyl chloride derivative (0.0375 mol), diluted with THF, was added dropwise and stirred for an hour. The reaction was monitored with TLC, and THF was removed under pressure. The remaining residue was washed with water, and the precipitated residue was filtered, dried, and crystallized from ethanol.

4.1.5. Synthesis of Target Compounds (3a–3u)

The appropriate N-(5-mercapto-1,3,4-thiadiazol-2-yl)­benzamide derivative (1 eq) was dissolved in THF. The appropriate 2-chloro-N-phenylacetamide derivative was added using K2CO3 as a catalyst, and stirring was allowed for 1 day. The reaction was completed using TLC, and the THF was evaporated under pressure. The remaining residue was washed with water and crystallized from ethanol to obtain the target compounds.

3,4-Dichloro-N-(5-((2-oxo-2-(phenylamino)­ethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3a)

Yield: 75%, M.P.: 250.3–253.2 °C. IR (cm–1 bands): 3255 (N–H), 3068 (C–H), 1658 (CO) 1H NMR (300 MHz, DMSO-d 6): δ = 4.25 (2H, s, −CH2), 7.07 (1H, t, Ar–H), 7.32 (2H, t, Ar–H), 7.57 (2H, d, J = 7.62 Hz, Ar–H), 7.85 (H, d, J = 8.47 Hz, Ar–H), 8.04 (1H, dd, J 1= 2.07 Hz, J 2= 8.47 Hz, Ar–H), 8.36 (H, d, J = 8.36 Hz, Ar–H),10.37 (1H, s, NH), 13.30 (1H, s, NH).13C NMR (75 MHz, DMSO-d 6): δ = 38.46, 119.59, 124.08, 128.12, 129.12, 129.15, 129.33, 130.35, 130.84, 131.48, 139.26, 150.32, 160.09, 165.90, 171.70. HRMS (m/z): [M + H]+ calcd for C17H12N4O2S2Cl2 [M + H]+: 438.9851; found: 438.9853.

3,4-Dichloro-N-(5-((2-oxo-2-((4-(trifluoromethyl)­phenyl)­amino)­ethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3b)

Yield: 82%, M.P.: 245.1–247.3 °C. IR (cm–1 bands): 3300 (N–H), 3169 (N–H), 1656 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 4.26 (2H, s, −CH2), 7.69 (2H, d, J = 8.65 Hz,Ar–H), 7.77–7.81 (3H, m, Ar–H), 8.04 (1H, dd, J 1 = 2.05 Hz, J 2 = 8.43 Hz, Ar–H), 8.33 (1H, d, J = 2.01 Hz, Ar–H), 10. 73 (1H, s, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 38.42, 119.51, 120.60, 122.64, 125.52, 126.64, 129.01, 130.12, 130.71, 131.27, 131.79, 142.76, 154.12, 157.75, 162.73, 166.82. HRMS (m/z): [M + H]+ calcd for C18 H11 N4 O2 F3 S2 Cl2 [M + H]+: 506.9725; found: 506.9723.

3,4-Dichloro-N-(5-((2-((4-fluorophenyl)­amino)-2-oxoethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3c)

Yield: 85%. M.P.: 255.8–258.5 °C. IR (cm–1 bands): 3300 (N–H), 3169 (N–H), 1650 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 4.31 (2H, s, −CH2), 7.15–7.19 (2H, m, Ar–H), 7.24–7.31 (1H, m, Ar–H), 7.85 (1H, d, J = 8.45 Hz, Ar–H), 7.88–7.94 (1H, m, Ar–H), 8.05 (1H, dd, J 1 = 2.13 Hz, J 2 = 8.44 Hz, Ar–H), 8.36 (1H, d, J = 2.07 Hz, Ar–H), 10.16 (1H, s, NH), 13.31 (1H, s, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 38.04, 115.89, 116.15, 124.18, 124.95, 126.03, 129.13, 130.83, 131.49, 132.05, 136.34, 152.54, 159.26, 161.22, 166.60. HRMS (m/z): [M + H]+ calcd for C17H11N4O2FS2Cl2 [M + H]+: 456.9757; found: 456.9770.

3,4-Dichloro-N-(5-((2-((4-chlorophenyl)­amino)-2-oxoethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3d)

Yield: 87%, M.P.: 242.5–245.4 °C. IR (cm–1 bands): 3288 (N–H), 3068 (C–H), 1651 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 4.21 (2H, s, −CH2), 7.38 (2H, d, J = 8.86 Hz, Ar–H), 7.68–7.63 (2H, m, −Ar–H), 7.80 (1H, d, J = 7.42 Hz, Ar–H), 8.04 (1H, dd, J 1 = 2.07 Hz, J 2 = 8.42 Hz, Ar–H), 8.33 (1H, d, J = 2.04 Hz, Ar–H),10. 50 (1H, s, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 38.39, 121.15, 127.59, 129.01, 129.23, 130.74, 131.27, 131.80, 134.04, 135.53, 138.18, 157.79, 158.73, 166.28, 166.46. HRMS (m/z): [M + H]+ calcd for C17H11N4O2S2Cl3 [M + H]+: 472.9462; found: 472.9464.

3,4-Dichloro-N-(5-((2-oxo-2-((4-sulfamoylphenyl)­amino)­ethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3e)

Yield: 83%, M.P.: 245.6–248.1 °C. IR (cm–1 bands): 3338 (N–H), 3244.2 (C–H), 1655 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 4.25 (2H, s, −CH2), 7.28–7.32 (3H, m, Ar–H), 7.72–7.80 (5H, m, Ar–H), 8.06 (1H, dd, J 1 = 2.06 Hz, J 2= 8.43 Hz, Ar–H), 10. 71 (1H, s, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 38.43, 119.19, 127.27, 129.00, 130.72, 131.26, 131.80, 134.07, 135.52, 139.13, 142.10, 164.73, 166.75, 166.92, 170.48. HRMS (m/z): [M + H]+ calcd for C17H13N5O4S3Cl2 [M + H]+: 517.9576; found: 517.9579.

4-Chloro-N-(5-((2-oxo-2-(phenylamino)­ethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3f)

Yield: 77%, M.P.: 253.2–255.3 °C. IR (cm–1 bands): 3296 (N–H), 3095 (C–H), 1660 (CO).1H NMR (300 MHz, DMSO-d 6): δ = 4.25 (2H, s, −CH2), 7.04–7.09 (1H, m, Ar–H), 7.32 (2H, t, J = 7.94 Hz, Ar–H), 7.57 (2H, d, J = 8.57 Hz, Ar–H), 7.64 (2H, d, J = 8.64 Hz, Ar–H), 7.92–8.12 (2H, m, Ar–H), 10.36 (1H, s, NH), 13.27 (1H, s, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 38.51, 119.61, 124.07, 129.25, 130.13, 130.82, 131.61, 138.42, 139.20, 159.15, 160.48, 165.93, 166.91. HRMS (m/z): [M + H]+ calcd for C17H13N4O2S2Cl [M + H]+: 405.0241; found: 405.0240.

4-Chloro-N-(5-((2-oxo-2-((4-(trifluoromethyl)­phenyl)­amino)­ethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3g)

Yield: 90%, M.P.: 257.7–259.9 °C. IR (cm–1 bands): 3307 (N–H), 3053 (C–H), 1656 (CO).1H NMR (300 MHz, DMSO-d 6): δ = 4.30 (2H, s, −CH2), 7.55–7.65 (2H, m, Ar–H), 7.69 (2H, d, J = 8.71 Hz, Ar–H), 7.80 (2H, d, J = 8.50 Hz, Ar–H), 7.92–8.12 (2H, m, Ar–H), 10.76 (1H, s, NH), 13.23 (1H, s, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 38.48, 112.15, 119.53, 124.40, 126.64, 129.25, 130.82, 131.60, 138.44, 142.74, 158.97, 164.87, 166.69, 166.92. HRMS (m/z): [M + H]+ calcd for C18H12N4O2F3S2Cl [M + H]+: 473.0115; found: 473.0124.

4-Chloro-N-(5-((2-((4-fluorophenyl)­amino)-2-oxoethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3h)

Yield: 83%, M.P.: 245.1–247.5 °C. IR (cm–1 bands): 3294 (N–H), 3045 (C–H), 1658 (CO).1H NMR (300 MHz, DMSO-d 6): δ = 4.31 (2H, s, −CH2), 7.15–7.18 (2H, m, Ar–H), 7.24–7.33 (1H, m, Ar–H), 7.64 (2H, d, J = 8.66 Hz, Ar–H), 7.88–7.96 (1H, m, Ar–H),8.11 (2H, d, J = 8.67 Hz, Ar–H), 10.16 (1H, s, NH), 13.23 (1H, s, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 38.06, 116.14, 120.56, 124.17, 124.94, 126.01, 129.25, 130.82, 138.40, 155.46, 159.02, 164.91, 166.64. HRMS (m/z): [M + H]+ calcd for C17 H12N4 O2FS2Cl [M + H]+: 423.0147; found: 423.0150.

4-Chloro-N-(5-((2-((4-chlorophenyl)­amino)-2-oxoethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3i)

Yield: 84%, M.P.: 260.4–263.0 °C. IR (cm–1 bands): 3282 (N–H), 3124 (N–H), 1651 (CO).1H NMR (300 MHz, DMSO-d 6): δ = 4.26 (2H, s, −CH2), 7.38 (2H, dd, J 1 = 2.04 Hz, J 2 = 8.55 Hz, Ar–H), 7.58–7.66 (4H, m, Ar–H), 7.92–8.12 (2H, m, Ar–H), 10.56 (1H, s, NH), 13.22 (1H, s, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 38.44, 121.16, 125.40, 127.62, 129.23, 130.82, 131.61, 138.16, 138.45, 159.09, 166.15, 166.46, 170.46. HRMS (m/z): [M + H]+ calcd for C17H12N4O2S2Cl2 [M + H]+: 438.9851; found: 438.9849.

4-Chloro-N-(5-((2-oxo-2-((4-sulfamoylphenyl)­amino)­ethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3j)

Yield: 84%, M.P.: 241.0–242.7 °C. IR (cm–1 bands): 3282 (N–H), 3124 (C–H), 1651 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 4.03 (2H, s, −CH2), 7.30 (4H, d, J = 11.75 Hz, Ar–H), 7.56–7.79 (6H, m, Ar–H), 10.60 (1H, s, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 38.45, 119.19, 126.47, 127.27, 128.95, 130.72, 137.51, 139.11, 142.10, 149.59, 166.82, 166.93, 170.49. HRMS (m/z): [M + H]+ calcd for C17H14N5O4S3Cl [M + H]+: 483.9964; found: 483.9969.

4-Fluoro-N-(5-((2-oxo-2-(phenylamino)­ethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3k)

Yield: 87%, M.P.: 241.4–244.8 °C. IR (cm–1 bands): 3253 (N–H), 3061 (C–H), 1681 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 4.07 (2H, s, −CH2), 7.05 (1H, t, J = 7.38 Hz, Ar–H), 7.18 (2H, t, J = 8.94 Hz, Ar–H), 7.31 (2H, t, J = 7.93 Hz, Ar–H), 7.59 (2H, d, J = 7.62 Hz, Ar–H), 8.13–8.17 (2H, m, Ar–H),10.39 (1H, s, NH).13C NMR (75 MHz, DMSO-d 6): δ = 38.40, 114.81 (d, J = 21.26 Hz), 119.56, 123.89, 129.25, 130.92­(d, J = 8.71 Hz), 132.26, 132.32, 135.75, 139.40, 152.37 163.82 (d, J = 246.30 Hz), 166.73, 168.95. HRMS (m/z): [M + H]+ calcd for C17H13N4O2FS2 [M + H]+: 389.0556; found: 389.0557.

4-Fluoro-N-(5-((2-oxo-2-((4-(trifluoromethyl)­phenyl)­amino)­ethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3l)

Yield: 87%, M.P.: 220.7–223.8 °C. IR (cm–1 bands): 3294 (N–H), 1658 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 4.02 (2H, s, −CH2), 7.68 (4H, d, J = 8.76 Hz, Ar–H), 7.77–7.81 (4H, m, Ar–H), 10.75 (1H, s, NH),10.66 (1H, s, NH).13C NMR (75 MHz, DMSO-d 6): δ = 38.44, 112.16, 115.65 (d, J = 21.22 Hz), 119.50, 123.78, 124.19, 126.39, 126.64 (d, J = 3.06 Hz),131.46 (d, J = 9.10 Hz), 142.80, 149.59, 164.74 (d, J = 249.55 Hz), 167.02, 170.47. HRMS (m/z): [M + H]+ calcd for C17H12N4O2F4S2 [M + H]+: 457.0411; found: 389.0556, 457.0413.

4-Fluoro-N-(5-((2-((4-fluorophenyl)­amino)-2-oxoethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3m)

Yield: 85%, M.P.: 221.7–224.6 °C. IR (cm–1 bands): 3269 (N–H), 3051 (C–H), 1664 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 4.14 (2H, s, −CH2), 7.27 (2H, t, J = 8.90 Hz, Ar–H), 7.35–7.39 (2H, m, Ar–H), 7.60–7.64 (2H, m, Ar–H), 8.14–8.18 (1H, m, Ar–H), 8.22–8.27 (1H, m, Ar–H),10.56 (1H, s, NH), 10.65–10.85 (1H, m, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 37.97, 115.31, 124.05, 124.47, 124.91, 125.86, 126.20, 131.39, 132.40, 155.39, 164.95, 166.22, 167.06. HRMS (m/z): [M + H]+ calcd for C17H12N4O2F2S2 [M + H]+: 407.0443; found: 407.0437.

4-Fluoro-N-(5-((2-((4-chlorophenyl)­amino)-2-oxoethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3n)

Yield: 82%, M.P.: 238.1–241.1 °C. IR (cm–1 bands): 3280 (N–H), 3057 (C–H), 1656 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 4.21 (2H, s, −CH2), 7.14–7.17 (2H, m, Ar–H), 7.25–7.31 (2H, m, Ar–H), 7.83–7.96 (2H, m, Ar–H), 8.14–8.19 (1H, m, Ar–H), 8.25–8.29 (1H, m, Ar–H),10.18 (1H, s, NH), 10.24–10.44 (1H, m, NH).13C NMR (75 MHz, DMSO-d 6): δ = 38.38, 115.51, 121.28, 127.49, 129.19, 131.34, 132.29, 138.04, 138.29, 162.79, 164.51, 165.76, 166.63. HRMS (m/z): [M + H]+ calcd for C17H12N4O2FS2Cl [M + H]+: 423.0147; found: 423.0156.

4-Fluoro-N-(5-((2-((4-chlorophenyl)­amino)-2-oxoethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3o)

Yield: 86%, M.P.: 224.9–227.2 °C. IR (cm–1 bands): 3277 (N–H), 2958 (C–H), 1681 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 4.05–4.09 (2H, m, −CH2), 7.16 (1H, t, J = 8.93 Hz, Ar–H), 7.26–7.38 (4H, m, Ar–H), 7.76 (3H, s, Ar–H), 7.99–8.04 (1H, m, Ar–H), 8.11–8.16 (1H, m, Ar–H). 13C NMR (75 MHz, DMSO-d 6): δ = 38.39, 114.68 (d, J = 21.04 Hz), 115.99, 116.21, 116.50, 119.16, 127.23, 130.84 (d, J = 8.34 Hz), 132.56, 142.31, 151.64, 163.54 (d, J = 163.54 Hz), 167.48, 170.50. HRMS (m/z): [M + H]+ calcd for C17H14N5O4FS3 [M + H]+: 468.0267; found: 468.0265.

4-Methoxy-N-(5-((2-oxo-2-(phenylamino)­ethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3p)

Yield: 83%, M.P.: 207.0–208.6 °C. IR (cm–1 bands): 3197 (N–H), 3055 (C–H), 1672 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 3.80 (3H,s, −OCH3), 4.11 (2H, s, −CH2), 6.97 (1H, d, J = 8.85 Hz, Ar–H), 7.03–7.13 (2H, m, Ar–H), 7.92 (3H, t, J = 7.91 Hz, Ar–H), 7.57–7.59–8.19 (2H, m, Ar–H), 8.07 (1H, d, J = 8.86 Hz, Ar–H). 13C NMR (75 MHz, DMSO-d 6): δ = 38.46, 55.74, 113.66, 115.33, 119.58, 123.94, 125.38, 129.28, 130.51, 139.33, 149.37, 166.26, 166.52, 170.47. HRMS (m/z): [M + H]+ calcd for C18H16N4O3S2 [M + H]+: 401.0737; found: 401.0737.

4-Methoxy-N-(5-((2-oxo-2-((4-(trifluoromethyl)­phenyl)­amino)­ethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3r)

Yield: 88%, M.P.: 250.5–253.3 °C. IR (cm–1 bands): 3304 (N–H), 2987 (C–H), 1674 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 3.84 (3H,s, -OCH3), 4.26 (2H, s, −CH2), 6.65–6.67 (H, m, Ar–H), 7.06 (H, d, J = 8.74 Hz, Ar–H), 7.32–7.42 (H, m, Ar–H), 7.69 (2H, d, J = 8.74 Hz, Ar–H), 7.77–7.81 (2H, m, Ar–H), 8.10 (H, d, J = 8.76 Hz, Ar–H), 10.64–10.90 (1H, m, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 30.87, 56.40, 112.16, 114.30, 119.51, 122.98, 125.85, 126.68, 130.92, 133.38, 142.78, 163.21, 166.85, 167.01, 170.46. HRMS (m/z): [M + H]+ calcd for C19H15N4F3O3S2 [M + H]+: 469.0616; found: 469.0610.

4-Methoxy-N-(5-((2-oxo-2-((4-fluorophenyl)­amino)­ethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3s)

Yield: 77%, M.P.: 187.6–191.0 °C. IR (cm–1 bands): 3286 (N–H), 2987 (C–H), 1668 (CO).1H NMR (300 MHz, DMSO-d 6): δ = 3.81 (3H,S, −OCH3), 4.19 (2H, s, −CH2), 6.98 (2H, d, J = 8.94 Hz, Ar–H), 7.14–7.18 (2H, m, Ar–H), 7.23–7.30 (1H, m, Ar–H), 7.90–7.97 (1H, m, Ar–H), 8.09 (2H, d, J = 8.90 Hz, Ar–H), 10.18 (1H, s, NH), 10.19 (1H, s, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 37.98, 55.80, 113.78, 116.08, 124.01, 124.87, 125.38, 125.84, 126.50, 130.61, 155.37, 162.18, 167.13, 170.54. HRMS (m/z): [M + H]+ calcd for C18H15N4O3FS2 [M + H]+: 419.0642; found: 419.0645.

4-Methoxy-N-(5-((2-oxo-2-((4-chlorophenyl)­amino)­ethyl)­thio)-1,3,4-thiadiazol-2-yl)­benzamide (3t)

Yield: 83%, M.P.: 233.5–235.8 °C. IR (cm–1 bands): 3283 (N–H), 3043 (C–H), 1655 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 3.82 (3H,s, −OCH3), 4.17 (2H, s, −CH2), 7.02 (2H, d, J = 8.94 Hz, Ar–H), 7.37 (2H, d, J = 8.86 Hz, Ar–H), 7.59–7.63 (2H, m, Ar–H), 8.14–8.09 (2H, d, J = 8.89 Hz Ar–H), 10.51 (1H, s, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 38.42, 55.90, 114.02, 121.15, 125.38, 126.71, 127.55, 129.21, 130.75, 138.23, 162.66, 166.49, 166.72, 170.46. HRMS (m/z): [M + H]+ calcd for C18H15N4O3S2Cl [M + H]+: 435.0347; found: 435.0362.

4-Methoxy-N-(5-(2-oxo-2-((4-sulfamoylphenyl)­amino)­ethyl)-1,3,4-thiadiazol-2-yl)­benzamide (3u)

Yield: 90%, M.P.: 214.5–216.7 °C. IR (cm–1 bands): 3296 (N–H), 2985 (C–H), 1678 (CO). 1H NMR (300 MHz, DMSO-d 6): δ = 3.80–3.82 (3H, m, −OCH3), 4.03 (2H, s, −CH2), 7.28–7.32 (4H, m, Ar–H), 7.71–7.79 (6H, m, Ar–H), 10.61 (1H, s, NH). 13C NMR (75 MHz, DMSO-d 6): δ = 39.11, 55.98, 113.26, 114.49, 119.19, 127.26, 130.26, 131.69, 139.11, 142.11, 149.59, 163.57, 166.93, 170.49. HRMS (m/z): [M + H]+ calcd for C18H17N5O5S3 [M + H]+: 480.0465; found: 480.0466.

4.2. Cytotoxicity Analysis

Cytotoxicity tests of all compounds (3a–3u) synthesized within the scope of the study were performed against doxorubicin using MCF-7, A549, and NIH3T3 cell lines with MTT test methods previously reported by our team. ,

4.3. Kinase Inhibition Assay

In this study, EGFR kinase inhibition was measured because the compounds were expected to exhibit EGFR-COX-2 inhibitory effects. The EGFR Kinase Test Kit (Catalog no. 40321) was used, and the test was performed according to the kit’s procedures (available from ref ).

4.4. COX-2 Enzyme Inhibition Assay

The BioVision COX-2 Inhibitor Screening Kit was used to measure the COX-2 inhibitory activities of all compounds (3a–3u) obtained in the study, and the procedure was followed (available from ref ). Solutions of all compounds prepared in DMSO at 10 and 10 M were applied to the plates according to the procedure. Incubation was carried out at 25 °C for 15 min, followed by the addition of 10 μL of arachidonic acid/NaOH solution to stop the reaction. Fluorometric readings were taken at 535/587 nm at 5 min intervals, and percent inhibition values were calculated. Ibuprofen, which inhibits COX-1 and COX-2 without any affinity, and the selective COX-2 inhibitors nimesulide and celecoxib were used as positive controls.

4.5. Flow Cytometric Analysis

Compounds 3o and 3j synthesized within the scope of this study were selected for their intended analyses. Annexin/EI method procedures were applied using erlotinib as a positive control. Finally, analysis was performed using the Apoptotic, Necrotic, and Healthy Cells Detection Kit (https://www.abpbio.com/product/apoptotic-necrotic-kit/, available from ref ) on a flow cytometry device to measure the apoptotic effects of the compounds.

4.6. Molecular Docking Studies

The binding mechanisms of substances 3j and 3o to the active site of the COX-2 and EGFR enzymes were determined using a structure-based in silico technique. From the Protein Data Bank Web site (www.pdb.org), the crystal structure of EGFR (PDB: 1M17) and COX-2 (PDB: 3LN1), was obtained. Utilizing the Schrödinger Maestro51 interface. Docking studies were carried out using Schrödinger program interfaces with standard docking procedure.

4.7. Molecular Dynamics Studies

100 ns molecular dynamics studies were performed using the POPE membrane model as previously described using the Maestro Desmond interface. −

Supplementary Material

ao5c09752_si_001.pdf (4.9MB, pdf)

Acknowledgments

As the authors of this study, we thank Anadolu University Faculty of Pharmacy Central Research Laboratory (MERLAB), for their support and contributions (YÖK thesis number: 888876).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c09752.

  • 1H NMR, 13C NMR, IR, HRMS, and HPLC spectra of compounds 3a–3u (PDF)

The authors declare no competing financial interest.

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Supplementary Materials

ao5c09752_si_001.pdf (4.9MB, pdf)

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