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Article

Electrochemical Synthesis of Versatile Pyrimidine and Oxadiazoles Tethered Triazoles as Inhibitors of VEGFR-2 in Human Breast Cancer Cells

by
Akshay Ravish
1,†,
Tejaswini P. Siddappa
1,†,
Zhang Xi
2,
Divakar Vishwanath
1,
Arunkumar Mohan
1,
Shreeja Basappa
3,
Niranjan Pattehalli Krishnamurthy
4,
Peter E. Lobie
2,5,6,*,
Vijay Pandey
5,6,* and
Basappa Basappa
1,*
1
Laboratory of Chemical Biology, Department of Studies in Organic Chemistry, University of Mysore, Manasagangotri, Mysore 570006, India
2
Shenzhen Bay Laboratory, Shenzhen 518055, China
3
Department of Chemistry, BITS-Pilani Hyderabad Campus, Jawahar Nagar, Medchal, Hyderabad 500078, India
4
NMR Research Centre, Indian Institute of Science, Bangalore 560012, India
5
Tsinghua Berkeley Shenzhen Institute, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China
6
Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen 518055, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Catalysts 2023, 13(10), 1353; https://doi.org/10.3390/catal13101353
Submission received: 28 August 2023 / Revised: 16 September 2023 / Accepted: 19 September 2023 / Published: 10 October 2023
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Versions Notes

Abstract

:
Metastasis, the dissemination of tumor cells, stands as the second most prominent contributor to mortality arising from breast cancer. To counteract this phenomenon, the molecular markers associated with angiogenesis, particularly vascular endothelial growth factor (VEGF) and its receptor (VEGFR), have emerged as promising strategies for impeding the progression of tumor cells. Compounds like pyrimidines, coumarins, oxadiazoles, and triazoles have undergone comprehensive investigations due to their notable anticancer potential, highlighting their encouraging capacities in inhibiting VEGFR-2, an essential mediator of angiogenesis signaling. Herein, we have synthesized pyrimidine–triazoles and oxadiazole–triazoles using electrochemical and conventional methods. The newly synthesized compounds were evaluated for anticancer activity against MCF-7 breast cancer cells, and it was found that the compounds 8a and 8b showed IC50 values of 5.29 and 15.54 μM, respectively. Our in silico mode of action revealed that these compounds could target VEGFR-2, which was further evidenced by our in silico structure-based bioinformatic analysis. In conclusion, we reported an electrochemical method to prepare novel drug-like compounds, based on triazole and other heterocyclic hybrids, that could be used to design VGFR-targeting drugs.

Graphical Abstract

1. Introduction

Metastatic breast cancer ranks among the primary causes of cancer-related deaths. In the United States, around 4.1 million women have had breast cancer. Among them, about 4% are grappling with metastatic disease, and a significant portion was initially diagnosed with early-stage (I-III) cancers [1]. Depending on the type, stage, and personal variables, treatment options for breast cancer include surgery, radiation therapy, chemotherapy, hormone therapy, or their combinations [2,3]. Heterocycles have been widely studied and integrated into medicinal chemistry due to their diverse biological activities, including targeting specific cellular pathways, DNA binding, alkylation, and apoptosis induction [4,5]. Presently, extensive research is focusing on various natural and synthetic heterocyclic compounds, such as pyrimidines [6,7,8], coumarins [9,10], pyrazoles [11,12,13], triazoles [14,15,16], oxadiazoles [17,18,19], and piperazines [20,21], among others, which have displayed promising biological properties.
Vascular endothelial growth factor (VEGF) is a pivotal protein in angiogenesis, and VEGF receptors (R) on cell surfaces bind VEGF, prompting angiogenesis, tumor growth, and metastasis. Targeting VEGF/VEGFRs with drugs inhibits cancer progression, disrupting blood vessel formation and metastasis [22,23,24]. The VEGF family comprises five growth factors: VEGFA, VEGFB, VEGFC, VEGFD, and PLGF. EGFA, VEGFB, and PLGF bind to VEGFR–1; VEGFA binds to VEGFR-2 [kinase domain receptor (KDR)], while VEGFC and VEGFD bind to VEGFR–3 receptors [25,26]. VEGFR is frequently overexpressed in breast cancer cells and is associated with more aggressive metastatic spread and a poorer prognosis [27,28,29]. Among the three VEGF receptors, VEGFR-2/KDR is a well-established and extensively investigated target for discovering novel anticancer drugs [30,31,32]. The binding of VEGF to VEGFR-2 on endothelial cells causes receptor phosphorylation and the activation of a cascade of events that leads to endothelial cell proliferation, migration, apoptosis suppression, and vascular structure maturation [33,34,35,36].
Therapies targeting VEGF and VEGFRs have been developed to inhibit angiogenesis and slow cancer progression, including metastasis [37,38]. Drugs that block VEGF or VEGFR signaling are used to treat various cancers, including metastatic breast cancer, disrupting the tumor’s ability to form new blood vessels and spread to other parts of the body [39]. Three inhibitor classes exist; type I binds to a specific area where adenine ring of ATP is accommodated (e.g., sunitinib). Type II targets HYD–II or the allosteric Phe pocket, and the type III inhibitor (e.g., vatalanib) binds covalently to cysteine, impeding ATP binding sites. FDA-approved drugs include pazopanib, cediranib, and sorafenib [40,41,42]. Pyrimidine, coumarins, triazoles and oxadiazoles compounds have often been utilized in medicinal chemistry research for drug discovery and reported as inhibitors of VEGFR (Figure 1A). Pyrimidine derivatives (1) significantly inhibit the cell proliferation and migration of VEGF–C-induced human dermal lymphatic endothelial cells, MDA–MB–231, and MDA–MB–436 cells by inactivating the VEGFR–3 signaling pathway [43]. Pyrimidines (2) are reported to act as ATP-competitive (type I) VEGFR-2 selective inhibitors [44]. Pyrimidine (3) demonstrate anticancer activity against A549 and HepG2 cancer cells, and is also a potent inhibitor of type I VEGFR-2 [45]. Pyridine carbamate derivatives (4) exhibit a dose-dependent inhibition of VEGFR-2 type II, with IC50 values in the nM range, and demonstrate good potency in in vitro assays against VEGF-induced HUVEC proliferation [46]. The piperazine and ester functional group derivative (5) was identified as an inhibitor of type III VEGFR-2 [47].
Coumarin-based derivatives (6, 7) exhibit notable VEGFR-2 inhibition and potent effects on MCF-7 cells [48,49]. Coumarin–acetohydrazide derivatives display anticancer activity by the dual inhibition of the VEGFR-2/AKT pathway. Some coumarin compounds, like (8), inhibit VEGFR-2 and DNA gyrase, while exhibiting potential anticancer and antibacterial properties. Coumarin-substituted acetohydrazide derivatives (9) act as robust anticancer agents against MCF-7 cells and exhibit potent VEGFR-2 kinase inhibitory activity [50,51]. Triazoles, known for their structural diversity and kinase interactions, are extensively studied for medicinal chemistry. Benzimidazole-substituted triazoles, e.g., compound (10), are potent inhibitors of VEGFR-2. Zebrafish studies demonstrate the more substantial anti-angiogenic potential of indole–2–one substituted 1,2,3–triazole (11) than that of sunitinib. Certain pyrimidine and pyridine-based motifs (12, 13, 14) inhibited VEGF–VEGFR signaling and angiogenesis, showing inhibitory effects against VEGFR-2. Quinazoline-substituted oxadiazoles are potential anticancer agents, with compound 15 showing potent VEGFR-2 inhibition among other kinases. Oxadiazole–naphthalene hybrids (compound 16) inhibit VEFGR–2 and display anticancer activity against MCF-7 and HepG2 cells. Oxadiazole–benzimidazole (compound 17) hybrids inhibit enzyme activity and exhibit anticancer properties against VEGFR-2 [52,53,54,55,56,57,58,59,60,61].
In recent years, electrochemistry has garnered increasing attention as a prominent method in synthetic chemistry, particularly within the context of green synthesis [62,63,64]. Electrochemical synthesis stands as a chemical procedure entailing an intricate interplay with electron transfer occurring at an electrode interface. This electron transfer, in turn, triggers a metamorphosis of the substrate into a reactive intermediate or a reagent of significance [65]. Compared with conventional methods of chemistry, electrochemical procedures exhibit the capacity to operate at elevated rates of reaction, expediting the production of desired molecules. Furthermore, these procedures possess exceptional selectivity and precision. Manipulating the applied potential or current offers the intriguing capability to selectively engineer desired products by homing in on specific chemical transformations. Due to several benefits it provides over conventional chemical synthesis routes, this electrochemical technique has emerged as a notably influential approach [66,67]. The groundwork for conventional triazoles synthesis was initially laid by Huisgen during the 1960s [68,69]. Building upon this foundation, a plethora of derivatives of triazoles were subsequently synthesized, mainly following the principles laid out in that seminal work. Electrochemical methods have also found their place in the synthesis of triazoles [70,71,72,73]. We have previously documented the electrochemical synthesis of triazoles adorned with pyrimidine substituents. This synthesis was achieved through electrolysis under a controlled potential of 0.3 V, facilitated by the presence of TBATFB as a catalyst and a solvent mixture of tert–butyl alcohol and water [74].
Based on the above-reported VEGFR-2 inhibitors, by incorporating the active functional motifs such as pyrimidines, coumarins, triazoles, alkyl chain, and oxadiazoles, we have synthesized novel conjugated coumarin–pyrimidine–triazoles 8(a–m), 17(a–f), and oxadiazole–triazoles 21(a–f) (Figure 1B). Among the synthesized compounds, 8a and 8b are more potent against MCF-7 cells with IC50 of 5.29 and 15.54 μM, respectively. Further, an in silico docking study of 8a and 8b with VEGFR-2 showed that the compounds have a binding affinity of −8.44 and −8.90 kcal/mol.

2. Results and Discussion

2.1. Synthesis of Coumarin–Pyrimidine–Alkynes

2–Thiouracil (1) was refluxed with propargyl bromide in ethanol:water under basic conditions, yielding (2). The substituted coumarins 3(a–c) were refluxed with dibromoethane under basic conditions in DMF, yielding 4(a–c). After purification of the product 4(a–c), it was treated with propargylated thiouracil (2) and refluxed in acetone to yield compounds 5(a–c), which were then purified through column chromatography (ethyl acetate:hexane). Substituted azides are created by treating different anilines with sodium nitrite and sodium azide in aqueous HCl at 0–5 °C, which are used to synthesize coumarin–pyrimidine–triazole derivatives 8(a–m) (Scheme 1).
Further, the propargylation of 6–methyl–2–thiouracil (9) was achieved in refluxing ethanol:water under basic conditions. 4–hydroxy–coumarins (3a/11), upon refluxing with bromochloropropane in DMF, yields compound (12). After purification, this compound was used for the next step by treating it with propargylated 6–methyl–2–thiouracil (10) in refluxing acetone, yielding compound (13). Ester substituted azides are synthesized from 3, and 4–aminobenzoic acids (14), refluxing with MeOH, EtOH, and n–BuOH in catalytical amounts of sulfuric acid, yielding esters 15(a–f), which on treating with sodium nitrite and sodium azide in aqueous HCl at 0–5 °C, yielding substituted phenylazides 16(a–f), which are used to synthesize derivatives 17(a–f) (Scheme 2).

2.2. Synthesis of Oxadiazole–Alkynes

Upon cyclizing with CS2 in ethanolic KOH, 4-methoxyphenyl acetic acid–hydrazide (18) yields oxadiazole–thiol (19), which on propargylation in refluxing acetone in presence of K2CO3 yields (20), which is used to synthesize oxadiazole–triazoles 21(a–f) (Scheme 3).

2.3. Synthesis of Triazoles

Method A: Using the electrochemical oxidation of copper as the working electrode and platinum as the counter electrode, azides, and alkynes were cyclized in ACN:EtOH:H2O (1:1:0.5), as a solvent, with a catalytic quantity of tetrabutylammonium tetrafluoroborate (TBATFB), yielding triazoles derivatives (Scheme 4) [73].
Method B: Using click chemistry, CuSO4.5H2O and sodium ascorbate as a catalyst, and THF:H2O (1:1) as a solvent, azide–alkyne cyclizes were used to form triazole derivatives.
In the conventional method, after completing the reaction, the resulting crude mixture was extracted to an ethyl acetate layer. CuI forms insoluble solid materials, making it difficult to separate the organic–aqueous layer, and the final yield was obtained to be 80–86% after purification by column chromatography.
In the electrochemical method, after the reaction is completed, no such insoluble solid materials are formed, and after purification, an 85–95% final yield was obtained.
The electrochemical method was well-suited for the currently synthesized molecules.

2.4. Efficacy of Triazoles in Breast Cancer Cells

All the newly synthesized triazoles were tested for cell viability against breast cancer cells (MCF-7) using alamarBlue assays (Figure 2). The resulting inhibition concentration of the synthesized compounds is shown in Table 1, Table 2 and Table 3, with the internal standard drugs tamoxifen and doxorubicin exhibiting an IC50 of 1.74 and 0.93 µM. The results of the alamarBlue assay revealed that compounds 8a and 8b are only active with IC50 of 5.29 and 15.25 μM, respectively, while the IC50 of other derivatives lies between 41–100 μM.
Our molecular docking analysis of tamoxifen and doxorubicin towards VEGFR-2 revealed the binding energies of −7.86 and −7.31 kcal/mol, respectively (see Supplementary File; Figures S1 and S2). Compared to these drugs, the compound 8a had a higher binding energy of −8.44 kcal/mol, which indicates the specificity of the titled compounds. The potency of Tamoxifen drug towards MCF-7 was due to its activity towards ER pathway, when compared to 8a which could target VEGFR. Compound 8a has similar structural motifs (pyrimidine) to those of pazopanib, but it is not structurally similar to tamoxifen or doxorubicin. This observation explains the lower affinity of tamoxifen and doxorubicin towards VEGFR-2. Further in silico studies of sorafenib (co-crystal ligand) and pazopanib (VEGFR-2 inhibitor) demonstrated the highest binding affinity of −11.15 and −9.25 kcal/mol with VEGFR-2, suggesting its potent inhibitory activity against VEGFR-2.

2.5. Swiss TargetPrediction and In Silico Molecular Interactions of Compound 8a or 8b with VEGFR-2

Swiss TargetPrediction was used to carry out an in silico target prediction [75]. Figure 3 depicts the outcome for the target prediction of compound 8a, which showed that it had a strong 80% affinity for kinases. Among the predicted kinases, VEGFR-2 kinase showed a higher probability of 0.110, with a known activity of 1387/0 (3D/2D) to the compound 8a.
Moreover, based on the above-reported VEGFR-2 inhibitors, we have incorporated the active motif in synthesizing new VEGFR-2 inhibitors. Swiss Target Prediction also showed that newly synthesized compounds could target VEGFR-2. Thus, herein, we conducted an in silico analysis to understand the binding affinity and critical interactions of two novel compounds, namely 8a or 8b, with the active site of VEGFR-2. By comparing the novel compounds with the standard drug (pazopanib) and co-crystal ligand (sorafenib), we aim to identify potential candidates for further development as VEGFR2 inhibitors. Initially, the protein structure was retrieved from the Protein Data Bank (PDB ID: 4ASD), and then an AutoDock tool was used to determine the binding energies of all the compounds. A molecular docking study shows the detailed key interactions of the compounds formed by pazopanib and sorafenib with the adenine pocket of VEGFR-2. Table 4 shows the detailed interactions of ligands with amino acid residues. Sorafenib, the co-crystal ligand, and pazopanib demonstrated the highest binding affinity with VEGFR-2 of −11.15 and −9.25 kcal/mol, suggesting its potent inhibitory activity. Compound 8a showed a binding energy of −8.44 kcal/mol, forming a hydrogen bond between S–pyrimidine and ASP–1046. Also, pyrimidine and triazole motifs showed π-cation and π-anion interaction with GLU–885 and LYS–868 amino acids, respectively. Compound 8b exhibited a slightly higher −8.90 kcal/mol binding energy than 8a. Coumarin 8b demonstrated a favorable interaction by forming hydrogen bonds with LYS–868 and LEU–1049 and also formed a π-cation interaction with LYS–868. However, 8a or 8b demonstrated competitive binding energies, indicating their potential as novel VEGFR-2 inhibitors. Thus, in silico analysis identifies 8a or 8b as promising novel compounds with comparable binding affinities to pazopanib and sorafenib (Figure 4).

2.6. ADMET Predictions of Compound 8a and 8b with Doxorubicin

We next utilized the vNN–ADMET platform to computationally predict the ADMET properties of compounds 8a, 8b, doxorubicin, and tamoxifen, and then compared the results [6,76]. The vNN–ADMET online tool was utilized to predict the in silico ADMET properties of compounds 8a, 8b, doxorubicin, or tamoxifen, and the predictions are tabulated in Table 5. The findings revealed that compounds 8a or 8b exhibited drug-induced liver toxicity, as they can be rapidly metabolized. In contrast, tamoxifen (TAM) showed resistance to metabolism. Additionally, unlike TAM, neither 8a nor 8b inhibited the standard drug metabolizing enzymes CYP450s isoforms. Similar to doxorubicin (DOX), compounds 8a or 8b can cross the blood–brain barrier (BBB). Notably, unlike TAM, 8a and 8b were not inhibitors of P–glycoprotein. Furthermore, neither 8a, 8b, nor DOX were found to be inhibitors of matrix metalloproteinases, indicating a lower likelihood of chemical mutagenicity. The maximum therapeutic doses predicted for compounds 8a or 8b were comparable to those of TAM/ DOX.

3. Materials and Methods

All chemicals and solvents were purchased from Sigma–Aldrich. Pre-coated silica gel TLC plates monitored the completion of the reaction. 1H and 13C NMR were recorded on an Agilent NMR spectrophotometer (400 MHz); TMS was used as an internal standard, and CDCl3 was used as a solvent; chemical shifts are expressed as ppm.

3.1. Synthesis of Coumarin–Pyrimidine–Alkynes Derivatives 5(a–c) and 13

In a comprehensive experimental procedure, 2–Thiouracil (1.0 mmol) (1), propargyl bromide (1.2 mmol), and KOH (1.4 mmol) were subjected to reflux conditions within a mixture of ethanol:water (in a 1:1 ratio) for duration of 45 min. After the completion of the reaction, the resulting solid precipitate was isolated by filtration, washed with NaHCO3 solution, and finally dried. This process yields the formation of compound 2. For the synthesis of 4(a–c), substituted–4–hydroxycoumarin (1.0 mmol), dibromoethane (1.2 mmol), and K2CO3 (1.5 mmol), reflux was carried out, using DMF as the solvent, for a period of 3 h. Following the completion of this 3 h reflux, the reaction mixture was allowed to cool to room temperature, after which water was introduced. The resultant solid was separated by filtration, and subsequent purification was achieved through column chromatography. Continuing the synthetic sequence, compound 2 (1.0 mmol), 4(a–c) (1.0 mmol), and K2CO3 (1.5 mmol) were subjected to reflux conditions in acetone for a duration of 3 h. After the completion of the reaction, the resulting crude mixture was purified using column chromatography resulting to form alkynes 5(a–c).
A reaction mixture consisting of 6–Methyl–2–thiouracil (1.0 mmol) (9), propargyl bromide (1.2 mmol), and KOH (1.4 mmol) was subjected to reflux conditions using a solvent mixture of ethanol:water (1:1) for a duration of 45 min. After the completion of the reaction, the resultant solid product was isolated through filtration, subsequently washed with a solution of NaHCO3 and then dried. This process yielded compound 10. For the synthesis of compound 12, 4–hydroxycoumarin (3a/11) (1.0 mmol), bromochloropropane (1.2 mmol), and K2CO3 (1.5 mmol) were subjected to reflux conditions using DMF as the solvent for a period of 3 h. After this 3 h reflux, the reaction mixture was cooled to room temperature, followed by adding water. The resulting solid precipitate was filtered off and subsequently purified using column chromatography, resulting in the formation of compound 12. Continuing the synthetic sequence, compound 10 (1.0 mmol) and compound 12 (1.0 mmol), along with K2CO3 (1.5 mmol), were refluxed in acetone for 3 h. Following the successful completion of the reaction, the crude product was subjected to purification via column chromatography, yielding compounds 13.
To synthesize the substituted azides 7(a–e) and 16(a–f), diverse anilines 6(a–e) and esterified anilines 15(a–f) were treated with sodium nitrite (1.2 mmol) and sodium azide (1.2 mmol) in aqueous HCl at a room temperature range of 0–5 °C. After the completion of the reaction, the mixture was extracted to the ethyl acetate layer and concentrated under reduced pressure [77].

3.2. Synthesis of Oxadiazole–Alkyne Derivatives 20

In a comprehensive experimental procedure, the transformation of 4-methoxyphenylacetic acid led to the generation of the corresponding hydrazide compound, referred to as compound 18. This hydrazide was subsequently treated with CS2 (1.2 mmol) in a refluxing solution of ethanolic KOH for a duration of 8 h. The progression of the reaction was tracked using thin-layer chromatography (TLC). Once the reaction reached completion, the reaction mixture was neutralized using aqueous HCl. The resulting solid product, identified as oxadiazole–thiol, referred to as compound 19, was precipitated, subsequently separated by filtration, and then dried. Upon subjecting oxadiazole–thiol (compound 19) to a reaction with propargyl bromide (1.2 mmol) and K2CO3 (1.5 mmol) under reflux conditions within acetone for a period of 1 h, the reaction was observed to culminate. After this reaction was completed, the resulting crude material was purified through column chromatography. This process yielded compound 20.

3.3. Synthesis of Triazoles 8(a–m), 17(a–f), and 21(a–f)

Method A: In the presence of copper (Cu) foil and a platinum (Pt) electrode, a mixture containing alkynes 5, 13, and 14, as well as and azides 7(a–e) and 16(a–f), dissolved in a solvent blend of ACN:EtOH:H2O (1:1:0.5), underwent treatment with a current of 0.3 volts. This reaction occurred in the presence of a catalytic quantity of TBATFB (0.1 mmol) for a duration of 1 h. The progress of the reaction was tracked using thin-layer chromatography (TLC). Upon reaching the reaction’s endpoint, the resultant crude mixture was extracted using ethyl acetate and subsequently purified through column chromatography.
Method B: Utilizing the click chemistry approach, a reaction mixture containing CuSO4.5H2O (0.2 mmol) and sodium ascorbate (0.2 mmol) as a catalyst, a solvent mixture of THF:H2O (1:1) facilitated the cyclization of azide–alkyne pairs to form triazole derivatives.

3.4. 4-(2-((2-(((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)pyrimidin-4-yl)oxy)ethoxy)-2H-chromen-2-one (8a)

White solid; MP: 120–122 °C; 1H NMR (500 MHz, CDCl3) δ 8.28 (d, J = 6.0 Hz, 1H), 7.94 (s, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.63–7.61 (m, 2H), 7.52 (t, J = 7.7 Hz, 1H), 7.45 (d, J = 8.7 Hz, 2H), 7.28 (d, J = 8.3 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 6.47 (d, J = 6.0 Hz, 1H), 5.71 (s, 1H), 4.88–4.86 (m, 2H), 4.53 (s, 2H), 4.43–4.41 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 170.37, 168.42, 165.37, 162.75, 157.92, 153.37, 146.78, 135.44, 134.59, 132.63, 130.00, 123.99, 123.12, 121.49, 120.31, 116.85, 115.47, 104.50, 90.92, 77.36, 77.31, 77.10, 76.85, 67.45, 63.82, 25.84; calculated for C24H18ClN5O4S = 507.08; obtained mass = 508.15.

3.5. 4-(2-((2-(((1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)pyrimidin-4-yl)oxy)ethoxy)2H-chromen-2-one (8b)

White solid; MP: 142–144 °C; 1H NMR (500 MHz, CDCl3) δ 8.07 (d, J = 6.0 Hz, 1H), 7.74 (s, 1H), 7.48 (dd, J = 8.0, 1.5 Hz, 1H), 7.41–7.37 (m, 2H), 7.36–7.33 (m, 2H), 7.32–7.28 (m, 1H), 7.09–7.05 (m, 1H), 7.01–6.97 (m, 1H), 6.25 (d, J = 6.0 Hz, 1H), 5.49 (s, 1H), 4.68–4.64 (m, 2H), 4.31 (s, 2H), 4.22–4.19 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 170.14, 168.20, 165.15, 162.55, 157.70, 153.15, 146.59, 135.70, 132.75, 132.41, 123.78, 122.91, 122.24, 121.50, 120.05, 116.63, 115.26, 104.29, 90.70, 67.24, 63.60, 25.62; calculated for C24H18BrN5O4S = 552.40; obtained mass = 554.12.

3.6. 4-(2-((2-(((1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)pyrimidin-4-yl)oxy)ethoxy)-2H-chromen-2-one (8c)

White solid; MP: 126–128 °C; 1H NMR (500 MHz, CDCl3) δ 8.28 (d, J = 6.0, 1H), 7.92 (s, 1H), 7.68 (d, J = 8.0 Hz), 7.53–7.51 (m, 3H), 7.28–7.24 (m, 3H), 7.20 (t, J = 7.5 Hz, 1H), 6.47–6.44 (m, 1H), 5.69 (s, 1H), 4.88–4.86 (m, 2H), 4.53 (s, 2H), 4.41–4.39 (m, 2H), 2.38 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 170.52, 168.42, 165.37, 162.76, 157.91, 153.36, 146.24, 138.96, 134.69, 132.60, 130.29, 123.99, 123.15, 120.40, 120.24, 116.80, 115.48, 104.41, 90.89, 67.47, 63.78, 25.97, 21.17.

3.7. 4-(2-((2-(((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)pyrimidin-4-yl)oxy)ethoxy)-7-methoxy-2H-chromen-2-one (8e)

White solid; MP: 130–132 °C; 1H NMR (500 MHz, CDCl3) δ 8.15 (d, J = 6.0 Hz, 1H), 7.82 (s, 1H), 7.52–7.47 (m, 2H), 7.43 (d, J = 8.5 Hz, 1H), 7.34–7.31 (m, 1H), 7.14–7.11 (m, 1H), 6.67–6.60 (m, 2H), 6.34 (dd, J = 5.5, 1.0 Hz, 1H), 5.44 (s, 1H), 4.76–4.71 (m, 2H), 4.40 (s, 2H), 4.26 (m, 2H), 3.71 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 170.21, 168.31, 165.64, 163.26, 163.15, 157.76, 155.07, 146.63, 135.30, 134.43, 129.84, 124.03, 121.35, 120.16, 112.19, 108.54, 104.36, 100.29, 88.22, 67.15, 63.70, 55.70, 25.70.

3.8. 4-(2-((2-(((1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)pyrimidin-4-yl)oxy)ethoxy)-7-methoxy-2H-chromen-2-one (8f)

White solid; MP: 162–164 °C; 1H NMR (500 MHz, CDCl3) δ 8.01 (d, J = 6.0 Hz, 1H), 7.69 (s, 1H), 7.35–7.34 (m, 1H), 7.33–7.32 (m, 1H), 7.30–7.29 (m, 2H), 7.29–7.28 (m, 1H), 6.99 (s, 1H), 6.48 (s, 1H), 6.20 (d, J = 6.0 Hz), 5.30 (s, 1H), 4.60–4.58 (m, 2H), 4.26 (s, 2H), 4.14–4.11 (m, 2H), 3.58 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 170.09, 168.20, 165.55, 163.15, 163.07, 157.66, 154.96, 146.56, 135.66, 132.70, 123.92, 121.78, 121.47, 120.01, 112.10, 108.44, 104.27, 100.19, 88.11, 67.05, 63.59, 55.60, 25.59.

3.9. 7-Methoxy-4-(2-((2-(((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)pyrim-idin-4-yl)oxy)ethoxy)-2H-chromen-2-one (8g)

White solid; MP: 124–126 °C; 1H NMR (500 MHz, CDCl3) δ 8.27 (d, J = 6.0 Hz, 1H), 7.87 (s, 1H), 7.57–7.54 (m, 3H), 6.96 (d, J = 8.0 Hz), 6.77–6.74 (m, 2H), 6.45 (d, J = 6.0 Hz, 1H), 5.56 (s, 1H), 4.87–4.83 (m, 2H), 4.52 (s, 2H), 4.39–4.37 (m, 2H), 3.83 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 170.52, 168.43, 165.79, 163.38, 163.31, 159.84, 157.89, 155.20, 130.44, 124.21, 122.32, 121.99, 120.57, 114.80, 112.28, 108.71, 104.41, 100.47, 88.34, 67.31, 63.84, 55.84, 55.72, 25.96.

3.10. 4-(2-((2-(((1-(4-Hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)pyrimidin-4-yl)oxy)ethoxy)-7-methoxy-2H-chromen-2-one (8h)

White solid; MP: 136–138 °C; 1H NMR (500 MHz, DMSO) δ 9.87 (s, 1H), 8.49 (s, 1H), 8.36 (d, J = 6.0 Hz, 1H), 7.58–7.54 (m, 2H), 7.49 (d, J = 9.0 Hz, 1H), 6.91 (d, J = 2.5 Hz, 1H), 6.86–6.82 (m, 2H), 6.84–6.83 (m, 1H), 6.69 (d, J = 6.0 Hz, 1H), 5.77 (s, 1H), 4.81–4.78 (m, 2H), 4.48 (s, 2H), 3.79 (s, 2H), 3.30 (s, 3H); 13C NMR (100 MHz, DMSO) δ 170.13, 168.75, 165.55, 163.43, 162.48, 158.92, 158.16, 155.14, 145.03, 129.21, 124.39, 122.30, 121.84, 116.43, 112.62, 108.60, 104.72, 101.04, 88.80, 68.23, 64.51, 56.44, 25.58.

3.11. 7-Methoxy-4-(2-((2-(((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl)thio)pyrimidin-4-yl)oxy)eth-oxy)-2H-chromen-2-one (8i)

White solid; MP: 120–122 °C; 1H NMR (500 MHz, CDCl3) δ 8.27 (d, J = 6.0 Hz, 1H), 7.92 (s, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.26–7.24 (m, 2H), 6.77–6.73 (m, 2H), 6.45 (d, J = 5.5 Hz, 1H), 5.55 (s, 1H), 4.86–4.83 (m, 2H), 4.52 (s, 2H), 4.39–4.36 (m, 2H), 3.83 (s, 3H), 2.38 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 170.51, 168.44, 165.79, 163.38, 163.29, 157.89, 155.19, 138.95, 134.70, 130.29, 124.21, 120.41, 120.26, 112.50, 112.27, 108.72, 104.41, 100.46, 88.34, 67.30, 63.83, 55.87, 55.84, 25.96.

3.12. 4-(2-((2-(((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)pyrimidin-4-yl)oxy)ethoxy)-6-fluoro-2H-chromen-2-one (8j)

White solid; MP: 110–112 °C; 1H NMR (500 MHz, CDCl3) δ 8.12 (d, J = 6.0 Hz, 1H), 7.80 (s, 1H), 7.48 (d, J = 9.0 Hz, 2H), 7.29 (d, J = 9.0 Hz, 2H), 7.23–7.20 (m, 1H), 7.08 (s, 2H), 6.31 (d, J = 5.0 Hz, 1H), 5.59 (s, 1H), 4.71–4.70 (m, 2H), 4.36 (s, 2H), 4.27–4.25 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 170.23, 168.17, 164.34, 162.19, 159.48, 157.79, 149.33, 134.43, 129.85, 121.35, 120.16, 120.05, 119.86, 118.35, 118.29, 108.91, 108.71, 104.31, 91.51, 67.51, 63.57, 25.67.

3.13. 4-(2-((2-(((1-(4-Bromophenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)pyrimidin-4-yl)oxy)ethoxy)-6-fluoro-2H-chromen-2-one (8k)

White solid; MP: 124–126 °C; 1H NMR (500 MHz, CDCl3) δ 8.28 (d, J = 6.0 Hz, 1H), 7.96 (s, 1H), 7.62–7.57 (m, 4H), 7.38 (dd, J = 8.0, 2.5 Hz, 1H), 7.26–7.22 (m, 2H), 6.47 (d, J = 6.0 Hz, 1H), 5.76 (s, 1H), 4.87–4.86 (m, 2H), 4.52 (s, 2H), 4.43–4.42 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 170.38, 168.34, 164.53, 162.37, 159.64, 157.96, 149.49, 135.93, 132.98, 122.47, 122.02, 121.74, 120.27, 120.03, 118.52, 109.08, 108.88, 104.48, 91.67, 67.68, 63.74, 25.83.

3.14. 6-Fluoro-4-(2-((2-(((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)thio)pyrimidin-4-yl)-oxy)ethoxy)-2H-chromen-2-one (8l)

White solid; MP: 106–108 °C; 1H NMR (500 MHz, CDCl3) δ 8.28 (d, J = 6.0, 1H), 7.89 (s, 1H), 7.58–7.55 (m, 2H), 7.38 (dd, J = 8.5, 3.0 Hz, 1H), 7.25–7.22 (m, 2H), 6.98–6.96 (m, 2H), 6.48–6.46 (m, 1H), 5.75 (s, 1H), 4.88–4.85 (m, 2H), 4.52 (s, 2H), 4.43–4.41 (m, 2H), 3.84 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 170.55, 168.32, 164.54, 162.37, 159.85, 157.96, 149.48, 130.45, 122.00, 120.56, 120.20, 120.00, 118.49, 118.42, 114.82, 109.10, 108.90, 104.38, 91.66, 67.70, 63.73, 55.72, 25.97.

3.15. 6-Fluoro-4-(2-((2-(((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)methyl)thio)pyrimidin-4-yl)oxy)ethoxy)-2H-chromen-2-one (8m)

White solid; MP: 112–114 °C; 1H NMR (500 MHz, CDCl3) δ 8.28 (d, J = 6.0 Hz, 1H), 7.93 (s, 1H), 7.54–7.52 (m, 2H), 7.37 (dd, J = 8.5, 3.0 Hz, 1H), 7.29–7.20 (m, 4H), 6.46 (d, J = 5.5 Hz, 1H), 5.74 (s, 1H), 4.87–4.85 (m, 2H), 4.52 (s, 2H), 4.42–4.40 (m, 2H), 2.38 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 170.53, 168.32, 164.51, 162.35, 159.64, 157.96, 149.48, 138.98, 134.70, 130.31, 120.43, 120.26, 120.19, 119.99, 118.47, 118.41, 109.10, 108.90, 104.39, 91.65, 67.69, 63.73, 25.97, 21.17; calculated for C25H20FN5O4S = 505.12; obtained mass = 506.10.

3.16. Ethyl-4-(4-(((4-methyl-6-(3-((2-oxo-2H-chromen-4-yl)oxy)propoxy)pyrimidin-2-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)benzoate (17a)

White solid; MP: 142–144 °C; 1H NMR (500 MHz) δ 8.15 (d, J = 8.5 Hz, 2H), 8.02 (s, 1H), 7.77 (t, J = 7.0 Hz, 3H), 7.52 (t, J = 7.7 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.23 (d, J = 7.6 Hz, 1H), 6.25 (s, 1H), 5.67 (s, 1H), 4.57 (t, J = 5.9 Hz, 2H), 4.52 (s, 2H), 4.39 (q, J = 7.1 Hz, 2H), 4.25 (t, J = 6.0 Hz, 2H), 2.36 (s, 3H), 2.35–2.29 (m, 2H), 1.59 (s, 4H), 1.40 (t, J = 7.1 Hz, 3H); 13C NMR (126 MHz) δ 169.52, 169.14, 168.17, 165.53, 165.45, 162.91, 153.39, 147.14, 140.03, 132.50, 131.34, 130.60, 123.99, 123.05, 120.25, 119.74, 116.84, 115.67, 102.89, 90.73, 65.96, 62.77, 61.51, 28.24, 25.76, 23.85, 14.37.

3.17. Ethyl-3-(4-(((4-methyl-6-(3-((2-oxo-2H-chromen-4-yl)oxy)propoxy)pyrimidin-2-yl)thio)-methyl)-1H-1,2,3-triazol-1-yl)benzoate (17b)

White solid; MP: 138–140 °C; 1H NMR (400 MHz, CDCl3) δ 8.16 (s, 2H), 8.04 (s, 1H), 7.79 (s, 3H), 7.53 (d, J = 4.9 Hz, 1H), 7.27 (d, J = 4.8 Hz, 2H), 6.27 (s, 1H), 5.69 (s, 1H), 4.56 (d, J = 16.2 Hz, 4H), 4.36 (s, 2H), 4.27 (s, 2H), 2.38 (s, 3H), 2.35 (s, 2H), 1.78 (s, 2H), 1.69 (s, 2H), 1.49 (s, 2H).

3.18. Methyl 3-(4-(((4-methyl-6-(3-((2-oxo-2H-chromen-4-yl)oxy)propoxy)pyrimidin-2-yl)thio)-methyl)-1H-1,2,3-triazol-1-yl)benzoate (17c)

White solid; MP: 136–138 °C; 1H NMR (400 MHz, CDCl3) δ 8.16 (s, 2H), 8.05 (s, 1H), 7.79 (s, 3H), 7.54 (s, 1H), 7.27 (s, 3H), 6.27 (s, 1H), 5.68 (s, 1H), 4.56 (d, J = 15.7 Hz, 4H), 4.27 (s, 2H), 3.96 (s, 3H), 2.38 (s, 3H), 2.35 (s, 2H), 1.66 (s, 3H).

3.19. Ethyl-3-(4-(((4-methyl-6-(3-((2-oxo-2H-chromen-4-yl)oxy)propoxy)pyrimidin-2-yl)thio)-methyl)-1H-1,2,3-triazol-1-yl)benzoate (17d)

White solid; MP: 126–128 °C; 1H NMR (400 MHz, DMSO) δ 8.81 (s, 1H), 8.36 (s, 1H), 8.14 (d, J = 7.8 Hz, 1H), 8.01 (d, J = 7.6 Hz, 1H), 7.72 (dd, J = 16.0, 7.9 Hz, 2H), 7.61 (t, J = 7.6 Hz, 1H), 7.39–7.23 (m, 2H), 6.51 (s, 1H), 5.87 (s, 1H), 4.57 (t, J = 5.6 Hz, 2H), 4.53 (s, 2H), 4.35 (dd, J = 13.0, 6.1 Hz, 2H), 3.33 (s, 2H), 2.33 (s, 3H), 2.30–2.21 (m, 2H), 1.35 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO) δ 169.27, 168.59, 165.24, 165.19, 162.02, 153.18, 145.80, 137.24, 133.09, 131.95, 130.91, 129.37, 124.84, 124.48, 123.27, 122.10, 120.61, 116.81, 115.59, 102.95, 91.03, 66.93, 63.61, 61.75, 28.05, 25.38, 23.70, 14.56.

3.20. Methyl-3-(4-(((4-methyl-6-(3-((2-oxo-2H-chromen-4-yl)oxy)propoxy)pyrimidin-2-yl)thio)-methyl)-1H-1,2,3-triazol-1-yl)benzoate (17e)

White solid; MP: 146–148 °C; 1H NMR (400 MHz, DMSO) δ 8.81 (s, 1H), 8.36 (s, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 7.7 Hz, 1H), 7.72 (dd, J = 14.3, 7.3 Hz, 2H), 7.61 (t, J = 7.7 Hz, 1H), 7.40–7.24 (m, 2H), 6.51 (s, 1H), 5.88 (s, 1H), 4.58 (t, J = 6.0 Hz, 2H), 4.53 (s, 2H), 4.32 (t, J = 6.3 Hz, 4H), 3.36 (s, 2H), 2.52 (s, 2H), 2.33 (s, 3H), 2.30–2.23 (m, 2H), 1.77–1.66 (m, 2H), 1.43 (dd, J = 14.9, 7.4 Hz, 2H), 1.22 (d, J = 7.0 Hz, 2H), 0.94 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, DMSO) δ 169.27, 169.25, 168.57, 165.22, 162.02, 153.16, 145.80, 137.24, 133.08, 131.90, 130.93, 129.36, 124.86, 124.47, 123.25, 122.10, 120.58, 116.80, 115.57, 102.94, 91.03, 66.91, 65.38, 63.59, 30.63, 28.03, 25.36, 23.69, 19.17, 14.05.

3.21. Butyl-3-(4-(((4-methyl-6-(3-((2-oxo-2H-chromen-4-yl)oxy)propoxy)pyrimidin-2-yl)thio)-methyl)-1H-1,2,3-triazol-1-yl)benzoate (17f)

White solid; MP: 130–132 °C; 1H NMR (400 MHz, CDCl3) δ 8.20 (s, 1H), 7.94 (d, J = 34.5 Hz, 3H), 7.71 (s, 1H), 7.47 (d, J = 19.5 Hz, 2H), 7.20 (s, 2H), 6.20 (s, 1H), 5.60 (s, 1H), 4.49 (s, 4H), 4.19 (s, 2H), 3.88 (s, 3H), 2.30 (s, 5H).

3.22. Butyl-4-(4-(((5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)-benzoate (21a)

Yellow solid; MP: 102–104 °C; 1H NMR (400 MHz, DMSO) δ 8.86 (s, 1H), 8.15 (d, J = 8.3 Hz, 2H), 8.05 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 8.1 Hz, 2H), 6.86 (d, J = 8.1 Hz, 2H), 4.66 (s, 2H), 4.32 (t, J = 6.4 Hz, 2H), 4.19 (s, 2H), 3.70 (s, 3H), 1.71 (dd, J = 14.1, 6.9 Hz, 2H), 1.44 (dd, J = 14.8, 7.4 Hz, 2H), 0.95 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, DMSO) δ 167.67, 165.31, 163.11, 158.86, 144.35, 140.02, 131.39, 130.41, 130.12, 126.42, 122.60, 120.38, 114.54, 65.22, 55.47, 30.66, 30.36, 26.98, 19.21, 14.07.

3.23. Ethyl-3-(4-(((5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)-benzoate (21b)

White solid; MP: 110–112 °C; 1H NMR (400 MHz, DMSO) δ 8.88 (s, 1H), 8.39 (s, 1H), 8.18–8.13 (m, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.21 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 8.5 Hz, 2H), 4.64 (s, 2H), 4.38 (q, J = 7.1 Hz, 2H), 4.18 (s, 2H), 3.70 (s, 3H), 1.36 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, DMSO) δ 167.67, 165.21, 163.12, 158.87, 144.14, 137.14, 132.02, 131.03, 130.41, 129.58, 126.43, 124.99, 122.74, 120.74, 114.54, 61.80, 55.48, 30.38, 27.05, 14.57.

3.24. Methyl-4-(4-(((5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)benzoate (21c)

White solid; MP: 108–110 °C; 1H NMR (400 MHz, DMSO) δ 8.87 (s, 1H), 8.15 (d, J = 8.2 Hz, 2H), 8.05 (d, J = 8.3 Hz, 2H), 7.22 (d, J = 8.1 Hz, 2H), 6.86 (d, J = 8.1 Hz, 2H), 4.66 (s, 2H), 4.19 (s, 2H), 3.90 (s, 3H), 3.71 (s, 3H); 13C NMR (100 MHz, DMSO) δ 167.67, 165.78, 163.11, 158.86, 144.35, 140.04, 131.44, 130.42, 129.86, 126.42, 122.58, 120.35, 114.54, 55.47, 52.89, 30.37, 26.98.

3.25. Methyl-3-(4-(((5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)benzoate (21e)

Yellow solid; MP: 110–112 °C; 1H NMR (400 MHz, DMSO) δ 8.88 (s, 1H), 8.40 (s, 1H), 8.16 (d, J = 8.1 Hz, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 7.21 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 8.5 Hz, 2H), 4.65 (s, 2H), 4.19 (s, 2H), 3.92 (s, 3H), 3.70 (s, 3H); 13C NMR (101 MHz, DMSO) δ 167.67, 165.72, 163.13, 158.86, 144.15, 137.14, 131.74, 131.07, 130.41, 129.59, 126.43, 125.00, 122.71, 120.75, 114.54, 55.48, 53.05, 40.59, 40.38, 40.17, 39.96, 39.75, 39.55, 39.34, 30.37, 27.04.

3.26. Butyl-3-(4-(((5-(4-methoxybenzyl)-1,3,4-oxadiazol-2-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)benzoate (21f)

Brown solid; MP: 102–104 °C; 1H NMR (400 MHz, DMSO) δ 8.87 (s, 1H), 8.39 (s, 1H), 8.15 (d, J = 8.1 Hz, 1H), 8.06 (d, J = 7.7 Hz, 1H), 7.76 (t, J = 7.9 Hz, 1H), 7.21 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 8.5 Hz, 2H), 4.65 (s, 2H), 4.34 (t, J = 6.5 Hz, 2H), 4.19 (s, 2H), 3.70 (s, 3H), 1.77–1.68 (m, 2H), 1.43 (dt, J = 14.7, 7.4 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H); 13C NMR (101 MHz, DMSO) δ 167.67, 165.25, 163.13, 158.87, 144.13, 137.15, 131.99, 131.07, 130.40, 129.59, 126.43, 125.03, 122.75, 120.73, 114.54, 65.44, 55.47, 30.64, 30.37, 27.04, 19.18, 14.06.

3.27. Cell Lines and Culture Conditions

A total of 4000 MCF-7 cells were cultured in either MEM or Leibovitz’s L-15 media supplemented with 2% FBS. These cell cultures were maintained at a temperature of 37 °C within a humidified environment containing 5% CO2 [78,79]. The compounds of interest were solubilized in DMSO and stored as stock solutions. These stock solutions were subsequently diluted to the required concentrations using a cell growth medium. The cells were treated for 72 h with triazoles at concentrations of 0, 0.01, 0.1, 10, 100, and 1000 M. The alamarBlue reagent was used to evaluated the inhibitory effect of cell viability.

3.28. Molecular Docking

In silico analysis was performed using AutoDock4Tools (v1.5.6) [80]. The initial stages involved preparing the ligands, namely, 8a, 8b, pazopanib, or sorafenib, which were designed and optimized using the appropriate software. The respective three-dimensional (3D) structures were saved in the PDB format. Subsequently, the crystal structure of VEGFR-2, in conjunction with the co-crystal ligand sorafenib (PDB ID: 4ASD), was retrieved from the Protein Data Bank (PDB). The protein was prepared using AutoDockTools, entailing the removal of water molecules and heteroatoms, while incorporating polar hydrogens. Gasteiger charges were assigned and saved in the PDBQT format. A grid box was defined, encompassing the active site of VEGFR-2 using AutoGrid. The grid dimensions spanned 40 Å × 40 Å × 40 Å, with a spacing of 0.640 Å, ensuring a thorough consideration of all interactions between the compounds and the active site. Subsequently, the prepared ligands were docked into the active site of VEGFR-2 using AutoDock4. The Lamarckian genetic algorithm (LGA) was used for docking, and each compound underwent a predetermined number of energy evaluations. Default docking parameters were employed, and each compound underwent 10 docking runs. The optimal docking pose for each compound was selected based on the lowest binding energy. The resulting complexes were visualized and analyzed using tools such as Biovia Discovery Studio [81], PyMOL [82], and UCSF Chimera [83]. These visualization tools enabled the examination of crucial interactions transpiring between the ligands and the active site of VEGFR-2, providing insights into the molecular binding mechanisms.

4. Conclusions

Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) are promising approaches for inhibiting tumor cells. Green chemistry principles prioritize processes that minimize waste, utilize non-toxic reagents and solvents, adhere to stringent safety protocols, and optimize the efficient utilization of resources and energy. In this study, we have utilized the electrochemical method to synthesize novel conjugated coumarin–pyrimidine–triazoles 8(a–m), 17(a–f), and oxadiazole–triazoles 21(a–f). Among the synthesized compounds, it was observed that compounds 8a and 8b demonstrated enhanced potency against MCF-7 cells, displaying IC50 values of 5.29 and 15.25 μM, respectively. Further enriching our understanding, an in silico docking study of 8a and 8b with VEGFR-2 unveiled binding affinities of −8.44 and −8.90 kcal/mol, respectively. These findings indicate that the newly synthesized compounds possess the potential to effectively target VEGFR-2 within breast cancer cells.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/catal13101353/s1. The Supplementary Data contains the Figures S1 and S2 in S2 contains Docking images of Tamoxifen and Doxorubicin; Figures in S3–S15 and S24–S32 contains the NMR spectra and the IC50 of 8(a–m); Figures in S16–S20 and S33–S34, contains the NMR spectra and the IC50 of 17(a–f); and Figures in S21–S23, S35–S36 contains NMR spectra and the IC50 of 21(a–f).

Author Contributions

A.R., T.P.S., Z.X., D.V., A.M., S.B. and B.B. are responsible for conceptualization, methodology, formal analysis, and writing; N.P.K. performed formal analysis and data curation; V.P., P.E.L. and B.B. contributed to conceptualization, methodology, software, data curation, the original draft, validation, writing, and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Vision Group on Science and Technology (CESEM), and the Government of Karnataka. This research was further supported by the National Natural Science Foundation of China (82172618); the Shenzhen Key Laboratory of Innovative Oncotherapeutics (ZDSYS20200820165400003) (Shenzhen Science and Technology Innovation Commission), China; the Universities Stable Funding Key Projects (WDZC20200821150704001), China; the Shenzhen Bay Laboratory, Oncotherapeutics (21310031), China; and the Cross-Disciplinary Research and Innovation Fund (JC2022) (Tsinghua Shenzhen International Graduate School), China. A.R. thanks the DST-PURSE Project, IOE, University of Mysore, Karnataka, India, for providing fellowship funding.

Data Availability Statement

All data are freely available in this article and its Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Catalysts 13 01353 g001aCatalysts 13 01353 g001b
Figure 1. (A) Drug molecules and reported VEGFR inhibitors with pyrimidine (red), coumarin (blue), triazole (pink), and oxadiazole (purple), carbamate (orange) and alkyl chain (green) heterocyclic motifs. (B) Newly synthesized coumarin–pyrimidine–triazoles and oxadiazole–triazoles derivatives.
Figure 1. (A) Drug molecules and reported VEGFR inhibitors with pyrimidine (red), coumarin (blue), triazole (pink), and oxadiazole (purple), carbamate (orange) and alkyl chain (green) heterocyclic motifs. (B) Newly synthesized coumarin–pyrimidine–triazoles and oxadiazole–triazoles derivatives.
Catalysts 13 01353 g001aCatalysts 13 01353 g001b
Catalysts 13 01353 sch001
Scheme 1. Synthesis of thiouracil–triazole derivatives 8(a–m). Reaction conditions: (i) propargyl bromide, KOH, EtOH:H2O (1:1), reflux; (ii) dibromoethane, K2CO3, DMF, reflux; (iii) K2CO3, acetone, reflux; (iv) (a) NaNO2, HCl(aq); (b) NaN3, HCl(aq) 0–5 °C; (v) Method A: Cu foil and Pt electrode, (1:1), ACN:EtOH:H2O, TBATFB, electrolysis; Method B: CuSO4.5H2O, sodium ascorbate, THF:H2O (1:1), rt.
Scheme 1. Synthesis of thiouracil–triazole derivatives 8(a–m). Reaction conditions: (i) propargyl bromide, KOH, EtOH:H2O (1:1), reflux; (ii) dibromoethane, K2CO3, DMF, reflux; (iii) K2CO3, acetone, reflux; (iv) (a) NaNO2, HCl(aq); (b) NaN3, HCl(aq) 0–5 °C; (v) Method A: Cu foil and Pt electrode, (1:1), ACN:EtOH:H2O, TBATFB, electrolysis; Method B: CuSO4.5H2O, sodium ascorbate, THF:H2O (1:1), rt.
Catalysts 13 01353 sch001
Catalysts 13 01353 sch002
Scheme 2. Synthesis of thiouracil–triazole derivatives 17(a–f). Reaction conditions: (vi) propargyl bromide, KOH, EtOH:H2O (1:1), reflux; (vii) bromochloropropane, K2CO3, DMF, reflux; (viii) K2CO3, acetone, reflux; (ix) ROH, Cat. H2SO4, reflux; (x) (a) NaNO2, HCl(aq); (b) NaN3, HCl(aq) 0–5 °C; (xi) Method A: Cu foil and Pt electrode, (1:1), ACN:EtOH:H2O, TBATFB, electrolysis; Method B: CuSO4.5H2O, sodium ascorbate, THF:H2O (1:1), rt.
Scheme 2. Synthesis of thiouracil–triazole derivatives 17(a–f). Reaction conditions: (vi) propargyl bromide, KOH, EtOH:H2O (1:1), reflux; (vii) bromochloropropane, K2CO3, DMF, reflux; (viii) K2CO3, acetone, reflux; (ix) ROH, Cat. H2SO4, reflux; (x) (a) NaNO2, HCl(aq); (b) NaN3, HCl(aq) 0–5 °C; (xi) Method A: Cu foil and Pt electrode, (1:1), ACN:EtOH:H2O, TBATFB, electrolysis; Method B: CuSO4.5H2O, sodium ascorbate, THF:H2O (1:1), rt.
Catalysts 13 01353 sch002
Catalysts 13 01353 sch003
Scheme 3. Synthesis of oxadiazole–triazole derivatives 21(a–f). Reaction conditions: (xii) CS2, KOH, EtOH, reflux; (xiii) propargyl bromide, K2CO3, reflux; (xiv) Method A: Cu foil and Pt electrode, (1:1), ACN:EtOH:H2O, TBATFB, electrolysis; Method B: CuSO4.5H2O, sodium ascorbate, THF:H2O (1:1), rt.
Scheme 3. Synthesis of oxadiazole–triazole derivatives 21(a–f). Reaction conditions: (xii) CS2, KOH, EtOH, reflux; (xiii) propargyl bromide, K2CO3, reflux; (xiv) Method A: Cu foil and Pt electrode, (1:1), ACN:EtOH:H2O, TBATFB, electrolysis; Method B: CuSO4.5H2O, sodium ascorbate, THF:H2O (1:1), rt.
Catalysts 13 01353 sch003
Catalysts 13 01353 sch004
Scheme 4. Proposed electrochemical reaction mechanism for 8a.
Scheme 4. Proposed electrochemical reaction mechanism for 8a.
Catalysts 13 01353 sch004
Catalysts 13 01353 g002
Figure 2. AlamarBlue viability assay, as described in the Section 3, against MCF-7 breast cancer cells for 72 h incubation of compounds. Error bars represent ±SD and points (0, 0.01, 0.1, 1, 10, or 100 µM).
Figure 2. AlamarBlue viability assay, as described in the Section 3, against MCF-7 breast cancer cells for 72 h incubation of compounds. Error bars represent ±SD and points (0, 0.01, 0.1, 1, 10, or 100 µM).
Catalysts 13 01353 g002
Catalysts 13 01353 g003
Figure 3. Prediction of the molecular target of compound 8a using Swiss Target Prediction.
Figure 3. Prediction of the molecular target of compound 8a using Swiss Target Prediction.
Catalysts 13 01353 g003
Catalysts 13 01353 g004aCatalysts 13 01353 g004b
Figure 4. Cartoon representation of Docked compounds inside the active site of VEGFR-2 (A). Representation of 2D structure showing interactions with the compound 8a (B1), 8b (B2), pazopanib (B3), and sorafenib (co-crystal ligand) (B4) with the active site residues of VEGFR-2. (C) The 3D surface view of all docked compounds 8a (black), 8b (red), pazopanib (yellow), and sorafenib (pink) inside the active groove of VEGFR-2, with an enlarged view for better visualization.
Figure 4. Cartoon representation of Docked compounds inside the active site of VEGFR-2 (A). Representation of 2D structure showing interactions with the compound 8a (B1), 8b (B2), pazopanib (B3), and sorafenib (co-crystal ligand) (B4) with the active site residues of VEGFR-2. (C) The 3D surface view of all docked compounds 8a (black), 8b (red), pazopanib (yellow), and sorafenib (pink) inside the active groove of VEGFR-2, with an enlarged view for better visualization.
Catalysts 13 01353 g004aCatalysts 13 01353 g004b
Table 1. List of thiouracil–triazoles 8(a–m) compounds synthesized.
Table 1. List of thiouracil–triazoles 8(a–m) compounds synthesized.
EntryRR1MCF-7
IC50 (μM)
8a–H–Cl5.29
8b–H–Br15.25
8c–H–OMe138.60
8d–H–Me126.80
8e–OMe–Cl172.30
8f–OMe–Br>100
8g–OMe–OMe214.90
8h–OMe–OH41.87
8i–OMe–Me71.89
8j–F–Cl>100
8k–F–Br>100
8l–F–OMe>100
8m–F–Me>100
Table 2. List of thiouracil–triazoles 17(a–f) compounds synthesized.
Table 2. List of thiouracil–triazoles 17(a–f) compounds synthesized.
EntryRMCF-7
IC50 (μM)
17a–4COOEt>100
17b–4COOBu>100
17c–4COOMe>100
17d–3COOEt>100
17e–3COOBu>100
17f–3COOMe>100
Table 3. List of oxadiazole–triazoles 21(a–f) compounds synthesized.
Table 3. List of oxadiazole–triazoles 21(a–f) compounds synthesized.
EntryRMCF-7
IC50 (μM)
21a–4COOBu>100
21b–4COOEt>100
21c–4COOMe>100
21d–3COOEt>100
21e–3COOMe>100
21f–3COOBu>100
Table 4. Docking results expressed as binding scores, amino acids interactions, and bond length of compounds 8a, 8b, pazopanib, or sorafenib (co-crystal ligand) within the active site of VEGFR2.
Table 4. Docking results expressed as binding scores, amino acids interactions, and bond length of compounds 8a, 8b, pazopanib, or sorafenib (co-crystal ligand) within the active site of VEGFR2.
CompoundBinding Scores
(kcal/mol)		InteractionsResidueBond Length
Å
8a−8.44Hydrogen
π-cation
π-anion		ASP-1046
GLU-885
LYS-868		2.71
3.27
4.05
HydrophobicLEU-840
VAL-848
ALA-866
VAL-899
VAL-916
LEU-1019
HIS-1026
CYS-1045
PHE-1047		5.10
4.62, 4.54
4.22
4.88
4.94.4.50
4.95
4.59
5.45
4.55
8b−8.90Hydrogen
π-cation
π-anion		LYS-868, LEU-1049
LYS-868
GLU-885		1.96, 2.14
3.95
3.64
HydrophobicALA-881
ILE-888
VAL-889
VAL-899
VAL-916
CYS-1045		4.77
4.58
3.68
5.00
4.48
5.49
Pazopanib−9.25Hydrogen
π-cation
π-anion		ASP-1028, ASP-1046
HIS-1026
HIS-1026		1.97, 2.29
4.73
4.90
HydrophobicLEU-889
LEU-899
LEU-1019
ARG-1027
ILE-1044
CYS-1045
TYR-1059
LEU-1067		4.86
4.39
4.73
4.49
4.58
5.01
4.79
4.86
Co-crystal ligand
(Sorafenib)		−11.15HydrogenASP-1046, GLU-885,
CYS-919		1.84, 1.93, and 2.42
2.20, and 2.48
Halogen (Fluorine)
Hydrophobic		ILE-1044
VAL-848
ALA-866
LYS-868
LEU-889
ILE-892
VAL-899
VAL-916
PHE-918
LEU-1019
ILE-1044
CYS-1045		2.82
5.13
3.94
5.33
3.45
4.78
5.05
4.81
4.73
4.20
5.13
4.64
Table 5. The vNN–ADMET predictions of compounds 8a, 8b, doxorubicin, and tamoxifen.
Table 5. The vNN–ADMET predictions of compounds 8a, 8b, doxorubicin, and tamoxifen.
QueryLiver
Toxicity		MetabolismMembrane TransportersOthers
CYP Inhibitors
DILICTHLM1A23A42D62C92C19BBBPgp InPgp SubhERGMMPAMESMRTD (mg/day)
8aYNYNNNNNNNNYNY244
8bYNYNNNNNYNNNNY274
TAMYNNNYNNNYYYYNN161
DOXNYYNNNNNNNYNYY116
Note: N, no; Y, yes; DILI, drug-induced liver injury; CT, cytotoxicity; CYP, cytochrome P450; HLM, human liver microsomes; BBB; blood–brain barrier; Pgp, P–glycoprotein; Sub, substrate; In, inhibition; hERG, human ether-a-go-go–related gene; MMP, matrix metalloproteins; AMES; salmonella/microsome mutagenicity; MRTD, maximum recommended therapeutic dose.
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Ravish, A.; Siddappa, T.P.; Xi, Z.; Vishwanath, D.; Mohan, A.; Basappa, S.; Krishnamurthy, N.P.; Lobie, P.E.; Pandey, V.; Basappa, B. Electrochemical Synthesis of Versatile Pyrimidine and Oxadiazoles Tethered Triazoles as Inhibitors of VEGFR-2 in Human Breast Cancer Cells. Catalysts 2023, 13, 1353. https://doi.org/10.3390/catal13101353

AMA Style

Ravish A, Siddappa TP, Xi Z, Vishwanath D, Mohan A, Basappa S, Krishnamurthy NP, Lobie PE, Pandey V, Basappa B. Electrochemical Synthesis of Versatile Pyrimidine and Oxadiazoles Tethered Triazoles as Inhibitors of VEGFR-2 in Human Breast Cancer Cells. Catalysts. 2023; 13(10):1353. https://doi.org/10.3390/catal13101353

Chicago/Turabian Style

Ravish, Akshay, Tejaswini P. Siddappa, Zhang Xi, Divakar Vishwanath, Arunkumar Mohan, Shreeja Basappa, Niranjan Pattehalli Krishnamurthy, Peter E. Lobie, Vijay Pandey, and Basappa Basappa. 2023. "Electrochemical Synthesis of Versatile Pyrimidine and Oxadiazoles Tethered Triazoles as Inhibitors of VEGFR-2 in Human Breast Cancer Cells" Catalysts 13, no. 10: 1353. https://doi.org/10.3390/catal13101353

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