Design, Synthesis and Biological Evaluation of 1,3,5-Triazine Derivatives Targeting hA1 and hA3 Adenosine Receptor

Adenosine mediates various physiological activities in the body. Adenosine receptors (ARs) are widely expressed in tumors and the tumor microenvironment (TME), and they induce tumor proliferation and suppress immune cell function. There are four types of human adenosine receptor (hARs): hA1, hA2A, hA2B, and hA3. Both hA1 and hA3 AR play an important role in tumor proliferation. We designed and synthesized novel 1,3,5-triazine derivatives through amination and Suzuki coupling, and evaluated them for binding affinities to each hAR subtype. Compounds 9a and 11b showed good binding affinity to both hA1 and hA3 AR, while 9c showed the highest binding affinity to hA1 AR. In this study, we discovered that 9c inhibits cell viability, leading to cell death in lung cancer cell lines. Flow cytometry analysis revealed that 9c caused an increase in intracellular reactive oxygen species (ROS) and a depolarization of the mitochondrial membrane potential. The binding mode of 1,3,5-triazine derivatives to hA1 and hA3 AR were predicted by a molecular docking study.


Introduction
Extracellular adenosines are important signal transmitters and mediate various physiological activities in the body [1,2]. Human adenosine receptors (hARs) can be classified into four subtypes: hA 1 , hA 2A , hA 2B , and hA 3. All four belong to the G protein-coupled receptor (GPCR) family, and each has a different pharmacological profile, tissue distribution, and function [3]. Although hARs have been known for a long time, new functions are continuously being discovered and novel ligands being developed. hA 1 AR is found in various tissues and cells and regulates many physiological activities in the body; for example, the activation of hA 1 AR leads to bradycardia [4], inhibits neurotransmitter release [5], lipolysis [6], and renal excretion [7] and induces smooth muscle contraction [8]. The hA 1 AR agonist has mainly been developed for treatment of cardiovascular diseases, such as heart failure, arrhythmia, and angina, or for neurological diseases, such as seizure, ischemia, and depression [9]. hA 3 AR is also important for physiological signaling in the body. hA 3 AR is expressed at a low level in most cells but is overexpressed in inflammatory and various neoplastic cells [10]. Therefore, it is an important drug target for developing therapeutic agents for glaucoma, stroke, asthma, inflammation, rheumatoid arthritis, and cancer [11].
Multiple studies have demonstrated that hA 1 AR regulates the proliferation of tumor cells and that hA 1 AR antagonists inhibit the proliferation and migration of tumor cells [12]. It is unclear exactly what function hA 3 AR has in tumor cell proliferation and death [13,14]. Numerous publications have shown that hA 3 AR is overexpressed in various types of cancer cells [15]. The activation of hA 3 AR induces the growth of tumor cells by increasing VEGF, HIF-1, MMP-9, angiogenesis, migration, and proliferation [16]. At the same time, it decreases cell proliferation by induction of G-CSF and IL-2 in the immune system [17].
Polypharmacology, a concept that controls multiple targets with one drug, is a new paradigm in drug discovery that has recently received attention [18]. Compared with combination therapy, polypharmacology has higher safety and lower risk of drug-drug interaction [19]. hA 1 and hA 3 AR are good targets for multitarget drugs because among hARs, hA 1 and hA 3 AR are quite similar, with 49% sequence similarity [3]. In addition, the two are activated by the same endogenous ligand, adenosine, and the downstream signal also inhibits cyclic adenosine monophosphate (cAMP) production in the same way.
Determining what alterations tumor cells undergo when simultaneously inhibiting both hA 1 and hA 3 AR that share a sub-signal transduction will be an important step in the development of anticancer drugs targeting adenosine receptors. Several studies on ligands that simultaneously regulate hA 1 and hA 3 AR have been published [20][21][22]. Compound 1 bearing a purine scaffold shows an inhibition constant (K i ) of 6.8 and 6.3 nM, and compound 2 shows a K i of 36.7 and 25.4 nM for hA 1 and hA 3 AR, respectively, indicating balanced binding ( Figure 1). To develop a novel scaffold of the hAR ligand in non-xanthine ligands, we analyzed previously reported compounds targeting adenosine receptors. Langmead et al., in an effort to find a novel hit through a docking study, reported that compounds containing 1,3,5-triazine bind to hA 1 and hA 2A AR (Figure 2) [23]. Compound 3 was found to be an hA 2A AR antagonist, with moderate selectivity (9.5-fold) against hA 1 AR. Compound 4 was discovered through virtual screening and bound more potently compared to compound 3, but the selectivity against hA 1 AR was 2.94, which was lower compared to compound 3 [24].
Compound 5 (1,3,5-triazine-thiadiazole) has been described as a potent hA 2A AR antagonist [25,26]. Compound 6 showed 319-fold selectivity against hA 2A AR compared to hA 1 AR; however, the selectivity index (hA 1 AR:hA 2A AR) of compound 5 was 11.66, indicating low selectivity. On the basis of previously published research, it was determined that derivatives of the 1,3,5-triazine scaffold were ligands that modulate adenosine receptors. In addition, the type of triazine substituents altered the subtype selectivity of adenosine receptors. This indicates that triazine modifications could be optimized to produce selective ligands for subtypes other than hA 2A AR. Therefore, we attempted to introduce various substitutions into 1,3,5-triazine in order to provide specific ligands for hA 1 and hA 3 AR. In this study, we developed a series of 1,3,5-triazine derivatives that bind to hA 1 and hA 3 AR by substituting at the 2, 4, and 6 positions of the 1,3,5-triazine scaffold. A novel 2-amino-1,3,5-triazine derivative was designed by introducing substituents at the 4 and 6 positions of 1,3,5-triazine. The designed derivatives were synthesized, and their binding affinities to hARs were evaluated using a radioligand. The binding mode of 1,3,5-triazine derivatives were predicted by a molecular docking study using the crystal structure of the hA 1 AR (PDB ID: 5N2S) and a homology model of the hA 3 AR [27].

Radioligand Binding Assays at Human Adenosine Receptors
All the synthesized compounds were screened with radioligand binding assays at hARs [28]. First, the percentage of inhibition was measured by treatment of each hAR subtype with 10 µM of each compound (Table 1). Except for 9c, all compounds showed <90% inhibition at hA 2A and hA 2B AR. Compounds 9a-c, 9e, and 9g showed >95% inhibition at hA 1 AR, and compounds 9a, 9c, and 9d showed >95% inhibition at hA 3 AR. In addition, 9d showed 69% inhibition at hA 1 AR whereas it showed 95% inhibition at hA 3 AR, showing significant selectivity. Compound 9c showed >95% inhibition at all subtypes except hA 2B AR. Compound 9f including 4-N-piperidine showed no binding with any of the subtypes, probably because the large piperidine interfered with binding to the hARs.
The binding affinities of compounds showing >95% inhibition were determined and are shown in Table 2. Compound 9a with 3-fluoro-4-methoxyaniline showed the best binding affinity to hA 3 AR (K i = 55.5 nM) and good binding affinity to hA 1 AR, with a 2.5-fold hA 1 AR:hA 3 AR selectivity index. Compound 9b with 3,5-dimethoxyaniline also showed potent and selective binding affinity to hA 1 AR (K i = 69.7 nM). Compound 9c with 3-methoxy-4-chloroaniline showed the best binding affinity to hA 1 AR (K i = 57.9 nM) and moderate binding affinity to hA 3 AR (K i = 661.1 nM). R 1 substitution in the aniline appeared to be well tolerated by hA 1 AR compared to hA 3 AR. The compounds substituted with methoxy in aniline generally showed good binding affinity to hA 1 AR, with higher affinity being obtained with compounds bearing meta-methoxy attached to the aniline core. Moreover, the compounds with methoxy substituted at the para-position of aniline showed the best binding affinity.
Since the most balanced binding to hA 1 AR:hA 3 AR was shown by 9a, we developed a series of 3-fluoro-4-methoxyaniline derivatives 11a-i for dual hA 1 -hA 3 AR ligands. Various substituents were introduced at the para-position of phenyl to replace the hydroxyl group, and the percentage inhibition was evaluated at the four hAR subtypes with 10 µM of the synthesized compounds (Table 3). Compounds 11a and 11b, which included methoxy and fluorine at the para-position of benzene, respectively, displayed >90% inhibition at both hA 1 and hA 3 AR and low percentage inhibition at hA 2A and hA 2B AR. Compounds 11c-e substituted with the electron-withdrawing groups OCF 3 , CF 3 , and CN, respectively, at the para-position of the phenyl core showed low percentage inhibition at all hAR subtypes. The compounds bearing a bulky group attached to the para-position of the phenyl core also showed low percentage inhibition at all hARs, indicating that the substituent at the para-position of the phenyl core is likely to be important for determining the binding affinity at hARs. Compounds 11h and 11i bearing two fluorine and nitro at the meta-position of the phenyl core, respectively, showed much lower percentage inhibition compared to 9a and 11b. We deduce that substituents at a position other than the para-position of the phenyl core negatively affect binding to hARs.  The binding affinities of 11a and 11b at hA 1 and hA 3 AR were determined and are shown in Table 4. Compound 11a with methoxy instead of a hydroxyl group showed improved binding affinity to both hA 1 and hA 3 AR, about 2-fold at hA 1 AR and 5-fold at hA 3 AR, with an hA 1 AR:hA 3 AR selectivity index of 5.87. For 11b bearing para-fluorine at the phenyl core, the binding affinity was less changed; however, the binding affinity to hA 1 AR slightly improved, indicating an hA 1 AR:hA 3 AR selectivity index of 1.7.

cAMP Assay at hA 1 and hA 3 AR Adenosine Receptors
To determine whether 1,3,5-triazine scaffold derivatives behave as agonists or antagonists, we performed cAMP accumulation assays with 11a and 11b at hA 1 and hA 3 AR. We confirmed whether the test compounds behave as antagonists by measuring the concentration change in cAMP in the presence of a full agonist 5-N-ethylcarboxamido adenosine (NECA) using antagonist mode assays. We also confirmed whether the test compounds could behave as agonists by comparing the relative percentage activation of NECA using agonist mode assays. Both 11a and 11b showed 77% and 88% inhibition at hA 3 AR in antagonist mode, respectively, and 17% and 6% percentage activation in agonist mode, respectively, indicating that 11a and 11b behave as antagonists at hA 3 AR.
Compound 11b showed 60% inhibition in antagonist mode and 23% activation in agonist mode, confirming that it acts as an antagonist and a partial agonist for hA 1 AR. However, the assay results of 11a at hA 1 AR were interesting. The percentage inhibition and percentage activation of 11a were 1% and 11% in antagonist mode and agonist mode, respectively, indicating that 11a is neither an antagonist nor an agonist for hA 1 AR. That is, 11a binds to hA 1 AR with high binding affinity, and the cAMP concentration does not change due to 11a binding. Thus, additional experiments are required to determine which signaling transduction occurs after 11a binds to hA 1 AR.
2.4. Cell Viability of 1,3,5-Triazine Derivatives 9a-c, 9g, and 11a-b To test the effect of the compounds in terms of cell growth regulation in lung cancer cell lines such as A549 and NCI-H1299 cells, these cells were treated in 96-well plates for 48 h with the compounds at concentrations ranging from 0 µM to 100 µM. Viability of A549 and NCI-H1299 cells was lowered by treatment with 1,3,5-triazine derivatives 9a-c, 9g, and 11a-b which showed good binding affinity at hA 1 AR. However, compound 9a and 11a-b, which bound to hA 1 and hA 3 AR, exhibited relatively low cell viability, whereas compound 9c, which had the highest binding affinity at hA 1 AR among the derivatives, exhibited the greatest inhibitory effect. To assess lung cancer cell viability, a cell viability assay was performed, and the results indicated a significant decrease in cell viability to 59.9% in A549 cells treated with 9c of 25 µM concentration, and to 68.8% in NCI-H1299 cells treated with 9c of 25 µM concentration ( Figure 3A). Therefore, we decided to perform an additional experiment with 9c. Microscopic analysis of A549 cells treated with 20 µM and 40 µM of 9c further showed that these cells exhibited gradual changes in cell growth in a dose-dependent manner relative to the concentration of 9c ( Figure 3B).

Compound 9c-Induced Intracellular ROS and Mitochondrial ROS
Although reactive oxygen species (ROS) play an important role in regulating normal cellular processes, abnormal ROS levels contribute to the development of a variety of human diseases, including cancer. Because of their accelerated metabolism, cancer cells have higher ROS levels than normal cells [29]. Nevertheless, the high ROS content of cancer cells makes them more susceptible to oxidative stress-induced cell death, which can be used to target cancer cells selectively [30]. Flow cytometry was used to determine whether 9c induces ROS generation in A549 cells by examining the results. The experiment was conducted with H 2 DCF-DA as a fluorescent probe. As shown in Figure 4A,B, A549 cells treated with 9c of 20 µM and 40 µM concentrations exhibited highly increased ROS of 7.31% and 22.6%, respectively. For comparison, the control sample exhibited a ROS level of 1.26%. Next, we determined the effect of 9c in regulating mitochondrial ROS. Our results show the 9c-treated A549 cells to have significantly increased mitochondrial ROS levels ( Figure 4C,D). These data indicate that 9c treatment makes lung cancer cells more sensitive to oxidative stress and makes them more vulnerable to ROS-mediated cell death.

Compound 9c-Induced Mitochondrial Membrane Dysfunction
ROS-induced oxidative stress can cause the rapid depolarization of the inner mitochondrial membrane potential (∆Ψm) and, as a result, impairment of oxidative phosphorylation [31]. Using tetramethylrhodamine methyl ester (TMRM) measurements, we investigated how mitochondrial membrane potential was affected by 9c. A549 cells were exposed to 9c following incubation with TMRM. Afterward, flow cytometry was employed to calculate the intensity of TMRM binding in the healthy membrane. Figure 5A,B show that, compared to the control value of 93.8%, the TMRM-positive intensity dramatically decreased to 74.6.% at 20 µM, and to 52.3% at 40 µM, respectively. The results suggest that 9c causes the A549 cell's mitochondrial membrane to depolarize, which leads to dysfunction.

Compound 9c Effects on Lung Cancer Cell Death
A live cell assay and a dead cell assay were carried out using a mixture of two fluorescent dyes: calcein (green dye for live cells) and ethidium homodimer-1 (EthD-1, red dye for dead cells). The cells were washed and stained with calcein and EthD-1 before conducting imaging fluorescence microscopy and flow cytometry experiments. When treated with 20 µM 9c, we observed a drastic decrease in the number of live cells (green) and a slight increase in the number of dead cells (red). This was supported by the flow cytometry results, which indicated an approximate increase in the number of dead cells (compared to the control group) by 34.4% following treatment with 20 µM 9c, and by 46.1% following treatment with 40 µM 9c ( Figure 6A,B), respectively. As a result, our findings suggest that 9c significantly reduces the viability of lung cancer cell lines.

Molecular Docking Study of 1,3,5-Triazine Derivatives
We attempted molecular docking to investigate how the triazine derivatives bind to hA 1 and hA 3 AR. Initially, the binding mode of 11b to hA 1 AR was predicted using x-ray structure (PDB; 5N2S). Consequently, two docking poses of 11b in hA 1 AR were proposed and depicted in Figure 7A. There was no significant difference between the two binding mode docking scores, −9.100 (red) and −8.624 (violet). 11b was stabilized by π-π interactions between the triazine and F171 5.29 in the two docking poses. By contrast, N254 6.5 formed hydrogen bonds with the nitrogen of aniline in the red-binding mode, or nitrogen of amino in the purple-binding mode, respectively. The aniline group of 11b was oriented toward the augmented TM2 region in hA 1 AR, adopting a purple-binding pose. Docking was also performed on 9a and 9c, which both exhibited strong binding affinity in hA 1 AR and were both predicted to bind in the same manner as 11b. (Supplementary Materials, Figure S3). In addition, 11h and 11i with a substituent at the meta position of the phenyl ring were also applied to a docking study ( Figure 7B). The phenyl rings of 11h and 11i were predicted to occupy the binding pocket at which triazine of 11b was located. Since the binding pocket for phenyl ring of 11b was narrow ( Figure 7B-red circle), it can be inferred that meta-substituents on a phenyl ring of triazine derivatives interfered with the binding to the receptor, which explained why 11h and 11i were inactive at hA 1 AR. We used the previously published homology model of hA 3 AR for docking [27], since the x-ray crystal structure of hA 3 AR has not yet been determined. The ligand used to generate the homology model is structurally distinct from triazine derivatives; therefore, induced-fit docking was utilized to predict the binding mode of 11b in hA 3 AR. The N250 6.55 of hA 3 AR generated hydrogen bonds with the nitrogen of aniline and triazine of 11b, and its triazine formed a π-π interaction with F168 5.29 ( Figure 7C). This was consistent with the binding mode in hA 1 AR, and the docking study explained how 11b binds to both hA 1 and hA 3 AR. 9a was predicted to bind in the same manner as 11b on the hA 3 AR model, which was generated from the 11b induced-fit docking ( Figure 7C).

General Chemical Synthesis
Reagents and solvents were purchased from commercial suppliers (Sigma-Aldrich, Seoul, Korea; Acros Organics, Seoul, Korea; TCI, Seoul, Korea, etc.) and used as provided, unless indicated otherwise. All reactions except Suzuki coupling that used boronic acids with palladium catalysts in a microwave were performed in a round-bottom flask under a nitrogen atmosphere with stirring at room temperature. Reactions were monitored with analytical thin-layer chromatography (TLC) using glass sheets pre-coated with silica gel 60 F 254 (Merck, Darmstadt, Germany), with visualization under ultraviolet (UV) light (254 nm).

General Procedure for the Synthesis of 8a-g
A cyanuric chloride (7) solution (1 equiv) in tetrahydrofuran (THF) was stirred and then cooled to −15 • C. Aniline (1 equiv) was added, and the mixture was stirred at the −15 • C for 0.5-1 h. Under TLC monitoring, ammonium hydroxide solution (25-28% NH 3 in water) was added, and the mixture was stirred at room temperature for 1-2 h. Finally, the solvent was removed under reduced pressure, and the resulting solid was collected by filtration and dried to obtain the desired product.

General Procedure for the Synthesis of 9a-g
Intermediates 8a-g (1 equiv), (4-hydroxyphenyl)boronic acid (2 equiv), tetrakis (triphenylphosphine)palladium (5 mol%), potassium carbonate (2 equiv), and a 4:1 dioxane:water mixture were added to a microwave tube. The mixture was heated to 120 • C for 1 h under microwave irradiation and then filtered through Celite with ethyl acetate as an eluent solvent. Next, the filtrate was washed with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo. Finally, the resulting residue was purified by column chromatography.   (7) solution (1 equiv) in tetrahydrofuran was stirred and then cooled to −15 • C. 3-Fluoro-4-methoxyaniline (1 equiv) was added, and the mixture was stirred at −15 • C for 0.5-1 h. Under TLC monitoring, ammonium hydroxide solution (25-28% NH 3 in water) was added, and the mixture was stirred at room temperature for 1-2 h. Finally, the solvent was removed under reduced pressure, and the resulting solid was collected by infiltration and dried to obtain the desired product.

General Procedure for the Synthesis of 11a-i
Intermediate 10 (1 equiv), R 2 -phenylboronic acid (2 equiv), tetrakis(triphenyl phosphine)palladium (5 mol%), potassium carbonate (2 equiv), and a 4:1 dioxane:water mixture were added to a microwave tube. The mixture was heated to 120 • C for 1 h under microwave irradiation and then filtered through Celite with ethyl acetate as an eluent solvent. Next, the filtrate was washed with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo. Finally, the resulting residue was purified by column chromatography.
Agonist Mode at hA 1 or hA 3 AR hA 1 and hA 3 AR functional experiments were carried out in CHO-A 1 and CHO-A 3 #18 cell line, respectively. The day before the assay, the cells were seeded on the 96-well culture plate (Falcon 353072). The cells were washed with wash buffer (Mixture F12 Ham's (Sigma N6658) for hA 1 AR; Dulbecco's modified eagle's medium nutrient mixture F-12 ham (Sigma D8062) for hA 3 AR; 25mM Hepes; pH = 7.4). Wash buffer was replaced by incubation buffer (Mixture F12 Ham's (Sigma N6658) for hA 1 AR; Dulbecco's modified eagle's medium nutrient mixture F-12 ham (Sigma D8062) for hA 3 AR; 25 mM Hepes, 20 µM Rolipram (Sigma R6520); pH = 7.4). The cells were pre-incubated at 37 • C for 15 min. Then, test compounds and 5 -(N-Ethilcarboxamido) adenosine (NECA) as reference compound (Sigma E2387) were added and incubated at 37 • C for 10 min. FSK (Sigma F3917) was added and incubated at 37 • C for 5 min. After incubation, the amount of cAMP was determined using cAMP Biotrak Enzyme immunoassay (EIA) System Kit (GE Healthcare RPN225).

Cell Culture
For our study, A549 and NCI-H1299 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, South Korea). CHO-A1, Hela-A2A, and HEK-293T-A2B (Euroscreen, Gosselies, Belgium) were also used in this study. The cell culture medium (Roswell Park Memorial Institute (RPMI) 1640, Thermo Fisher Scientific, Waltham, MA, USA) contained 10% fetal bovine serum (FBS) and 1% Antibiotic-Antimycotic (Thermo Fisher Scientific, Waltham, MA, USA) and was used in accordance with guidelines provided by KCLB. The cells were cultured at 37 • C in an incubator with 5% CO 2 . When the cell density reached 90%, subcultures were generated using a trypsin-EDTA solution.

Cell Viability Assay
A549 and NCI-H1299 cells were seeded in 96-well plates at a density of 1 × 10 5 cells per well for 24 h before being exposed to compounds at various concentrations. After 48 h of compound treatment, the CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA) was used to conduct a cell viability assay. The cells were incubated with solution reagents for 2 h at 37 • C before being measured for absorbance at 490 nm using a Synergy HTX Multi-Mode microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

Microscopy
A549 cells were seeded in 6-well plates at a density of 1 × 10 6 cells per well for microscopy analysis and then treated with compounds (20 and 40 µM). Cell morphology was examined after 48 h and images of the cells were acquired using an EVOS M5000 Imaging System (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Live-Dead Assay
The A549 cells were seeded in 6-well plates at a density of 1 × 10 6 cells per well, and the cells underwent treatment with the compound (20 and 40 µM) for 48 h. The A549 cells were analyzed using fluorescent dyes for both living and dead cells with the LIVE/DEAD kit (Thermo Fisher Scientific, Waltham, MA, USA). For cell staining, we used EthD-1 and calcein by referring to the manufacturer's instructions. Images were captured with an EVOS M5000 Imaging System (Thermo Fisher Scientific Inc., Waltham, MA, USA).
The structure of 1,3,5-triazine derivatives were drawn using Chemdraw [33] and its 3D conformation was generated using the Schrödinger LigPrep programme [34]. LigPrep generated all possible tautomers and states at pH 7.0 using Epik [35,36] for 1,3,5-triazine derivatives. The crystal structure of hA 1 AR co-crystallized with PSB36 (PDB ID: 5N2S) was acquired from the Protein Data Bank (PDB). The homology model of hA 3 AR was obtained from the research work by Lee et al. [27]. The protein was prepared using the Protein Preparation Wizard [37] to assign bond orders, add hydrogens at pH 7.0, and remove water molecules. Prime was used to complete missing side chains and loops. Finally, a restrained minimization was performed using the default constraint of 0.30 Å RMSD and the OPLS 2005 force field in order to complete the protein preparation. Molecular docking simulations were performed using the Glide ligand docking module for hA 1 AR and the Glide induced fit docking module [38][39][40] for hA 3 AR in standard protocol (standard precision) mode. The binding conformations of 1,3,5-triazine derivatives were analyzed in order to identify the important interactions with the active site residues of hA 1 and hA 3 AR.

Conclusions
We synthesized 1,3,5-triazine derivatives from cyanuric chloride and evaluated their binding affinity toward hARs. Of these derivatives, 11b showed good binding affinity to both hA 1 and hA 3 AR (K i =98.3 and 56.6 nM, respectively; selectivity index = 1.74). 11b was found to be a hA 1 and hA 3 AR dual antagonist in cAMP accumulation assays at hA 1 and hA 3 AR. Compound 9c showed the highest binding affinity to hA 1 AR (K i = 57.9 nM), and we demonstrated that 9c exhibits cytotoxic activity in lung cancer cells. According to our findings, 9c increased the expression of ROS, and this accumulation led to mitochondrial membrane dysfunction, which caused cell death. Thus, 9c is a promising therapeutic agent for lung cancer, and further research efforts should focus on elucidating the mechanisms involved in detail.The binding modes of triazine derivatives were identified through a molecular docking study at hA 1 and hA 3 AR. The 1,3,5-triazine derivatives were predicted to bind to both hA 1 and hA 3 AR. We demonstrated that 1,3,5-triazine derivatives have the potential to be developed as hA 1 and hA 3 AR antagonists. As a result, further SAR efforts on the 1,3,5-triazine derivatives are already underway to improve their efficacy and selectivity to hA 1 or hA 3 AR, or to both hA 1 and hA 3 AR as dual ligands.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules27134016/s1, Figure S1: 1 H and 13 C NMR Copies of 9a-g and 11a-i; Figure S2. HR-MS Copies of 9a-g, and 11a-i; Figure   Data Availability Statement: All data that generated or analyzed during this study are available from the corresponding authors upon justified request.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.