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Article

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

by
Sujin Park
1,2,
Yujin Ahn
3,4,
Yongchan Kim
2,
Eun Joo Roh
2,
Yoonji Lee
5,
Chaebin Han
1,2,
Hee Min Yoo
3,4,* and
Jinha Yu
1,*
1
College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Korea
2
Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea
3
Biometrology Group, Korea Research Institute of Standards and Science (KRISS), Daejeon 34113, Korea
4
Department of Precision Measurement, University of Science and Technology (UST), Daejeon 34113, Korea
5
College of Pharmacy, Chung-Ang University, Seoul 06974, Korea
*
Authors to whom correspondence should be addressed.
Molecules 2022, 27(13), 4016; https://doi.org/10.3390/molecules27134016
Submission received: 13 May 2022 / Revised: 16 June 2022 / Accepted: 20 June 2022 / Published: 22 June 2022
(This article belongs to the Special Issue Purinergic Signaling: Targeting GPCRs, Ion Channels and Enzymes—A Theme Issue in Honor of Dr. Kenneth A. Jacobson)
Browse Figures
Versions Notes

Abstract

:
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.

1. 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: hA1, hA2A, hA2B, and hA3. 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.
hA1 AR is found in various tissues and cells and regulates many physiological activities in the body; for example, the activation of hA1 AR leads to bradycardia [4], inhibits neurotransmitter release [5], lipolysis [6], and renal excretion [7] and induces smooth muscle contraction [8]. The hA1 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].
hA3 AR is also important for physiological signaling in the body. hA3 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 hA1 AR regulates the proliferation of tumor cells and that hA1 AR antagonists inhibit the proliferation and migration of tumor cells [12]. It is unclear exactly what function hA3 AR has in tumor cell proliferation and death [13,14]. Numerous publications have shown that hA3 AR is overexpressed in various types of cancer cells [15]. The activation of hA3 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]. hA1 and hA3 AR are good targets for multitarget drugs because among hARs, hA1 and hA3 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 hA1 and hA3 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 hA1 and hA3 AR have been published [20,21,22]. Compound 1 bearing a purine scaffold shows an inhibition constant (Ki) of 6.8 and 6.3 nM, and compound 2 shows a Ki of 36.7 and 25.4 nM for hA1 and hA3 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 hA1 and hA2A AR (Figure 2) [23]. Compound 3 was found to be an hA2A AR antagonist, with moderate selectivity (9.5-fold) against hA1 AR. Compound 4 was discovered through virtual screening and bound more potently compared to compound 3, but the selectivity against hA1 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 hA2A AR antagonist [25,26]. Compound 6 showed 319-fold selectivity against hA2A AR compared to hA1 AR; however, the selectivity index (hA1 AR:hA2A 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 hA2A AR. Therefore, we attempted to introduce various substitutions into 1,3,5-triazine in order to provide specific ligands for hA1 and hA3 AR.
In this study, we developed a series of 1,3,5-triazine derivatives that bind to hA1 and hA3 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 hA1 AR (PDB ID: 5N2S) and a homology model of the hA3 AR [27].

2. Results and Discussion

2.1. Synthesis of 1,3,5-Triazine Derivatives

2-Amino-1,3,5-triazine derivatives were prepared as described in Scheme 1. Briefly, cyanuric chloride (7) was reacted with various types of anilines to obtain 8a–g, followed by amination using aqueous ammonia (25–28% in water), synthesizing 9a–g. Suzuki coupling with (4-hydroxyphenyl) boronic acid and 8a–g in the presence of Pd(PPh3)4 yielded 9a–g in moderate-to-good yield (42–82%).
Based on compound 8a, we synthesized derivatives including 3-fluoro-4-methoxyaniline at the 4 position of 1,3,5-triazine through amination and Suzuki coupling (Scheme 2). Stepwise amination of cyanuric chloride using 3-fluoro-4-methoxyaniline and ammonium hydroxide yielded 10, and Suzuki coupling with 10 and various boronic acids yielded the condensed compounds (11a–i).

2.2. 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 hA2A and hA2B AR. Compounds 9a–c, 9e, and 9g showed >95% inhibition at hA1 AR, and compounds 9a, 9c, and 9d showed >95% inhibition at hA3 AR. In addition, 9d showed 69% inhibition at hA1 AR whereas it showed 95% inhibition at hA3 AR, showing significant selectivity. Compound 9c showed >95% inhibition at all subtypes except hA2B 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 hA3 AR (Ki = 55.5 nM) and good binding affinity to hA1 AR, with a 2.5-fold hA1 AR:hA3 AR selectivity index. Compound 9b with 3,5-dimethoxyaniline also showed potent and selective binding affinity to hA1 AR (Ki = 69.7 nM). Compound 9c with 3-methoxy-4-chloroaniline showed the best binding affinity to hA1 AR (Ki = 57.9 nM) and moderate binding affinity to hA3 AR (Ki = 661.1 nM). R1 substitution in the aniline appeared to be well tolerated by hA1 AR compared to hA3 AR. The compounds substituted with methoxy in aniline generally showed good binding affinity to hA1 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 hA1 AR:hA3 AR was shown by 9a, we developed a series of 3-fluoro-4-methoxyaniline derivatives 11a–i for dual hA1–hA3 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 hA1 and hA3 AR and low percentage inhibition at hA2A and hA2B AR. Compounds 11c–e substituted with the electron-withdrawing groups OCF3, CF3, 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 hA1 and hA3 AR were determined and are shown in Table 4. Compound 11a with methoxy instead of a hydroxyl group showed improved binding affinity to both hA1 and hA3 AR, about 2-fold at hA1 AR and 5-fold at hA3 AR, with an hA1 AR:hA3 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 hA1 AR slightly improved, indicating an hA1 AR:hA3 AR selectivity index of 1.7.

2.3. cAMP Assay at hA1 and hA3 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 hA1 and hA3 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 hA3 AR in antagonist mode, respectively, and 17% and 6% percentage activation in agonist mode, respectively, indicating that 11a and 11b behave as antagonists at hA3 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 hA1 AR. However, the assay results of 11a at hA1 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 hA1 AR. That is, 11a binds to hA1 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 hA1 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 hA1 AR. However, compound 9a and 11a–b, which bound to hA1 and hA3 AR, exhibited relatively low cell viability, whereas compound 9c, which had the highest binding affinity at hA1 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).

2.5. 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 H2DCF-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.

2.6. Compound 9c-Induced Mitochondrial Membrane Dysfunction

ROS-induced oxidative stress can cause the rapid depolarization of the inner mitochondrial membrane potential (ΔΨm) 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.

2.7. 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.

2.8. Molecular Docking Study of 1,3,5-Triazine Derivatives

We attempted molecular docking to investigate how the triazine derivatives bind to hA1 and hA3 AR. Initially, the binding mode of 11b to hA1 AR was predicted using x-ray structure (PDB; 5N2S). Consequently, two docking poses of 11b in hA1 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 F1715.29 in the two docking poses. By contrast, N2546.5 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 hA1 AR, adopting a purple-binding pose. Docking was also performed on 9a and 9c, which both exhibited strong binding affinity in hA1 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 hA1 AR.
We used the previously published homology model of hA3 AR for docking [27], since the x-ray crystal structure of hA3 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 hA3 AR. The N2506.55 of hA3 AR generated hydrogen bonds with the nitrogen of aniline and triazine of 11b, and its triazine formed a π–π interaction with F1685.29 (Figure 7C). This was consistent with the binding mode in hA1 AR, and the docking study explained how 11b binds to both hA1 and hA3 AR. 9a was predicted to bind in the same manner as 11b on the hA3 AR model, which was generated from the 11b induced-fit docking (Figure 7C).

3. Materials and Methods

3.1. Chemical Synthesis

3.1.1. 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 F254 (Merck, Darmstadt, Germany), with visualization under ultraviolet (UV) light (254 nm).
Proton nuclear magnetic resonance (1H-NMR) spectra of the compounds dissolved in CDCl3, deuterated dimethyl sulfoxide (DMSO-d6), or D2O, were recorded on a Bruker Avance 400 MHz (Bruker Corporation, Billerica, MA, USA). The chemical shifts were expressed as δ-values in parts per million (ppm) using residual solvent peaks (CDCl3: 1H, 7.26 ppm; DMSO: 1H, 2.50 ppm) as a reference. Coupling constants were given in hertz (Hz). The peak patterns are indicated by the following abbreviations: bs = broad singlet, d = doublet, dd = doublet of doublet, m = multiplet, q = quadruplet, s = singlet, and t = triplet. High-resolution spectra were obtained using Waters ACQUITY UPLC® BEH C18 1.7μ-Q-TOF SYNAPT G2-Si (Waters Corporation, Milford, MA, USA) high-resolution mass spectrometry (HRMS). Column chromatography was performed on silica gel 60 (230–400 mesh). Eluent solvents for all chromatographic methods are noted as appropriate mixed solvents with given volume-to-volume ratios.

3.1.2. 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% NH3 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.

3.1.3. 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 Na2SO4, filtered, and concentrated in vacuo. Finally, the resulting residue was purified by column chromatography.

Compound 9a

The 4-{4-amino-6-[(3-fluoro-4-methoxyphenyl)amino]-1,3,5-triazin-2-yl}phenol (9a) yield was 62%: 1H-NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 9.41 (s, 1H), 8.15 (d, J = 8.8 Hz, 2H), 7.87 (d, J = 12.5 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.09 (t, J = 9.4 Hz, 1H), 7.00 (s, 2H), 6.85 (d, J = 8.8 Hz, 2H), 3.80 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 170.01, 166.98, 164.32, 160.61, 150.95 (d, J = 240.5 Hz); 141.87 (d, J = 10.9 Hz); 133.81 (d, J = 9.8 Hz); 129.71, 127.48, 115.44, 114.99, 114.02 (d, J = 2.9 Hz); 108.22 (d, J = 22.9 Hz); 56.26; HRMS (ES+): m/z calculated for C16H14FN5O2: 328.1210 [M + H]+; found 328.1221.

Compound 9b

The 4-{4-amino-6-[(3,5-dimethoxyphenyl)amino]-1,3,5-triazin-2-yl}phenol (9b) yield was 56%: 1H-NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 9.33 (s, 1H), 8.17 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 2.3 Hz, 2H), 7.00 (s, 2H), 6.84 (d, J = 8.7 Hz, 2H), 6.14 (t, J = 2.2 Hz, 1H), 3.74 (s, 6H); 13C-NMR (100 MHz, DMSO-d6) δ 170.02, 166.97, 164.55, 160.66, 160.32, 141.84, 129.70, 127.47, 114.99, 98.05, 94.00, 55.02; HRMS (ES+): m/z calculated for C17H17N5O3: 340.1410 [M + H]+; found 340.1436.

Compound 9c

The 4-{4-amino-6-[(4-chloro-3-methoxyphenyl)amino]-1,3,5-triazin-2-yl}phenol (9c) yield was 55%: 1H-NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H), 9.53 (s, 1H), 8.18 (d, J = 8.8 Hz, 2H), 7.92 (d, J = 2.2 Hz, 1H), 7.44–7.24 (m, 2H), 7.06 (s, 2H), 6.85 (d, J = 8.8 Hz, 2H), 3.88 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 170.13, 166.97, 164.45, 160.71, 154.27, 140.50, 129.78, 129.27, 127.41, 115.04, 113.27, 112.27, 104.51, 55.86; HRMS (ES+): m/z calculated for C16H14ClN5O2: 344.0914 [M + H]+; found 344.0938.

Compound 9d

The 4-(4-amino-6-{[3-(trifluoromethyl)phenyl]amino}-1,3,5-triazin-2-yl)phenol (9d) yield was 82%: 1H-NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 9.75 (s, 1H), 8.35 (s, 1H), 8.18 (d, J = 8.9 Hz, 2H), 8.07 (d, J = 6.6 Hz, 1H), 7.52 (t, J = 7.8 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.08 (s, 2H), 6.85 (d, J = 8.9 Hz, 2H); 13C-NMR (100 MHz, DMSO-d6) δ 170.23, 167.07, 164.58, 160.83, 141.08, 130.03–128.68 (m); 129.56, 127.30, 124.38 (q, J = 272.3 Hz); 123.08, 117.86, 117.82, 115.83, 115.08; HRMS (ES+): m/z calculated for C16H12F3N5O: 348.1072 [M + H]+; found 348.1100.

Compound 9e

The 4-{4-amino-6-[(3,5-dimethylphenyl)amino]-1,3,5-triazin-2-yl}phenol (9e) yield was 69%: 1H-NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 9.22 (s, 1H), 8.16 (d, J = 8.7 Hz, 2H), 7.46 (s, 2H), 6.93 (s, 2H), 6.84 (d, J = 8.8 Hz, 2H), 6.62 (s, 1H), 2.26 (s, 6H); 13C-NMR (100 MHz, DMSO-d6) δ 170.00, 167.06, 164.57, 160.60, 139.96, 137.24, 129.74, 127.59, 123.47, 117.73, 115.01, 21.28; HRMS (ES+): m/z calculated for C17H17N5O: 308.1511 [M + H]+; found 308.1553.

Compound 9f

The 4-(4-amino-6-{[4-(piperidin-1-yl)phenyl]amino}-1,3,5-triazin-2-yl)phenol (9f) yield was 58%: 1H-NMR (400 MHz, DMSO-d6) δ 9.99 (s, 1H), 9.13 (s, 1H), 8.15 (d, J = 8.8 Hz, 2H), 7.60 (d, J = 9.0 Hz, 2H), 7.11–6.49 (m, 6H), 3.22–2.86 (m, 4H), 1.79–1.58 (m, 4H), 1.57–1.42 (m, 2H); 13C-NMR (100 MHz, DMSO-d6) δ 169.87, 167.05, 164.41, 160.49, 147.32, 131.82, 129.70, 127.69, 121.22, 116.33, 114.94, 50.43, 25.44, 23.89; HRMS (ES+): m/z calculated for C20H22N6O: 363.1933 [M + H]+; found 363.1973.

Compound 9g

The 4-{4-amino-6-[(2,4-dimethylphenyl)amino]-1,3,5-triazin-2-yl}phenol (9g) yield was 45%: 1H-NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H), 8.57 (s, 1H), 8.09 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 7.9 Hz, 1H), 7.03 (s, 1H), 6.98 (dd, J = 8.2, 2.3 Hz, 1H), 6.81 (d, J = 8.7 Hz, 2H), 6.75 (s, 2H), 2.27 (s, 3H), 2.18 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 169.88, 167.25, 165.45, 160.40, 134.67, 133.92, 133.05, 130.70, 129.62, 127.66, 126.36, 114.86, 20.50, 18.06; HRMS (ES+): m/z calculated for C17H17N5O: 308.1511 [M + H]+; found 308.1548.

3.1.4. Procedure for the Synthesis of Intermediate 10

A cyanuric chloride (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% NH3 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.

3.1.5. General Procedure for the Synthesis of 11a–i

Intermediate 10 (1 equiv), R2-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 Na2SO4, filtered, and concentrated in vacuo. Finally, the resulting residue was purified by column chromatography.

Compound 11a

The N2-(3-fluoro-4-methoxyphenyl)-6-(4-methoxyphenyl)-1,3,5-triazine-2,4-diamine (compound 11a) yield was 76%: 1H-NMR (400 MHz, CDCl3) δ 8.33 (d, J = 8.9 Hz, 2H), 7.69 (dd, J = 13.2, 2.7 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 7.03 (s, 1H), 7.01–6.87 (m, 3H), 5.23 (s, 2H), 3.89 (d, J = 5.7 Hz, 6H); 13C-NMR (100 MHz, DMSO-d6) δ 169.81, 167.02, 164.33, 162.01, 150.96 (d, J = 240.5 Hz); 141.94 (d, J = 10.9 Hz); 133.73 (d, J = 9.8 Hz); 129.54, 129.05, 115.51, 114.01 (d, J = 2.9 Hz); 113.66, 108.29 (d, J = 22.9 Hz); 56.26, 55.33; HRMS (ES+): m/z calculated for C17H16FN5O2: 342.1361 [M + H]+; found 342.1368.

Compound 11b

The N2-(3-fluoro-4-methoxyphenyl)-6-(4-fluorophenyl)-1,3,5-triazine-2,4-diamine (11b) yield was 81%: 1H-NMR (400 MHz, CDCl3) δ 8.47–8.30 (m, 2H), 7.67 (dd, J = 13.2, 2.7 Hz, 1H), 7.21–7.06 (m, 3H), 7.04 (s, 1H), 6.94 (t, J = 9.0 Hz, 1H), 5.25 (s, 2H), 3.90 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 169.21, 167.06, 164.35, 164.27 (d, J = 248.9 Hz); 150.95 (d, J = 240.9 Hz); 142.10 (d, J = 10.9 Hz); 133.51 (d, J = 9.8 Hz); 133.22 (d, J = 2.9 Hz); 130.17 (d, J = 9.1 Hz); 115.70, 115.31 (d, J = 21.4 Hz); 114.00 (d, J = 2.9 Hz); 108.44 (d, J = 22.9 Hz); 56.25; HRMS (ES+): m/z calculated for C16H13F2N5O: 328.1010 [M − H]−; found 328.1013.

Compound 11c

The N2-(3-fluoro-4-methoxyphenyl)-6-[4-(trifluoromethoxy)phenyl]-1,3,5-triazine-2, 4-diamine (11c) yield was 84%: 1H-NMR (400 MHz, CDCl3) δ 8.40 (d, J = 8.9 Hz, 2H), 7.67 (dd, J = 13.2, 2.6 Hz, 1H), 7.30 (d, J = 7.9 Hz, 2H), 7.13 (d, J = 8.6 Hz, 1H), 7.01 (s, 1H), 6.95 (t, J = 9.1 Hz, 1H), 5.25 (s, 2H), 3.90 (s, 3H); 13C-NMR (100 MHz, CDCl3) δ 167.37, 164.89, 152.25 (d, J = 244.9 Hz); 152.06, 144.15 (d, J = 10.6 Hz); 134.81 (d, J = 4.9 Hz); 131.87 (d, J = 10.6 Hz); 131.32–118.83 (m); 130.30, 120.51, 116.30 (d, J = 3.6 Hz); 113.88 (d, J = 2.9 Hz); 110.30, 110.07, 56.82; HRMS (ES+): m/z calculated for C17H13F4N5O2: 394.0927 [M − H]−; found 394.0929.

Compound 11d

The N2-(3-fluoro-4-methoxyphenyl)-6-[4-(trifluoromethyl)phenyl]-1,3,5-triazine-2,4-diamine (11d) yield was 48%: 1H-NMR (400 MHz, chloroform-d) δ 8.50 (d, J = 8.1 Hz, 2H), 7.74 (d, J = 8.3 Hz, 2H), 7.66 (dd, J = 13.0, 2.5 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 6.96 (t, J = 9.0 Hz, 1H), 5.47 (s, 2H), 3.91 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 168.94, 167.11, 164.35, 150.94 (d, J = 241.0 Hz); 142.21 (d, J = 11.0 Hz); 140.68, 133.34 (d, J = 9.5 Hz); 131.17 (d, J = 32.3 Hz); 128.42, 125.36 (d, J = 3.9 Hz); 124.15 (d, J = 272.2 Hz); 115.78, 114.00 (d, J = 2.9 Hz); 108.51 (d, J = 22.4 Hz); 56.25; HRMS (ES+): m/z calculated for C17H13F4N5O: 378.0978 [M − H]−; found 378.0970.

Compound 11e

The 4-{4-amino-6-[(3-fluoro-4-methoxyphenyl)amino]-1,3,5-triazin-2-yl}benzonitrile (11e) yield was 85%: 1H-NMR (400 MHz, DMSO-d6) δ 9.67 (s, 1H), 8.41 (d, J = 8.8 Hz, 2H), 8.00 (d, J = 8.7 Hz, 2H), 7.88 (s, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.32 (s, 2H), 7.11 (t, J = 9.4 Hz, 1H), 3.81 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 168.75, 167.08, 164.33, 150.93 (d, J = 240.9 Hz); 142.25 (d, J = 10.9 Hz); 141.08, 133.25 (d, J = 9.8 Hz); 132.46, 128.32, 118.60, 115.83, 113.98 (d, J = 2.9 Hz); 113.54, 108.56 (d, J = 22.5 Hz); 56.24; HRMS (ES+): m/z calculated for C17H13FN6O: 335.1057 [M − H]−; found 335.1051.

Compound 11f

The methyl-4-{4-amino-6-[(3-fluoro-4-methoxyphenyl)amino]-1,3,5-triazin-2-yl}benzoate (11f) yield was 71%: 1H-NMR (400 MHz, chloroform-d) δ 8.42 (d, J = 8.8 Hz, 2H), 8.13 (d, J = 8.8 Hz, 2H), 7.68 (dd, J = 13.1, 2.6 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.04 (s, 1H), 6.95 (t, J = 9.0 Hz, 1H), 5.27 (s, 2H), 3.95 (s, 3H), 3.90 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 169.27, 167.11, 165.91, 164.33, 150.94 (d, J = 241.0 Hz); 142.15 (d, J = 10.6 Hz); 141.11, 133.40 (d, J = 9.5 Hz); 131.87, 129.22, 127.97, 115.71, 114.01 (d, J = 2.9 Hz); 108.44 (d, J = 22.7 Hz); 56.25, 52.29; HRMS (ES+): m/z calculated for C18H16FN5O3: 368.1159 [M − H]−; found 368.1145.

Compound 11g

The ethyl-4-{4-amino-6-[(3-fluoro-4-methoxyphenyl)amino]-1,3,5-triazin-2-yl}benzoate (11g) yield was 86%: 1H-NMR (400 MHz, chloroform-d) δ 8.41 (d, J = 8.8 Hz, 2H), 8.14 (d, J = 8.8 Hz, 2H), 7.67 (dd, J = 13.1, 2.6 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 7.10 (s, 1H), 6.95 (t, J = 9.0 Hz, 1H), 5.33 (s, 2H), 4.41 (q, J = 7.1 Hz, 2H), 3.90 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 169.31, 167.12, 165.43, 164.35, 150.95 (d, J = 241.2 Hz); 142.16 (d, J = 10.9 Hz); 141.07, 133.42 (d, J = 9.4 Hz); 132.16, 129.18, 127.96, 115.73, 114.00 (d, J = 2.9 Hz); 108.44 (d, J = 22.5 Hz); 60.98, 56.25, 14.17; HRMS (ES+): m/z calculated for C19H18FN5O3: 384.1466 [M + H]+; found 384.1468.

Compound 11h

The 4-{4-amino-6-[(3-fluoro-4-methoxyphenyl)amino]-1,3,5-triazin-2-yl}-2,6-difluorophenol (11h) yield was 68%: 1H-NMR (400 MHz, DMSO-d6) δ 10.91 (s, 1H), 9.52 (s, 1H), 8.38–7.68 (m, 3H), 7.42 (d, J = 8.2 Hz, 1H), 7.29–6.99 (m, 3H), 3.81 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 168.14, 166.98, 164.23, 151.91 (dd, J = 241.2, 6.9 Hz); 150.93 (d, J = 240.9 Hz); 142.13 (d, J = 10.9 Hz); 136.94 (t, J = 15.4 Hz); 133.42 (d, J = 9.4 Hz); 127.19 (t, J = 7.7 Hz); 115.70, 114.01 (d, J = 2.9 Hz); 111.01 (dd, J = 14.9, 7.6 Hz); 108.48 (d, J = 19.6 Hz); 56.25; HRMS (ES+): m/z calculated for C16H12F3N5O2: 362.0865 [[M − H]−; found 362.0877.

Compound 11i

The 6-(4-fluoro-3-nitrophenyl)-N2-(3-fluoro-4-methoxyphenyl)-1,3,5-triazine-2,4-diamine (11i) yield was 66%: 1H-NMR (400 MHz, DMSO-d6) δ 9.72 (s, 1H), 9.01 (dd, J = 7.6, 2.0 Hz, 1H), 8.74–8.52 (m, 1H), 7.91 (s, 1H), 7.76 (dd, J = 11.2, 8.7 Hz, 1H), 7.47–7.31 (m, 3H), 7.11 (t, J = 9.4 Hz, 1H), 3.81 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) δ 167.38, 167.04, 164.22, 156.59 (d, J = 266.3 Hz); 150.94 (d, J = 241.2 Hz); 142.30 (d, J = 11.3 Hz); 136.79 (d, J = 7.6 Hz); 135.10 (d, J = 9.8 Hz); 133.83 (d, J = 3.6 Hz); 133.21 (d, J = 10.2 Hz); 125.47, 118.96 (d, J = 21.4 Hz); 115.82, 113.98 (d, J = 2.9 Hz); 108.60 (d, J = 20.3 Hz); 56.26; HRMS (ES+): m/z calculated for C16H12F2N6O3: 373.0861 [M − H]−; found 373.0866.

3.2. Biological Evaluation

3.2.1. Binding Assay at Human Adenosine Receptors

Binding Assay at hA1, hA2A, and hA3 AR

hA1, hA2A, and hA3 AR competition binding experiments were carried out in a multiscreen GF/C 96-well plate (Millipore, Madrid, Spain) pretreated with binding buffer (Hepes 20 mM, NaCl 100 mM, MgCl2 10 mM, 2 U/mL adenosine deaminase, pH = 7.4 for hA1 AR; Tris-HCl 50 mM, EDTA 1 mM, MgCl2 10 mM, 2 U/mL adenosine deaminase, pH = 7.4 for hA2A AR; and Tris-HCl 50 mM, EDTA 1 mM, MgCl2 5 mM, 2 U/mL adenosine deaminase, pH = 7.4 for hA3 AR, respectively). In each well was incubated 5 µg of membranes from Euroscreen CHO-A1 cell line, 5µg of membranes from Hela-A2A cell line, or 30µg of membranes from Hela-A3 cell line and prepared in laboratory: 1 nM [3H]-DPCPX (140 Ci/mmol, 1 mCi/Ml, Perkin Elmer NET974001MC) for hA1 AR; 1 nM [3H]-ZM241385 (50 Ci/mmol, 1 mCi/mL, ARC-ITISA 0884) for hA2A AR; and 10 nM [3H]-NECA (29.6 Ci/mmol, 1 mCi/mL, Perkin Elmer NET811250UC) for hA3 AR, respectively; and the compounds studied according to standard protocol. Non-specific binding was determined in the presence of R-PIA 10 µM (Sigma P4532) for hA1 AR; of NECA 50 µM (Sigma E2387) for hA2A AR; and of R-PIA 100 µM (Sigma P4532) for hA3 AR, respectively. The reaction mixture (Vt: 200 µL/well) was incubated at 25 °C for 60 min (hA1 AR), 30min (hA2A AR), or 180min (hA3 AR); whereafter it was filtered and washed four times (hA1 and hA2A AR) or six times (hA3 AR) with 250 μL wash buffer (Hepes 20 mM, NaCl 100 mM, MgCl2 10 mM pH = 7.4, for hA1 AR; Tris-HCl 50 mM, EDTA 1 mM, MgCl2 10 mM, pH = 7.4, for hA2A AR; and Tris-HCl 50mM pH = 7.4, for hA3 AR, respectively) before being measured in a microplate beta scintillation counter (MicrobetaTrilux, PerkinElmer, Madrid, Spain).

Binding Assay at hA2B AR

hA2B AR competition binding experiments were carried out in a multiscreen GF/C 96-well plate. In each well was incubated 25 µg of membranes from Euroscreen HEK-A2B cell line and prepared in laboratory, 25 nM [3H]-DPCPX (140 Ci/mmol, 1 mCi/mL, Perkin Elmer NET974001MC) and compounds studied according to standard protocol. Non-specific binding was determined in the presence of NECA 1000 µM (Sigma E2397). The reaction mixture (Vt: 250 µL/well) was incubated at 25°C for 30 min, 200 µL was transferred to GF/C 96-well plate (Millipore, Madrid, Spain), and pretreated with binding buffer (Tris-HCl 50 Mm, EDTA 1 mM, MgCl2 5 mM, Bacitracin 100 µg/µL, adenosine deaminase 2 U/Ml, pH = 6.5). It was then filtered and washed four times with 250 μL wash buffer (Tris-HCl 50 mM, EDTA 1 mM, MgCl2 5 mM, pH = 6.5), before being measured in a microplate beta scintillation counter (MicrobetaTrilux, PerkinElmer, Madrid, Spain).

3.2.2. cAMP Accumulation Assay

Antagonist Mode at hA1 or hA3 AR

hA1 and hA3 AR functional experiments were carried out in CHO-A1 and CHO-A3#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 (Nutrient Mixture F12 Ham’s (Sigma N6658) for hA1 AR; Dulbecco’s modified eagle’s medium nutrient mixture F-12 ham (Sigma D8062) for hA3 AR; 25 mM Hepes; pH = 7.4). Wash buffer was replaced by incubation buffer (Mixture F12 Ham’s (Sigma N6658) for hA1 AR; Dulbecco’s modified eagle’s medium nutrient mixture F-12 ham (Sigma D8062) for hA3 AR; 25 mM Hepes, 20 µM Rolipram (Sigma R6520); pH = 7.4). Test compounds and XAC (Sigma X103) or MRS1220 (Sigma M228) as reference compound for hA1 and hA3 AR, respectively, were added and incubated at 37 °C for 15 min. Afterward, 0.1 µM of 5′-(N-Ethilcarboxamido) adenosine (NECA) (Sigma E2387) was 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).

Agonist Mode at hA1 or hA3 AR

hA1 and hA3 AR functional experiments were carried out in CHO-A1 and CHO-A3#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 hA1 AR; Dulbecco’s modified eagle’s medium nutrient mixture F-12 ham (Sigma D8062) for hA3 AR; 25mM Hepes; pH = 7.4). Wash buffer was replaced by incubation buffer (Mixture F12 Ham’s (Sigma N6658) for hA1 AR; Dulbecco’s modified eagle’s medium nutrient mixture F-12 ham (Sigma D8062) for hA3 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).

3.2.3. 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% CO2. When the cell density reached 90%, subcultures were generated using a trypsin-EDTA solution.

3.2.4. Cell Viability Assay

A549 and NCI-H1299 cells were seeded in 96-well plates at a density of 1 × 105 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).

3.2.5. Microscopy

A549 cells were seeded in 6-well plates at a density of 1 × 106 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).

3.2.6. Mitochondrial Membrane Potential (MMP) and Reactive Oxygen Species (ROS) Assay

A549 cells were incubated with a fluorescent indicator, specifically 100 nM tetramethylrhodamine methyl ester perchlorate (TMRM, Thermo Fisher Scientific, Waltham, MA, USA), to determine the mitochondrial membrane potential. The A549 cells treated with the compound were harvested after 48 h, washed in PBS, and re-suspended in FACS buffer (PBS supplemented with 2 percent fetal bovine serum). A flow cytometer (BD FACSVerse, BD Biosciences, San Jose, CA, USA) and the FlowJo software (FlowJo LLC, Ashland, OR, USA) were used to analyze the cells. For the reactive oxygen species measurements, we began by seeding A549 cells in 6-well plates up to a density of 1 × 106 cells in each well, then we treated the cells with the compound (20 and 40 μM) for 48 h. 2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (DCFDA) or MitoSOX-red mitochondrial superoxide indicator (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect intracellular and mitochondrial ROS levels. DCFDA (1 μM) or MitoSOX (5 µM) was added to the cells and then incubated at room temperature for 20 min. PBS was then used to wash the cells, and the cells were subsequently re-suspended in FACS buffer (PBS supplemented with 1% FBS). Intracellular fluorescence measurements involved the use of a flow cytometer (BD FACSVerse, BD Biosciences, San Jose, CA, USA) and the FlowJo software (Version 10, TreeStar, Ashland, OR, USA).

3.2.7. Live–Dead Assay

The A549 cells were seeded in 6-well plates at a density of 1 × 106 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).

3.2.8. Statistical Analysis

GraphPad Prism (GraphPad Software, Inc., version 7, San Diego, CA, USA) was used for statistical analysis, and the results were presented as means ± SEM. The Student’s t-test was used to further analyze the data. The resulting p-values were considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p ≤ 0.0001).

3.3. Molecular Modeling

Schrödinger Maestro, version 13.1 (Release 2021-2, Schrödinger, LLC, New York, NY, USA) was used to perform the molecular docking of 1,3,5-triazine derivatives [32]. 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 hA1 AR co-crystallized with PSB36 (PDB ID: 5N2S) was acquired from the Protein Data Bank (PDB). The homology model of hA3 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 hA1 AR and the Glide induced fit docking module [38,39,40] for hA3 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 hA1 and hA3 AR.

4. 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 hA1 and hA3 AR (Ki =98.3 and 56.6 nM, respectively; selectivity index = 1.74). 11b was found to be a hA1 and hA3 AR dual antagonist in cAMP accumulation assays at hA1 and hA3 AR. Compound 9c showed the highest binding affinity to hA1 AR (Ki = 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 hA1 and hA3 AR. The 1,3,5-triazine derivatives were predicted to bind to both hA1 and hA3 AR. We demonstrated that 1,3,5-triazine derivatives have the potential to be developed as hA1 and hA3 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 hA1 or hA3 AR, or to both hA1 and hA3 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: 1H and 13C NMR Copies of 9a–g and 11a–i; Figure S2. HR-MS Copies of 9a–g, and 11a–i; Figure S3: Molecular Docking of 9a, 11b, 11h and 11i in hA1 AR.

Author Contributions

Conceptualization, J.Y.; data curation, Y.L., S.P. and Y.A.; investigation, H.M.Y., S.P., Y.A., Y.K. and C.H.; methodology, S.P., Y.A. and Y.K.; supervision, H.M.Y., J.Y. and E.J.R.; writing—original draft, H.M.Y., J.Y. and S.P.; writing—review & editing, H.M.Y. and J.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Ministry of Science and ICT (MSIT), National Research Foundation of Korea (NRF-2022R1C1C1004804 and 2020R1A4A4079494) and the Korea Institute of Science and Technology (KIST) to J.Y. In addition, this work was also supported by the “Establishment of measurement standards for Chemistry and Radiation”, grant number KRISS-2022-GP2022-0003 funded by the Korea Research Institute of Standards and Science and Ministry of Science and ICT (MSIT), National Research Foundation of Korea (NRF-2021M3C1C3097638) to H.M.Y.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

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.

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Molecules 27 04016 g001 550
Figure 1. The structure of dual hA1–hA3 AR antagonists. AR: adenosine receptor.
Figure 1. The structure of dual hA1–hA3 AR antagonists. AR: adenosine receptor.
Molecules 27 04016 g001
Molecules 27 04016 g002 550
Figure 2. 1,3,5-Triazine derivatives as hAR ligands. AR: adenosine receptor.
Figure 2. 1,3,5-Triazine derivatives as hAR ligands. AR: adenosine receptor.
Molecules 27 04016 g002
Molecules 27 04016 sch001 550
Scheme 1. Synthesis of 2-(4-hydroxyphenyl)-4-amino-1,3,5-triazine derivatives. THF: tetrahydrofuran.
Scheme 1. Synthesis of 2-(4-hydroxyphenyl)-4-amino-1,3,5-triazine derivatives. THF: tetrahydrofuran.
Molecules 27 04016 sch001
Molecules 27 04016 sch002 550
Scheme 2. Synthesis of 6-(3-fluoro-4-methoxyaniline)-4-amino-1,3,5-triazine derivatives. THF: tetrahydrofuran.
Scheme 2. Synthesis of 6-(3-fluoro-4-methoxyaniline)-4-amino-1,3,5-triazine derivatives. THF: tetrahydrofuran.
Molecules 27 04016 sch002
Molecules 27 04016 g003 550
Figure 3. Compound 9c decreases cell viability in lung cancer cell lines. (A) Cell viability as measured through MTS assays in which A549 and NCI-H1299 cells were exposed to various concentrations of the compounds. (B) Cell morphologies were taken after 48 h of treatment with 9c (scale bar: 750 μm).
Figure 3. Compound 9c decreases cell viability in lung cancer cell lines. (A) Cell viability as measured through MTS assays in which A549 and NCI-H1299 cells were exposed to various concentrations of the compounds. (B) Cell morphologies were taken after 48 h of treatment with 9c (scale bar: 750 μm).
Molecules 27 04016 g003
Molecules 27 04016 g004 550
Figure 4. Compound 9c-induced reactive oxygen species (ROS) generation in A549 cell lines. (A) Changes in ROS levels following treatment with 20 μM and 40 μM 9c, respectively, for 48 h as measured using H2DCFDA and a flow cytometer. (B) Quantification of ROS levels. (C) A549 cells were treated with 9c and mitochondrial ROS was indicated by MitoSOX Red. Flow cytometry was employed to analyze the A549 cells. (D) Quantification of mitochondrial ROS levels. Values indicate means ± SEM. (n = 3, ** p ≤ 0.01, **** p ≤ 0.0001).
Figure 4. Compound 9c-induced reactive oxygen species (ROS) generation in A549 cell lines. (A) Changes in ROS levels following treatment with 20 μM and 40 μM 9c, respectively, for 48 h as measured using H2DCFDA and a flow cytometer. (B) Quantification of ROS levels. (C) A549 cells were treated with 9c and mitochondrial ROS was indicated by MitoSOX Red. Flow cytometry was employed to analyze the A549 cells. (D) Quantification of mitochondrial ROS levels. Values indicate means ± SEM. (n = 3, ** p ≤ 0.01, **** p ≤ 0.0001).
Molecules 27 04016 g004
Molecules 27 04016 g005 550
Figure 5. 9c decreases mitochondrial membrane potential in the A549 cell line. (A) Effects of treatment with 20 μM and 40 μM 9c for 48 h on the mitochondrial membrane potential of A549 cells as measured using the TMRM reagent (100 nM) and a flow cytometer. (B) Quantification of mitochondrial membrane potential. Values indicate the means ± SEM. (n = 3, *** p ≤ 0.001).
Figure 5. 9c decreases mitochondrial membrane potential in the A549 cell line. (A) Effects of treatment with 20 μM and 40 μM 9c for 48 h on the mitochondrial membrane potential of A549 cells as measured using the TMRM reagent (100 nM) and a flow cytometer. (B) Quantification of mitochondrial membrane potential. Values indicate the means ± SEM. (n = 3, *** p ≤ 0.001).
Molecules 27 04016 g005
Molecules 27 04016 g006 550
Figure 6. Compound 9c-induced cytotoxic effects on lung cancer cell lines. (A) Live and dead assay performed using a flow cytometer following 9c treatment of A549 cells. (B) Quantification of dead cells. Values indicate means ± SEM. (n = 3, **** p ≤ 0.0001).
Figure 6. Compound 9c-induced cytotoxic effects on lung cancer cell lines. (A) Live and dead assay performed using a flow cytometer following 9c treatment of A549 cells. (B) Quantification of dead cells. Values indicate means ± SEM. (n = 3, **** p ≤ 0.0001).
Molecules 27 04016 g006
Molecules 27 04016 g007 550
Figure 7. (A) Superimposition of two predicted docking poses in hA1 AR of 11b. (B) Superimposition of docking poses in hA1 AR of 11b (red carbon atom), 11h (salmon pink carbon atom), and 11i (yellow-green carbon atom). The narrow binding pocket for para-phenol ring of 11b is highlighted with a red circle. (C) Superimposition of docking poses in hA3 AR of 9a (blue carbon atom) and 11b (green carbon atom). Hydrogen-bonding and π–π stacking interaction are pictured as yellow and cyan dashed lines, respectively.
Figure 7. (A) Superimposition of two predicted docking poses in hA1 AR of 11b. (B) Superimposition of docking poses in hA1 AR of 11b (red carbon atom), 11h (salmon pink carbon atom), and 11i (yellow-green carbon atom). The narrow binding pocket for para-phenol ring of 11b is highlighted with a red circle. (C) Superimposition of docking poses in hA3 AR of 9a (blue carbon atom) and 11b (green carbon atom). Hydrogen-bonding and π–π stacking interaction are pictured as yellow and cyan dashed lines, respectively.
Molecules 27 04016 g007
Table
Table 1. Percentage inhibition of 2-(p-phenol)-4-amino-1,3,5-triazine derivatives 9a–g at hA1, hA2A, hA2B, and hA3 ARs.
Table 1. Percentage inhibition of 2-(p-phenol)-4-amino-1,3,5-triazine derivatives 9a–g at hA1, hA2A, hA2B, and hA3 ARs.
Molecules 27 04016 i001
CompoundR1Percentage Inhibition (%) at 10 μM a
hA1 ARhA2A ARhA2B ARhA3 AR
9a3-F-4-OCH399 ± 158 ± 172 ± 297 ± 1
9b3,5-(OCH3)299 ± 184 ± 462 ± 293 ± 1
9c3-OCH3-4-Cl99 ± 194 ± 390 ± 195 ± 1
9d3-CF369 ± 240 ± 574 ± 195 ± 1
9e3,5-(CH3)296 ± 358 ± 342 ± 292 ± 1
9f4-N-piperidine28 ± 128 ± 344 ± 438 ± 1
9g2,4-(CH3)299 ± 180 ± 447 ± 291 ± 1
a All binding experiments were performed using adherent mammalian cells stably transfected with cDNA encoding the appropriate hAR (hA1 and hA3 AR in CHO cells, hA2A AR in Hela cells, and hA2B AR in HEK-293 cells). Binding was carried out using 2 nM [3H]DPCPX, 3 nM [3H]ZM241385, 25 nM [3H]DPCPX, and 0.5 nM [3H]NECA as radioligands for hA1, hA2A, hA2B, and hA3 AR, respectively. Values are expressed as the percentage inhibition of specific radioligand binding at 10 μM, with nonspecific binding defined using 10 μM NECA. AR: adenosine receptor; cDNA: complementary DNA; CHO: Chinese hamster ovary; DPCPX: 8-cyclopentyl-1,3-dipropylxanthine; hAR: human adenosine receptor; HEK: human embryonic kidney; NECA: 5-N-ethylcarboxamido adenosine.
Table
Table 2. Binding affinity to hA1 and hA3 ARs.
Table 2. Binding affinity to hA1 and hA3 ARs.
CompoundR1Ki(nM) aSelectivity
(hA1 AR:hA3 AR)
hA1 ARhA3 AR
9a3-F-4-OCH3139.355.52.51
9b3,5-(OCH3)269.7ND-
9c3-OCH3-4-Cl57.9661.10.0875
9d3-CF3ND1258-
9e3,5-(CH3)2872.1ND-
9f4-N-piperidineNDND-
9g2,4-(CH3)2116.5ND-
a All binding experiments were performed using adherent mammalian cells stably transfected with cDNA encoding the appropriate hAR (hA1 and hA3 in CHO cells). Binding was carried out using 2 nM [3H]DPCPX and 0.5 nM [3H]NECA as radioligands for hA1 and hA3 AR, respectively. AR: adenosine receptor; cDNA: complementary DNA; CHO: Chinese hamster ovary; DPCPX: 8-cyclopentyl-1,3-dipropylxanthine; hAR: human adenosine receptor; ND: not determined; NECA: 5-N-ethylcarboxamido adenosine.
Table
Table 3. Percentage inhibition of 6-(3-fluoro-4-methoxyaniline)-4-amino-1,3,5-triazine derivatives 11a–i at hA1, hA2A, hA2B, and hA3 ARs.
Table 3. Percentage inhibition of 6-(3-fluoro-4-methoxyaniline)-4-amino-1,3,5-triazine derivatives 11a–i at hA1, hA2A, hA2B, and hA3 ARs.
Molecules 27 04016 i002
CompoundR2Percentage Inhibition (%) at 10 μM a
hA1 ARhA2A ARhA2B ARhA3 AR
9a4-OH99 ± 156 ± 366 ± 397 ± 1
11a4-OCH395 ± 183 ± 165 ± 297 ± 1
11b4-F98 ± 149 ± 156 ± 195 ± 4
11c4-OCF348 ± 481 ± 127 ± 660 ± 1
11d4-CF374 ± 130 ± 193 ± 379 ± 1
11e4-CN45 ± 550 ± 252 ± 172 ± 3
11f4-C(O)OCH352 ± 135 ± 655 ± 285 ± 1
11g4-C(O)OCH2CH356 ± 148 ± 635 ± 278 ± 3
11h3,5-diF-4-OH88 ± 126 ± 566 ± 679 ± 1
11i3-NO2-4-F53 ± 157 ± 272 ± 683 ± 1
a All binding experiments were performed using adherent mammalian cells stably transfected with cDNA encoding the appropriate hAR (hA1 and hA3 in CHO cells, hA2A in Hela cells, and hA2B in HEK-293 cells). Binding was carried out using 2 nM [3H]DPCPX, 3 nM [3H]ZM241385, 25 nM [3H]DPCPX, and 0.5 nM [3H]NECA as radioligands for hA1, hA2A, hA2B, and hA3 AR, respectively. Values are expressed as the percentage inhibition of a specific radioligand binding at 10 μM, with nonspecific binding defined using 10 μM NECA. AR: adenosine receptor; cDNA: complementary DNA; CHO: Chinese hamster ovary; DPCPX: 8-cyclopentyl-1,3-dipropylxanthine; hAR: human adenosine receptor; HEK: human embryonic kidney; NECA: 5-N-ethylcarboxamido adenosine.
Table
Table 4. Binding affinity to hA1 and hA3 ARs.
Table 4. Binding affinity to hA1 and hA3 ARs.
CompoundR2Ki (nM) aSelectivity
(hA1 AR:hA3 AR)
hA1 ARhA3 AR
9a4-OH139.3 55.5 2.51
11a4-OCH378.1 13.3 5.87
11b4-F98.3 56.6 1.74
a All binding experiments were performed using adherent mammalian cells stably transfected with cDNA encoding the appropriate hAR (hA1 and hA3 in CHO cells). Binding was carried out using 2 nM [3H]DPCPX and 0.5 nM [3H]NECA as radioligands for hA1 and hA3 AR, respectively. AR: adenosine receptor; cDNA: complementary DNA; CHO: Chinese hamster ovary; DPCPX: 8-cyclopentyl-1,3-dipropylxanthine; hAR: human adenosine receptor; NECA: 5-N-ethylcarboxamido adenosine.
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Park, S.; Ahn, Y.; Kim, Y.; Roh, E.J.; Lee, Y.; Han, C.; Yoo, H.M.; Yu, J. Design, Synthesis and Biological Evaluation of 1,3,5-Triazine Derivatives Targeting hA1 and hA3 Adenosine Receptor. Molecules 2022, 27, 4016. https://doi.org/10.3390/molecules27134016

AMA Style

Park S, Ahn Y, Kim Y, Roh EJ, Lee Y, Han C, Yoo HM, Yu J. Design, Synthesis and Biological Evaluation of 1,3,5-Triazine Derivatives Targeting hA1 and hA3 Adenosine Receptor. Molecules. 2022; 27(13):4016. https://doi.org/10.3390/molecules27134016

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Park, Sujin, Yujin Ahn, Yongchan Kim, Eun Joo Roh, Yoonji Lee, Chaebin Han, Hee Min Yoo, and Jinha Yu. 2022. "Design, Synthesis and Biological Evaluation of 1,3,5-Triazine Derivatives Targeting hA1 and hA3 Adenosine Receptor" Molecules 27, no. 13: 4016. https://doi.org/10.3390/molecules27134016

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