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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 17  |  Issue : 5  |  Page : 482-492

Design and synthesis of some novel triazine-tyrosine hybrids as potential agents for the treatment of multiple sclerosis


1 Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R. Iran
2 Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences; Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan, I.R. Iran
3 Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences; Department of Pharmacology and Toxicology, Isfahan, I.R. Iran

Date of Submission12-Feb-2022
Date of Decision08-May-2022
Date of Acceptance09-Jul-2022
Date of Web Publication08-Sep-2022

Correspondence Address:
Ghadam Ali Khodarahmi
Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R. Iran
I.R. Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1735-5362.355208

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  Abstract 

Background and purpose: One of the most noteworthy methods to slow down multiple sclerosis (MS) progress is a decrease of lymphocyte cells via S1P1 receptor modulating. Here, a series of S1P1 receptor modulators were designed and investigated for their ability to decrease lymphocytes in a rat model.
Experimental approach: Molecular docking was performed to compare the binding mode of desired compounds 5a-f with fingolimod to the active site of the S1P1 receptor, theoretically. To prepare desired compounds, 5a-f, cyanuric chloride was reacted with different amines, a-f, which then converted to 4a-f compounds through reaction with N-boc-Tyr-OMe ester. Finally, deprotection of the carboxyl and amino groups was carried out to obtain 5a-f as final products. Lymphocyte counting in the rat model was carried out using flow cytometry to evaluate the efficacy of the suggested compounds.
Findings / Results: All compounds exhibited lower binding energy than fingolimod. Compound 5e with ΔG= -8.10 kcal/mol was the best compound. The structure of the compounds was confirmed spectroscopically. The in vivo study proved that compounds 5b and 5a decreased the lymphocytes level at 0.3 and 3 mg/kg, respectively.
Conclusion and implications: The desired compounds were well fitted in the receptor active site following molecular docking studies. The results of lymphocyte count revealed that compounds 5a and 5b with propyl and ethyl substitutes showed the maximum activity in vivo. Finally, the results of the present project can be used for forthcoming investigations towards the design and synthesis of novel potential agents for MS treatment.

Keywords: Lymphocyte counts; Molecular docking; Multiple sclerosis; S1P1R modulator


How to cite this article:
Saeidi S, Asadi P, Hassanzadeh F, Aliomrani M, Khodarahmi G. Design and synthesis of some novel triazine-tyrosine hybrids as potential agents for the treatment of multiple sclerosis. Res Pharma Sci 2022;17:482-92

How to cite this URL:
Saeidi S, Asadi P, Hassanzadeh F, Aliomrani M, Khodarahmi G. Design and synthesis of some novel triazine-tyrosine hybrids as potential agents for the treatment of multiple sclerosis. Res Pharma Sci [serial online] 2022 [cited 2022 Sep 25];17:482-92. Available from: https://www.rpsjournal.net/text.asp?2022/17/5/482/355208


  Introduction Top


Multiple sclerosis (MS), the most common immune-mediated disorder [1], is an unpredictable, chronic autoimmune, inflammatory neurological disorder affecting the central nervous system [2],[3]. MS interrupts the communication between the brain and body by attacking the myelin covering nerve fibers [4]. It is supposed that environmental and genetic factors are contributors to the risk of MS; however, its etiology is still unidentified.

Sphingosine-1-phosphate (S1P), a signaling lipid, has significant regulatory roles in the body, such as proliferation, survival and migration of cells, inflammation, vascular permeability, and immune response through five subfamilies of the receptors [5],[6],[7],[8],[9]. S1P1, as a first subtype of the S1P receptor (S1PR), has a key role in lymphocyte trafficking regulation, which makes it a therapeutic target for the treatment of MS.

This subtype (S1P1) is expressed on the surfaces of different cells like neural cells, lymphocytes, and endothelial cells [10],[11].

Fingolimod (FTY720), a compound that interacts with S1PR, was approved in 2010 for the treatment of MS [12]. Fingolimod slows down the progression of disability and prevents episodes of signs of relapsing-remitting MS. Fingolimod and sphingosine are structurally analogous, but in vivo phosphorylation must happen for activation of fingolimod to produce fingolimod-phosphate (fingolimod-P) [13]. Then fingolimod-P, as an S1PR modulator, competes with S1P for the receptor and exerts its therapeutic effects [14]. However, hypertension, headache, bradycardia, and atrioventricular block are the most common side effects of fingolimod [11].

According to the previous studies, all S1P1 modulators have three main parts including [1] a polar head group, [2] a linker which commonly consists of a heterocycle or aromatic ring, and [3] a nonpolar chain located in the tail of the molecules Scheme 1 [15],[16],[17],[18]. The structure-activity relationship (SAR) studies indicate that minor changes to the linker core could lead to selective, potent, and novel new S1P1 modulators [17]. For example, the study by Chen et al. showed that different isoforms of oxadiazole ring as a core could demonstrate a 10-fold increase in S1P1 activation than fingolimod [18].

Considering the perivenous studies [15],[16],[17],[18], our aim was the development of structurally novel S1P1 modulators based on the s-triazine ring and tyrosine structure with potential use as a therapeutic agent against MS. To this end, a series of compounds containing tyrosine as a polar head group, triazine moiety as a linker, and aliphatic amine as a lipophilic tail have been designed Scheme 1. Subsequently, using the crystallographic structure of hS1P1R, docking studies have been performed to identify possible interactions of derivatives with this receptor, and these compounds were then synthesized. After confirming the purity and identity of the synthesized compounds, the efficacy of the desired compounds was evaluated throughlymphocyte counting in a rat model using flow cytometry.




  Materials and methods Top


All chemicals and reagents were obtained commercially and used without any purification. Reactions were followed by thin-layer chromatography (TLC Silica gel 60 F254 pre-coated plates, E. Merck, Germany), and spots were detected by CAMAG UV Cabinet 4 instrument at λ 254 nm. Melting points were measured by an electrothermal melting point apparatus (Electrothermal 9200, USA). Infrared (IR) spectra were recorded on a Rey light WQF-510 IR/Perkin-Elmer1420 Ratio Recording Infrared instrument (USA). The compounds were dissolved in deuterated dimethyl sulfoxide (DMSO-d6) for proton nuclear magnetic resonance (1H NMR), and the NMR apparatus was Bruker-Ultrashield 400 MHz (USA). Chemical shifts are reported as δ (parts per million, ppm) from tetramethylsilane (TMS) as the internal standard.

In silico procedure

The molecular docking study of six designed compounds 5a-5f with an S1P1 binding pocket was evaluated by employing protein-ligand docking [19]. The RCSB Protein Data Bank (https://www.rcsb.org/) was used to obtain the crystallography file of the S1P1 protein (PDB ID: 5A86). Co-crystal ligand (fingolimod) and all of the irrelevant compartments were deleted using Discovery Studio Visualizer 4.5 (BIOVIA, San Diego, CA, USA) [20]. By using AutoDockTools 1.5.6 (ADT), polar hydrogen and Kollman charges were inserted and a PDBQT file of the protein was generated. The structure of the ligands (5a-5f) was drawn and optimized by PM3 semi-empirical force field using Hyperchem software (Version 8Hyperchem, Hypercube, Inc., and Auto Desk, Inc. [21]. For the preparation of ligands (5a-5f) for docking, Gasteiger charges and polar hydrogens were added and non-polar hydrogens merged. In all docking procedures, a grid box with 70 × 70 × 70 dimensions and 0.375 Å spacing was applied. The center of the grid box was assigned (27.366, 12.163, and 28.710) according to the centroid of the co-crystal ligand. AutoDock 4.2 was used for each docking procedure [22],[23] with 50 runs without changing the defaults of the program, such as search and docking parameters. To validate the procedure, the co-crystal ligand was extracted and re-docked into the protein using the same parameters of docking for designed compounds. All graphical representations of the interactions were produced using PyMOL Molecular Graphics System [24].

Synthesis of compounds

The desired compounds were prepared according to the three following steps:

Step 1: cyanuric chloride 1 (4.3 mmol) was dissolved in acetone (5 mL) in an ice bath. After that, appropriate amine a-f (4.3 mmol) was added dropwise for 10 min. The mixture was stirred at room temperature for 3 h and the progress of the reaction was checked using TLC. After completion of the reaction, the mixture was poured on crushed ice and a white precipitate was obtained. The precipitate was filtered and recrystallized from an ethanol/water (100/1 mL) mixture [24],[25],[26],[27].

Step 2: a mixture of 2a-2f (5 mmol), protected tyrosine 3 (5 mmol), potassium carbonate (7 mmol), and dimethylformamide (5 mL) was stirred at 10-25 °C for 24 h. After completion of the reaction by TLC, the reaction mixture was poured on crushed ice, filtered, washed with water, and recrystallized from the ethanol/water mixture to give compounds 4a-4f [Table 1].

Table 1: The structure of compounds 4a-4f.


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Step 3: in this step, 4a-4f (0.1 g), was deprotected by dissolving in dioxane and then HCL (2 mL, 7N) and stirredat room temperature for 24 h. After that, the mixture was concentrated and washed with diethyl ether to produce the final products.

In vivo study (counting lymphocyte cells)

In this study, 6-8-week-old male Wistar rats (160-180 mg) were used. The rats were housed at 22 ± 5 °C on a 12/12-h light/dark cycle and kept for three days to get used to the environment with free access to water and food. The animal experimental procedures and protocols were accepted by the Isfahan University of Medical Sciences, Ethics Committee for Laboratory AnimalsEthical No. IR.MUI.RESEARCH.REC.1398.198). These rats were intraperitoneally dosed with either vehicle control (normal saline and DMSO) or fingolimod and the proposed compounds 5a-5f (0.3, 1, and 3 mg/kg/day). After 24 h, the rats were anesthetized with the help of an anesthetic, and then retro-orbital blood sampling was performed. The blood samples were evaluated by flow cytometry to count lymphocyte cells.

Statistical analysis

Repeated measure one-way ANOVA was performed in Graphpad Prism software to compare the statistical alterations among groups, and followed up with Tukey post hoc tests. P-values ≤ 0.05 were considered significant. Data are presented as mean ± SD.

Results

Molecular docking study

The free binding energies (ΔG), H-bonding, and hydrophobic interactions of the compounds with the binding pocket of S1P1 are depicted in [Table 2]. Validation of docking procedure was performed by redocking of cocrystal ligand and measurement of root mean square deviation (RMSD). The RMSD value for the re-docking step was 1.3 Å.

Table 2: binding energy (ΔG), hydrophobic and H-bonding interactions of proposed compounds and fingolimod in the sphingosine-1-phosphate first subtype active site.


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Synthesis of compounds

According to Scheme 2, the preparation of compounds included three steps.

Characterization of synthesized compounds

4,6-dichloro-N-propyl-1, 3, 5-triazin-2-amine

(2a)

Yield: 85%, white powder, melting point (MP): 145 °C, lit MP: 146 °C [25]. Fourier-transform infrared spectroscopy (FT-IR, KBr, cm-1) ν: 3276 (NH), 2970 (CH aliphatic), 1626 (C=N), 1318 (CN), 812 (CCl).

4,6-dichloro-N-ethyl-1, 3, 5-triazin-2-amine

(2b)

Yield: 80%, white powder, MP: 109 °C, lit MP: 110 °C [26], FT-IR (KBr, cm-1) ν: 3274 (NH), 2998 (CH aliphatic), 1556 (C=N), 1318 (CN), 834 (CCl).

N-butyl-4,6-dichloro-1, 3, 5-triazin-2-amine

(2c)

Yield: 80%, white powder, MP: 49 °C, lit MP: 50 °C [27], FT-IR (KBr, cm-1) ν: 3269 (NH), 2948 (CH aliphatic), 1622 (C=N), 1322 (CN), 813 (CCl).

4,6-dichloro-N-isobutyl-1, 3, 5-triazin-2-amine

(2d)

Yield: 85%, white powder, MP: 93 °C, lit MP: 93 °C [27], FT-IR (KBr, cm-1) ν: 3273(NH), 2963 (CH aliphatic), 1626 (C=N), 1321 (CN), 839 (CCl).

4-(4,6-dichloro-1, 3, 5-triazin-2-yl) morpholine

(2e)

Yield: 90%, white powder, MP: 127 °C, lit MP:127 °C [26], FT-IR (KBr, cm-1) ν: 2974 (CH aliphatic), 1582 (C=N), 1336 (C-O), 1296 (C-N), 821 (CCl).

4,6-dichloro-N-hexyl-1, 3, 5-triazin-2-amine

(2f)

Yield: 70%, white powder, MP: 57 °C, lit MP: 56 °C [28]. FT-IR (KBr, cm-1) ν: 3275 (NH), 2939 (CH aliphatic), 1631 (C=N), 1322(CN), 819 (CCl).



Methyl 2-((tert-butoxycarbonyl)amino)-3-(4-(( 4-chloro-6-(propylamino)-1, 3, 5-triazin-2-yl)oxy) phenyl)propanoate (4a)

Yield: 75%, white powder, MP: 100 °C, IR (KBr, cm-1) ν: 3365 (NH), 2980 (CH aliphatic), 1716 (C=O), 1586 (CC aromatic), 1291 (C-N), 1190 (C-O), 789 (CCl). 1H NMR (400 MHz, DMSO-d6) δ: 0.79 (t, 3H, J = 3 Hz, H-C21), 1.20 (m, 2H, H-C22), 1.43 (s, 9H, Boc), 2.97 (d, 2H, J = 4.1 Hz, H-C16), 3.64 (s, 3H, H-C25), 3.83 (m, 2H, H-C23), 3.85 (br, 1H, H-N8), 4.18 (m, 1H, H-C17), 5.2 (br, H, H-N20), 7.16 (d, 2H, J = 7.2 Hz, H-C12, H-C14), 7.31 (d, 2H, J = 7.2 Hz, H-C11, H-C15)

Methyl 2-((tert-butoxycarbonyl)amino)-3-(4-(( 4-chloro-6-(ethylamino)-1, 3, 5-triazin-2-yl) oxy) phenyl)propanoate (4b)

Yield: 65%, white powder, MP: 79 °C, IR (KBr, cm-1) ν: 3417 (NH), 2987 (CH aliphatic), 1734 (C=O), 1612 (C=N), 1573 (CC aromatic), 1299 (CN), 1293 (C-O), 777 (CCl). 1H NMR (400 MHz, DMSO-d6) δ: 0.70 (t, 3H, H-C21), 1.40 (s, 9H, Boc), 2.98 (d, 2H, H-C16), 3.81 (s, 3H, H-C24), 3.81 (m, 2H, H-C22), 3.90 (br, H, H-N8), 4.21 (m, H, H-C17), 4.92 (br, H, H-N20), 7.10 (d, 2H, J = 7.5 Hz H-C12, H-C14), 7.42 (d, 2H, J = 7.2 Hz, H-C11, H-C15).

Methyl 2-((tert-butoxycarbonyl)amino)-3-(4-((4-(butylamino)-6-chloro-1, 3, 5-triazin-2-yl) oxy) phenyl)propanoate (4c)

Yield: 85%, white powder, MP: 81 °C, IR (KBr, cm-1) ν: 3357 (NH), 2961 (CH aliphatic), 1727 (C=O), 1575 (C=N), 1540 (CC aromatic), 1295 (C-N), 1211 (C-O), 709 (CCl). 1H NMR (400 MHz, DMSO-d6) δ: 0.79 (t, 3H, J = 3 Hz, H-C24), 1.20 (m, 4H, H-C21, H-C22), 1.40 (s, 9H, Boc), 2.99 (d, 2H, J = 4.1 Hz, H-C16), 3.04 (q, 2H, J = 5.4 Hz, H-C23), 3.09 (s, 3H, H-C26), 4.12 (br, H, H-N8), 4.18 (m, H, H-C17), 5.2 (br, H, H-N20), 7.16 (d, 2H, J = 7.2 Hz, H-C12, H-C14), 7.32 (d, 2H, J = 7.2 Hz, H-C11, H-C15).

Methyl 2-((tert-butoxycarbonyl)amino)-3-(4-((4-chloro-6-(isobutylamino)-1, 3, 5-triazin-2-yl)oxy) phenyl)propanoate (4d)

Yield: 70%, white powder, MP: 95 °C, IR (KBr, cm-1) ν: 3268 (NH), 2967 (CH aliphatic), 1728 (C=O), 1624 (C=N), 1570 (CC aromatic), 1352 (C-N), 1232 (C-O), 701 (CCl). 1H NMR (400 MHz, DMSO-d6) δ: 0.88 (d, 6H, J = 4 Hz, H-C21, H-C24), 1.33 (s, 9H, Boc), 1.74 (m, H, H-C22), 2.89 (d, 2H, J = 4.16 Hz, H-C16), 3.04 (t, 2H, J = 5.4 Hz, H-C23), 3.11 (s, 3H, H-C26), 4.07 (br, H, H-N8), 4.18 (m, H, H-C17), 5.18 (br, H, H-N20), 7.16 (d, 2H, J = 8 Hz, H-C12, H-C14), 7.29 (d, 2H, J = 8 Hz, H-C11, H-C15).

Methyl 2-((tert-butoxycarbonyl)amino)-3-(4-((4-chloro-6- morpholino-1, 3, 5-triazin-2-yl )oxy) phenyl)propanoate (4e)

Yield: 90%, white powder, MP:80 °C, IR (KBr, cm-1) ν: 3356 (NH), 1714 (C=O), 1587 (C=N), 1532 (CC aromatic), 1364 (C-N), 1249 (C-O), 795 (CCl). 1H NMR (400 MHz, DMSO-d6) δ: 1.33 (s, 9H, Boc), 2.76 (t, 4H, J = 3 Hz, H-C21, H-C25), 2.87(d, 2H, J = 4 Hz, H-C16), 3.61 (s, 3H, H-C27), 3.73 (t, 4H, J = 3 Hz, H-C22, H-C24), 4.22 (m, H, H-C17), 4.95 (br, H, H-N20), 7.15 (d, 2H, J = 7 Hz, H-C12, H-C14), 7.32 (d, 2H, J = 7 Hz, H-C11, H-C15).

Methyl 2-((tert-butoxycarbonyl)amino) -3-(4-((4-chloro-6-(hexylamino) -1, 3, 5-triazin-2-yl)oxy) phenyl)propanoate (4f)

Yield: 85%, white powder, MP: 87 °C, IR (KBr, cm-1) ν: 3318 (NH), 2962(CH aliphatic), 1720 (C=O), 1586 (C=N), 1509 (CC aromatic), 1293 (C-N), 1197 (C-O), 870 (CCl). 1H NMR (400 MHz, DMSO-d6) δ: 0.84 (t, 3H, J = 3 Hz, H-C26), 1.19 (m, 8H, H-C22, H-C23, H-C24, H-C25), 1.39 (s, 9H, Boc), 2.84 (d, 2H, J = 3 Hz, H-C16), 3.05 (q, 2H, J = 4 Hz, H-C21), 3.61 (s, 3H, H-C28), 3.83 (br, H, H-N8), 4.18 (m, H, H-C17), 5.2 (br, H, H-N20), 7.15 (d, 2H, J = 8 Hz, H-C12, H-C14), 7.31 (d, 2H, J = 8 Hz, H-C11, H-C15).

2-amino-3-(4-((4-chloro-6-(propylamino)-1, 3, 5-triazin-2-yl)oxy)phenyl)propanoic acid (5a)

Yield: 65%, white powder, MP: 160 °C, IR (KBr, cm-1) ν: 2976 (CH aliphatic), 2593-3300 (OH), 1737 (C=O), 1619 (C=N), 1519 (CC aromatic), 1221 (C-O), 717 (CCl). 1H NMR (400 MHz, DMSO-d6) δ: 0.85 (t, 3H, J = 4Hz, H-C24), 1.43 (m, 2H, H-C23), 2.97 (d, 2H, J = 4 Hz, H-C16), 3.23 (m, 2H, H-C22), 3.68 (br, 2H, H-N21), 4.05 (br, H, H-N8), 4.11 (m, H, H-C17), 6.72 (d, 2H, J = 7Hz H-C12, H-C14), 7.07 (d, 2H, J = 7 Hz, H-C11, H-C15), 11.19 (s, H, H-O19).

2-amino-3-(4-((4-chloro-6-(ethylamino)-1, 3, 5-triazin-2-yl)oxy)phenyl)propanoic acid (5b)

Yield: 70%, white powder, MP: 170 °C, IR (KBr, cm-1) ν: 3487 (NH), 2318-3165(OH), 1725 (C=O), 1613 (C=N), 1558 (CC aromatic), 1234 (C-O), 747(CCl). 1H NMR (400 MHz, DMSO-d6) δ: 1.07 (t, 3H, J = 3 Hz, H-C23), 2.96 (d, 2H, J = 4 Hz, H-C16), 3.07 (m, 2H, H-C22), 3.24 (br, 2H, H-N21), 3.69 (br, H, H-N8), 4.13 (m H, H-C17), 6.72 (d, 2H, J = 8 Hz, H-C12, H-C14), 7.17 (d, 2H, J = 8 Hz, H-C11, H-C15), 11.19 (s, H, H-O19).

2-amino-3-(4-((4-(butylamino)-6-chloro-1, 3, 5-triazin-2-yl)oxy)phenyl)propanoic acid (5c)

Yield: 85%, white powder, MP: 150 °C, IR (KBr, cm-1) ν: 3536 (NH), 2627-3400 (OH), 1719 (C=O), 1604 (CC aromatic), 1236 (C-O), 771 (CCl). 1H NMR (400 MHz, DMSO-d6) δ: 0.85 (t, 3H, J = 3 Hz, H-C25), 1.25 (m, 2H, H-C21), 1.33 (m, 2H, H-C22), 2.93 (d, 2H, J = 4 Hz, H-C16), 3.24 (m, 2H, H-C23), 3.65 (br, 2H, H-N20), 4.06 (br, H, H-N8), 4.11 (m, H, H-C17), 6.72 (d, 2H, J = 7 Hz, H-C12, H-C14), 7.07 (d, 2H, J = 7 Hz, H-C11, H-C15), 10.85 (s, H, H-O24).

2-amino-3-(4-((4-chloro-6-(isobutylamino)-1, 3, 5-triazin-2-yl)oxy)phenyl)propanoic acid (5d)

Yield: 75%, white powder, MP: 150 °C, IR (KBr, cm-1) ν: 3473 (NH), 2500-3500 (OH), 1743 (C=O), 1257 (C-O), 905 (CCl). 1H NMR (400 MHz, DMSO-d6) δ: 0.83 (d, 6H, J = 3.5 Hz, H-C21 , H-C24), 1.73 (m, H, H-C22), 3.01 (d, 2H, J = 3 Hz, H-C16), 3.10 (q, 2H, J = 3 Hz, H-C23), 3.67 (br, 2H, H-N20), 3.70 (br, H, H-N8), 4.04 (m, H, H-C17), 6.71 (d, 2H, J = 7 Hz, H-C12, H-C14), 7.06 (d, 2H, J = 7 Hz, H-C11, H-C15), 10.96 (s, H, H-O25).

2-amino-3-(4-((4-chloro-6-morpholino-1, 3, 5-triazin-2-yl)oxy)phenyl)propanoic acid (5e)

Yield: 60%, white powder, MP: 175 °C, IR (KBr, cm-1) ν: 3404 (NH), 2508-3500 (OH), 1734 (C=O), 1557 (CC aromatic), 1260 (CN), 1212 (C-O), 814 (CCl). 1H NMR (400 MHz, DMSO-d6) δ: 2.60 (t, 4H, J = 3 Hz, H-C21, H-C25), 3.10 (d, 2H, J = 3 Hz, H-C16), 3.57 (t, 4H, J = 3 Hz, H-C22, H-C24), 3.87 (br, 2H, N20), 4.17 (q, H, J = 3 Hz, H-C17), 6.72 (d, 2H, J = 8 Hz, H-C12, H-C14), 7.07 (d, 2H, J = 8 Hz, H-C11, H-C15), 10.62 (s, H, H-O26).

2-amino-3-(4-((4-chloro-6-(hexylamino)-1, 3, 5-triazin-2-yl)oxy)phenyl)propanoic acid (5f)

Yield: 65%, white powder, MP: 165 °C, IR (KBr, cm-1) ν: 3020(CH aromatic), 2304-3500 (OH), 1724 (C=O), 1620 (C=N), 1518 (CC aromatic), 1211 (C-O), 809 (CCl). 1H NMR (400 MHz, DMSO-d6) δ: 0.85 (t, 3H, J = 3 Hz, H-C27), 1.13-1-34 (m, 8H, H-C25, H-C24, H-C23, H-C22), 2.94 (d, 2H, J = 3 Hz, H-C16), 3.34(q, 2H, J = 3 Hz, H-C21), 3.41 (br, 2H, H-N20), 3.91(br, H, H-N8), 4.30 (q, H, J = 3 Hz, H-C17), 7.22 (d, 2H, J = 8 Hz, H-C12, H-C14), 7.33 (d, 2H, J = 8 Hz, H-C11, H-C15), 11.02 (s, H, H-O26).

In vivo study

As mentioned above, the compounds 5a-5f were injected at 0.3, 1, and 3 mg/kg doses. Each group contained six male Wistar rats (180-200 mg). Lymphocyte percentage diagrams are illustrated in [Figure 1].

Figure 1: Lymphocytes percentage before and after the injection of 5a-5f and fingolimod at (A) 0.3 mg/kg, (B) 1 mg/kg, and (C) 3 mg/kg. Lymphocyte percentage was determined using the flow cytometry method. Data are shown as mean ± SD. **P < 0.01 and ***P < 0.001 indicate significant differences before and after injection of the same compound.


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  Discussion Top


The binding of novel synthesized triazine-tyrosine hybrid compounds (5a-5f) to S1P1R was investigated with a docking procedure. Fingolimod was used as a reference and according to the docking results; the novel designed compounds could be fitted well within the binding site cavity of the S1P1R, showing acceptable ΔG values [Table 1]. The docking results demonstrated that the amino group of all compounds, except 5b, interacts electrostatically with GLU 321 residue, mimicking the amino group of fingolimod. Triazine ring has a role in π-π interactions with TYR 306 and TRP 299 residues in 5a, 5b, and 5d similar to the benzene ring of fingolimod. In compound 5c, the triazine ring interacts hydrophobically with MET 243 and VAL211. N-alkyl substitute acts like the aliphatic chain of fingolimod and is responsible for the hydrophobic interactions with TYR306, Met243, PHE288, and TRP299. The N-morpholine substituted compound, 5e, has the best free energy of binding. In this compound similar to fingolimod, 5a, 5b, and 5d, the amino group interacts electrostatically with GLU 321 residue and the morpholine ring interacts hydrophobically with key residues, PHE 288 and TRP 299. The interactions of fingolimod, 5a, and 5b, as representative structures, with binding residues of the S1P1R, are shown in [Figure 2]. As shown in [Table 1], ΔG binding of the suggested derivatives was as follows; 5e > 5d > 5f > 5a > 5c > 5b > fingolimod.

Figure 2: Graphical representation of (A) fingolimod, (B) 5a, and (C) 5b interactions with binding residues of the sphingosine-1-phosphate receptor.


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In the present work, six triazine-tyrosine compounds 5a-5f were synthesized according to the procedure shown in Scheme 2. Since one site of triazine can be substituted at low temperature, in the first step, different amines a-f was reacted with cyanuric chloride at low temperature (0-5 °C). Both melting point and the IR spectrum of 2a-2f were according to the reported values in the literature [24],[25],[26],[27].

The disubstituted s-triazine compounds (4a-4f) were obtained by the reaction of 2a-2f with N-Boc-tyr-OMe ester. In IR spectra of these compounds, the peaks around 1730 cm-1 confirm the presence of esteric C=O groups in their framework. After de-protection of the tyrosine moiety by HCl, an acidic C=O group was observed around 1710 cm-1 which confirmed the conversion of ester to an acid group. Additionally, a new band, observed at 2500-3500 cm-1 was assigned to the vibration of a hydroxyl group of acid in the IR spectrum, confirming the formation of the deprotected final products. The 1H-NMR spectral data of the intermediates and final products recorded in DMSO-d6 along with their assignments were reported in the experimental part. All the aromatic and aliphatic H-atoms were found in their expected region. The peaks around 0.8-3.7 ppm are attributable to the aliphatic H-atoms of these compounds. The NH of these intermediates exhibited a broad singlet around 4-5 ppm. Also, four aromatic hydrogens of the tyrosine ring were observed around 7-8 ppm. Finally, observation of a peak around 11 ppm and also the absence of a peak related to the tert-butoxy group around 1 ppm confirmed the structure of the final synthesized compounds.

According to the in vivo results of this project, fingolimod at 0.3, 1, and 3 mg/kg has reducing effects on the percentage of the lymphocytes i.e., dose 0.3 mg/kg from 71.5% to 21.5%, dose 1 mg/kg from 61.2% to 12.9%, and dose 3 mg/kg from 58.8% to 10.6%, before and after treatment, respectively. These results are in accordance with the studies conducted by Skidmore et al [29]. Studies have shown that the ability of fingolimod to reduce the number of lymphocytes is due to its interaction with the S1P1R subunit [30],[31].

The results of our experiments demonstrated that compound 5a, with propylamine substitution, at 3 mg/kg, has an acceptable activity and reduced the average percentage of lymphocytes from 90.4% to 6.9%. Ethylamine substitution on compound 5b at 0.3 mg/kg, resulted in a significant reduction of lymphocytes from 76.3% to 14.0%. Interestingly, both 5a and 5b compounds have short-chain substitutions. Additionally, compound 5c with butylamine substitution, significantly increased the percentage of lymphocytes at 3 and 0.3 mg/kg while it was expected to be more effective due to the lipophilicity of the butyl amine chain. Other compounds such as 5e with morpholine, 5f with hexylamine, and 5d with isobutyl amine substitution did not show a significant effect on lymphocyte number. In addition, the reports obtained from the control and sham groups showed that the effects of the carrier and injection are negligible and have not caused any problems in the study process.

In this study the modification was carried out on the three dominant segments of the representative S1P1 modulator, fingolimod; modification of the polar-head group, replacement of the triazine as a linker and different aliphatic amine moieties as the hydrophobic tail. The insertion of moieties possessing both amine and carboxyl functional groups as the polar head of the structure of S1P1 modulators, linearly or cyclically, has shown good effects [32],[33]. To the best of our knowledge, the simultaneous use of tyrosine amino acid as polar head and triazine as a linker has not been reported in the design of S1P1 modulators. Similar to earlier studies reported on different hybrid structures, the results of this study showed a dose-dependent lymphocyte depletion manner [29],[34],[35]. But unlike others [36],[37], compounds possessing larger hydrophobic tails showed weaker effects while compounds with smaller hydrophobic groups, such as ethyl and propyl, exhibited better effects on lymphocyte depletion which may be due to the large size of the total structure of these new compounds, perhaps less entry into the receptor site. Therefore, only two compounds 5a and 5b are suggested for future studies.


  Conclusion Top


In this study, a series of novel hybrid derivatives bearing s-triazine and tyrosine moieties were designed and synthesized as an S1P1 modulator. According to the structural requirements of S1P1 modulators, these compounds contain tyrosine as a polar-head group, triazine moiety as a linker, and aliphatic amine as a lipophilic tail. The docking study of the designed compounds with S1P1 as a key receptor in MS treatment revealed that these compounds could be fitted well in the receptor active site through electrostatic, π-π, and hydrophobic interactions. Structures of the new compounds were characterized by 1H NMR and IR spectra. The results of lymphocyte count revealed that compounds 5a and 5b with propyl and ethyl substitutes showed the maximum activity in vivo and other compounds with longer alkyl and also morpholine substitutions did not show good activity. These results were fair in terms of a triggering point for further research on the triazine-tyrosine hybrid, but it appears that better candidates must be developed and evaluated more accurately in vivo.

Acknowledgments

This project was financially supported by the Vice Chancellery of Research of Isfahan University of Medical Sciences, Isfahan, I.R. Iran under Grant No. 298018

Conflicts of interest statement

All authors declared no conflict of interest in this study.

Authors’ contribution

G.A. Khodarahmi conceived and supervised the project; S. Saeidi performed the experiments, analyzed and interpreted the data; P. Asadi assisted in the docking study, synthesis of compounds, and writing the manuscript, M. Aliomrani advised the biological study of compounds; F. Hassanzadeh helped in the synthesis of compounds and interpretation of the spectra. The final version of the manuscript was approved by all authors.



 
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