2024 Volume 13 Issue 4
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Design and Synthesis of Functionalized 2,4-Diamino-1,3,5-Triazines, Potential Inhibitors Involved in Immune and Inflammatory Response


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  1. Cancer and Immunity Targets and Drugs, IICiMed-UR1155, Health Research Institute 2, Nantes University, Nantes, France.
  2. Laboratory of Pharmaceutical Sciences, Analytical Sciences and Public Health, Training and research unit of Pharmaceutical and Biological Sciences, Félix Houphouët-Boigny University, Abidjan, Ivory Coast.
  3. Laboratory of Constitution and Reaction of Matter, Training and research unit of Matter Structures Sciences and Technology, Félix Houphouët-Boigny University, Ivory Coast.
  4. Roscoff biological station, USR 3151, CNRS-UPMC, Kinase Inhibitory Specialized Screening facility, KISSf, Place George Teissier, BP 74, 29682 Roscoff, France.
Abstract

A two-step synthesis method, initially using a microwave reactor for the preparation of reaction intermediates, allowed us to synthesize eleven 6-aryl-2,4-diamino-1,3,5-triazines 5a-k. The intermediates were biguanide derivatives 3a-f which were synthesized under microwave irradiation for 10 min at 130°C. The 2,4-diamino-1,3,5-triazine derivatives (5a-k) were obtained with yields from 16% to 86%. The compounds synthesized were submitted to biological evaluation on twelve protein kinases, through the implementation of a dose-response method which allowed the determination of median inhibitory concentration or IC50. Indeed, enzymatic activities were carried out in the presence of 10 µM of ATP, in a final volume of 6 µl for 30 min at 30°C in ADP-Glo buffer. Among the triazines synthesized, results of enzymatic activity showed that the most active molecule was compound 5b which inhibited PIM1 kinase with IC50 = 1.18 µg/mL This result is the starting point of a larger research program for our group which could investigate the introduction of the substituted group on triazine’s terminal amino group.


Keywords: Protein kinases, Inhibitors, Immunity, Inflammation, Triazines

INTRODUCTION

Protein kinases (PK) major role in cell survival and regulation processes, mainly at the level of proliferation mechanisms and immune response, was currently confirmed [1-5]. As a result, they constituted a set of increasingly sought-after targets in the treatment of cancers, and inflammatory and autoimmune diseases [6-12]. Thus, as part of the development of new so-called targeted therapies, we carried out the design and synthesis, in two steps, of new compounds with 2,4-diamino-1,3,5-triazine moiety. The literature described several routes to access these compounds [13-15] from arylamine or aryl nitrile derivatives with some ester derivates using a microwave reactor [16, 17]. We reported here the results of syntheses and biological evaluation of these compounds on several protein kinases.

MATERIALS AND METHODS

All commercial reagents were used for our syntheses without any further purification. All solvents were reagent or HPLC grade. Analytical TLC was performed on silica gel 60 F254 plates. Column chromatography was carried out on silica gel Merck 60 (70-230 mesh ASTM) and flash Chromatography Grace Reveleris X2TM. Melting points were determined on the electrothermal IA 9000 melting point apparatus and are uncorrected. 1H and 13C NMR spectra were recorded in CDCl3 or DMSO-d6 using a Bruker AVANCE 400 MHz spectrometer. Chemical shift values were reported in parts per million (ppm) and coupling constants (J) were given in hertz (Hz). Multiplicities were reported as follows: br s = broad singlet, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. LC-MS analyses were run on Waters ACQUITY UPLC-MS system composed of a single quadrupole detector (SQD) mass spectrometer (MS) equipped with an electrospray ionization interface (ESI) and a photodiode array detector (PDA).

General procedure for synthesis of biguanides

To a solution of dicyandiamide (252 mg, 3 mmol, 1 eq) in 5 mL of acetonitrile, was added the corresponding amine derivative (1 eq). Trimethylsilyl chloride or TMSCl (1.1 eq) was added dropwise to the reaction mixture. Stirring was maintained for a few min to obtain the most homogeneous mixture. This mixture, contained in a borosilicate glass vial of 35 ml equipped with snap caps, was put in a CEM Discover SP single-mode microwave reactor for 10 min at 130°C, under pressure of 18 bar and power of 60 Watt. After 10 min, the reaction mixture was cooled in ice crushed and the precipitate formed was filtered, washed with acetonitrile or isopropanol, and dried. The biguanide derivatives (3a-f) were obtained as powders and were used with further purification for the next step of synthesis.

4-methylthio-phenyl-biguanide (3a)

Compound 3a was obtained according to the general procedure, as a purple powder from dicyandiamide 2 (252 mg, 3 mmol), 4-methylthioaniline 1a (0.373 mL, 3 mmol), and TMSCl (0.417 mL, 3.3 mmol).

4-Ethymorpholinyl-biguanide (3b)

Compound 3b was obtained according to the general procedure, as a coral powder from dicyandiamide 2 (504 mg, 6 mmol), N-ethylamino-morpholine 1b (0.8 mL, 6 mmol), and TMSCl (0.834 mL, 6.6 mmol).

4-Cyano-phenyl-biguanide (3c)

Compound 3c was obtained according to the general procedure, as an orange powder from dicyandiamide 2 (504 mg, 6 mmol), 4-aminobenzonitrile 1c (708.84 mg, 6 mmol), and TMSCl (0.834 mL, 6.6 mmol).

Benzo[d][1,3]dioxol-5-biguanide (3d)

Compound 3d was obtained according to the general procedure, as a purple powder from dicyandiamide 2 (613 mg, 7.29 mmol), methylene-dioxianiline 1d (1g, 7.29 mmol), and TMSCl (1.01 mL, 8.02 mmol).

2,4-Dichloro-phenyl-biguanide (3e)

Compound 3e was obtained according to the general procedure, as a white powder from dicyandiamide 2 (311.38 mg, 3.70 mmol), 2,4-dichloroaniline 1e (600 mg, 3.70 mmol), and TMSCl (0.51 mL, 4.07 mmol).

3,4-Dichloro-phenyl-biguanide (3f)

Compound 3f was obtained according to the general procedure, as a beige powder from dicyandiamide 2 (518.77 mg, 6.17 mmol), 3,4-dichloroaniline 1f (1 g, 6.17 mmol) and TMSCl (0.86 mL, 6.79 mmol).

General procedure for the synthesis of 2,4-diamino-1,3,5-triazines

To a solution of corresponding biguanide, 3 (1 eq) in 15 ml of anhydrous methanol (MeOH) cooled at 0°C under nitrogen, were added sodium methanolate or sodium ethanolate (10 to 15 eq). To the cooled mixture, the corresponding ester derivative 4 (1.3 to 1.5 eq) was added. The reaction mixture was brought to room temperature and stirring was maintained for 30 min. Heating was conducted at reflux for 3h to 8h, depending on the nature of biguanide derivative 3. After refluxing, the mixture was cooled in ice crushed and the precipitate formed was filtered. The product was purified either by extraction or washing in organic solvent recrystallization or column chromatography. After purification, the 2,4-diamino-1,3,5-triazine derivatives (5a-k) were obtained as colored powders with yields from 16% to 86%.

6-(4-chlorophenyl)-N2-(4-(methylthio)phenyl)-1,3,5-triazine-2,4-diamine (5a)

Compound 5a was synthesized according to the general procedure from 4-methylthio-phenyl-biguanide hydrochloride 3a (1.05 g, 4.70 mmol), sodium methanolate (3 mL, 53.3 mmol), and 4-chlorobenzoate 4a (0.87 mL, 5.56 mmol). The precipitate formed was washed with dichloromethane (DCM), dried, and obtained as a beige powder with a yield of 16%. Mp 168°C. 1H NMR (400 MHz, DMSO-d6), δ : 2.45 (s, 3H, SCH3), 7.24 (d, J = 8.3 Hz, 4H, H-2’’, H-6’’, NH2), 7.58 (d, J = 8.4 Hz, 2H, H-3’’, H-5’’), 7.79 (d, J = 8.1 Hz, 2H, H-3’, H-5’), 8.30 (d, J = 8.3 Hz, 2H, H-2’, H-6’), 9.60 (s, 1H, NH). 13C NMR (101 MHz, DMSO) :15.89 (SCH3), 120.69 (C-2’’, C-6’’), 127.29 (C-2’, C-6’), 128.40 (C-3’, C-5’), 129.49 (C-4’’), 130.33 (C-3’’, C-5’’), 135.61 (C-1’), 136.13 (C-4’), 137.49 (C-1’’), 164.45 (C-4), 167.08 (C-6), 169.20 (C-2). IR, n(cm-1): 3317.56 (N-H), 1361.74 (CH3), 1658.78 (C=Car), 802.39 (C-Har). UPLC ESI/MS, [M+H]+: 344.8.

6-(4-chlorophenyl)-N2-(2-morpholinoethyl)-1,3,5-triazine-2,4-diamine (5b)

Compound 5b was synthesized according to the general procedure from 4-ethylmorpholinyl-biguanide hydrochloride 3b (1 g, 3.99 mmol), sodium methanolate (3 mL, 53.3 mmol) and 4-chlorobenzoate 4a (0.75 mL, 4.79 mmol). The reaction mixture was cooled, filtered, and evaporated under vacuum. The residue was put in water and extracted several times with diethyl ether. The organic phase was dried and evaporated to yield the expected product as a white powder with a yield of 34%. Mp 187°C. 1H NMR (400 MHz, DMSO-d6), δ: 2.44 (m, 6H, H-2’, H-3’’’, H-5’’’), 3.36 - 3.50 (m, 2H, H-1’), 3.51 - 3.60 (m, 4H, H-2’’’, H-6’’’), 6.80 (br s, 1H, NH), 6.93 (s, 2H, NH2), 7.54 (m, 2H, H-3’’, H-5’’), 8.25 (m, 2H, H-2’’, H-6’’). 13C NMR (101 MHz, DMSO-d6), δ : 53.29 (C-1’), 57.41 (C-3’’’, C-5’’’, C-2’), 66.17 (C-2’’, C-6’’), 128.23 (C-2’’, C-6’’), 129.34 (C-3’’, C-5’’), 135.75 (C-1’’), 135.88 (C-4’’), 166.00 (C-6), 167.23 (C-4), 168.55 (C-2). IR, n(cm-1): 3267.41 (N-H), 2976.16 (C=CH2), 1604.77 (C=Car), 813.96 (C-Har). UPLC ESI/MS, [M+H]+: 335.8.

4-((4-amino-6-(4-chlorophenyl)-1,3,5-triazin-2-yl)amino)benzonitrile (5c)

Compound 5c was synthesized according to the general procedure from 4-cyanophenyl-biguanide hydrochloride 3c (1.05 g, 5.2 mmol), sodium methanolate (3 mL, 53.3 mmol), and 4-chlorobenzoate 4a (0.84 mL, 5.37 mmol). The precipitate formed was washed with water, and methanol and recrystallized from MeOH/H2O mixture (50/50). After filtration, the precipitate was dried and obtained as a white powder with a yield of 30%. Mp 298°C. 1H NMR (400 MHz, DMSO-d6), δ : 7.42 (br s, 2H, NH2), 7.54 - 7.66 (m, 2H, H-2’’, H-6’’), 7.75 (d, J = 8.8 Hz, 2H, H-3’’, H-5’’), 8.06 (d, J = 8.8 Hz, 2H, H-3’, H-5’), 8.24 - 8.37 (m, 2H, H-2’, H-6’), 10.11 (s, 1H, NH), 13C NMR (101 MHz, DMSO-d6), δ : 103.24 (C-4’’), 119.41 (CN), 119.48 (C-2’’, C-6’’), 128.49 (C-2’, C-6’), 129.57 (C-3’, C-5’), 132.84 (C-3’’, C-5’’), 135.29 (C-1’), 136.39 (C-4’), 144.4 (C-1’’), 164.51 (C-6), 167.06 (C-4), 169.54 (C-2). IR, n(cm-1): 3317.56 (N-H), 1662.64 (C=Car), 802.39 (C-Har). UPLC ESI/MS, [M+H]+: 323.8.

N2-(benzo[d][1,3]dioxol-5-yl)-6-(4-chlorophenyl)-1,3,5-triazine-2,4-diamine (5d)

Compound 5d was synthesized according to the general procedure from benzo[d][1,3]dioxol-5-biguanide hydrochloride 3d (800 mg, 3.62 mmol), sodium methanolate (3 mL, 53.3 mmol) and 4-chlorobenzoate 4a (85 mL, 5.43 mmol). The precipitate formed was successively washed with methanol, water, diethyl ether and dried. It was obtained as a green powder with a yield of 52%. Mp 206°C. 1H NMR (400 MHz, DMSO-d6), δ: 5.98 (s, 2H, OCH2O), 6.85 (d, J = 8.4 Hz, 1H, H-5’), 7.14 (m, 3H, NH2, H-2’), 7.58 (m, 3H, H-3’’, H-5’’, H-6’), 8.28 (d, J = 8.4 Hz, 2H, H-2’’, H-6’’), 9.48 (s, 1H, NH). 13C NMR (101 MHz, DMSO-d6), δ : 100.75 (OCH2O), 102.48 (C-2’), 107.77 (C-5’), 112.80 (C-6’), 128.38 (C-2’’, C-6’’), 129.42 (C-3’’, C-5’’), 134.13 (C-1’’), 135.68 (C-4’’), 136.07 (C-1’), 142.21 (C-4’), 146.91 (C-3’), 164.38 (C-6), 167.05 (C-4), 169.09 (C-2). IR, n(cm-1): 3325.28 (N-H), 1658 (C=Car), 1473.62 (C=CH2), 808.17 (C-Har). UPLC ESI/MS, [M+H]+: 342.8.

6-(4-chlorophenyl)-N2-(2,4-dichlorophenyl)-2,4-diamino-1,3,5-triazine (5e)

Compound 5e was synthesized according to the general procedure from 2,4-dichloro-phenyl-biguanide hydrochloride 3e (450 mg, 1.83 mmol), sodium methanolate (3 mL, 53.3 mmol) and 4-chlorobenzoate 4a (0.43 mL, 2.74 mmol). The precipitate formed was successively washed with methanol, water, and cyclohexane and dried. It was obtained as a white powder with a yield of 40%. Mp. 239°C. 1H NMR (400 MHz, DMSO-d6), δ : 7.18 (br s, 2H, NH2), 7.44 (dd, J = 8.7, 2.4 Hz, 1H, H-6’’), 7.56 (d, J = 8.6 Hz, 2H, H-3’, H-5’), 7.68 (d, J = 2.4 Hz, 1H, H-5’’), 7.78 (d, J = 8.7 Hz, 1H, H-3’’), 8.23 (d, J = 8.5 Hz, 2H, H-2’, H-6’), 8.97 (s, 1H, NH). 13C NMR (101 MHz, DMSO-d6), δ: 127.37 (C-6’’), 128.48 (C-4’’), 128.58 (C-2’’), 128.79 (C-5’’), 129.16 (C-2’, C-6’), 129.48 (C-3’, C-5’), 129.59 (C-3’’), 134.95 (C-1’), 135.36 (C-4’), 136.21 (C-1’’), 165.11(C-6), 167.21 (C-4), 169.33 (C-2). IR, n(cm-1): 3342.64 (N-H), 1591.27 (C=Car, 804.32 (C-Har). UPLC ESI/MS, [M+H]+: 367.6.

6-(4-chlorophenyl)-N2-(3,4-dichlorophenyl)-2,4-diamino-1,3,5-triazine (5f)

Compound 5f was synthesized according to the general procedure from 3,4-dichloro-phenyl-biguanide hydrochloride 3f (500 mg, 2.03 mmol), sodium methanolate (3 mL, 53.3 mmol) and 4-chlorobenzoate 4a (0.48 mL, 3.045 mmol). The precipitate formed was washed with methanol, water, and cyclohexane and dried. It was obtained as a white powder with a yield of 45%. Mp. 252°C 1H NMR (400 MHz, DMSO-d6), δ: 7.37 (sl, 2H, NH2), 7.54 (d, J = 8.8 Hz, 1H, H-2’’), 7.60 (d, J = 8.5 Hz, 2H, H-3’, H-5’), 7.80 (dd, J = 8.8, 2.3 Hz, 1H, H-2’’), 8.22 (d, J = 1.8 Hz, 1H, H-5’’), 8.29 (d, J = 8.5 Hz, 2H, H-2’, H-6’), 9.90 (s, 1H, NH). 13C NMR (101 MHz, DMSO-d6), δ : 119.67 (C-6”), 120.78 (C-2’’), 123.23 (C-4’’), 128.47 (C-2’, C-6’), 129.48 (C-3’, C-5’), 130.18 (C-5’’), 130.72 (C-3’’), 135.37 (C-1’), 136.33 (C-4’), 140. 14 (C-1’’), 164.39 (C-6), 167.02 (C-4), 169.39 (C-2). IR, n(cm-1): 3342.64 (N-H), 1591.27 (C= Car), 804.32 (C-Har). UPLC ESI/MS, [M+H]+: 367.6.

N-2-(4-(methylthio)phenyl)-6-(pyridin-2-yl)-1,3,5-triazine-2,4-diamine (5g)

Compound 5g was synthesized according to the general procedure from 4-thiomethyl-phenyl-biguanide hydrochloride 3g (525 mg, 2.35 mmol), sodium ethanolate (3 mL, 38.26 mmol), and ethyl-2-picolinate 4b (0.41 mL, 3.05 mmol). The reaction mixture was evaporated and the solid obtained was recrystallized from a MeOH/H2O mixture (1/2). It was obtained as a beige powder with a yield of 23%. Mp 172°C. 1H NMR (400 MHz, DMSO-d6), δ : 2.45 (s, 3H, CH3), 7.26 (m, 4H, H-2’, H-6’, NH2), 7.54 (m, 1H, H-4’’), 7.83 (d, J = 8.7 Hz, 2H, H-3’, H-5’), 7.96 (td, J = 7.7, 1.7 Hz, 1H, H-5’’), 8.26 (d, J = 7.9 Hz, 1H, H-6’’), 8.71 (d, J = 4.6 Hz, 1H, H-3’’), 9.78 (s, 1H, NH). 13C NMR (101 MHz, DMSO-d6), δ : 15.91 (CH3), 120.50 (C-2’, C-6’), 123.23 (C-4’’), 125.41 (C-6’’), 127.31 (C-4’), 130.17 (C-3’, C-5’), 136.70 (C-1’), 137.63 (C-5’’), 149.23 (C-3’’), 154.34 (C-1’’), 164.67 (C-6), 167.33 (C-2), 170.17 (C-4). IR, n(cm-1): 3180.62 (N-H), 1606.70 (C=Car), 1369.46 (CH3), 825.33 (C-Har). UPLC ESI/MS, [M+H]+: 311.4

N2-(2,4-dichlorophenyl)-6-(pyridin-2-yl)-2,4-diamino-1,3,5-triazine (5h)

Compound 5h was synthesized according to the general procedure from 2,4-dichloro-phenyl-biguanide hydrochloride 3h (400 mg, 1.625 mmol), 3 mL of sodium ethanolate (38.26 mmol) and ethyl-2-picolinate 4b (0.44 mL, 3.25 mmol). The reaction mixture was evaporated. Then, the solid formed was purified on column chromatography with a DCM/MeOH mixture (9/1). After evaporation under vacuum, the dry residue was washed with DCM and was obtained as a pink powder with a yield of 45%. Mp 230°C. 1H NMR (400 MHz, DMSO-d6), δ: 7.02-8.04 (m, 7H, H-3’, H-5’, H-6’, H-4’’, H-5’’, NH2), 8.20 (d, J = 7.7 Hz, 1H, H-6’’), 8.69 (d, J = 3.8 Hz, 1H, H-3’’), 9.07 (s, 1H, NH). 13C NMR (101 MHz, DMSO-d6), δ: 123.31 (C-6’), 125.47 (C-4’’), 127.39 (C-6’’), 128.50 (C-4’), 128.80 (C-5’), 129.23 (C-2’), 135.00 (C-3’), 136.72 (C-1’), 149.25 (C-5’’), 149.25 (C-3’’), 154.17 (C-1’’), 165.38 (C-6), 167.50 (C-2),  170.40 (C-4). IR, n (cm-1): 3113.11 (N-H), 1606,70 (C=Car), 812,03 (C-Har). UPLC ESI/MS, [M+H]+: 334.2.

N2-(3,4-dichlorophenyl)-6-(pyridin-2-yl)-2,4-diamino-1,3,5-triazine (5i)

Compound 5i was synthesized according to the general procedure from 3,4-dichloro-phenyl-biguanide hydrochloride 4i (510 mg, 2.07 mmol), 3 mL of sodium ethanolate (38.26 mmol), and ethyl-2-picolinate 4b (0.56 mL, 4.14 mmol). The precipitate formed was successively washed with methanol, water, and diethyl ether and dried.  It was obtained as a pink powder with a yield of 86%. Mp 243°C. 1H NMR (400 MHz, DMSO-d6), δ : 7.22-7.67 (m, 4H, H-2’, H-5’, NH2), 7.84 (d, J = 8.5 Hz, 1H, H-4’’), 7.98 (t, J = 7.2 Hz, 1H, H-6’), 8.29 (m, 2H, H-5’’, H-6’’), 8.72 (d, J = 3.3 Hz, 1H, H-3’’), 10.11 (s, 1H, NH). 13C NMR (101 MHz, DMSO-d6), δ : 119.55 (C-6’), 120.66 (C-2’), 123.18 (C-4’), 123.27 (C-4’’), 125.67 (C-6’’), 130.16 (C-5’), 130.75 (C-3’), 136.88 (C-5’’), 140.23 (C-1’), 149.36 (C-3’’), 153.94 (C-1’’), 164.63 (C-6), 167.19 (C-2), 170.20 (C-4). UPLC ESI/MS, [M+H]+: 334.03. IR, n(cm-1): 3147.83 (N-H), 1598.99 (C=Car), 790.81 (C-Har). UPLC ESI/MS, [M+H]+: 334.2.

N2-(benzo[d][1,3]dioxol-5-yl)-6-(pyridin-4-yl)-2,4-diamino-1,3,5-triazine (5j)

Compound 5j was synthesized according to the general procedure from benzo[d][1,3]dioxol-5-biguanide hydrochloride 4j (650 mg, 2.94 mmol), sodium ethanolate (3 mL, 38.26 mmol) and ethyl isonicotinate 4c (0.57 mL, 3.822 mmol). The precipitate formed was successively washed with methanol, water and DCM then dried. It was obtained as a grey powder with a yield of 24%. Mp 218°C. 1H NMR (400 MHz, DMSO-d6), δ: 5.99 (s, 2H, CH2), 6.86 (d, J = 8.4 Hz, 1H, Hb), 7.15 (dd, J = 8.4, 1.8 Hz, 1H, Ha), 7.31 (s, 2H, NH2), 7.58 (s, 1H, He), 8.11 (d, J = 5.5 Hz, 2H, Hg), 8.76 (d, J = 5.7 Hz, 2H, Hh), 9.60 (s, 1H, NH). 13C NMR (101 MHz, DMSO-d6), δ: 100.79 (OCH2O), 102.59 (C-2’), 107.79 (C-5’), 112.97 (C-6’), 121.46 (C-2’’, C-6’’), 133.93 (C-1’), 142.37 (C-1’’), 144.21 (C-3’’), 146.93 (C-5’’), 150.18 (C-6), 164.42 (C-4), 167.13 (C6), 168.64 (C4). IR, n(cm-1): 3180.62 (N-H), 1591.27 (C= Car), 1487.12 (C=CH2), 802.39 (C- Har). UPLC ESI/MS, [M+H]+: 309.10.

N2-(3,4-dichlorophenyl)-6-(pyridin-4-yl)-1,3,5-triazine-2,4-diamine (5k)

Compound 5k was synthesized according to the general procedure from 3,4-dichlorophenylbiguanide hydrochloride 3k (500 mg, 2.03 mmol), sodium ethanolate (3 mL, 38.26 mmol), and ethyl isonicotinate 4c (0.39 mL, 2.64 mmol). The precipitate formed was successively washed with methanol, water, and diethyl ether and then dried. It was obtained as a pink powder with a yield of 36%. Mp 276°C. 1H NMR (400 MHz, DMSO-d6), δ: 7.55 (m, 3H, H-2’, NH2), 7.81 (dd, J = 8.9, 2.4 Hz, 1H, H-5’), 8.12 (d, J = 5.9 Hz, 2H, H-2’’, H-6’’), 8.22 (d, J = 1.7 Hz, 1H, H-6’), 8.77 (d, J = 5.8 Hz, 2H, H-3’’, H-5’’), 10.01 (s, 1H, NH). 13C NMR (101 MHz, DMSO-d6), δ: 119.81 (C-6’), 120.92 (C-2’), 121.49 (C-6’’, C-2’’), 123.47 (C-4’), 130.23 (C-5’), 130.75 (C-3’), 139.98 (C-1’), 143.91 (C-1’’), 150.27 (C-3’’, C-5’’), 164.46 (C-6), 167.11 (C-4), 168.96 (C-2). IR, n(cm-1): 3307.92 (N-H), 1600.32 (C=Car), 804.32 (C-Har). UPLC ESI/MS, [M+H]+: 334.03.

RESULTS AND DISCUSSION

Chemistry

In this work, novel triazines were synthesized in two steps. The first step consisted of adding primary amines 1 with dicyandiamide 2 in the presence of chlorotrimethylsilane in acetonitrile, under microwave irradiation for 10 min at 130°C (18 bar, 60 W). The intermediates resulting from this first step were biguanides 3a-f (Scheme 1).

 

Scheme 1. Synthesis of biguanides

At the end of the reaction, the biguanide formed was cooled in ice crushed, filtered, and washed with acetonitrile and isopropanol. The resulting biguanide in powdery form was dried and used without further purification for the next step [18]. The yields of synthesized biguanides ranged from 61 to 95% (Table 1).

Table 1. Synthesized biguanides.

Molecules

Structures

Yields (%)

3a

61

3b

95

3c

92

3d

76

3e

70

3f

50

 

Then, biguanides previously obtained were condensed with various esters of interest according to the reaction scheme below (Scheme 2).

 

Scheme 2. Synthesis of 6-aryl-2,4-diamino-1,3,5-triazines

The reaction was carried out under an inert atmosphere, at reflux of ethanol or methanol according to the chosen base (EtONa or MeONa [19]). The reaction was followed by TLC and UPLC-MS. After completion of the reaction, synthesized triazines 5a-k precipitated. The precipitate obtained was recovered by filtration, and washed successively with methanol and dichloromethane. Depending on its solubility, it was rinsed with ethyl acetate, diethyl ether, or cyclohexane. If necessary, it was either recrystallized in a methanol/water mixture 1:1 or 1:2 or purified on a silica gel column with a convenient solvent. The yields of 6-aryl-2,4-diamino-1,3,5-triazines (5a-k) ranged from 16 to 86% (Table 2).

Table 2. Synthesized triazines.

Molecules

R1

R2

Yields (%)

5a

16

5b

34

5c

30

5d

52

5e

40

5f

45

5g

23

5h

45

5i

86

5j

24

5k

36

 

Biological section

Table 3. Experimental conditions used for protein kinase assays.

Protein Kinase

(Family)

Enzyme Description

Substrate*

(Working concentration)

Buffer used**

GSK3α

(CMGC)

Human, recombinant, expressed by baculovirus in Sf9 insect cells

GS-1 peptide:

YRRAAVPPSPSLSRHSSPHQSpEDEEE ***

(20µM)

A

GSK3β

(CMGC)

Human, recombinant, expressed by baculovirus in Sf9 insect cells

GS-1 peptide:

YRRAAVPPSPSLSRHSSPHQSpEDEEE ***

(20µM)

A

CDK5/p25

(CMGC)

Human, recombinant, expressed in bacteria

Histone H1

(37.2µM)

A

EGFR

(TK)

Human, recombinant, expressed by baculovirus in Sf9 insect cells

Poly(L-glutamic acid – L-tyrosine) sodium salt

(0.17µg/µL)

A

VEGFR2

(TK)

Human, recombinant, expressed by baculovirus in Sf9 insect cells

Poly(L-glutamic acid – L-tyrosine) sodium salt

(0.17µg/µL)

A

EphB1

(TK)

Human, recombinant, expressed by baculovirus in Sf9 insect cells

Poly(L-glutamic acid – L-tyrosine) sodium salt

(0.17µg/µL)

A

JAK3

(TK)

Human, recombinant, expressed by baculovirus in Sf9 insect cells

Peptide: GGEEEEYFELVKKKK

(94µM)

A

ABL1

(TK)

Human, recombinant, expressed by baculovirus in Sf9 insect cells

Peptide: EAIYAAPFAKKK

(127 µM)

A

DYRK1A

(CMGC)

Human recombinant, expressed by baculovirus in Sf9 insect cells

Peptide: KKISGRLSPIMTEQ

(10.7µM)

A

CLK1

(CMGC)

Human, recombinant, expressed by baculovirus in Sf9 insect cells

Peptide: GRSRSRSRSRSR

(57.3µM)

A

CK1ε

(CK1)

Human, recombinant, expressed by baculovirus in Sf9 insect cells

Peptide: RRKHAAIGSpAYSITA ***

(170µM)

A

PIM1

(CAMK)

Human proto-oncogene, recombinant, expressed in bacteria

PimTide: ARKRRRHPSGPPTA

(630 µM)

A

* Peptide substrates were obtained from ProteoGenix (Schiltigheim, France) or Sigma for Histone H1, Poly (L-glutamic acid – L-tyrosine) sodium salt

** Composition of the buffers: Buffer A: 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM Tris-HCl pH 7.5, 50 µg/mL heparin

*** “Sp’’ stands for phosphorylated serine

Primary screening and IC50 determination

In this study, synthesized triazines were tested on twelve (12) protein kinases (GSK3-a/b, CDK5/p25, EGFR, VEGFR2, EphB1, JAK3, ABL1, DYRK1A, CLK1, CK1ε, and PIM1), (Table 3) to evaluate their potential enzymatic activity. Kinase activities were measured using the ADP-GloTM bioluminescent kinase assay kit (Promega, Madison, WI) according to the recommendations of the manufacturer, described according to the protocol of Zegzouti et al. [20]. These enzymatic activities were detected in the presence of 10 µM ATP.  Reactions were carried out in the presence of 10µM of ATP, in a final volume of 6 µl for 30 min at 30°C in ADP-Glo buffer (25 mM Tris-HCl pH 7.5, 10mM MgCl2, 1mM EGTA, 1mM DTT, 50 µg/mL heparin and 0.1mg/ml of BSA). After that, 6 µl of ADP-GloTM Kinase Reagent was added to stop the kinase reaction. After an incubation time of 50 min at room temperature, 12 µl of ADP-GloTM Kinase Detection Reagent was added for one hour at room temperature. The transmitted signal was measured using the Envision (PerkinElmer, Waltham, MA) microplate luminometer and expressed in Relative Light Unit (RLU). Kinase activities were expressed in percentage (%) of maximal activity, i.e. measured in the absence of an inhibitor. In primary screening, chemical compounds, dissolved in DMSO, are tested at 10 µM and 1 µM final in kinase assay. To determine the half-maximal inhibitory concentration (IC50), the assays were performed in duplicate in the absence or presence of increasing doses of the tested compounds. Positive controls (for total activity) and negative controls (for background noise) were performed with appropriate dilutions of dimethylsulfoxide (DMSO). Note that a value ≥100 indicates that the compound tested can not inhibit the enzymatic activity of the kinase at the concentration tested (Table 4).

Table 4. Primary screening of triazines.

Compound

Concentration

GSK3α

GSK3β

CDK5/p25

EGFR

VEGFR2

EphB1

JAK3

ABL1

DYRK1A

CLK1

CK1ε

PIM1

5a

10 µM

90

86

89

≥100

≥100

42

55

≥100

85

71

55

43

1 µM

≥100

87

≥100

89

≥100

70

71

≥100

102

99

≥100

≥100

5b

10 µM

≥100

≥100

≥100

≥100

78

≥100

≥100

≥100

92

65

≥100

27

1 µM

≥100

≥100

89

≥100

85

90

≥100

≥100

≥100

100

≥100

55

5c

10 µM

100

≥100

67

79

96

48

18

≥100

86

67

97

74

1 µM

97

97

≥100

100

100

91

81

≥100

92

99

≥100

97

5d

10 µM

68

71

67

70

66

44

59

87

90

71

44

46

1 µM

83

85

87

78

70

74

59

86

≥100

≥100

93

≥100

5e

10 µM

94

89

96

≥100

≥100

95

77

86

≥100

≥100

87

62

1 µM

100

82

99

≥100

≥100

73

86

74

98

≥100

≥100

≥100

5f

10 µM

≥100

91

86

97

≥100

48

74

57

≥100

≥100

90

82

1 µM

≥100

91

90

97

≥100

61

51

50

96

≥100

100

≥100

5g

10 µM

98

80

≥100

90

≥100

29

76

77

91

≥100

78

77

1 µM

≥100

81

98

99

≥100

66

78

73

92

≥100

95

≥100

5h

10 µM

79

78

83

81

72

53

70

60

≥100

98

97

78

1 µM

78

96

≥100

70

62

56

73

53

≥100

≥100

110

99

5i

10 µM

76

≥100

≥100

69

50

55

83

56

≥100

97

93

86

1 µM

66

60

≥100

70

51

70

45

54

≥100

≥100

≥100

≥100

5j

10 µM

68

85

68

74

49

73

57

58

72

51

71

≥100

1 µM

96

≥100

≥100

93

80

82

76

87

96

89

≥100

≥100

5k

10 µM

96

97

83

≥100

91

58

≥100

124

49

50

57

77

1 µM

≥100

93

≥100

≥100

104

43

≥100

99

62

89

90

89

 

Primary screening results highlighted seven (7) compounds 5a, 5b, 5c, 5d, 5g, 5i and 5k, which inhibited kinases EphB1, JAK3, CK1Ɛ and PIM1. Enzymatic activity percentages of these compounds, at 10 and 1 μM concentrations, were noticed in Table 4. Dose-response studies were subsequently undertaken with IC50 determination. IC50 of active molecules which obtained the best results were determined in Table 5.

Table 5. IC50 determination at 10 µM for active triazines

Compounds

IC50 (µg/mL)

EphB1

PIM1

JAK3

5a

3.07

-

-

5b

-

1.18

-

5c

-

-

1.56

5d

-

1.76

-

5g

15.43

-

-

 

From these different results, we were able to identify two chemical cores favorable to the inhibition of enzymatic activity. These are 6-chlorophenyl-1,3,5-triazine and 6-(pyridin-2-yl)-1,3,5-triazine which carry an aminophenyl group in position 2. The latter must be monosubstituted in the para position of amine function by a thiomethyl or nitrile group (compounds 5a, 5c, and 5g). On the one hand, polysubstitution in the ortho and para position or the meta and para position by chlorine atoms, led to inactivity on kinases. On the other hand, introduction into position 2, on 6-chlorophenyl-2,4-diamino-1,3,5-triazine of ethylamino-morpholinyl substituent (compound 5b) or 5-aminobenzodioxole (compound 5d) caused inhibition of protein kinases. Moreover, the most active molecule was compound 5b which inhibited PIM1 kinase (IC50 = 1.18 µg/mL), Table 5. However, introduction at position 6 of piridin-4-yl group on 1,3,5-triazine moiety resulted in lack of enzymatic activity, even though this structural variation was coupled with the presence, in position 2, of the group previously favorable to inhibition such as 5-aminobenzodioxole (5j).

CONCLUSION

Here, we have reported the synthesis in two steps and the biological evaluation of a series of new triazines 5a-k. We successfully developed a library of eleven (11) new compounds with overall yields ranging from 16 to 86%. For the first step, we used microwave irradiation for the synthesis of biguanides 3a-e. This approach allowed structural diversity of intermediates biguanides 3a-e which led to final triazines. Enzymatic inhibition was measured on all final compounds. Results on protein kinase assays showed that the most active molecule was compound 5b which inhibited PIM1 kinase with the value of IC50 = 1.18 µg/mL. This result is the starting point of a larger research program within our group that can investigate the introduction of substituted groups on triazine’s terminal amino group. Therefore, the perspectives identified will concern the search for new pharmaco-modulations and structural optimization favorable to the increase of protein kinases inhibitory activity involved in immune pathophysiology, inflammatory or cancerous diseases, to generate lead compounds and be able to conduct studies on relevant in vivo models.

ACKNOWLEDGMENTS : We would like to thank Cancéropôle Grand Ouest for its support for this work as well as the different teams of the network with whom we have had constructive exchanges.

CONFLICT OF INTEREST : None

FINANCIAL SUPPORT : This work was financially supported by the Cooperation and Cultural Action Service of the French Embassy in Côte d'Ivoire (SCAC).

ETHICS STATEMENT : None

References
  1. Reys V, Labesse G. In silico profiling of protein kinase inhibitors. Med Sci. 2020;36:38‑41.
  2. Wilson LJ, Linley A, Hammond DE, Hood FE, Coulson JM, MacEwan DJ, et al. New perspectives, opportunities, and challenges in exploring the human protein kinome. Cancer Res. 2018;78(1):15‑29.
  3. Quesada GY. The regulation of Staufen1 in cell cycle and proliferation [PhD thesis]. Department of Biochemistry and Molecular Medicine, Faculty of Medicine, University of Montreal. 2023, 1-211.
  4. El Jammal T, Gerfaud-Valentin M, Seve P, Jamilloux Y. Inhibitors of JAK/STAT signaling during rheumatological diseases: A growing spectrum. Rheum Rev. 2020;87(4):261‑72.
  5. Sailliet N, Brosseau C, Robert JM, Brouard S. Role of JAK inhibitors and immune cells in transplantation. Cytokine Growth Factor Rev. 2019;47:62‑73.
  6. Ayala-Aguilera CC, Valero T, Lorente-Macías Á, Baillache DJ, Croke S, Unciti-Broceta A. Small molecule kinase inhibitor drugs (1995–2021): Medical indication, pharmacology, and synthesis. J Med Chem. 2022;65(2):1047‑131.
  7. Geier CB, Sauerwein KMT, Leiss-Piller A, Zmek I, Fischer MB, Eibl MM, et al. Hypomorphic mutations in the BCR signalosome lead to selective immunoglobulin M deficiency and impaired B-cell homeostasis. Front Immunol. 2018; 9:2984.
  8. Chen X, Jin J, Wang Q, Xue H, Zhang N, Du Y, et al. A de novo pathogenic CSNK1E mutation identified by exome sequencing in family trios with epileptic encephalopathy. Hum Mutat. 2019;40(3):281‑7.
  9. Narasimamurthy R, Hunt SR, Lu Y, Fustin JM, Okamurae H, Partch CL, et al. CK1δ/ε protein kinase primes the PER2 circadian phosphoswitch. Proc Natl Acad Sci USA. 2018;115(23):5986‑91.
  10. Abramov D, Guiberson NGL, Burré J. STXBP1 encephalopathies: Clinical spectrum, disease mechanisms, and therapeutic strategies. J Neurochem. 2021;157(2):165‑78.
  11. McInnes IB, Gravallese EM. Immune-mediated inflammatory disease therapeutics: Past, present and future. Nat Rev Immunol. 2021;21(10):680‑6.  
  12. David T, Ling SF, Barton A. Genetics of immune-mediated inflammatory diseases. Clin Exp Immunol. 2018;193(1):3‑12.
  13. Yan W, Zhao Y, He J. Anti‑breast cancer activity of selected 1,3,5‑triazines via modulation of EGFR‑TK. Mol Med Rep. 2018;18(5):4175‑84.
  14. Junaid A, Lim FPL, Chuah LH, Dolzhenko AV. N2 -Diaryl-1,3,5-triazine-2,4-diamines: Synthesis, antiproliferative activity and 3D-QSAR modeling. RSC Adv. 2020;10(21):12135‑44.
  15. Gucev Z, Tasic V, Plaseska‐Karanfilska D, Dimishkovska M, Laban N, Bozinovski Z, et al. Severe digital malformations in a rare variant of fibrodysplasia ossificans progressiva. Am J Med Genet A. 2019;179(7):1310‑4.
  16. Kawahata W, Asami T, Kiyoi T, Irie T, Taniguchi H, Asamitsu Y, et al. Design and synthesis of novel amino-triazine analogues as selective bruton's tyrosine kinase inhibitors for treatment of rheumatoid arthritis. J Med Chem. 2018;61(19):8917‑33.
  17. Díaz-Ortiz Á, Elguero J, Foces-Foces C, De La Hoz A, Moreno A, Del Carmen Mateo M, et al. Green synthesis and self-association of 2,4-diamino-1,3,5-triazine derivatives. New J Chem. 2004;28(8):952‑8.
  18. Nagasawa H, Narise K, Okuda K, Hirayama T, Enomoto Y. Optimization of biguanide derivatives as selective antitumor agents blocking adaptive stress responses in the tumor microenvironment. Drug Des Dev Ther. 2014:701-17.
  19. Tsitsa P, Antoniadou-Vyza E, Hamodrakas S, Eliopoulos E, Tsantili-Kakoulidou A, Lada-Hytiroglou E, et al. Synthesis, crystal structure and biological properties of a new series of lipophilic s-triazines, dihydrofolate reductase inhibitors. Eur J Med Chem. 1993;28(2):149-58.
  20. Zegzouti H, Zdanovskaia M, Hsiao K, Goueli SA. ADP-Glo: A Bioluminescent and homogeneous ADP monitoring assay for kinases. Assay Drug Dev Technol. 2009;7(6):560‑72.

 


How to cite this article
Vancouver
Diakité AS, Ambeu-Loko CNM, Yapi AD, Logé C, Kacou A, Kra S, et al. Design and Synthesis of Functionalized 2,4-Diamino-1,3,5-Triazines, Potential Inhibitors Involved in Immune and Inflammatory Response. Int J Pharm Res Allied Sci. 2024;13(4):1-11. https://doi.org/10.51847/hsT2C61XWx
APA
Diakité, A. S., Ambeu-Loko, C. N. M., Yapi, A. D., Logé, C., Kacou, A., Kra, S., Baratte, B., Bach, S., Ruchaud, S., Sissouma, D., et al. (2024). Design and Synthesis of Functionalized 2,4-Diamino-1,3,5-Triazines, Potential Inhibitors Involved in Immune and Inflammatory Response. International Journal of Pharmaceutical Research and Allied Sciences, 13(4), 1-11. https://doi.org/10.51847/hsT2C61XWx
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