HETEROCYCLES

Paper | Regular issue | Vol. 81, No. 4, 2010, pp. 867-882
Received, 2nd December, 2009, Accepted, 8th February, 2010, Published online, 9th February, 2010.
DOI: 10.3987/COM-09-11877

■ Synthesis of Bis-Aryl Phosphates Based on Triazine Scaffold

Caroline Courme, Nohad Gresh, Christine Lenoir, Michel Vidal, Christiane Garbay, Jean-Claude Florent, and Emmanuel Bertounesque*

Medicinal Chemistry, C.N.R.S. UMR 176, 26 rue d’Ulm, Paris 75005, France

Abstract

In view of targeting the Grb2-SH2 signaling protein, the synthesis of potential ligands based on the 1,3,5-triazine scaffold bearing two phenylphosphate groups is described. The triazine functionality was introduced using an orthogonal strategy via the sulfone chemistry.

INTRODUCTION
The 1,3,5-triazine scaffold has occupied an important position in drug discovery programs, especially since Fesik identified it as a privileged structure using screening by NMR.1 Recent examples illustrate the value of 1,3,5-triazine derivatives for cytotoxic activity,2 antitumoral activity,3 inhibition of angiogenesis,4 antimicrobial activity5 and anti-HIV activity.6 The use of this molecular scaffold would be also of interest to design inhibitors of the Grb2 (growth factor receptor bound-protein 2) adaptor protein. Grb2 is involved in RTK signal transduction via the binding of the SH2 domain to phosphotyrosines on the C-terminal end of the activated receptors. Thus, it is implicated in Ras activation but can also play a role in other signaling pathways in mammalian cells (e.g., PI3K signaling pathway). There has been tremendous interest in the development of inhibitors of the SH2 domain of Grb2 as therapeutic agents in treating cancers.7 Much of the effort in designing active site directed Grb2-SH2 inhibitors has focused on the synthesis of peptidic compounds containing phosphotyrosine (pTyr) or phosphotyrosine mimics.8 To date, only a limited number of non-peptidic antagonists of Grb-SH2 was reported from the low micromolar range to the high micromolar range, i.e., urea 1,9 thiazole 2,10 tetrahydropyrimidinone 311 and triazine 412 (Figure 1). The discovery of the latter class of compounds should facilitate the development of more drug-like inhibitors for the treatment of cancers.

In the context of our ongoing research on Grb2 targeting,12-14 we report herein the synthesis of novel triazine scaffold-based bis-aryl phosphates 5-7 (Figure 2), the design of which was inspired by the structure of triazine 4, using a flexible synthetic strategy. We have sought to replace the 4-phenoxyphenyl substructure by the simpler 4-aminomethylphenyl one. For the three compounds, MD simulations showed that this modification enabled to dock, on the one hand, its linked phosphate group into the pTyr-binding pocket encompassing residues Arg 86, Ser 88, Ser 90, Ser 96 and Lys 109; and, on the other hand, the aniline-bound phosphate into the second pTyr pocket, involving residues Arg 142, Asn 143, and Ser 141. In particular, it appeared interesting to introduce a urea functionality on the triazine framework so as to obtain hydrogen-bonding interactions with the main-chain carbonyl of Leu 120 and amide of Lys 109 in the specificity pocket.

RESULTS AND DISCUSSION
The bis-aryl phosphates 5-7 were synthetized using the orthogonal strategy developped by Chang et al.15 from cyanuric chloride (Scheme 1), wherein the last substitution step takes place via an oxidation-activation of the benzylthioether group of 2-(benzylthio)-4,6-dichloro-1,3,5-triazine 9.

The synthesis of aminotriazine 5 began with a one-pot sequential procedure, involving addition of benzyl mercaptan to cyanuric chloride followed by that of 4-aminophenol to give 10 in 82% yield (Scheme 2).

Subsequent addition of 4-hydroxybenzylamine furnished 11 (75% overall yield). The latter has also been synthesized in a one-pot synthesis from cyanuric chloride but in a lower overall yield (61%). Phosphorylation of 11 with dibenzylchlorophosphate (in situ prepared from dibenzylphosphite) gave 12 in 54% yield.16 Oxidation of the benzylsulfanyl group with MCPBA led to the desired sulfone 13 in 79% yield. Then, a two-step sequence (i. e., substitution of ammonia and hydrogenation over Pd/C) allowed to obtain amino-triazine 5 of very low solubility which was finally converted into the water-soluble sodium salt 15. In order to access to urea-triazine 6, preliminary investigation showed that the formation of the urea group from 14 failed, due to its unreactivity, under classical conditions (KOCN 1-2 eq, AcOH/H2O or MeOH/1M HCl, rt or 50 °C, 2 h). The clivage of a dibenzylphosphate group was observed when prolonging the reaction time (4 h) with 2.5 eq of KOCN.

Additionally, benzylation prior to the phosphorylation reaction led us to the synthesis of N2-(4-(benzyloxy)benzyl)-N4-(4-(benzyloxy)phenyl)-1,3,5-triazine-2,4,6-triamine 19 (Scheme 3). The latter was accessible via a four-step sequence from 10, involving the preparation of (4-(benzyloxy)phenyl)methanamine.17 Triazine 19 was also found unaffected, even under strong conditions (KOCN 2.4 eq, AcOH/H2O, reflux, 16 h). Moreover, formation of the urea 20 from this protected aminotriazine was also unsuccessful using 1-nitrourea (H2SO4 cc 0.05%, EtOH, reflux, 2 h or H2SO4 cc 4-8%, EtOH, 80 °C, 16 h). Finally, the sulfone derivative 18 was allowed to react with cyanamide in refluxing tetrahydrofuran, affording the desired compound 21. Subsequent hydrolysis using sulfuric acid at high temperature led to the corresponding urea 20 which underwent deprotection of the two benzyl groups by hydrogenolysis to give 22 as a precursor of 6.

The introduction of the carboxamide group was also carried out starting from sulfonyle 18 (Scheme 3). Treatment of 18 with potassium cyanide in dimethylformamide at 80 °C led to nitrile 23. Note that cyanation from 13 was possible but much less satisfying in terms of yield (KCN 2.5 eq, DMF, 80 °C, 3 h, 43%). Hydrolysis of 23 using the method described by Dragovich et al.18 (sodium hydroxide and hydrogen peroxide at 60 °C) gave the corresponding carboxamide derivative 24. Hydrogenation over Pd/C then gave compound 25.

Phosphorylation of both derivatives 22 and 25 gave 26 and 27, respectively (Scheme 4). Following catalytic hydrogenation, the resulting bis-phosphates (6 and 7, resp.) of very low solublity were directly transformed into the sodium salts 28 and 29, respectively.

Bis-aryl phosphate sodium salts 15, 28 and 29 were assayed for binding to Grb2 using an ELISA test.19 The phosphorylated peptide PSpYVNVPQ (Kd = 9 nM) was used for this competitive binding assay. None of the designed compounds were found to be antagonists of the SH2 domain of Grb2 contrary to earlier predictions from MD simulations of their complexes (unpublished) in the Grb2 SH2 domain structure with implicit solvent. These results reassert the importance of accounting for explicit water molecules in the MD simulations. Water probably plays a critical role in the stability of such Grb2 complexes with peptide and non-peptidic inhibitors as shown recently by Leroux et al.20

We have synthesized novel bis-aryl phosphates based on the privileged structure triazine, using an orthogonal strategy from cyanuric chloride, via the sulfone chemistry. In order to strengthen the 1,3,5-trisubstituted triazine scaffold-based ligand design strategy that exploits the use of mimics of phosphates (e.g., keto-acid, IZD),12 further molecular modeling of such compounds through MD simulations in water should be also considered.

EXPERIMENTAL
All commercial reagents were used without purification and all solvents were reaction grade. When necessary, solvents were previously dried over molecular sieves. Tetrahydrofuran was also dried over molecular sieves 4 Å unless otherwise stated (distilled from sodium/benzophenone under argon). All reactions were performed under an inert atmosphere of argon unless otherwise stated. All reaction mixtures were stirred magnetically and monitored by thin-layer chromatography using Merck silica gel 60 F254, visualized with UV light. Flash chromatography were performed using SDS silica gel 60 (35-70 µm). Melting points (uncorrected) were determined on a Kofler bench. Mass spectra were recorded on a ZQ 2000 MS (ES) or a spectrometer (LC) HRMS ESI/TOF (LCT, Waters).

1H, 13C and 31P NMR spectra were recorded on a Bruker AM 300 spectrometer. Chemical shift values are reported in parts per million (ppm) and coupling constants in Hertz (Hz). In order to simplify the reading of the NMR spectra interpretations (COSY, HMQC, HMBC), the following attributions were chosen (Scheme 5): Letter "A" will always refer to the heterocyclic core – whether it is a pyrimidine or a triazine – and letter "B" to the aryl group linked to this heterocycle through an NH bridge. "C" and "D" will refer to the diaryl ether and finally "E" will refer to the benzyl groups, either benzyloxy or dibenzylphosphates. Two AA’BB’ systems were observed for the two para-substituted aromatic rings B and D. Compounds 11, 12 and 17 are described below as examples.

4-(4-Benzylthio-6-chloro-1,3,5-triazin-2-ylamino)phenol (10)
Benzylthiol (6 mL, 51.0 mmol) and diisopropylethylamine (9 mL, 51.7 mmol) were added to a solution of cyanuric chloride (10 g, 54.2 mmol) in dry THF (200 mL) at 0 °C. After 2 h of stirring at 0 °C, 4-aminophenol (5.9 g, 54.1 mmol) and diisopropylethylamine (9.5 mL, 54.5 mmol) were added. The mixture was then stirred at room temperature for 3 h and the solvent was removed under reduce pressure. Purification by flash chromatography with cyclohexane/EtOAc (4:1) gave 10 as a white solid (14.4 g, 82%). Mp 179 °C; 1H NMR (300 MHz; MeOD): δ 4.28 (2H, s, CH2), 6.75-6.78 (2H, m, 3C, 5C), 7.21-7.23 (3H, m, 3B, 4B, 5B), 7.28-7.31 (2H, m, 2C, 6C), 7.35-7.43 (2H, m, 2B, 6B); 13C NMR (75 MHz; MeOD): δ 34.8 (CH2), 115.6 (3C, 5C), 123.5 (4B), 124.1 (3B, 5B), 127.5 (1C), 128.6 (2C, 6C), 129.0 (2B, 6B), 136.4 (1B), 154.6 (4C), 162.7 (4A), 168.2 (6A), 183.2 (2A); MS (ES+) m/z 345 [M+H]+, 367 [M+Na]+; MS (ES–) m/z 343 [M–H]–; HRMS (ES+) m/z: calcd for C16H13ClN4OS 345.0577 [M+H]+, 367.0396 [M+Na]+; found 345.0583, 367.0404.

4-[4-Benzylthio-6-(4-hydroxybenzylamino)-1,3,5-triazin-2-ylamino]phenol (11)
Compound 10 (234 mg, 0.679 mmol) and 4-(aminomethyl)phenol (91 mg, 0.739 mmol) were dissolved in dry THF. Diisopropylethylamine (140 µL, 0.804 mmol) was added and the mixture was refluxed for 36 h. The solvent was removed under reduce pressure. Purification by flash chromatography with cyclohexane/EtOAc (gradient from 4:1 to 1:1) gave 11 as a white solid (270 mg, 92%). Mp 94-97 °C; 1H NMR (300 MHz; MeOD): δ 4.31 (2H, s, S-CH2), 4.42 (2H, s, NH-CH2), 6.72 (4H, br s, 3C, 5C, 3D, 5D), 7.11-7.14 (2H, m, 2D, 6D), 7.20-7.28 (5H, m, 2B, 3B, 4B, 5B, 6B), 7.31-7.33 (2H, m, 2C, 6C); 13C NMR (75 MHz; MeOD): δ 34.7 (S-CH2), 44.8 (NH-CH2), 116.1 (3C, 5C, 3D, 5D), 124.5 (2C, 6C), 129.4, 129.7, 130.0 (2B, 3B, 4B, 5B, 6B, 2D, 6D), 131.5 (1C), 132.1 (1D), 139.5 (1B), 154.8 (4C), 157.4 (4D), 164.2 (4A), 165.6 (6A), 180.9 (2A); MS (ES+) m/z 432 [M+H]+, 454 [M+Na]+; MS (ES–) m/z 430 [M–H]–.

Dibenzyl 4-[4-benzylthio-6-(4-dibenzylphosphoryloxybenzylamino)-1,3,5-triazin-2-ylamino]phenyl phosphate (12)
To a solution of 11 (4 g, 9.27 mmol) in dry MeCN (200 mL) cooled at –15 °C was added dry CCl4 (40 mL) and the mixture was stirred at –15 °C for 5 min. Diisopropylethylamine (12.9 mL, 74.1 mmol) and N,N-dimethylaminopyridine (1.13 g, 9.27 mmol) were then added and the mixture was stirred at –15 °C for 30 min. Dibenzylphosphite (8.2 mL, 37.1 mmol) was added dropwise and the mixture was stirred at –15 °C for 1 h 30. A 0.5 M solution of potassium dihydrogen phosphate (50 mL) was added, the mixture was warmed up to room temperature and extracted with EtOAc. The combined extracts were then washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. Purification by flash chromatography with CH2Cl2/MeOH (99:1) afforded 12 as a colourless oil (4.74 g, 54%). 1H NMR (300 MHz; CDCl3): δ 4.33 (2H, s, S-CH2), 4.55 (2H, m, NH-CH2), 5.10 (m, 4H, 2 × P-O-CH2), 5.12 (m, 4H, 2 × P-O-CH2), 7.04-7.11 (4H, m, 3C, 5C, 3D, 5D), 7.20-7.26 (2H, m, 2D, 6D), 7.24-7.31 (25H, m, 4 × 2E, 4 × 3E, 4 × 4E, 4 × 5E, 4 × 6E, 2B, 3B, 4B, 5B, 6B), 7.40-7.43 (m, 2H, 2C, 6C); 13C NMR (75 MHz; CDCl3): δ 34.1 (S-CH2), 44.1 (NH-CH2), 69.9 (P-O-CH2), 120.1, 120.2 (3C, 5C, 3D, 5D), 121.7 (2C, 6C), 127.1, 128.0, 128.4, 128.5, 128.6, 128.7, 128.8 (2E, 3E, 4E, 5E, 6E, 2B, 3B, 4B, 5B, 6B, 2D, 6D), 135.2, 135.3, 135.4 (1C, 1D, 1E), 137.4 (1B), 146.0 (4C), 149.7 (4D), 162.7 (4A), 164.5 (6A), 180.3 (2A).

Dibenzyl 4-[4-benzylsulfonyl-6-(4-dibenzylphosphoryloxybenzylamino)-1,3,5-triazin-2-ylamino]phenyl phosphate (13)
MCPBA (430 mg, 2.49 mmol) was added portionwise over 20 min to a solution of thioether 12 (1 g, 1.05 mmol) in THF (50 mL) at 0 °C under air. The mixture was stirred at 0 °C for 2 h and at room temperature for 15 min and a saturated aqueous sodium hydrogen carbonate solution was added (50 mL). The product was extracted with EtOAc, the combined extracts were then washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. Purification by flash chromatography with CH2Cl2/MeOH (100:1.5) afforded 13 as a beige powder (820 mg, 79%). Mp 47 °C; 1H NMR (300 MHz; CDCl3): δ 4.54 (2H, s, NH-CH2), 4.61 (2H, s, SO2-CH2), 5.10 (4H, s, 2 × P-O-CH2), 5.12 (4H, s, 2 × P-O-CH2), 7.11 (4H, m, 3C, 5C, 3D, 5D), 7.17 (2H, m, 2D, 6D), 7.31 (25H, br s, 4 × 2E, 4 × 3E, 4 × 4E, 4 × 5E, 4 × 6E, 2B, 3B, 4B, 5B, 6B), 7.37-7.45 (4H, m, 2C, 6C); 13C NMR (75 MHz; CDCl3): δ 44.6 (NH-CH2), 56.7 (SO2-CH2), 70.0 (P-O-CH2), 70.1 (P-O-CH2), 120.4, 120.5 (3C, 5C, 3D, 5D), 122.0 (2C, 6C), 126.6 (2B, 6B), 128.0 (2E, 6E), 128.6, 128.7, 128.8 (3E, 4E, 5E, 2D, 6D, 3B, 4B, 5B), 131.4 (1B), 134.0 (1D), 134.3 (1C), 135.4 (1E), 147.0 (4C), 150.0 (4D), 163.7 (4A), 165.5 (6A), 172.4 (2A); MS (ES+) m/z 1001 [M+NH4]+, 1006 [M+Na]+.

4-[4-Amino-6-(4-dibenzylphosphoryloxybenzylamino)-1,3,5-triazin-2-ylamino]phenyl dibenzyl phosphate (14)
Ammonia was bubbled for 1 h into a solution of compound 13 (298 mg, 0.303 mmol) in CH2Cl2 (15 mL) at room temperature. The flask was then sealed and the mixture was stirred for 15 h. The solvent was removed under reduce pressure and purification by flash chromatography with CH2Cl2/MeOH (100:4) gave 14 as a white solid (184 mg, 72%). Mp 114-117 °C; 1H NMR (300 MHz; CDCl3): δ 4.53 (2H, s, NH-CH2), 5.09 (4H, s, 2 × P-O-CH2), 5.11 (4H, s, 2 × P-O-CH2), 7.04-7.10 (4H, m, 3C, 5C, 3D, 5D), 7.21-7.24 (2H, m, 2D, 6D), 7.31 (20H, br s, 4 × 2E, 4 × 3E, 4 × 4E, 4 × 5E, 4 × 6E), 7.42-7.45 (2H, m, 2C, 6C); 13C NMR (75 MHz; CDCl3): δ 44.1 (NH-CH2), 70.0 (P-O-CH2), 70.1 (P-O-CH2), 120.3, 120.4 (3C, 5C, 3D, 5D), 121.5 (2C, 6C), 128.1 (2E, 6E), 128.7 (3E, 4E, 5E), 128.8 (2D, 6D), 135.5 (1E), 135.9, 136.1 (1D, 1C), 146.0 (4C), 149.8 (4D), 164.7 (4A), 166.5 (6A, 2A); MS (ES+) m/z 844 [M+H]+, 867 [M+Na]+.

4-[4-Amino-6-(4-phosphoryloxybenzylamino)-1,3,5-triazin-2-ylamino]phenyl phosphate (5)
10% palladium on carbon (60 mg) was added to a solution of compound 14 (121 mg, 0.143 mmol) in MeOH (50 mL). The mixture was stirred under H2 at room temperature for 15 h. The catalyst was filtered using two filter papers and washed with hot MeOH, affording 5 as a white solid (25 mg, 36%). Mp 216-218 °C with decomposition; MS (ES–) m/z 241 [M–2H]2–.

Tetrasodium 4-[4-amino-6-(4-phosphonatooxybenzyl amino)-[1,3,5]-triazin-2-ylamino]phenyl phosphate (15)
A fresh 0.539 M sodium methoxide solution (276 µL, 149 µmol) was added to a suspension of compound 5 (18 mg, 37.2 µmol) in dry MeOH (2.5 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h and the MeOH was removed under reduced pressure to afford 15 as a water-soluble white powder (21 mg, 99%). 1H NMR (300 MHz; D2O): δ 4.37 (2H, s, NH-CH2), 7.06 (4H, br s, 3C, 5C, 3D, 5D), 7.16-7.19 (4H, m, 2C, 6C, 2D, 6D); 13C NMR (75 MHz; D2O): δ 43.3 (NH-CH2), 120.5, 120.6 (3C, 5C, 3D, 5D), 124.4 (2C, 6C), 127.9 (2D, 6D), 132.0 (1C), 132.7 (1D), 150.8, 152.9 (4C, 4D), 164.6 (4A), 165.6 (6A), 166.8 (2A); 31P NMR (121 MHz; D2O): δ 0.51; HRMS (ES–) m/z: calcd for C16H14N6Na4O8P2 527.0222 [M–2Na+H]–, 505.0403 [M–3Na+2H] –; found 527.0247, 505.0428.

4-[4-(4-Benzyloxybenzylamino)-6-benzylthio-1,3,5-triazin-2-ylamino]phenol (16)
Compound 10 (664 mg, 1.93 mmol) and (4-benzyloxyphenyl)methanamine (821 mg, 3.85 mmol) were dissolved in a THF/MeOH 5:1 mixture (18 mL). Diisopropylethylamine (670 µL, 3.85 mmol) was added and the mixture was refluxed for 15 h. The solvent was removed under reduce pressure and purification by flash chromatography with cyclohexane/EtOAc (4:1) gave 16 as a white solid (926 mg, 92%). Mp 117 °C; 1H NMR (300 MHz; CDCl3): δ 4.32 (2H, s, S-CH2), 4.51 (2H, s, NH-CH2), 5.03 (2H, s, O-CH2), 5.60 (1H, br s, NH-CH2), 6.71 (2H, s, 3C, 5C), 6.60-6.92 (2H, m, 3D, 5D), 7.17-7.19 (2H, m, 2D, 6D), 7.23-7.40 (12H, m, 2C, 6C, 2B, 3B, 4B, 5B, 6B, 2E, 3E, 4E, 5E, 6E); 13C NMR (75 MHz; CDCl3): δ 34.1 (S-CH2), 44.3 (NH-CH2), 70.1 (O-CH2), 115.0 (3D, 5D), 115.6 (3C, 5C), 123.0 (2C, 6C), 127.1 (4B), 127.5 (2E, 6E), 128.0 (4E), 128.5, 128.6 (3B, 5B, 3E, 5E), 128.8, 128.9 (2D, 6D, 2B, 6B), 130.7 (1D), 131.0 (1C), 136.9 (1E), 136.6 (1B), 152.2 (4C), 158.1 (4D), 162.9 (4A), 164.2 (6A), 179.5 (2A); MS (ES+) m/z 522 [M+H]+, 544 [M+Na]+, MS (ES–) m/z 520 [M–H]–.

N2-(4-Benzyloxybenzyl)-N4-(4-benzyloxyphenyl)-6-benzylthio-1,3,5-triazine-2,4-diamine (17)
Potassium carbonate (1.9 g, 13.7 mmol) was added to a solution of 16 (4.72 g, 9.05 mmol) in dry DMF (200 mL). Benzyl bromide (1.6 mL, 13.5 mmol) was then added dropwise at 0 °C and the mixture was stirred at 0 °C for 1 h and then at room temperature for 1 h. Water was added and the mixture was extracted with Et2O. The combined extracts were washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. Purification by flash chromatography with cyclohexane/EtOAc (5:1) gave 17 as a white powder (4.18 g, 75%). Mp 45-47 °C; 1H NMR (300 MHz; CDCl3): δ 4.33 (2H, s, S-CH2), 4.54 (2H, s, NH-CH2), 5.04 (4H, s, O-CH2), 5.61 (1H, br s, NH-CH2), 6.89-6.94 (4H, m, 3C, 5C, 3D, 5D), 7.02 (1H, br s, NH), 7.23-7.26 (5H, m, 2D, 6D, 3B, 4B, 5B), 7.35-7.41 (14H, m, 2C, 6C, 2B, 6B, 2 × 2E, 2 × 3E, 2 × 4E, 2 × 5E, 2 × 6E); 13C NMR (75 MHz; CDCl3): δ 34.3 (S-CH2), 44.5 (NH-CH2), 70.2 (O-CH2), 115.2 (3C, 5C, 3D, 5D), 122.8 (2C, 6C), 127.2 (4B), 127.6 (2E, 6E), 128.1 (4E), 128.6, 128.7 (2B, 6B, 2D, 6D, 3E, 5E), 129.1 (3B, 5B), 130.9 (1D), 131.8 (1C), 137.1 (1E), 137.5 (1B), 155.3 (4C), 158.3 (4D), 162.9 (4A), 164.6 (6A), 178.7 (2A).

N2-(4-Benzyloxybenzyl)-N4-(4-benzyloxyphenyl)-6-benzylsulfonyl-1,3,5-triazine-2,4-diamine (18)
Sulfone 18 was prepared as sulfone 13, from compound 17 (4.2 g, 6.87 mmol) and MCPBA (3.6 g, 20.9 mmol). Purification via flash chromatography with cyclohexane/EtOAc (5:1) afforded 18 as an off-white solid (3.5 g, 79%). Mp 144-151 °C; 1H NMR (300 MHz; CDCl3): δ 4.53 (2H, s, NH-CH2), 4.62 (2H, s, SO2-CH2), 5.06 (4H, s, O-CH2), 5.86 (1H, br s, NH-CH2), 6.95 (4H, s, 3C, 5C, 3D, 5D), 7.19-7.22 (2H, m, 2D, 6D), 7.31-7.41 (17H, m, 2C, 6C, 2B, 3B, 4B, 5B, 6B, 2 × 2E, 2 × 3E, 2 × 4E, 2 × 5E, 2 × 6E); 13C NMR (75 MHz; CDCl3): δ 44.9 (NH-CH2), 56.4 (SO2-CH2), 70.1 (O-CH2), 115.1, 115.2 (3C, 5C, 3D, 5D), 122.5 (2C, 6C), 126.6 (1B), 127.4 (2E, 6E), 128.0 (4E), 128.6, 128.8 (3B, 4B, 5B, 2D, 6D, 3E, 5E), 129.1, 129.5 (1C, 1D), 131.4 (2B, 6B), 136.8 (1E), 156.0 (4C), 158.4 (4D), 163.8 (4A), 165.6 (6A), 172.0 (2A); MS (ES+) m/z 666 [M+Na]+.

N2-(4-Benzyloxybenzyl)-N4-(4-benzyloxyphenyl)-1,3,5-triazine-2,4,6-triamine (19)
Ammonia was bubbled for 1 h into a solution of sulfone 18 (2 g, 3.11 mmol) in CH2Cl2 (100 mL) at room temperature. The flask was then sealed and the mixture was stirred for 15 h. The solvent was removed under reduce pressure and purification by flash chromatography with CH2Cl2/MeOH (gradient from 100:2 to 100:5) gave 19 as a white powder (1.12 g, 72%). Mp 77 °C; 1H NMR (300 MHz; CDCl3): δ 4.48 (2H, s, NH-CH2), 5.02 (4H, s, O-CH2), 5.81 (1H, br s, NH-CH2), 6.87-6.93 (4H, m, 3C, 5C, 3D, 5D), 7.19-7.22 (2H, m, 2D, 6D), 7,29-7,43 (12H, m, 2C, 6C, 2 × 2E, 2 × 3E, 2 × 4E, 2 × 5E, 2 × 6E); 13C NMR (75 MHz; CDCl3): δ 44.1 (N-CH2), 70.0 (O-CH2), 114.9, 115.0 (3C, 5C, 3D, 5D), 122.4 (2C, 6C), 127.4 (2E, 6E), 127.9 (4E), 128.5 (3E, 5E), 128.7 (2D, 6D), 131.2 (1D), 132.0 (1C), 136.9 (1E), 154.9 (4C), 158.0 (4D), 164.5 (4A), 166.1 (6A, 2A); MS (ES+) m/z 505 [M+H]+, 527 [M+Na]+; MS (ES–) m/z 503 [M–H]–.

N-[4-(4-Benzyloxybenzylamino)-6-(4-benzyloxyphenylamino)-1,3,5-triazin-2-yl]cyanamide (21)
Sulfone 18 (707 mg, 1.10 mmol) was dissolved in THF (100 mL). Cyanamide (461 mg, 11.0 mmol) and diisopropylethylamine (380 µL, 2.18 mmol) were added and the mixture was refluxed overnight. THF was removed under reduce pressure, water was added (50 mL) and the product was extracted with EtOAc. The combined extracts were then washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure to give an off-white powder (1.05 g). This crude product (21) proved to be highly insoluble and was hence used in the next step without purification. MS (ES+) m/z 530 [M+H]+, 552 [M+Na]+, MS (ES–) m/z 528 [M–H]–.

1-[4-(4-Benzyloxybenzylamino)-6-(4-benzyloxyphenylamino)-1,3,5-triazin-2-yl]urea (20)
The crude product 21 (826 mg) was placed into a 4 N HCl/EtOH mixture (1:1, 50 mL). The mixture was heated at 90 °C for 16 h then neutralized with 1 N NaOH. The product was extracted with EtOAc. The combined extracts were then washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure to give a white powder (477 mg). As in the previous reaction, compound 20 was insoluble and therefore used in the next step without purification. MS (ES+) m/z 548 [M+H]+.

1-[4-(4-Hydroxybenzylamino)-6-(4-hydroxyphenylamino)-1,3,5-triazin-2-yl]urea (22)
10% Palladium on carbon (230 mg) was added to a suspension of crude compound 20 (477 mg) in a MeOH/AcOH mixture (1:1, 250 mL). The mixture was stirred under an hydrogen atmosphere at room temperature for 16 h. The catalyst was filtered and the product was purified by flash chromatography with CH2Cl2/MeOH (100:8) to give 22 as a white powder (133 mg, 42% yield over 3 steps). Mp 264 °C; 1H NMR (300 MHz; DMSO-d6): δ 4.32 (2H, s, NH-CH2), 6.64-6.67 (2H, m, 3C, 5C), 6.68-6.71 (2H, m, 3D, 5D), 7.09-7.12 (2H, m, 2D, 6D), 7.38-7.41 (2H, m, 2C, 6C), 9.13 (1H, br s, 4C-OH), 9.28 (1H, br s, 4D-OH); 13C NMR (75 MHz; DMSO-d6): δ 43.1 (NH-CH2), 114.9 (3C, 5C, 3D, 5D), 121.7 (2C, 6C), 128.5 (2D, 6D), 129.8 (1D), 131.0 (1C), 152.9 (4C), 156.0 (CO), 156.2 (4D), 163.0, 164.9, 165.1 (2A, 4A, 6A); MS (ES+) m/z 368 [M+H]+, 390 [M+Na]+.

1-[4-(4-Dibenzylphosphoryloxybenzylamino)-6-(4-dibenzylphosphoryloxyphenylamino)-1,3,5- triazin-2-yl]urea (26)
To a solution of 22 (60 mg, 0.163 mmol) in dry MeCN (10 mL) cooled at –15 °C was added dry CCl4 (2 mL) and the mixture was stirred at –15 °C for 5 min. Diisopropylethylamine (230 µL, 1.32 mmol) and N,N-dimethylaminopyridine (20 mg, 0.164 mmol) were then added and the mixture was stirred at –15 °C for 30 min. Dibenzylphosphite (180 µL, 0.815 mmol) was added dropwise and the mixture was stirred at –15 °C for 2 h. A 0.5 M solution of potassium dihydrogen phosphate (5 mL) was added, the mixture was warmed up to room temperature and extracted with EtOAc. The combined extracts were then washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. Purification by flash chromatography with CH2Cl2/MeOH (gradient from 100:2 to 100:5) afforded 26 as a colorless oil (89 mg, 61%). 1H NMR (300 MHz; CDCl3): δ 4.44 (2H, s, NH-CH2), 5.08 (4H, s, P-O-CH2), 5.10 (4H, s, P-O-CH2), 7.03-7.10 (4H, m, 3C, 5C, 3D, 5D), 7.22-7.26 (2H, m, 2D, 6D), 7.30 (20H, br s, 4 × 2E, 4 × 3E, 4 × 4E, 4 × 5E, 4 × 6E), 7.56-7.67 (m, 2H, 2C, 6C); 13C NMR (75 MHz; CDCl3): δ 44.3 (NH-CH2), 69.9 (P-O-CH2), 70.0 (P-O-CH2), 120.1, 120.5 (3C, 5C, 3D, 5D), 122.4 (2C, 6C), 128.0, 128.3, 128.6 (2E, 3E, 4E, 5E, 6E, 2D, 6D), 135.4 (1C, 1D, 1E), 147.0 (4C), 149.9 (4D), 157.1 (CO), 163.0, 163.3 (2A, 4A, 6A); MS (ES+) m/z 888 [M+H]+, 910 [M+Na]+.

1-[4-(4-Phosphoryloxybenzylamino)-6-(4-phosphoryloxyphenylamino)-1,3,5-triazin-2-yl]urea (6)
10% Palladium on carbon (41 mg) was added to a solution of compound 26 (83 mg, 93.4 µmol) in MeOH (30 mL). The mixture was stirred under an hydrogen atmosphere at room temperature overnight. The catalyst was filtered using two filter papers, then washed with both hot MeOH and hot water. Purification by flash chromatography on reverse phase silica gel C18 with water/MeCN (95:5) gave 6 as a white solid (30 mg, 61%). Mp 209-211 °C with decomposition; MS (ES–) m/z 262 [M–2H]2–.

Sodium 1-[4-(4-phosphonatooxybenzylamino)-6-(4-phosphonatooxyphenylamino)-1,3,5-triazin-2-yl]urea (28)
A fresh 0.539 M sodium methoxide solution (205 µL, 111 µmol) was added to a suspension of compound 6 (14 mg, 27.7 µmol) in dry MeOH (3 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h and the MeOH was removed under reduced pressure to afford 28 as a water-soluble white powder (17 mg, 100%). 1H NMR (300 MHz; D2O): δ 4.37 (2H, s, NH-CH2), 7.03 (4H, br s, 3C, 5C, 3D, 5D), 7.15 (2H, br s, 2D, 6D), 7.19-7.22 (2H, m, 2C, 6C); 13C NMR (75 MHz; D2O): δ 43.5 (NH-CH2), 120.6 (3C, 5C, 3D, 5D), 124.1 (2C, 6C), 127.4 (2D, 6D), 130.6 (1C), 132.6 (1D), 150.4 (4C), 152.9 (4D), 157.5, 159.0 (CO, 2A, 4A), 164.4 (6A) 31P NMR (121 MHz; D2O): δ 0.52; HRMS (ES–) m/z: calcd for C17H15N7Na4O9P2 592.0100 [M–Na]–; found 592.0108.

4-(4-Benzyloxybenzylamino)-6-(4-benzyloxyphenylamino)-1,3,5-triazine-2-carbonitrile (23) Potassium cyanide (455 mg, 6.99 mmol) was cautiously added to a solution of compound 18 (1.5 g, 2.33 mmol) in dry DMF (80 mL). The mixture was heated at 80 °C for 3 h and the solvent was removed under high vacuum. Water was added and the mixture was extracted with Et2O. The combined extracts were washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure to afford 23 as a yellow powder (1.15 g, 96%). This product was used in the next step without purification. Mp 147 °C; 1H NMR (300 MHz; CDCl3): δ 4.29 (2H, s, NH-CH2), 5.06 (4H, s, O-CH2), 6.95 (4H, s, 3C, 5C, 3D, 5D), 7.22-7.24 (2H, m, 2D, 6D), 7.36-742 (12H, m, 2C, 6C, 2 × 2E, 2 × 3E, 2 × 4E, 2 × 5E, 2 × 6E); 13C NMR (75 MHz; CDCl3): δ 44.7 (NH-CH2), 70.2 (O-CH2), 115.3 (3C, 5C, 3D, 5D), 123.0, 123.3 (2C, 6C, CN), 127.6 (2E, 6E), 128.2 (4E), 128.8 (3E, 5E), 129.0 (2D, 6D), 129.4 (1D), 130.3 (1C), 136.9 (1E), 156.0 (4C), 158.5 (4D), 163.5 (4A), 165.1 (6A), 176.4 (2A) ; MS (ES+) m/z 515 [M+Na]+. HRMS (ES+) m/z: calcd for C31H26N6O2 537.2015 [M+Na]+; found 537.2014.

4-(4-Benzyloxybenzylamino)-6-(4-benzyloxyphenylamino)-1,3,5-triazine-2-carboxamide (24)
A solution of compound 23 (150 mg, 0.291 mmol) in a 1 N NaOH/ EtOH/ 30% H2O2 mixture (6:3:1, 10 mL) was heated at 60 °C overnight. EtOH was removed under reduce pressure, the mixture was neutralized with 1 N HCl and the product was extracted with CH2Cl2. The combined extracts were washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. Purification by flash chromatography with CH2Cl2/MeOH (gradient from 100:2 to 100:9) gave 24 as a beige solid (100 mg, 64%). Mp 222 °C; 1H NMR (300 MHz; CDCl3): δ 4.43 (2H, m, NH-CH2), 5.07 (4H, s, O-CH2), 6.94 (4H, m, 3C, 5C, 3D, 5D), 7.22-7.43 (12H, m, 2D, 6D, 2 × 2E, 2 × 3E, 2 × 4E, 2 × 5E, 2 × 6E), 7.57 (2H, m, 2C, 6C); 13C NMR (75 MHz; CDCl3): δ 42.9 (NH-CH2), 69.2 (O-CH2), 114.6 (3C, 5C, 3D, 5D), 121.4 (2C, 6C), 127.6 (2E, 6E), 127.7 (4E), 128.4 (3E, 5E), 128.8 (2D, 6D), 131.6 (1D), 132.5 (1C), 137.1 (1E), 153.9 (4C), 157.2 (4D), 163.9, 165.0 (4A, CONH2), 165.7 (6A), 166.1 (2A); MS (ES+) m/z 532 [M+H]+, 555 [M+Na]+. HRMS (ES+) m/z: calcd for C31H28N6O3 555.2121 [M+Na]+; found 555.2115.
4-(4-Hydroxybenzylamino)-6-(4-hydroxyphenylamino)-1,3,5-triazine-2-carboxamide (25)
10% Palladium on carbon (77 mg) was added to a solution of compound 24 (770 mg, 1.45 mmol) in a MeOH/AcOH mixture (1:1, 150 mL). The mixture was stirred under H2 at room temperature for 36 h. The catalyst was filtered and the product was purified by flash chromatography with MeCN/water (100:2.5) to afford 25 as pale yellow oil (322 mg, 63%). 1H NMR (300 MHz; MeOD): δ 4.46 (2H, s, NH-CH2), 6.71-6.75 (4H, m, 3C, 5C, 3D, 5D), 7.14-7.19 (2H, m, 2D, 6D), 7.37-7.40 (2H, m, 2C, 6C); 13C NMR (75 MHz; MeOD): δ 45.1 (NH-CH2), 116.2, 116.3 (3C, 5C, 3D, 5D), 123.5 (2C, 6C), 129.8 (2D, 6D), 131.0, 132.0 (1D, 1C), 154.8 (4C), 157.6 (4D), 165.7 (4A, CONH2), 167.5 (6A), 167.8 (2A); MS (ES+) m/z 353 [M+H]+, 375 [M+Na]+.

Dibenzyl 4-[4-(4-(dibenzylphosphoryloxy)benzylamino)-6-carbamoyl-1,3,5-triazin-2-ylamino]phenyl phosphate (27)
To a solution of 25 (322 g, 0.914 mmol) in dry MeCN (50 mL) cooled at –15 °C was added dry CCl4 (10 mL) and the mixture was stirred at –15 °C for 5 min. Diisopropylethylamine (1.4 mL, 8.04 mmol) and N,N-dimethylaminopyridine (111 mg, 0.909 mmol) were then added and the mixture was stirred at –15 °C for 30 min. Dibenzylphosphite (1 mL, 4.53 mmol) was added dropwise and the mixture was stirred at –15 °C for 2 h. A 0.5 M solution of potassium dihydrogen phosphate (20 mL) was added, the mixture was warmed up to room temperature and extracted with EtOAc. The combined extracts were then washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. Purification by flash chromatography with CH2Cl2/MeOH (gradient from 100:2 to 100:8) afforded 27 as a colorless oil (574 mg, 72%). 1H NMR (300 MHz; CDCl3): δ 4.57 (2H, m, NH-CH2), 5.06-5.10 (8H, m, P-O-CH2), 7.08-7.11 (4H, m, 3C, 5C, 3D, 5D), 7.21-7.24 (2H, m, 2D, 6D), 7.30 (20H, br s, 4 × 2E, 4 × 3E, 4 × 4E, 4 × 5E, 4 × 6E), 7.45-7.48 (2H, m, 2C, 6C); 13C NMR (75 MHz; CDCl3): δ 44.3 (NH-CH2), 69.9 (P-O-CH2), 70.0 (P-O-CH2), 120.2 (3C, 5C, 3D, 5D), 121.7 (2C, 6C), 128.0 (2E, 6E), 128.5 (3E, 4E, 5E), 128.9 (2D, 6D), 134.9 (1C, 1D), 135.2 (1E), 146.3 (4C), 149.8 (4D), 164.3, 165.1 (CONH2, 4A), 166.2 (6A), 169.6 (2A); MS (ES+) m/z 873 [M+H]+, 895 [M+Na]+.

4-[4-(4-Phosphoryloxybenzylamino)-6-carbamoyl-1,3,5-triazin-2-ylamino]phenyl phosphate (7) 10% Palladium on carbon (75 mg) was added to a solution of compound 27 (153 mg, 0.175 mmol) in MeOH (50 mL). The mixture was stirred under an hydrogen atmosphere at room temperature overnight. The catalyst was filtered on two filter papers and washed with a hot MeOH/AcOH 98:2 mixture. Purification by column chromatography on reverse phase silica gel C18 using a water/MeCN 95:5 mixture gave 7 as a yellow solid (39 mg, 43%). Mp 196-199 °C; MS (ES–) m/z 255 [M–2H]2–.

Tetrasodium 4-[4-(4-phosphonatooxybenzylamino)-6-carbamoyl[1,3,5]triazin-2-ylamino]phenyl phosphate (29)
A fresh 0.539 M sodium methoxide solution (304 µL, 164 µmol) was added to a suspension of compound 7 (21 mg, 41.0 µmol) in dry MeOH (3 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h and the MeOH was removed under reduced pressure to afford 29 as a watersoluble beige powder (24 mg, 98%). 1H NMR (300 MHz; D2O): δ 4.40 (2H, s, NH-CH2), 7.03-7.05 (4H, m, 3C, 5C, 3D, 5D), 7.15-7.17 (2H, m, 2D, 6D), 7.27-7.29 (2H, m, 2C, 6C); 13C NMR (75 MHz; D2O): δ 43.4 (NH-CH2), 120.4, 120.6 (3C, 5C, 3D, 5D), 123.7 (2C, 6C), 128.3 (2D, 6D), 131.7 (1C), 132.0 (1D), 150.8 (4C), 152.8 (4D), 164.3, 164.4 (4A, CONH2), 165.4 (6A), 166.8 (2A); 31P NMR (121 MHz; D2O): δ 0.43; HRMS (ES–) m/z: calcd for C17H14N6Na4O9P2 576.9991 [M–Na]–, 533.0352 [M–3Na+2H]–; found 576.9979, 533.0339.

ACKNOWLEDGEMENTS
We thank CNRS, Institut Curie and Ministère de la Recherche (ACI “Molécules et Cibles Thérapeutiques” 2002 N° 02L0521) for financial support. Fondation pour la Recherche Médicale (FRM) is gratefully acknowledged for a fellowship granted to Caroline Courme.

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