Structure activity relationship studies on Amb639752: toward the identification of a common pharmacophoric structure for DGKα inhibitors

Abstract A series of analogues of Amb639752, a novel diacylglycerol kinase (DGK) inhibitor recently discovered by us via virtual screening, have been tested. The compounds were evaluated as DGK inhibitors on α, θ, and ζ isoforms, and as antagonists on serotonin receptors. From these assays emerged two novel compounds, namely 11 and 20, which with an IC50 respectively of 1.6 and 1.8 µM are the most potent inhibitors of DGKα discovered to date. Both compounds demonstrated the ability to restore apoptosis in a cellular model of X-linked lymphoproliferative disease as well as the capacity to reduce the migration of cancer cells, suggesting their potential utility in preventing metastasis. Finally, relying on experimental biological data, molecular modelling studies allow us to set a three-point pharmacophore model for DGK inhibitors.

S3 heated at reflux for 2h, than was cooled at room temperature and a solution containing 1 eq of methyl 2-(4-oxocyclohexyl)acetate and 1 eq of bis(4-fluorophenyl)methanone was added dropwise. The reaction was heated at reflux for 3 h. The reaction was then worked up by dilution with diethylether and NaHCO3 sol. sat, filtered on a celite pad. The aqueous phase was washed with diethylether (x3) and the combined organic phases washed with brine (x1) and dried over sodium sulphate. The crude was finally purified by column chromatography using PE/EtOAc 98:2 and 95:5 as eluants to give C as white solid.

Synthesis 2-(4-(bis(4-fluorophenyl)methylene)cyclohexyl)ethan-1-ol (D)
Under a nitrogen atmosphere, the corresponding ester (C) was dissolved in THF dry (0.3 M). The resulting solution was cooled at 0 °C and 1.1. eq of LiAlH4 were added portionwise. After 2 h at room temperature, the reaction was quenched adding a saturated aqueous solution of Na2SO4. The resultion solution was extracted with EtOAc (x3) and the combined organic phases were washed with brine (x1) and dried over sodium sulphate. The crude was purified by column chromatography using PE/EtOAc 8:2 as eluent to give the desired compound as colorless oil.

Synthesis of 2-(4-(bis(4-fluorophenyl)methylene)cyclohexyl)ethyl methanesulfonate (E)
1 eq of alcohol D was dissolved in dichloromethane dry (0.3 M), the resulting solution was cooled at 0 °C and 2 eq of TEA and 1.5 eq of MsCl were subsequently added. The reaction was stirred for 1 h at room temperature under a nitrogen atmosphere, then was diluted with water and extracted with dichloromethane (x3). The combined organic phases were washed with brine (x1) and dried over sodium sulphate. The crude was purified by column chromatography using PE/EtOAc 8:2 as eluant to give the desired product as yellowish oil.

Synthesis of 4,4'-((4-(2-azidoethyl)cyclohexylidene)methylene)bis(fluorobenzene) (F)
The mesylate E was dissolved in DMF (0.4 M) and 1.2 eq of sodium azide were added. The solution was heated at 80 °C overnight. The reaction was then diluted with water and extracted with EtOAc (x3). The combined organic phases were washed with water (x3), brine (x1) and dried over sodium sulphate. The crude obtained as yellowish oil was sufficiently pure to be used in the next step.

Synthesis 2-(4-(bis(4-fluorophenyl)methylene)cyclohexyl)ethan-1-amine (G)
The azide F was dissolved in THF (0.3 M). At the resulting solution 1.5 eq of triphenylphosphine and 6 eq of water were added. The reaction was heated at reflux for 2 h. After evaporation of the solvent, the crude was purified by column chromatography using PE/EtOAc 2:8 and EtOAc/MeOH 8.2 as eluants to give the desired amine as white solid.  37.6, 34.8, 33.9, 31.0. IR: ν (KBr) 3440, 3195, 1505, 1223, 1155 Melting point: 185.5-188.6 °C (1) The amine G was dissolved in dioxane (0.3 M) and 2 eq of methyl 2-isothiocyanatobenzoate were added. The reaction was heated at reflux overnight. After dilution with water, the solution was extracted with EtOAc (x3). The combined organic phases were washed with brine (x1) and dried over sodium sulphate. The crude was purified by column chromatography using PE/EtOAc 95:5 and 9:1 as eluants to give compound 1 as white solid.

Synthesis of 4-(furan-2-carbonyl)piperazin-1-ium trifluoroacetate (a)
The Boc-piperazine derivative was dissolved in dichloromethane dry (0.3 M). To the resulting solution, cooled at 0 °C, 16 eq of trifluoroacetic acid were added. The reaction was stirred for 3 h at 0 °C. Evaporation of the solvent, gave a solid, which was used as such for the following step.