Diversity-oriented synthetic strategy for developing a chemical modulator of protein–protein interaction

Diversity-oriented synthesis (DOS) can provide a collection of diverse and complex drug-like small molecules, which is critical in the development of new chemical probes for biological research of undruggable targets. However, the design and synthesis of small-molecule libraries with improved biological relevance as well as maximized molecular diversity represent a key challenge. Herein, we employ functional group-pairing strategy for the DOS of a chemical library containing privileged substructures, pyrimidodiazepine or pyrimidine moieties, as chemical navigators towards unexplored bioactive chemical space. To validate the utility of this DOS library, we identify a new small-molecule inhibitor of leucyl-tRNA synthetase–RagD protein–protein interaction, which regulates the amino acid-dependent activation of mechanistic target of rapamycin complex 1 signalling pathway. This work highlights that privileged substructure-based DOS strategy can be a powerful research tool for the construction of drug-like compounds to address challenging biological targets.


Synthetic procedure for the preparation of tert-butyl (S)-(1-((6-chloro-5formylpyrimidin-4-yl)amino)-3-((triisopropylsilyl)oxy)pro-pan-2-yl)carbamate (SI-2).
To a solution of SI-  was quenched with saturated NH4Cl(aq) and extracted twice with ethyl acetate. The combined organic layer was dried over anhydrous Na2SO4(s) and filtered. The organic solvent was evaporated under vacuum to afford a crude oil, which was purified by silica-gel flash column chromatography to afford SI-3. To obtain the cyclic imine product 1, SI-3 was first treated with 10% trifluoroacetic acid (TFA) in DCM (20.0 ml) at r.t. After the starting material was consumed as indicated by TLC, any excess TFA was removed by azeotropic evaporation with toluene under reduced pressure, and the crude resultant was dissolved in 1% acetic acid (AcOH) in CHCl3 (100.0 ml). The resulting mixture was stirred at 40 o C. After completion of the reaction as indicated by TLC, the resultant was quenched with saturated NaHCO3(aq) and extracted twice with CHCl3. The combined organic layer was dried over anhydrous Na2SO4(s) and filtered. The filtrate was condensed under reduced pressure, followed by silica-gel flash column chromatography to afford the desired product 1 ( Supplementary Fig. 2).
Synthetic procedure for the preparation of 13f and 13f́.

Compound SI-7
To and extracted twice with DCM. The combined organic layer was dried over anhydrous Na2SO4(s) and the filtrate was condensed under reduced pressure, followed by silica-gel flash column chromatography to afford SI-7 (70.68 mg, 72% yield, d.r. 9:1) as a yellow oil ( Supplementary Fig. 4). The diastereomeric ratio was determined by 1 H NMR spectroscopy ( Supplementary Fig. 52).

Compound 13f
To a solution of SI-7 (70.68 mg, 0.143 mmol) in DCM (14.3 ml) was added 3-chloroperbenzoic acid (m-CPBA, 32.08 mg, 0.186 mmol) and the mixture was left to stir at reflux. After 1h, S108 solvent was removed under reduced pressure and the residue was purified by silica-gel flash column chromatography to obtain the desired product 13f (42.45 mg, 58% yield, 42% overall yield) as a colorless oil (Supplementary Fig. 4).

Compound 13f́
To a solution of SI-7 (70.68 mg, 0.143 mmol) in DCM (14.3 ml) was added 3-chloroperbenzoic acid (m-CPBA, 32.08 mg, 0.186 mmol) and the mixture was left to stir at reflux. After 1 h, 3chlorobenzoic acid (22.39mg, 0.143 mmol) was added and left to stir at r.t. After completion of the reaction as indicated by TLC, solvent was removed under reduced pressure and the residue was purified by silica-gel flash column chromatography to obtain the desired product 13f́(70.72mg, 74% yield, 53% overall yield) as a pale yellow oil ( Supplementary Fig. 4). The E/Z ratio (99:1) was determined by LC-MS analysis of the crude reaction mixtures ( Supplementary Fig. 12).

Synthetic procedure for the preparation of 14f
Compound 14f. 1f (90.00 mg, 0.198 mmol) was treated with HF/pyridine/THF (5/5/90) solution (2 ml) and then, the mixture was stirred at r.t. After the starting material was consumed as indicated by TLC, ethoxytrimethylsilane (2 ml) was added and allowed to react for 1 h to quench any excess HF (HF/pyridine protocol). The reaction mixture was condensed in vacuo to afford the crude product. To a solution of crude product in DCM (4 ml) was added Boc2O (56.09 mg, 0.257 mmol) and then, the mixture was stirred at r.t. After completion of the reaction as indicated by TLC, solvent was removed under reduced pressure and the residue was purified through the recrystallization in hexane to obtain desired product 14f (68.47 mg, 87% overall yield) as a white solid.  at 0 o C. After 30min, the resulting aldehyde product (148.3 mg, 0.339 mmol) was added. Then the reaction mixture was allowed to slowly warm to r.t over a 12 h period. The reaction mixture was quenched with saturated NH4Cl(aq). The resultant was extracted twice with ethyl acetate, dried with anhydrous Na2SO4(s), filtered, and concentrated in vacuo, followed by silica-gel flash column chromatography to afford SI-11 (130.0 mg, 88% yield). To a solution of SI-11 (130.0 mg, 0.298 mmol) in toluene was added second generation Grubbs' catalyst (50.94 mg, 0.060 mmol, 20 mol%) and the mixture was left to stir at reflux. After 4h, solvent was removed under reduced pressure and the residue was purified by silica-gel flash column chromatography to obtain desired product 23f (44.83 mg, 52% recovery yield, 31% overall yield) as a yellow oil. As a running buffer, 1× PBS (pH 7.3) containing 3% DMSO and 0.005% P20 solution were used. The binding events were measured at 25 °C. Data analysis were done by using Biacore S126 T100 Evaluation software from GE Healthcare. Final sensorgrams were obtained after the elimination of responses from flow cell 1 and buffer-only control. The dissociation constant (KD) was calculated by fitting the sensorgrams to the 1:1 binding model.

Transfection
Cells were seeded in chambered coverglass from Nunc, 24 h prior to transfection. mCherry-GFP-LC3 plasmid was transfected to HeLa cell using Lipofectamine TM 2000 reagent.
Transfection was preceded according to manufacturer's protocol.

Cell proliferation assay
HEK293T cells were seeded in transparent 96-well plate from CORNING. 24 h after seeding, leucine-free DMEM and compound-DMEM solution (final compound concentration of 5 μM) were treated in each well after the removal of culture media. Cell proliferation assay was done with WSTs assay and cell proliferation ELISA, BrdU (colorimetric) kit, following the manufacturer's protocol.