TP-064, a potent and selective small molecule inhibitor of PRMT4 for multiple myeloma

Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as a candidate oncogene owing to its overexpression in several types of cancer. Selective PRMT4 inhibitors are useful tools for clarifying the molecular events regulated by PRMT4 and for validating PRMT4 as a therapeutic target. Here, we report the discovery of TP-064, a potent, selective, and cell-active chemical probe of human PRMT4 and its co-crystal structure with PRMT4. TP-064 inhibited the methyltransferase activity of PRMT4 with high potency (half-maximal inhibitory concentration, IC50 < 10 nM) and selectivity over other PRMT family proteins, and reduced arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155; IC50= 340 ± 30 nM) and Mediator complex subunit 12 (MED12; IC50 = 43 ± 10 nM). TP-064 treatment inhibited the proliferation of a subset of multiple myeloma cell lines, with affected cells arrested in G1 phase of the cell cycle. TP-064 and its negative control (TP-064N) will be valuable tools to further investigate the biology of PRMT4 and the therapeutic potential of PRMT4 inhibition.


Experimental procedure for 4
The stirred and degassed solution of 3 (8.5 g, 29.274 mmol, 1 eq.) in methanol (200 ml) was combined with 10% Pd/C (0.311 g, 2.927 mmol, 0.1 eq.) and subjected to hydrogenation under balloon pressure for 18 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the solution was filtered through a celite bed and washed with methanol, and the filtrate was concentrated under reduced vacuum pressure to obtain 4 (7.1 g, yield: 97 %) as an off-white gummy liquid.

Experimental procedure for 5
The stirred solution of 4 (5 g, 17.1 mmol, 1 eq.) in tetrahydrofuran (40 ml) was combined with triethylamine (5.18 g, 51.304 mmol, 3 eq.) and thionyl chloride (2.35 g, 20.52 mmol, 1.2 eq.) at 0°C. The resultant reaction mixture was allowed to stir at room temperature for 1 h before adding 40 ml methylamine (2 M in tetra-nbutylammonium fluoride) at room temperature. The mixture was stirred at room temperature for 18 h, with the progress of the reaction monitored by TLC. After completion of the reaction, the mixture was concentrated under reduced vacuum pressure to obtain the crude compound, which was purified by silica gel (60-120 mesh) column chromatography, eluted with 5% methanol/ dichloromethane to obtain 5 (5.09 g, yield: 62 %) as a pale yellow gummy liquid.

Tert-butyl-4-(4-((N-methyl-3-phenoxybenzamido) methyl)pyridin-2-yl)piperidine-1-carboxylate
The progress of the reaction was monitored by TLC. After completion of the reaction, the solution was diluted with water and extracted with ethyl acetate. The Ethyl acetate layer was washed with water and brine solution and dried over anhydrous Na 2 SO 4 , then filtered and evaporated under reduced pressure to obtain the crude compound. This was purified by silica gel (60-120 mesh) column chromatography, eluted with 60% ethyl acetate/pet ether to obtain compound 7 (2.9 g, Yield: 38.6 %) as a pale yellow gummy liquid. Experimental procedure for 8 1,4-Dioxane in HCl (2M) (10 ml) was added to the stirred solution of 7 (2.5 g, 5.186 mmol, 1 eq.) in 1,4 dioxane (10 ml) at 0°C and the resultant mixture was stirred at room temperature for 1 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the solution was concentrated under reduced pressure to obtain the crude compound, which was co-distilled with dichloromethane and washed with npentane to obtain 8 (1.91 g, yield: 79.5%) as a pale yellow solid.

Experimental procedure for 10
Sodium triacetoxyborohydride (0.527 g, 2.490 mmol, 5 eq.) was added to the stirred solution of 8 (0.2 g, 0.498 mmol, 1 eq.) and 9 (0.172 g, 0.996 mmol, 2 eq.) in ethanol (20 ml) at 0°C; the resultant reaction mixture was allowed to stir at room temperature for 18 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the solution was basified with saturated sodium bicarbonate solution and extracted with ethyl acetate. The ethyl acetate layer was washed with water and brine solution, dried over anhydrous Na 2 SO 4 , filtered, and evaporated under reduced pressure to obtain the crude compound, which was purified by silica gel (60-120 mesh) column chromatography and eluted with 3% methanol/ dichloromethane to obtain 10 (0.14 g, yield: 50.3 %) as a brown gummy liquid, which was confirmed by LC-MS. LC-MS (M+H): 559.38.

Experimental procedure for TP-064
1,4-Dioxane in HCl (2M) (2 ml) was added to the stirred solution of 10 (0.14 g, 0.882 mmol, 1 eq.) in 1,4 dioxane (5 ml) was added at 0°C and the resulting reaction mixture was allowed to stir at room temperature for 1 h. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was