Small-molecule inhibitor of AF9/ENL-DOT1L/AF4/AFF4 interactions suppresses malignant gene expression and tumor growth

Chromosome translocations involving mixed lineage leukemia (MLL) gene cause acute leukemia with a poor prognosis. MLL is frequently fused with transcription cofactors AF4 (~35%), AF9 (25%) or its paralog ENL (10%). The AHD domain of AF9/ENL binds to AF4, its paralog AFF4, or histone-H3 lysine-79 (H3K79) methyltransferase DOT1L. Formation of AF9/ENL/AF4/AFF4-containing super elongation complexes (SEC) and the catalytic activity of DOT1L are essential for MLL-rearranged leukemia. Protein-protein interactions (PPI) between AF9/ENL and DOT1L/AF4/AFF4 are therefore a potential drug target. Methods: Compound screening followed by medicinal chemistry was used to find inhibitors of such PPIs, which were examined for their biological activities against MLL-rearranged leukemia and other cancer cells. Results: Compound-1 was identified to be a novel small-molecule inhibitor of the AF9/ENL-DOT1L/AF4/AFF4 interaction with IC50s of 0.9-3.5 µM. Pharmacological inhibition of the PPIs significantly reduced SEC and DOT1L-mediated H3K79 methylation in the leukemia cells. Gene profiling shows compound-1 significantly suppressed the gene signatures related to onco-MLL, DOT1L, HoxA9 and Myc. It selectively inhibited proliferation of onco-MLL- or Myc-driven cancer cells and induced cell differentiation and apoptosis. Compound-1 exhibited strong antitumor activity in a mouse model of MLL-rearranged leukemia. Conclusions: The AF9/ENL-DOT1L/AF4/AFF4 interactions are validated to be an anticancer target and compound-1 is a useful in vivo probe for biological studies as well as a pharmacological lead for further drug development.


Synthesis of inhibitors
All chemicals for synthesis were purchased from Alfa Aesar (Ward Hill, MA), Aldrich (Milwaukee, WI) or Combi-Blcoks (San Diego, CA). The identity of the synthesized compounds was characterized by 1 H and 13 C NMR on a Varian (Palo Alto, CA) 400-MR spectrometer and mass spectrometer (Shimadzu LCMS-2020). The identity of compound 1 was confirmed with high resolution mass spectra (HRMS) using an Agilent 6550 iFunnel quadrupole-time-of-flight (Q-TOF) mass spectrometer with electrospray ionization (ESI). The purities of the final compounds were determined to be >95% with a Shimadzu Prominence HPLC using a Zorbax C18 (or C8) column To a solution of 4-tert-butylaniline (10, 7.5 g, 50 mmol) in DMSO (50 mL) was slowly added NaH (2.4 g, 60 mmol, 60% dispersion in mineral oil) at 0 o C. After stirring at 0 o C for 1 h, benzonitrile (5.69 g, 55 mmol) in DMSO (30 mL) was added. The resulting mixture was warmed to room temperature and stirred for another 12 h.
To a solution of compound 11 (8 g, 62 mmol) in acetonitrile (60 mL) was added ethyl bromopyruvate (4.2 g, 68 mmol) and tetrabromomethane (8.29 g, 25.0 mmol). After stirring at room temperature for 12 h, the solvent was removed under vacuum. The mixture was extracted with ethyl acetate (3 × 100 mL) and the combined organic layers were washed with water and brine and dried over Na2SO4. The volatiles were removed in vacuo to afford a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 5:1 to 3:1) to afford ethyl 1-(4-(tert-butyl)phenyl)-2-phenyl-1H-imidazole-4-carboxylate 12 (2.92, g, 50%) as colorless oil. 1  To a solution of compound 12 (2.2 g, 6.3 mmol) in THF/H2O (9/3 mL) was added sodium hydroxide (632 mg, 15.8 mmol). The resulting mixture was refluxed for 5 h. The solvent was removed under vacuum and acidified with 3N HCl, extracted with ethyl acetate (3 × 50 mL) and the combined organic layers were washed with water and brine and dried over Na2SO4. The volatiles were removed in vacuo to afford a crude product, which was used for the next step without further purification.
To a solution of compound 13a (1.5 g, 2.98 mmol) in dicholromethane (20 mL) was added dropwise HCl (2.2 mL, 4 N in p-dioxane) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 5 h.
The volatiles were removed in vacuo to afford an oil, which was triturated in diether ether and solidified to give the final product as a hydrochloric salt (1, 1.18 g, 90%) as a white powder.

S18
The reaction was then cooled and quenched with brine (10 mL). The product was extracted with ethyl acetate (3 × 20 mL) and the combined organic layers were washed with water and brine and dried over Na2SO4. The volatiles were removed in vacuo to give a crude oil, which was purified by column chromatography (silica gel, hexanes: ethyl acetate from 2:1 to 1:1) to afford the product 18a (90 mg, 82%) as a white foam.
To a solution of compound 18a (90 mg, 0.16 mmol) in dichloromethane (6 mL) was added dropwise HCl (0.2 mL, 4 N in p-dioxane) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 3 h.