Stereoselective synthesis of allele-specific BET inhibitors

Abstract

Developing stereoselective synthetic routes that are efficient and cost-effective allows easy access to allele-selective bumped BET inhibitors.

Development of BromoTag: A “Bump-and-Hole”–PROTAC System to Induce Potent, Rapid, and Selective Degradation of Tagged Target Proteins

“…To achieve enantiomerically pure PROTACs, we developed a new stereoselective synthesis to bumped BET ligands, which we disclosed recently. 21 In brief, our novel stereoselective route allowed us to incorporate the alkyl bump much earlier in the synthesis of the BET-ligand scaffold. To achieve this, we optimized a lithium hexamethyldisilazane (LHMDS)-mediated, diastereoselective alkylation of a di-protected aspartate derivative and took this through to final bumped JQ1 acid analogues with complete retention of stereochemistry and in >99% ee.…”

“…To achieve this, we optimized a lithium hexamethyldisilazane (LHMDS)-mediated, diastereoselective alkylation of a di-protected aspartate derivative and took this through to final bumped JQ1 acid analogues with complete retention of stereochemistry and in >99% ee. 21 At this stage in the project, we therefore decided to use the enantiomerically pure ET-JQ1-OH ( 45 ) to prepare new B&H–PROTACs, AGB1 ( 46 ), AGB2 ( 47 ), and AGB3 ( 48 ) ( Scheme 5 ).…”

“… 37 In addition, to demonstrate that the on -target activity on BromoTag-Brd2 is due to the recruitment of the BromoTag, we pretreated with ET-JQ1-OMe, which binds with high affinity to the Brd4 BD2 L387A variant ( K d = 65 nM) but exhibits undetectable binding to the wild-type domain. 21 In this experiment, we separately exposed our heterozygous BromoTag-Brd2 HEK293 cells to the different inhibitors for a short 1 h prior to subsequent treatment with 200 nM 46 before continuing treatment for a further 3 h to minimize potential confounding effects due to inhibitor cytotoxicity. As expected, the on -target degradation activity by 46 was completely abrogated upon pretreatments with MLN4924 or VH298, demonstrating dependency on CRL2 VHL activity ( Figure 8 A and see Supporting Information Figure S17).…”

“…A catalytic amount of 10 wt % Pd/C was added, and the reaction was stirred under an atmosphere of hydrogen for 3 h. The reaction mixture was then filtered through PTFE syringe filters and evaporated to dryness to obtain the desired amine quantitative yields. The resulting amine (7.4 mg, 12 μmol) was dissolved in DMF (96 μL) and added to a solution of ET-JQ1-OH ( 45 , synthesized according to the literature 21 ) (5 mg, 12 μmol), COMU (5.1 mg, 12 μmol) and DIPEA (4.18 μL, 12 μmol) in DMF (96 μL) and stirred at r.t. for 2 h. The mixtures were then concentrated in vacuo , and the residues were purified by HPLC using a linear gradient of 5–95% MeCN in 0.1% formic acid in water over 12 min to afford AGB3 ( 48 ). Yield: 2.2 mg (18%); 1 H NMR (400 MHz, CDCl 3 ): δ 8.68 (s, 1H), 8.18 (t, J = 5.5 Hz, 1H), 7.36 (d, J = 8.3 Hz, 2H), 7.31–7.24 (m, 5H), 7.17–7.12 (m, 3H), 4.99 (d, J = 4.9 Hz, 1H), 4.85 (t, J = 8.2 Hz, 1H), 4.80 (d, J = 9.7 Hz, 1H), 4.51 (br s, 1H), 4.46 (dd, J = 7.2, 15.8 Hz, 1H), 4.26 (d, J = 10.5 Hz, 1H), 4.19–4.11 (m, 2H), 4.09 (d, J = 15.9 Hz, 1H), 3.83–3.63 (m, 15H), 3.50–3.42 (m, 1H), 2.64 (s, 3H), 2.53 (s, 3H), 2.39 (s, 3H), 2.34–2.27 (m, 1H), 2.16 (dd, J = 7.5, 13.5 Hz, 1H), 1.96–1.89 (m, 1H), 1.64–1.56 (m, 4H), 1.03–0.96 ppm (m, 12H); 13 C NMR (101 MHz, CDCl 3 ): δ 173.8, 171.7, 171.6, 170.3, 163.9, 155.0, 150.3, 149.9, 148.5, 138.6, 136.9, 136.7, 132.0, 131.9, 131.31, 131.28, 130.9, 130.6, 130.1, 129.4, 129.0, 127.5, 71.3, 71.1, 70.75, 70.69, 70.4, 70.3, 59.8, 59.4, 57.7, 56.7, 50.3, 42.9, 39.8, 36.9, 36.0, 26.6, 23.1, 16.2, 14.6, 13.3, 12.0, 11.9; HRMS m / z : calcd for C 51 H 65 ClN 9 O 8 S 2 [M + H] + , 1030.4081; found, 1030.4589.…”

“…In this work, we describe the development of a novel degron tag called the “BromoTag”. The new tag system leverages significant developments and discoveries in the past decade from our laboratory on both PROTAC degraders − and allele-selective bump-and-hole (B&H) targeting − of the bromo and extra terminal (BET) bromodomain proteins Brd2, Brd3, and Brd4. We describe the structure-based design and development of cooperative bumped PROTAC compounds designed to target a mutant Brd4 bromodomain degron tag for the degradation of an endogenously CRISPR-tagged target protein.…”