Design and characterization of bivalent BET inhibitors

Journal name:
Nature Chemical Biology
Volume:
12,
Pages:
1089–1096
Year published:
DOI:
doi:10.1038/nchembio.2209
Received
Accepted
Published online

Abstract

Cellular signaling is often propagated by multivalent interactions. Multivalency creates avidity, allowing stable biophysical recognition. Multivalency is an attractive strategy for achieving potent binding to protein targets, as the affinity of bivalent ligands is often greater than the sum of monovalent affinities. The bromodomain and extraterminal domain (BET) family of transcriptional coactivators features tandem bromodomains through which BET proteins bind acetylated histones and transcription factors. All reported antagonists of the BET protein BRD4 bind in a monovalent fashion. Here we describe, to our knowledge for the first time, a bivalent BET bromodomain inhibitor—MT1—which has unprecedented potency. Biophysical and biochemical studies suggest MT1 is an intramolecular bivalent BRD4 binder that is more than 100-fold more potent, in cellular assays, than the corresponding monovalent antagonist, JQ1. MT1 significantly (P < 0.05) delayed leukemia progression in mice, as compared to JQ1. These data qualify a powerful chemical probe for BET bromodomains and a rationale for further development of multidomain inhibitors of epigenetic reader proteins.

At a glance

Figures

  1. Rational design of bivalent BET bromodomain inhibitors.
    Figure 1: Rational design of bivalent BET bromodomain inhibitors.

    (a) Structure and activity of JQ1 and its close analog MS417. General chemical structure and design of dimeric JQ1-like molecules are also shown. Linkage sites at the C6 (6) position of the diazepine and the C2 (2) position of the thiophene are indicated. t-Bu, tert-butyl; Me, methyl. (b,c) BRD4(1) biochemical and MV4;11 cellular activity of the (6+6) homodimer (b) and the (2+2) homodimer (c). Data represent mean ± s.d. of at least 2 (biochemical data) and 4 (cellular data) replicates.

  2. Heterodimeric bivalent inhibitors and activity optimization.
    Figure 2: Heterodimeric bivalent inhibitors and activity optimization.

    (a,b) General chemical structures of heterodimeric bivalent inhibitors with changes in linker length (a) and stereochemistry (b). (c) Heat map of dimeric compound activity (pIC50) in biochemical (BRD4(1) and BRDT(1)) and cellular (MV4;11 and NMC797) assays compared to that of JQ1 or I-BET151 (black). Data represent mean of at least 2 (biochemical data) or 4 (cellular data) replicates. (d) Biochemical and cellular activity of the different (6+2)-PEG1 diastereomers measured by BRD4(1) AlphaScreen and ATPLite. Data represent mean ± s.d. of at least 2 (biochemical data) or 4 (cellular data) replicates.

  3. Biochemical and biophysical properties of bivalent inhibitors.
    Figure 3: Biochemical and biophysical properties of bivalent inhibitors.

    (a) SEC assay of complex formation after incubation of (6S+2S)-PEG1, (6R+2R)-PEG1 or JQ1 with BRD4(1). (b) Stoichiometry of (6S+2S)-PEG1 or JQ1 binding to BRD4(1) as analyzed by ITC (experiments were performed in duplicate). (c) Immunoblot analysis of BRD4, MYC and actin expression after treatment of MV4;11 cells with (6S+2S)-PEG1 or JQ1 at the indicated concentrations for 2 h. (d) Immunoblot analysis of BRD4, MYC, HEXIM1 and actin expression after treatment of MV4;11 cells with 10 nM (6S+2S)-PEG1 for indicated times. Full blots can be found in Supplementary Figure 15. Each experiment was repeated twice.

  4. MT1: an intramolecular bivalent chemical probe of BET bromodomains.
    Figure 4: MT1: an intramolecular bivalent chemical probe of BET bromodomains.

    (a) Chemical structure of MT1. (b) Biochemical (BRD4(1) AlphaScreen) and cellular (MV4;11 and NMC797) activity of MT1. Data represent mean ± s.d. of at least 2 (biochemical data) or 4 (cellular data) replicates. (c) Cocrystal structure of MT1 bound to two BRD4(2) monomers (PDB 5JWM). (d) Cocrystal structure showing two bromodomain monomers (red, blue and white) creating a hydrophobic pocket (middle) that surrounds MT1 (yellow). (e) SEC analysis of wild-type and mutant (N140A or N433A) tandem bromodomain constructs with MT1.

  5. Kinetic and antitumor advantage of dual-BET bromodomain inhibition.
    Figure 5: Kinetic and antitumor advantage of dual-BET bromodomain inhibition.

    (a) CETSA of MT1 and JQ1 in MV4;11 cells; data are mean ± s.e.m. of technical triplicates. Blots can be found in Supplementary Figure 11. (b) Immunoblot analysis of BRD4, PARP cleavage, cleaved caspase 3, MYC, HEXIM1 and actin in MV4;11 cells treated with 100 nM MT1 for the indicated times. Full blots can be found in Supplementary Figure 15. (c) Percentage of early (annexin V) and late (annexin V and propidium iodide) apoptotic and healthy (unstained) MV4;11 cells after treatment with MT1, JQ1 or DMSO for 24 h, as analyzed by annexin V and propidium iodide staining and flow cytometric analysis. Error bars represent mean ± s.d. of 3 biological replicates.

  6. Efficacy of dual-BET bromodomain inhibition in vivo.
    Figure 6: Efficacy of dual-BET bromodomain inhibition in vivo.

    (a) Luminal radiance (mean ± s.e.m.) in mice treated with MT1 at 44.2 μmol/kg (n = 8) or 22.1 μmol/kg (n = 9), JQ1 at 44.2 μmol/kg (n = 8) or vehicle (n = 8) from day 7 to day 21 after initial leukemic cell injection of MV4;11 cells in a disseminated xenograft model. (b) Percentage of mCherry+ leukemic cells (mean ± s.e.m.) in flushed bone marrow from mice in a. (c) Images of luminal radiance in mice before (day 11) and after (day 45) treatment (the same mice are shown for each time point). MT1 (n = 11), JQ1 (n = 11) or vehicle (n = 11) was administered once daily at 44.2 μmol/kg from day 17 to day 28 and day 31 to day 35 after injection of MV4;11 cells as in a. (d) Luminescence (mean ± s.e.m.) in mice from c. (e) Kaplan–Meier survival plot for MT1-, JQ1- and vehicle-treated mice from c. ns, not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001, Mantel-Cox test (e) or unpaired two-tailed Welch's t-test with no correction for multiple comparisons (a,b,d).

Compounds

35 compounds View all compounds
  1. tert-butyl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 1 tert-butyl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  2. methyl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 2 methyl (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  3. tert-butyl (R)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 3 tert-butyl (R)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  4. N,N'-(oxybis(ethane-2,1-diyl))bis(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide)
    Compound 4 N,N'-(oxybis(ethane-2,1-diyl))bis(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide)
  5. dimethyl 2,2'-((6S,6'S)-(((oxybis(ethane-2,1-diyl))bis(azanediyl))bis(carbonyl))bis(4-(4-chlorophenyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2,6-diyl))diacetate
    Compound 5 dimethyl 2,2'-((6S,6'S)-(((oxybis(ethane-2,1-diyl))bis(azanediyl))bis(carbonyl))bis(4-(4-chlorophenyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2,6-diyl))diacetate
  6. methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 6 methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  7. methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 7 methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  8. methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-(2-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 8 methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-(2-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  9. methyl 2-((S)-4-(4-chlorophenyl)-2-((1-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 9 methyl 2-((S)-4-(4-chlorophenyl)-2-((1-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  10. methyl 2-((S)-4-(4-chlorophenyl)-2-((1-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-yl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 10 methyl 2-((S)-4-(4-chlorophenyl)-2-((1-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-yl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  11. methyl 2-((S)-4-(4-chlorophenyl)-2-((1-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2-oxo-6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 11 methyl 2-((S)-4-(4-chlorophenyl)-2-((1-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-2-oxo-6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  12. methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-(2-((R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 12 methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-(2-((R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  13. methyl 2-((R)-4-(4-chlorophenyl)-2-((2-(2-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 13 methyl 2-((R)-4-(4-chlorophenyl)-2-((2-(2-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  14. methyl 2-((R)-4-(4-chlorophenyl)-2-((2-(2-(2-((R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 14 methyl 2-((R)-4-(4-chlorophenyl)-2-((2-(2-(2-((R)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamido)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  15. methyl (S)-2-(2-((2-(2-acetamidoethoxy)ethyl)carbamoyl)-4-(4-chlorophenyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 15 methyl (S)-2-(2-((2-(2-acetamidoethoxy)ethyl)carbamoyl)-4-(4-chlorophenyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  16. (R)-7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-1-(1-(pyridin-2-yl)ethyl)-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one
    Compound 16 (R)-7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-1-(1-(pyridin-2-yl)ethyl)-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one
  17. 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(2-(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamido)ethyl)acetamide
    Compound 17 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(2-(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamido)ethyl)acetamide
  18. 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(2-(2-(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamido)ethoxy)ethyl)acetamide
    Compound 18 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(2-(2-(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamido)ethoxy)ethyl)acetamide
  19. 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(2-(2-(2-(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamido)ethoxy)ethoxy)ethyl)acetamide
    Compound 19 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(2-(2-(2-(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamido)ethoxy)ethoxy)ethyl)acetamide
  20. 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(1-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)acetamide
    Compound 20 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(1-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)acetamide
  21. 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(1-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-yl)acetamide
    Compound 21 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(1-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-yl)acetamide
  22. 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(1-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)-2-oxo-6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)acetamide
    Compound 22 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(1-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)-2-oxo-6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)acetamide
  23. methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamido)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 23 methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamido)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  24. methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamido)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 24 methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamido)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  25. methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-(2-(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamido)ethoxy)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 25 methyl 2-((S)-4-(4-chlorophenyl)-2-((2-(2-(2-(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamido)ethoxy)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  26. methyl 2-((S)-4-(4-chlorophenyl)-2-((1-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 26 methyl 2-((S)-4-(4-chlorophenyl)-2-((1-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  27. methyl 2-((S)-4-(4-chlorophenyl)-2-((1-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-yl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 27 methyl 2-((S)-4-(4-chlorophenyl)-2-((1-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-yl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  28. methyl 2-((S)-4-(4-chlorophenyl)-2-((1-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)-2-oxo-6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
    Compound 28 methyl 2-((S)-4-(4-chlorophenyl)-2-((1-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)-2-oxo-6,9,12,15,18,21,24-heptaoxa-3-azahexacosan-26-yl)carbamoyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate
  29. N,N'-(ethane-1,2-diyl)bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamide)
    Compound 29 N,N'-(ethane-1,2-diyl)bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamide)
  30. N,N'-(oxybis(ethane-2,1-diyl))bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamide)
    Compound 30 N,N'-(oxybis(ethane-2,1-diyl))bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamide)
  31. N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamide)
    Compound 31 N,N'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamide)
  32. N,N'-(((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamide)
    Compound 32 N,N'-(((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamide)
  33. N,N'-(3,6,9,12-tetraoxatetradecane-1,14-diyl)bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamide)
    Compound 33 N,N'-(3,6,9,12-tetraoxatetradecane-1,14-diyl)bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamide)
  34. N,N'-(3,6,9,12,15,18,21-heptaoxatricosane-1,23-diyl)bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamide)
    Compound 34 N,N'-(3,6,9,12,15,18,21-heptaoxatricosane-1,23-diyl)bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3H-imidazo[4,5-c]quinolin-3-yl)acetamide)
  35. N,N'-(3,6,9,12,15,18,21-heptaoxatricosane-1,23-diyl)bis(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide)
    Compound 35 N,N'-(3,6,9,12,15,18,21-heptaoxatricosane-1,23-diyl)bis(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetamide)

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Referenced accessions

Protein Data Bank

References

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Author information

  1. Present address: Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.

    • James E Bradner
  2. These authors contributed equally to this work.

    • Minoru Tanaka &
    • Justin M Roberts

Affiliations

  1. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Minoru Tanaka,
    • Justin M Roberts,
    • Amanda Souza,
    • Joshiawa Paulk,
    • Thomas G Scott,
    • Stephen L DeAngelo &
    • James E Bradner
  2. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.

    • Minoru Tanaka &
    • James E Bradner
  3. Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Hyuk-Soo Seo,
    • Stephen L DeAngelo &
    • Sirano Dhe-Paganon

Contributions

M.T. and J.M.R. designed and led the study under the supervision of J.E.B. M.T. designed bivalent molecules and performed organic chemistry and medicinal chemistry experiments. J.M.R. designed and performed biochemical, cellular, and in vivo experiments. J.P. performed BRD4(1) FP experiments. S.D.-P., H.-S.S. and S.L.D. performed crystallographic studies. H.S.S. performed SEC and ITC experiments. A.S. and T.G.S. performed in vivo studies. M.T., J.M.R. and J.E.B. wrote the manuscript with input from all authors.

Competing financial interests

M.T. is a visiting scientist from Mitsubishi Tanabe Pharma Corporation and is supported by the company for nonresearch funds. J.E.B. is a founder of Tensha Therapeutics, a biotechnology company that develops drug-like derivatives of JQ1 as investigational cancer therapies. Dana-Farber Cancer Institute and M.T., J.M.R. and J.E.B. have filed patent applications (62/259,797, 62/261,703 and 62/338,968) that include MT1 and the analogs described in this manuscript. J.E.B. is an employee of the Novartis Institutes of BioMedical Research (effective 1 January 2016).

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