Minoru Tanaka 1,2,#, Justin M. Roberts 1,#, Hyuk-Soo Seo 3, Amanda Souza 1, Joshiawa Paulk 1,

Transcription

1 Design and Characterization of Bivalent BET Inhibitors Minoru Tanaka 1,2,#, Justin M. Roberts 1,#, Hyuk-Soo Seo 3, Amanda Souza 1, Joshiawa Paulk 1, Thomas G. Scott 1, Stephen L. DeAngelo 1,3, Sirano Dhe-Paganon 3, and James E. Bradner 1,2,4 * 1 Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, Massachusetts 02215, USA. 2 Department of Medicine, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115, USA. 3 Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, Massachusetts 02215, USA. 4 Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139, USA # These authors contributed equally to this work. *Corresponding author james_bradner@dfci.harvard.edu 1

2 General procedure for the synthesis of (6S+2S)-PEG derivatives. (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)acetic acid (CAS # ) (40 mg, 0.1 mmol), corresponding mono-boc-amino-peg-amine (1.5 eq), HATU (1.5 eq), and N,N-diisopropylethylamine (1.5 eq) were added to DMF (0.1 M). The mixture was stirred for 4 hours at room temperature. The mixture was diluted with EtOAc, washed with 1N NaOH and brine, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The residue was purified by flash chromatography to give mono-amides (49 to 99%). The mono-amide was dissolved into dichloromethane (0.5 M). Trifluoroacetic acid (0.5 M) was added to the solution. The mixture was stirred for 1 hour at room temperature. The mixture was diluted with dichloromethane, washed with 1N NaOH, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The free amine was used for the next step without purification. The free amine, (S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[ 4,3-a][1,4]diazepine-2-carboxylic acid (CAS # ) (1.1 eq), HATU (1.5 eq), and N,N-diisopropylethylamine (1.5 eq) were added to DMF (0.1 M). The mixture was stirred for 4 hours at room temperature. The mixture was diluted with ethyl acetate, washed with 1N NaOH and brine, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The residue was purified by flash chromatography to give the titled compounds (50 to 81%, for 2 steps). 2

3 (6S+2S)-PEG0 (6) 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-thi eno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.54 (s, 3 H) 1.97 (s, 3 H) 2.32 (s, 3 H) 2.55 (s, 6 H) (m, 8 H) 3.68 (s, 3 H) 4.54 (ddd, J=9.68, 7.92, 6.26 Hz, 2 H) (m, 4 H) (m, 4 H) 7.73 (br. s., 1 H) 7.86 (br. s., 1 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 11.71, 11.78, 13.03, 14.30, 16.55, 36.55, 38.88, 39.18, 41.61, 51.81, 53.72, 54.17, , , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 869 (M+H) + HRMS (ESI) m/z: C 41 H 38 Cl 2 N 10 O 4 S 2 requires m/z , found m/z (M+H) + (6S+2S)-PEG1 (7) 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-dimet hyl-6h-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate 3

4 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.59 (s, 3 H) 2.03 (s, 3 H) 2.34 (s, 3 H) 2.50 (s, 3 H) 2.57 (s, 3 H) 3.15 (dd, J=13.69, 4.30 Hz, 1 H) (m, 11 H) 3.67 (s, 3 H) (m, 2 H) (m, 4 H) (m, 4 H) (m, 2 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 11.71, 13.06, 14.30, 16.61, 36.54, 39.34, 39.61, 40.27, 51.86, 53.74, 54.63, 69.20, 69.43, , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 913 (M+H) + HRMS (ESI) m/z: C 43 H 42 Cl 2 N 10 O 5 S 2 requires m/z , found m/z (M+H) + (6S+2S)-PEG2 (8) 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 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.68 (m, 3 H) 2.07 (s, 3 H) 2.41 (s, 3 H) 2.56 (s, 3 H) 2.61 (s, 3 H) (m, 16 H) 3.76 (s, 3 H) 4.65 (ddd, J=18.00, 8.22, 5.87 Hz, 2 H) 7.18 (br. s., 2 H) (m, 4 H) (m, 4 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 11.68, 13.13, 14.39, 15.26, 16.52, 36.58, 39.32, 40.12, 53.79, 54.31, 69.65, 69.80, 70.28, , , , , , 4

5 131.14, , , , , , , MS (ESI) m/z: 957 (M+H) + (6S+2S)-PEG3 (9) 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)carbam oyl)-3,9-dimethyl-6h-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.60 (s, 3 H) 1.98 (s, 3 H) 2.33 (s, 3 H) 2.55 (s, 3 H) (m, 3 H) (m, 20 H) (m, 3 H) (m, 2 H) (m, 2 H) (m, 4 H) (m, 4 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 11.70, 13.03, 14.13, 14.33, 16.44, 36.53, 38.89, 39.31, 39.95, 51.88, 53.78, 54.25, 69.34, 69.65, 70.10, 70.39, , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 1001 (M+H) + (6S+2S)-PEG4 (10) 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)c 5

6 arbamoyl)-3,9-dimethyl-6h-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.60 (br. s., 3 H) 1.98 (br. s., 3 H) 2.32 (br. s., 3 H) (m, 3 H) 2.59 (br. s., 3 H) (m, 24 H) 3.69 (br. s., 3 H) (m, 2 H) 5.22 (br. s., 2 H) (m, 5 H) (m, 4 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 11.69, 11.75, 12.99, 14.30, 16.42, 36.51, 38.78, 39.27, 39.80, 51.82, 53.74, 54.20, 69.37, 69.80, 69.97, 70.11, 70.31, 70.39, , , , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 1045 (M+H) + (6S+2S)-PEG7 (11) 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-azahexacosa n-26-yl)carbamoyl)-3,9-dimethyl-6h-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl) acetate 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.60 (s, 3 H) 1.99 (s, 3 H) 2.33 (s, 3 H) 2.58 (s, 3 H) 2.63 (s, 3 H) (m, 28 H) 3.70 (s, 3 H) (m, 2 H) (m, 1 H) 6.86 (t, J=5.09 Hz, 1 H) (m, 4 H) (m, 4 H) 6

7 13 C NMR (100 MHz, CHLOROFORM-d) ppm 11.80, 12.97, 14.29, 16.41, 36.48, 38.96, 39.33, 39.92, 51.82, 53.77, 54.24, 69.24, 69.69, 70.13, 70.27, 70.41, , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 1177 (M+H) + General procedure for the synthesis of (2S+2S)-PEG derivatives. (S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[ 4,3-a][1,4]diazepine-2-carboxylic acid (CAS # ) (44 mg, 0.1 mmol), mono-boc-amino-peg-amine (1.5 eq), HATU (1.5 eq), and N,N-diisopropylethylamine (1.5 eq) were added to DMF (0.1 M). The mixture was stirred for 4 hours at room temperature. The mixture was diluted with ethyl acetate, washed with 1N NaOH and brine, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The residue was purified by flash chromatography to give mono-amides (93%). The mono-amide was dissolved into dichloromethane (0.5 M). Trifluoroacetic acid (0.5 M) was added to the solution. The mixture was stirred for 1 hour at room temperature. The mixture was diluted with dichloromethane, washed with 1N NaOH, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The free amine was used for the next step without purification. The free amine, (S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[ 4,3-a][1,4]diazepine-2-carboxylic acid (CAS # ) (1.1 eq), HATU (1.5 eq), and 7

8 N,N-diisopropylethylamine (1.5 eq) were added to DMF (0.1 M). The mixture was stirred for 4 hours at room temperature. The mixture was diluted with ethyl acetate, washed with 1N NaOH and brine, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The residue was purified by flash chromatography to give the titled compounds (55%, for 2 steps). (2S+2S)-PEG1 (5) Dimethyl 2,2'-((6S,6'S)-(((oxybis(ethane-2,1-diyl))bis(azanediyl))bis(carbonyl))bis(4-(4-chlorophe nyl)-3,9-dimethyl-6h-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-2,6-diyl))diacetate 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.98 (s, 6 H) 2.59 (s, 6 H) (m, 12 H) 3.68 (s, 6 H) 4.53 (t, J=6.85 Hz, 2 H) 6.54 (br. s., 2 H) (m, 4 H) (m, 4 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 11.78, 16.49, 36.44, 39.94, 51.88, 53.77, 69.43, , , , , , , , , , , , MS (ESI) m/z: 957 (M+H) + General procedure for the synthesis of (6S+6S)-PEG derivatives. (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)acetic acid (CAS # ) (40 mg, 0.1 mmol), mono-boc-amino-peg-amine (1.5 eq), HATU (1.5 eq), and N,N-diisopropylethylamine (1.5 eq) were added to DMF (0.1 M). The mixture was stirred for 4 hours at room temperature. The mixture was diluted with ethyl 8

9 acetate, washed with 1N NaOH and brine, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The residue was purified by flash chromatography to give mono-amides (78 to 99%). The mono-amide was dissolved into dichloromethane (0.5 M). Trifluoroacetic acid (0.5 M) was added to the solution. The mixture was stirred for 1 hour at room temperature. The mixture was diluted with dichloromethane, washed with 1N NaOH, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The free amine was used for the next step without purification. The free amine, (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)acetic acid (CAS # ) (1.1 eq), HATU (1.5 eq), and N,N-diisopropylethylamine (1.5 eq) were added to DMF (0.1 M). The mixture was stirred for 4 hours at room temperature. The mixture was diluted with ethyl acetate, washed with 1N NaOH and brine, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The residue was purified by flash chromatography to give the titled compounds (69 to 72%, for 2 steps). (6S+6S)-PEG1 (4) 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-thi eno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.66 (br. s., 6 H) 2.38 (br. s., 6 H) 2.65 (br. s., 6 9

10 H) 3.25 (d, J=10.96 Hz, 2 H) 3.49 (br. s., 2 H) 3.59 (br. s., 2 H) 3.79 (br. s., 2 H) (m, 2 H) 4.10 (d, J=7.04 Hz, 2 H) 4.77 (d, J=7.04 Hz, 2 H) 7.26 (d, J=7.04 Hz, 5 H) 7.37 (d, J=7.43 Hz, 4 H) (m, 2 H) 13 C NMR (125 MHz, DMSO-d 6 ) ppm 11.73, 13.00, 14.28, 38.86, 39.58, 54.68, 68.85, , , , , , , , , , , MS (ESI) m/z: 869 (M+H) + (6S+6S)-PEG7 (MT1, 35) 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-thi eno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate (MT1) 1 H NMR (500 MHz, DMSO-d 6 ) ppm 1.61 (s, 6 H) 2.40 (s, 6 H) 2.58 (s, 6 H) (m, 10 H) (m, 28 H) (m, 2 H) 7.42 (s, 4 H) (m, 4 H) (m, 2 H) 13 C NMR (125 MHz, DMSO-d 6 ) ppm 11.31, 12.71, 14.08, 37.52, 38.65, 53.86, 69.22, 69.63, 69.78, , , , , , , , , , , , MS (ESI) m/z: 1133 (M+H) + HRMS (ESI) m/z: C 54 H 66 Cl 2 N 10 O 9 S 2 requires m/z , found m/z (M+H) + 10

11 General procedure for the synthesis of 6S+IBET derivatives. I-BET151 (CAS # ) (42 mg, 0.1 mmol) was dissolved to DMF (0.5 M). Sodium hydride (1.2 eq) was added to the solution. The mixture was stirred for 30 minutes at room temperature. bromoacetate (1.3 eq) was added to the mixture. The mixture was stirred for 4 hours at room temperature. The mixture was diluted with EtOAc, washed with water and brine, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The methyl ester was used for the next step without purification. The methyl ester was dissolved to THF (0.5 M) and methanol (0.5 M). 1N NaOH (4 eq) was added to the mixture. The mixture was stirred for 1 hour at room temperature. The mixture was diluted with EtOAc, washed with 1N HCl and brine, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure to afford I-BET151 acetate. I-BET151 acetate was used for the next step without purification. I-BET151 acetate, corresponding JQ1-PEG-amine (1.5 eq), HATU (1.5 eq), and N,N-diisopropylethylamine (1.5 eq) were added to DMF (0.1 M). The mixture was stirred for 4 hours at room temperature. The mixture was diluted with EtOAc, washed with 1N NaOH and brine, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The residue was purified by flash chromatography to give the titled compounds (17 to 52%, for 3 steps). 6S+IBET-PEG0 (17) 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazep in-6-yl)-n-(2-(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((r)-1-(pyridin-2-yl)eth 11

12 yl)-1,2-dihydro-3h-imidazo[4,5-c]quinolin-3-yl)acetamido)ethyl)acetamide 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.68 (s, 3 H) (m, 5 H) (m, 3 H) (m, 3 H) (m, 3 H) (m, 4 H) (m, 6 H) 4.64 (dd, J=9.39, 5.48 Hz, 1 H) (m, 2 H) 6.41 (d, J=7.04 Hz, 1 H) (m, 1 H) (m, 2 H) (m, 2 H) (m, 2 H) 7.84 (s, 1 H) 7.92 (br. s., 1 H) 8.61 (d, J=4.70 Hz, 1 H) 8.78 (s, 1 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 10.7, 11.7, 11.8, 13.1, 14.4, 38.8, 39.1, 39.6, 54.5, 55.7, 76.7, 77.0, 77.3, 112.3, 115.8, 121.6, 122.7, 123.7, 128.8, 129.8, 130.4, 130.9, 131.1, 132.1, 136.4, 137.0, 137.3, 149.3, 150.0, 155.5, 155.9, 159.6, 164.2, 166.4, 166.8, MS (ESI) m/z: 898 (M+H) + 6S+IBET-PEG1 (18) 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazep in-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 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.66 (s, 3 H) 2.05 (s, 3 H) 2.16 (s, 3 H) 2.32 (s, 3 H) 2.42 (s, 3 H) 2.72 (s, 3 H) (m, 4 H) (m, 6 H) (m, 2 H) (m, 1 H) (m, 1 H) 4.69 (dd, J=11.35, 3.91 Hz, 1 H) 4.96 (s, 2 H) 6.42 (d, J=7.43 Hz, 1 H) (m, 1 H) 7.21 (dd, J=7.24, 4.89 Hz, 1 H) (m, 4 H)

13 (td, J=7.63, 1.57 Hz, 1 H) 7.86 (s, 1 H) 8.55 (br. s., 1 H) 8.63 (d, J=4.30 Hz, 1 H) 8.86 (br. s., 1 H) 9.19 (s, 1 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 10.8, 11.7, 11.9, 13.1, 14.3, 39.2, 39.6, 39.9, 44.9, 54.9, 55.8, 55.8, 69.1, 69.2, 112.4, 115.9, 121.7, 122.6, 128.8, 129.8, 130.5, 131.0, 131.1, 131.9, 136.6, 136.8, 137.3, 149.2, 149.8, 155.7, 159.6, 164.2, 166.3, 167.0, MS (ESI) m/z: 942 (M+H) + 6S+IBET-PEG2 (19) 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazep in-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)ac etamide 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.18 (br. s., 2 H) 1.59 (br. s., 3 H) 1.97 (br. s., 1 H) (m, 4 H) 2.22 (br. s., 3 H) 2.33 (br. s., 3 H) 2.61 (br. s., 3 H) (m, 10 H) (m, 1 H) 4.64 (br. s., 1 H) (m, 2 H) 6.36 (br. s., 1 H) 6.76 (br. s., 1 H) (m, 5 H) 7.52 (br. s., 1 H) (m, 2 H) 8.20 (br. s., 1 H) (m, 1 H) 8.76 (br. s., 1 H) MS (ESI) m/z: 986 (M+H) + 6S+IBET-PEG3 (20) 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazep 13

14 in-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 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.66 (s, 3 H) 2.12 (s, 3 H) 2.14 (s, 3 H) 2.29 (s, 3 H) 2.41 (s, 3 H) (m, 3 H) (m, 4 H) (m, 3 H) (m, 11 H) (m, 2 H) (m, 3 H) 6.40 (br. s., 1 H) 6.79 (br. s., 1 H) (m, 2 H) (m, 3 H) (m, 1 H) 7.78 (s, 1 H) 8.08 (br. s., 1 H) (m, 1 H) 8.54 (br. s., 1 H) 8.64 (d, J=4.30 Hz, 1 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 10.73, 11.65, 11.83, 13.08, 14.37, 29.67, 38.68, 39.27, 39.33, 54.44, 55.7, 69.69, 69.88, 70.05, 70.19, 112.5, 115.9, , 122.6, , , , , 131.0, 131.3, , , , , , , , , , , , , , , MS (ESI) m/z: 1030 (M+H) + 6S+IBET-PEG4 (21) 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazep in-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 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.55 (s, 3 H) 1.93 (s, 3 H) (m, 3 H) 14

15 (m, 3 H) 2.28 (s, 3 H) (m, 2 H) (m, 3 H) (m, 8 H) (m, 15 H) 4.60 (t, J=6.85 Hz, 1 H) 4.74 (s, 2 H) 6.32 (d, J=6.65 Hz, 1 H) (m, 1 H) (m, 2 H) 7.29 (d, J=8.61 Hz, 2 H) 7.48 (t, J=7.04 Hz, 1 H) 7.57 (br. s., 1 H) 7.69 (s, 1 H) 7.80 (br. s., 1 H) 8.52 (d, J=4.30 Hz, 1 H) 8.58 (s, 1 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 14.38, , , , , , , , MS (ESI) m/z: 1074 (M+H) + 6S+IBET-PEG7 (22) 2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazep in-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 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.56 (s, 3 H) (m, 5 H) 2.18 (s, 3 H) 2.29 (s, 3 H) 2.55 (s, 3 H) 3.28 (dd, J=14.67, 7.24 Hz, 1 H) (m, 5 H) (m, 32 H) 4.56 (t, J=7.04 Hz, 1 H) (m, 2 H) 6.33 (d, J=7.43 Hz, 1 H) 7.12 (dd, J=7.24, 4.89 Hz, 1 H) (m, 3 H) 7.30 (d, J=8.61 Hz, 2 H) 7.51 (t, J=7.04 Hz, 1 H) 7.70 (s, 2 H) 8.54 (d, J=4.70 Hz, 1 H) 8.58 (s, 1 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 10.64, 10.71, 11.55, 11.64, 11.69, 11.79, 12.96, 13.05, 14.32, 38.88, 39.30, 39.47, 54.25, 54.34, 70.15, 70.21, 70.36, , , 15

16 121.63, , , , , , , , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 1206 (M+H) + General procedure for the synthesis of 2S+IBET derivatives. I-BET151 acetate, corresponding mono-boc-amino-peg-amine (1.5 eq), HATU (1.5 eq), and N,N-diisopropylethylamine (1.5 eq) were added to DMF (0.1 M). The mixture was stirred for 4 hours at room temperature. The mixture was diluted with EtOAc, washed with 1N NaOH and brine, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The residue was purified by flash chromatography to give mono-amide (54 to 88%, for 3 steps). The mono-amide was dissolved into dichloromethane (0.5 M). Trifluoroacetic acid (0.5 M) was added to the solution. The mixture was stirred for 1 hour at room temperature. The mixture was diluted with dichloromethane, washed with 1N NaOH, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The amine was used for the next step without purification. The amine, (S)-4-(4-chlorophenyl)-6-(2-methoxy-2-oxoethyl)-3,9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[ 4,3-a][1,4]diazepine-2-carboxylic acid (CAS # ) (1.1 eq), HATU (1.5 eq), and N,N-diisopropylethylamine (1.5 eq) were added to DMF (0.1 M). The mixture was stirred for 4 hours at room temperature. The mixture was diluted with EtOAc, washed with 1N NaOH and 16

17 brine, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The residue was purified by flash chromatography to give the titled compounds (38 to 64%, for 2 steps). 2S+IBET-PEG0 (23) 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)carba moyl)-3,9-dimethyl-6h-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetate 1 H NMR (400 MHz, METHANOL-d4) ppm 1.90 (s, 3 H) 1.94 (s, 3 H) (m, 5 H) 2.61 (s, 3 H) 3.08 (s, 3 H) 3.27 (dt, J=3.23, 1.71 Hz, 2 H) (m, 2 H) 3.69 (s, 3 H) 3.72 (s, 1 H) 4.60 (t, J=7.24 Hz, 1 H) (m, 3 H) 4.86 (d, J=4.70 Hz, 2 H) 6.33 (d, J=7.43 Hz, 1 H) 6.73 (br. s., 1 H) (m, 2 H) (m, 2 H) 7.29 (dd, J=7.43, 5.09 Hz, 1 H) (m, 1 H) 7.43 (s, 2 H) (m, 1 H) 7.72 (td, J=7.83, 1.57 Hz, 1 H) 8.53 (d, J=4.30 Hz, 1 H) 8.59 (s, 1 H) 13 C NMR (100 MHz, METHANOL-d4) ppm 10.76, 11.63, 11.76, 11.87, 15.79, 16.92, 18.52, 37.33, 40.56, 41.45, 45.14, 52.58, 54.99, 56.7, , , , 123.2, , , , , , 130.3, , , , , , , 135.5, , , , , , , , , , , , , , , , 173.1, MS (ESI) m/z: 942 (M+H) + 17

18 2S+IBET-PEG1 (24) 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)e thyl)carbamoyl)-3,9-dimethyl-6h-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)ace tate 1 H NMR (400 MHz, METHANOL-d4) ppm 1.94 (s, 3 H) 2.00 (s, 3 H) (m, 3 H) 2.18 (s, 3 H) 2.61 (s, 3 H) 3.04 (s, 2 H) 3.24 (dt, J=3.13, 1.57 Hz, 2 H) (m, 6 H) 3.63 (s, 3 H) 3.69 (s, 1 H) (m, 1 H) (m, 5 H) 6.30 (q, J=7.30 Hz, 1 H) 6.75 (br. s., 1 H) (m, 3 H) (m, 3 H) (m, 2 H) (m, 1 H) 7.70 (td, J=7.73, 1.76 Hz, 1 H) 8.50 (d, J=4.30 Hz, 1 H) 8.59 (s, 1 H) 13 C NMR (100 MHz, METHANOL-d4) ppm 10.77, 11.65, 11.74, 11.83, 14.61, 15.78, 16.81, 18.36, 37.27, 40.85, 41.26, 52.54, 52.56, 55.04, 56.73, 70.41, 70.49, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 986 (M+H) + 2S+IBET-PEG2 (25) 18

19 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)ethox y)ethoxy)ethyl)carbamoyl)-3,9-dimethyl-6h-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazep in-6-yl)acetate 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.94 (d, J=8.22 Hz, 3 H) 2.01 (s, 3 H) 2.09 (m, 2 H) (s, 3 H) 2.55 (s, 3 H) (m, 18 H) 3.64 (s, 3 H) 4.53 (dd, J=7.83, 6.26 Hz, 1 H) (m, 2 H) 6.29 (q, J=6.78 Hz, 1 H) (m, 1 H) 6.85 (br. s., 1 H) 6.93 (br. s., 1 H) (m, 2 H) (m, 2 H) (m, 2 H) 7.54 (td, J=7.63, 1.57 Hz, 1 H) 7.69 (s, 1 H) 8.49 (d, J=4.70 Hz, 1 H) 8.56 (s, 1 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 10.68, 10.76, 11.60, 11.77, 16.49, 36.53, 45.04, 52.02, 53.90, 69.45, 70.09, , , , , , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 1030 (M+H) + 2S+IBET-PEG3 (26) 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-aza tetradecan-14-yl)carbamoyl)-3,9-dimethyl-6h-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaz 19

20 epin-6-yl)acetate 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.98 (d, J=8.22 Hz, 3 H) (m, 5 H) 2.21 (s, 3 H) 2.38 (br. s., 2 H) (m, 3 H) 3.04 (s, 2 H) (m, 16 H) (m, 3 H) (m, 1 H) (m, 2 H) 6.32 (d, J=7.04 Hz, 1 H) 6.71 (br. s., 1 H) 7.04 (br. s., 1 H) (m, 6 H) 7.56 (t, J=7.43 Hz, 1 H) 7.74 (s, 1 H) 8.55 (d, J=4.30 Hz, 1 H) 8.61 (s, 1 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 10.64, 10.72, 11.65, 11.82, 14.10, 15.62, 16.53, 36.50, 39.43, 40.04, 44.69, 51.84, 51.98, 53.74, 53.83, 69.52, 69.93, 70.04, 70.20, 70.31, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 1074 (M+H) + 2S+IBET-PEG4 (27) 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 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.93 (s, 3 H) 1.96 (s, 3 H) (m, 3 H) 20

21 2.17 (s, 3 H) (m, 2 H) 2.56 (s, 3 H) (m, 23 H) (m, 3 H) (m, 1 H) (m, 2 H) 6.28 (d, J=7.04 Hz, 1 H) 6.69 (br. s., 1 H) (m, 5 H) 7.29 (d, J=8.22 Hz, 2 H) 7.51 (t, J=7.04 Hz, 2 H) (m, 1 H) 8.51 (d, J=4.30 Hz, 1 H) 8.55 (s, 1 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 16.51, 16.58, 36.54, 53.77, 53.84, 70.13, 70.28, , , , , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 1118 (M+H) + 2S+IBET-PEG7 (28) 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 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.93 (s, 3 H) 1.95 (s, 3 H) 2.04 (d, J=7.43 Hz, 2 H) 2.17 (s, 3 H) 2.49 (br. s., 2 H) 2.58 (s, 3 H) (m, 36 H) (m, 3 H) 4.52 (dd, J=7.83, 6.26 Hz, 1 H) (m, 2 H) 6.32 (d, J=7.04 Hz, 1 H) (m, 2 H) 7.04 (br. s., 1 H) 7.12 (dd, J=7.04, 5.09 Hz, 1 H) (m, 2 H) (m, 2 H)

22 (m, 2 H) 7.68 (s, 1 H) 8.53 (d, J=4.70 Hz, 1 H) 8.57 (s, 1 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 36.52, 39.53, 39.99, 44.46, 53.84, 55.60, 59.38, 70.26, , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 1250 (M+H) + General procedure for the synthesis of IBETx2 derivatives. I-BET151 acetate, corresponding mono-boc-amino-peg-amine (1.5 eq), HATU (1.5 eq), and N,N-diisopropylethylamine (1.5 eq) were added to DMF (0.1 M). The mixture was stirred for 4 hours at room temperature. The mixture was diluted with EtOAc, washed with 1N NaOH and brine, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The residue was purified by flash chromatography to give mono-amide (54 to 88%, for 3 steps). The mono-amide was dissolved into dichloromethane (0.5 M). Trifluoroacetic acid (0.5 M) was added to the solution. The mixture was stirred for 1 hour at room temperature. The mixture was diluted with dichloromethane, washed with 1N NaOH, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The amine was used for the next step without purification. The amine, I-BET151 acetate (1.1 eq), HATU (1.5 eq), and N,N-diisopropylethylamine (1.5 eq) were added to DMF (0.1 M). The mixture was stirred for 4 hours at room temperature. The mixture was diluted with EtOAc, washed with 1N NaOH and brine, dried over Na 2 SO 4, filtrated and concentrated under reduced pressure. The residue 22

23 was purified by flash chromatography to give the titled compounds (26 to 45%, for 2 steps). IBETx2-PEG0 (29) N,N'-(ethane-1,2-diyl)bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-methoxy-2-oxo-1-((R)-1-(pyri din-2-yl)ethyl)-1,2-dihydro-3h-imidazo[4,5-c]quinolin-3-yl)acetamide) 1 H NMR (400 MHz, CHLOROFORM-d) ppm (m, 12 H) (m, 6 H) 3.48 (d, J=17.22 Hz, 10 H) (m, 4 H) 6.42 (q, J=6.65 Hz, 2 H) (m, 2 H) 7.22 (dd, J=7.24, 4.89 Hz, 2 H) (m, 4 H) 7.63 (td, J=7.73, 1.76 Hz, 2 H) (m, 2 H) (m, 2 H) 8.68 (s, 2 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm , , , , , , , , , , , MS (ESI) m/z: 971 (M+H) + IBETx2-PEG1 (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) 1 H NMR (400 MHz, CHLOROFORM-d) ppm (m, 12 H) 2.30 (s, 6 H) (m, 14 H) 4.79 (q, J=16.04 Hz, 4 H) 6.42 (d, J=7.04 Hz, 2 H) (m, 2 H) 7.23 (dd, J=7.43, 4.70 Hz, 2 H) 7.35 (br. s., 2 H) 7.62 (td, J=7.83, 1.57 Hz, 2 H) (m, 2 H) 8.04 (t, J=5.28 Hz, 2 H) 8.62 (d, J=4.70 Hz, 2 H) 8.77 (s, 2 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 10.69, 10.76, 11.62, 11.71, 17.74, 39.71, 23

24 45.04, 55.71, 55.78, 69.21, , , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 1015 (M+H) + IBETx2-PEG2 (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) 1 H NMR (400 MHz, CHLOROFORM-d) ppm 1.97 (s, 6 H) 2.21 (br. s, 10 H) 2.47 (br. s., 4 H) 3.41 (d, J=17.22 Hz, 10 H) (m, 6 H) (m, 4 H) 6.36 (d, J=7.04 Hz, 2 H) (m, 2 H) (m, 2 H) 7.31 (br. s., 2 H) (m, 4 H) 7.74 (s, 2 H) 8.53 (d, J=4.70 Hz, 2 H) 8.62 (s, 2 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 10.67, 10.75, 11.59, 11.69, 39.54, 55.75, 69.49, 70.11, , , , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 1059 (M+H) + IBETx2-PEG3 (32) N,N'-(((oxybis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl))bis(2-(7-(3,5-dimethylisoxa 24

25 zol-4-yl)-8-methoxy-2-oxo-1-((r)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3h-imidazo[4,5-c]qui nolin-3-yl)acetamide) 1 H NMR (400 MHz, CHLOROFORM-d) ppm (m, 12 H) 2.28 (s, 6 H) 2.58 (br. s., 4 H) (m, 8 H) (m, 10 H) (m, 4 H) 6.41 (d, J=7.04 Hz, 2 H) (m, 2 H) 7.21 (dd, J=7.43, 5.09 Hz, 2 H) 7.35 (br. s., 2 H) (m, 4 H) 7.80 (s, 2 H) 8.61 (d, J=4.30 Hz, 2 H) 8.68 (s, 2 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 10.66, 10.73, 11.57, 11.66, 14.12, 39.58, 44.58, 55.71, 60.33, 69.61, 69.96, 70.17, , , , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 1103 (M+H) + IBETx2-PEG4 (33) N,N'-(3,6,9,12-tetraoxatetradecane-1,14-diyl)bis(2-(7-(3,5-dimethylisoxazol-4-yl)-8-meth oxy-2-oxo-1-((r)-1-(pyridin-2-yl)ethyl)-1,2-dihydro-3h-imidazo[4,5-c]quinolin-3-yl)aceta mide) 1 H NMR (400 MHz, CHLOROFORM-d) ppm (m, 6 H) 2.28 (s, 6 H) 2.53 (br. s., 4 H) 3.52 (d, J=3.52 Hz, 4 H) 3.63 (t, J=4.89 Hz, 4 H) (m, 10 H) (m, 4 H) 6.43 (d, J=7.04 Hz, 2 H) (m, 2 H) (m, 2 H) 7.34 (br. s., 2 H) 7.60 (t, J=7.24 Hz, 2 H) (m, 4 H) 8.62 (d, J=4.70 Hz, 2 H) 8.68 (s, 2 H) 25

26 13 C NMR (100 MHz, CHLOROFORM-d) ppm 10.66, 10.73, 11.58, 11.66, 14.13, 17.66, 39.62, 44.41, 55.61, 55.70, 69.76, 70.14, 70.35, , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 1147 (M+H) + IBETx2-PEG7 (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-y l)acetamide) 1 H NMR (400 MHz, CHLOROFORM-d) ppm (m, 12 H) 2.27 (s, 6 H) (m, 34 H) (m, 4 H) 6.43 (d, J=6.65 Hz, 2 H) (m, 2 H) 7.21 (dd, J=7.04, 5.09 Hz, 2 H) 7.32 (br. s., 2 H) (m, 4 H) 7.79 (s, 2 H) 8.63 (d, J=4.69 Hz, 2 H) 8.66 (s, 2 H) 13 C NMR (100 MHz, CHLOROFORM-d) ppm 10.64, 10.72, 11.55, 11.66, 39.57, 44.43, 55.60, 55.71, 69.61, 70.26, 70.36, , , , , , , , , , , , , , , , , , , , , , , , MS (ESI) m/z: 1279 (M+H) + 26

27 Synthetic schemes of MT1. 27