Xanthine derivative inhibitors of BET proteins

Abstract

This invention relates to xanthine derivative compounds that are inhibitors of BET bromodomains proteins, the method of preparation thereof and applications thereof. ##STR00001##

Claims

1. An inhibitor of a BET protein comprising a xanthine compound of formula (I) ##STR00244## wherein: X represents an oxygen atom; Y represents an oxygen atom, an amino group, or NHR.sub.o; wherein R.sub.o represents —C(O)OR.sub.p wherein R.sub.p represents a C.sub.1-C.sub.4 alkyl; R.sub.1 represents a hydrogen atom; R.sub.3 is selected from the group consisting of: ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl or a benzyl optionally substituted by: a halogen atom, or a C.sub.1-C.sub.4alkyl; R.sub.7 represents a C.sub.1-C.sub.4 alkyl; R.sub.8 represents —SH, —CH.sub.2—O—R.sub.r or —CH.sub.2—S—R, wherein R.sub.r represents one of the following groups optionally substituted by one or more C.sub.1-C.sub.6 alkyl, amino groups, halogen atoms, (C.sub.1-C.sub.4)alkanoic acid, —S(O.sub.2)—C.sub.1-C.sub.4)alkyl, —S (O.sub.2)-piperidine, —S(O.sub.2)—(N,N)dimethylamine, —S(O.sub.2)-morpholine, nitro groups, —C(═O)—O—(C.sub.1-C.sub.4)alkyl, —S(O.sub.2)—N(H)—(C.sub.1-C.sub.4)alkyl, oxo-pyrazole optionally substituted by one or more (C.sub.1-C.sub.4)alkanoic acid, pyrazole optionally substituted by one or more (C.sub.1-C.sub.4)alkanoic acid or (C.sub.1—C.sub.4)alkyl, phenyl, oxy-phenyl, pyrrolidine optionally substituted by one or more (C.sub.1-C.sub.4)alkanoic acid, thiazolidin optionally substituted by one or more (C.sub.1-C.sub.4)alkanoic acid, —C(═O)—N(H)-benzyl, —N(H)-quinazolinone, —OH, thiophenyl optionally substituted by one or more (C.sub.1-C.sub.4)alkanoic acid, methyl-tetrahydrofuran or —CH.sub.2-pyrazole optionally substituted by one or more (C.sub.1-C.sub.4) alkyl: ##STR00245## wherein * is the linking point to the sulfur atom or oxygen atom, or a pharmaceutically acceptable salt thereof and/or tautomeric form thereof.

2. An inhibitor of a BET protein which is a xanthine compound of formula (I) ##STR00246## wherein: X represents an oxygen atom; Y represents an oxygen atom, an amino group, or NHR.sub.o; wherein R.sub.o represents —C(O)OR.sub.p wherein R.sub.p represents a C.sub.1-C.sub.4 alkyl; R.sub.1 represents a hydrogen atom; R.sub.3 is selected from the group consisting of: ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, n-hexyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, or a benzyl, optionally substituted by: a halogen atom, or a C.sub.1-C.sub.4 alkyl; R.sub.7 represents a C.sub.1-C.sub.4 alkyl; R.sub.8 represents —CH.sub.2—O—R.sub.s or —CH.sub.2—S—R.sub.s; wherein R.sub.s represents one of the following groups optionally substituted by one or more methyl, ethyl, butyl or amino groups, halogen atoms, (C.sub.1-C.sub.4)alkanoic acid, —S(O.sub.2)—(C.sub.1-C.sub.4)alkyl, —S(O.sub.2)-piperidine, —S(O.sub.2)—(N,N)dimethylamine, —S(O.sub.2)-morpholine, nitro groups, —C(═O)—O—(C.sub.1-C.sub.4)alkyl, —S(O.sub.2)—N(H)—(C.sub.1-C.sub.4)alkyl, oxo-pyrazole optionally substituted by one or more (C.sub.1-C.sub.4)alkanoic acid, pyrazole optionally substituted by one or more (C.sub.1-C.sub.4)alkanoic acid or (C.sub.1-C.sub.4)alkyl, thiazolidin optionally substituted by one or more (C.sub.1-C.sub.4)alkanoic acid, —C(═O)—N(H)-benzyl, —OH, thiophenyl optionally substituted by one or more (C.sub.1-C.sub.4)alkanoic acid, methyl-tetrahydrofuran or —CH.sub.2-pyrazole optionally substituted by one or more (C.sub.1-C.sub.4) alkyl: ##STR00247## wherein * is the linking point to the sulfur atom or oxygen atom; or a pharmaceutically acceptable salt thereof and/or tautomeric form thereof.

3. A pharmaceutical composition comprising as active principle, the inhibitor according to claim 1 and a pharmaceutically acceptable excipient.

4. A method for treating leukemia comprising administering to a mammal in need thereof a therapeutically effective amount of the inhibitor according to claim 1.

5. The inhibitor according to claim 1 wherein said inhibitor has an IC.sub.50 for BRD4 (BD1) equal to or less than 50 μM.

6. The inhibitor according to claim 1 wherein said inhibitor has an IC.sub.50 equal to or less than 20 μM for BRD4 (BD1).

7. The inhibitor according to claim 1 wherein said inhibitor has an IC.sub.50 equal to or less than 10 μM for BRD4 (BD1).

8. A non-therapeutic method for inhibiting a bromodomain of a BET protein comprising contacting said bromodomain of a BET protein with the inhibitor according to claim 1, wherein said BET protein is selected from the group consisting of BRD2, BRD3, BRD4 and BRDT.

9. A non-therapeutic method for inhibiting the BRD4 protein, wherein said inhibitor binds to BD1 of BRD4, and wherein said inhibitor has an IC.sub.50 for BRD4 (BD1) equal to or less than 50 μM, comprising contacting the inhibitor according to claim 1 with a BRD4 protein.

10. A non-therapeutic in vivo method for degrading a BET protein by using the inhibitor according claim 1 in the presence of cells which express the E3 ubiquitin ligase, wherein the inhibitor comprises either Y, R.sub.3 or R.sub.8 as a linker-ligand for the E3 ubiquitin ligase, said ligand being a cereblon ligand or a VHL ligand having the following formulas: ##STR00248##

11. A non-therapeutic method for inhibiting a bromodomain of a BET protein comprising contacting said bromodomain of a BET protein with the pharmaceutical composition according to claim 3, wherein the BET protein is selected from the group consisting of BRD2, BRD3, BRD4 and BRDT.

12. A pharmaceutical composition comprising as active principle, the inhibitor according to claim 2 and a pharmaceutically acceptable excipient.

13. A method for treating leukemia comprising administering to a mammal in need thereof a therapeutically effective amount of the inhibitor according to claim 2.

14. The inhibitor according to claim 2 wherein said inhibitor has an IC50 for BRD4 (BD1) equal to or less than 50 μM.

15. The inhibitor according to claim 2 wherein said inhibitor has an IC50 equal to or less than 20 μM for BRD4 (BD1).

16. The inhibitor according to claim 2 wherein said inhibitor has an IC50 equal to or less than 10 μM for BRD4 (BD1).

17. A non-therapeutic method for inhibiting a bromodomain of a BET protein comprising contacting said bromodomain of a BET protein with the inhibitor according to claim 2, wherein said BET protein is selected from the group consisting of BRD2, BRD3, BRD4 and BRDT.

18. A non-therapeutic method for inhibiting the BRD4 protein, wherein said inhibitor binds to BD1 of BRD4, and wherein said inhibitor has an IC.sub.50 for BRD4 (BD1) equal to or less than 50 μM, comprising contacting the inhibitor according to claim 2 with a BRD4 protein.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1. Cell-based assays (A) Effect of 26b on Jurkat cell viability as a function of the compound concentration after 72 h incubation at 37° C. (B) C-myc pro-oncogene downregulation profile in the presence of different concentrations of 26b at 0.5% DMSO after 24 h incubation (50 μg of loaded protein).

EXAMPLES

(2) Preparation of the Compounds According to the Invention

(3) General Synthesis of the Compounds According to the Invention

(4) Proton NMR spectra, 1H and 13C NMR, were recorded by using a Bruker AC400 or AC250 spectrometer. Chemical shifts, 6 are expressed in ppm and coupling values, J, in hertz. Abbreviations for peaks are, br: broad, s: singlet, d: doublet, t: triplet, q: quadruplet, quint: quintuplet, sex: sextuplet and m: multiplet. The spectra recorded are consistent with the proposed structures. Reaction monitoring and purity of compounds were recorded by using analytical Agilent Infinity high performance liquid chromatography (Column Zorbax SB-C18 1.8 μM (2.1×50 mm); Mobile phase (A: 0.1% FA H.sub.2O, B: 0.1% FA MeCN, Time/% B 0/10, 4/90, 7/90, 9/10, 10/10); Flow rate 0.3 mL/min; Diluent MeOH) with DAD at 230 nM. All tested compounds yielded data consistent with a purity of ≥95%.

Example 1: 3-Butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (26b)

(5) ##STR00223##

(6) To a solution of 6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-thiol (35 mg, 0.15 mmol) in dimethylformamide (2 mL) was injected diisopropylethylamine (26 μL, 0.15 mmol). After 5 min under stirring, a solution of 3-butyl-8-chloromethyl-7-ethylxanthine (43 mg, 0.15 mmol) in dimethylformamide (2 mL) was added dropwise. The resulting mixture was stirred at room temperature for 30 min. The solvent was distillated off under reduced pressure and the residue purified by column chromatography (CH.sub.2Cl.sub.2-MeOH 10:0.5) to afford the 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (64 mg, 88%) as white solid. Rf=0.29. 1H NMR (250 MHz, CDCl.sub.3) δ 7.88 (1H, sbroad), 4.73 (2H, s), 4.49 (2H, q, J=7.1 Hz), 4.04 (2H, t, J=7.4 Hz), 2.76 (3H, s), 2.73-2.69 (2H, m), 2.69 (3H, s), 1.73 (2H, quint, J=7.4 Hz), 1.56-1.34 (6H, m), 1.40-1.28 (2H, m), 1.45 (3H, t, J=7.4 Hz), 1.01 (3H, t, J=7.1 Hz) and 0.95 (3H, t, J=7.4 Hz); 13C NMR (100 MHz, CDCl3) δ 165.0, 164.1, 154.1, 153.8, 150.6, 150.1, 149.2, 143.5, 121.8, 42.7, 41.4, 31.9, 30.2, 28.0, 26.8, 23.8, 22.9, 20.0, 16.6, 14.0, 13.9 and 13.8. LCMS C.sub.23H.sub.32N.sub.8O.sub.2S Rt=6.755 min, m/z=484.6, purity >99%. HRMS (ESI+) for C.sub.23H.sub.33N.sub.8O.sub.2S (M+H) calcd, 485.2442; found, 485.2442.

3-butyl-8-chloromethyl-7-ethylxanthine (19c)

(7) ##STR00224##

(8) To a solution of 3-butyl-7-ethyl-8-hydroxymethylxanthine (345 mg, 1.3 mmol) in dichloromethane (5 mL) was injected thionyl chloride (283 μL, 3.9 mmol). The reaction mixture was refluxed 15 min. then the solvent was distillated off under reduced pressure. The residue was extended with Et.sub.2O (20 mL) to allow crystallization and the precipitate was collected by filtration affording 3-butyl-8-chloromethyl-7-ethylxanthine (325 mg, 88%) as a light yellow powder. .sup.1H NMR (250 MHz, MeOD) δ 4.77 (2H, s), 4.38 (2H, q, J=7.1 Hz), 4.00 (2H, t, J=7.3 Hz), 1.70 (2H, quint, J=7.3 Hz), 1.50 (3H, t, J=7.1 Hz) 1.39 (2H, sex, J=7.3 Hz) and 0.94 (3H, t, J=7.3 Hz).

3-Butyl-7-ethyl-8-hydroxymethylxanthine (18a)

(9) ##STR00225##

(10) Under argon, at 0° C., to a mixture of 3-butyl-8-hydroxymethylxanthine (238 mg, 1 mmol) and potassium carbonate (152 mg, 1.1 mmol) in dimethylformamide (20 mL) was injected a solution of ethyl iodide (80 μL, 1 mmol) in dimethylformamide (5 mL). The resulting mixture was allowed to warm at room temperature and stirred at this temperature overnight. The solvent was distillated off under reduce pressure and the residue successively diluted with H.sub.2O (10 mL), acidified with aqueous 10% HCl (10 mL) and extracted with CH.sub.2Cl.sub.2 (3×5 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 and the solvent was distillated off to afford 3-butyl-7-ethyl-8-hydroxymethylxanthine (182 mg, 72%) as a light yellow solid. .sup.1H NMR (250 MHz, MeOD) δ 4.72 (2H, s), 4.40 (2H, q, J=7.2 Hz), 3.98 (2H, t, J=7.3 Hz), 1.69 (2H, quint, J=7.3 Hz), 1.45 (3H, t, J=7.2 Hz) 1.37 (2H, sex, J=7.3 Hz) and 0.93 (3H, t, J=7.3 Hz); .sup.13C NMR (63 MHz, MeOD) δ 156.4, 153.7, 152.7, 150.4, 109.0, 56.9, 43.4, 42.1, 31.2, 20.9, 16.7 and 14.1.

3-Butyl-8-hydroxymethylxanthine (17a)

(11) ##STR00226##

(12) At 85° C., to a solution of N-(6-amino-1-butyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-2-hydroxyacetamide (1.83 g, 7.1 mmol) in a mixture of ethanol (10 mL) and water (5 mL) was added dropwise a 10% aqueous sodium hydroxide (5 mL) over 30 min. After addition the reaction mixture was stirred at 85° C. for 2 h, allowed to cool at room temperature, diluted with H.sub.2O (10 mL) and then acidified with aqueous 1N HCl until pH=1. After cooling in ice bath for few min. the precipitate was collected by filtration and generously washed with H.sub.2O. The solid was dried overnight in oven at 80° C. affording 3-butyl-8-hydroxymethylxanthine (1.45 g, 81%) as a light yellow powder. .sup.1H NMR (250 MHz, DMSO-d6) δ 4.49 (2H, s), 3.89 (2H, t, J=7.3 Hz), 1.61 (2H, quint, J=7.3 Hz), 1.28 (2H, sex, J=7.3 Hz) and 0.90 (3H, t, J=7.3 Hz). .sup.13C NMR (63 MHz, DMSO-d6) δ 154.5, 153.9, 150.9, 149.4, 106.8, 56.9, 41.7, 19.5 and 13.0.

N-(6-Amino-1-butyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-2-hydroxyacetamide

(13) ##STR00227##

(14) Under stirring and solvent free condition, a mixture of 5,6-diamino-1-butyluracil (2.75 g, 13.9 mmol) and glycolic acid (2.116 g, 28 mmol) was heated at 120° C. for 30 min. To the resulting solid, allowed to cool at room temperature, was successively added EtOH (20 mL), H.sub.2O (4 mL) and Et.sub.2O (60 mL). The precipitate was filtrated off and washed with Et.sub.2O to afford N-(6-amino-1-butyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-2-hydroxyacetamide (3.29 g, 93%) as light yellow powder. .sup.1H NMR (250 MHz, DMSO-d6) δ 10.57 (1H, sbroad), 8.09 (1H, s), 6.57 (2H, s), 5.34 (1H, sbroad), 3.93 (2H, s), 3.78 (2H, t, J=7.2 Hz), 1.49 (2H, quint, J=7.2 Hz), 1.30 (2H, sex, J=7.2 Hz) and 0.89 (3H, t, J=7.2 Hz).

5,6-diamino-1-butyluracil

(15) ##STR00228##

(16) At 70° C., to a solution of 6-amino-5-nitroso-1-butyluracil (7.68 g, 36 mmol) in 12% aqueous solution of ammonia (150 mL) was added sodium hydrosulfite (19 g, 0.11 mol) in solid fraction over 15 min. After addition, the reaction mixture was allowed to cool at room temperature and kept at 4° C. for 1 h. The precipitate was collected by filtration, washed with H.sub.2O, and dried under reduced pressure to afford 5,6-diamino-1-butyluracil 18 (4.9 g, 68%) as light green powder. .sup.1H NMR (250 MHz, DMSO-d6) δ 10.51 (1H, sbroad), 6.13 (2H, sbroad), 3.76 (2H, t, J=7.3 Hz), 2.85 (2H, sbroad), 1.48 (2H, quint, J=7.3 Hz), 1.28 (2H, sex, J=7.3 Hz) and 0.88 (3H, t, J=7.3 Hz). .sup.13C NMR (63 MHz, MeOD) δ 159.3, 152.9, 152.3, 124.4, 44.5, 30.8, 20.7 and 14.1.

6-Amino-5-nitroso-1-butyluracil

(17) ##STR00229##

(18) At 70° C., to a solution of 6-amino-1-butyluracil (7.7 g, 42 mmol) in a mixture of N,N-dimethylformamide (70 mL) and water (25 mL) was added sodium nitrite (1.27 g, 69 mmol) then concentrated hydrochloric acid (5 mL). After 10 min. under stirring at 70° C., the mixture was allowed to cool at room temperature and kept at 4° C. overnight. The precipitate was filtered off, washed with H.sub.2O, and dried under reduced pressure, to afford 6-amino-5-nitroso-1-butyluracil 16 (7.68 g, 86%) as purple powder. .sup.1H NMR (250 MHz, DMSO-d6) δ 12.62 (2H, sbroad), 10.89 (1H, sbroad), 3.76 (2H, t, J=7.4 Hz), 1.48 (2H, quint, J=7.4 Hz), 1.29 (2H, sex, J=7.4 Hz) and 0.91 (3H, t, J=7.4 Hz).

6-Amino-1-butyluracil

(19) ##STR00230##

(20) At 85° C., to a suspension of N-butyl-N′-cyanoacetylurea (13.5 g, 73.8 mmol) in a mixture of water (30 mL) and ethanol (50 mL) was added dropwise a 10% aqueous sodium hydroxide (10 mL) over a period of 15 min. After addition, the resulting mixture was stirred at 85° C. for 45 min. The reaction solution was concentrated under reduced pressure to allow crystallization. The solid was collected by filtration and fully washed with Et.sub.2O to afford 6-amino-1-butyluracil (13 g, 96%) as white powder. .sup.1H NMR (250 MHz, DMSO-d6) δ 10.29 (1H, sbroad), 6.77 (2H, sbroad), 4.52 (1H, s), 3.71 (2H, t, J=7.3 Hz), 1.46 (2H, quint, J=7.3 Hz), 1.27 (2H, sex, J=7.3 Hz) and 0.88 (3H, t, J=7.3 Hz).

N-Butyl-N′-cyanoacetylurea

(21) ##STR00231##

(22) At 0° C., to a solution of cyanoacetic acid (10.18 g, 0.120 mol) in acetic anhydride (22.3 mL) was added butylurea (13.88 g, 0.12 mol) by solid fraction over 5 min. The mixture was successively stirred at 0° C. for 15 min., allowed to warm at room temperature, heated at 60° C. for 30 min, until completion of the reaction, then allowed to cool at room temperature. To the resulting precipitate was added Et.sub.2O (60 mL) and the solid was collected by filtration to afford N-butyl-N′-cyanoacetylurea (18.5 g, 84%) as fine white needles. .sup.1H NMR (250 MHz, DMSO-d6) δ 10.53 (1H, sbroad), 7.96 (1H, t, J=7.4 Hz), 3.91 (2H, s), 3.14 (2H, q, J=7.4 Hz), 1.44 (2H, quint, J=7.4 Hz), 1.28 (2H, sex, J=7.4 Hz), and 0.88 (3H, t, J=7.4 Hz).

Butylurea

(23) ##STR00232##

(24) At 0° C., to butylamine (10.73 g, 0.147 mol) was added concentrated hydrochloric acid (14.7 ml, 0.176 mol) and the mixture was poured into hot ethanol (150 mL). The resulting solution was then added to a solution of potassium cyanate (14.61 g, 0.176 mol) in water (150 mL) and stirring was maintained at room temperature overnight. The reaction mixture was concentrated under reduced pressure until few volume to allow butylurea to crystallize (15 g, 88%) as white plates. .sup.1H NMR (250 MHz, DMSO-d6) δ 6.02 (1H, t, J=6.7 Hz), 5.41 (2H, sbroad), 2.92 (2H, q, J=6.7 Hz), 1.34-1.19 (4H, m) and 0.84 (3H, t, J=6.7 Hz); .sup.13C NMR (63 MHz, DMSO-d6) δ 160.0, 39.5, 32.4, 20.1 and 14.3.

Example 2: 3-Butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylmethylsulfanyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (32a)

(25) ##STR00233##

(26) To a solution of 3-butyl-7-ethyl-8-mercaptoxanthine (54 mg, 0.4 mmol) in dimethylformamide (2 mL) was injected diisopropylethylamine (70 μL, 0.4 mmol). After 5 min under stirring, a solution of 6-butyl-2-chloromethyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine (50 mg, 0.4 mmol) in dimethylformamide (2 mL) was added dropwise. The resulting mixture was stirred at room temperature for 1 h, until the substrate was consumed. The solvent was distillated off under reduced pressure and the residue purified by column chromatography (CH.sub.2Cl.sub.2-MeOH 10:0.5) to afford the 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylmethylsulfanyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (63 mg, 65%) as white solid. Rf=0.44. .sup.1H NMR (250 MHz, CDCl.sub.3) δ 9.22 (1H, sbroad), 4.72 (2H, s), 4.25 (2H, q, J=7.1 Hz), 4.02 (2H, t, J=7.4 Hz), 2.73 (3H, s), 2.73-2.64 (2H, m), 2.64 (3H, s), 1.69 (2H, quint, J=7.4 Hz), 1.52-1.42 (4H, m), 1.40-1.28 (2H, m), 1.35 (3H, t, J=7.4 Hz), 0.96 (3H, t, J=7.1 Hz) and 0.89 (3H, t, J=7.4 Hz); .sup.13C NMR (100 MHz, CDCl3) δ 164.5, 163.4, 162.5, 153.5, 150.9, 150.5, 149.9, 143.6, 122.0, 108.5, 46.3, 36.5, 31.7, 31.4, 31.0, 30.0, 29.0, 28.4, 27.9, 23.7, 22.8, 22.2, 13.9, 13.8 and 13.7. LCMS C.sub.23H.sub.32N.sub.8O.sub.2S Rt=6.197 min, m/z=484.6, purity >96%.

3-butyl-7-ethyl-8-mercaptoxanthine (15a)

(27) ##STR00234##

(28) Under argon, a suspension of 3-butyl-8-chloro-7-ethylxanthine (270 mg, 1 mmol) and sodium sulfide (390 mg, 5 mmol) in dimethylformamide (20 mL) was heated at 80° C. for 1 h. The solvent was distillated off under reduced pressure and the residue was dissolved in water (30 mL), and then acidified with aqueous solution of 1N HCl until pH=4-5. The precipitate was collected by filtration and successively washed with H.sub.2O and Et.sub.2O to afford the 3-butyl-7-ethyl-8-mercaptoxanthine (238 mg, 89%) as white powder. .sup.1H NMR (250 MHz, DMSO-d6) δ 11.28 (1H, sbroad), 4.23 (2H, q, J=7.2 Hz), 3.79 (2H, t, J=7.4 Hz), 1.56-1.42 (2H, m), 1.38-1.27 (2H, m), 1.22 (3H, t, J=7.2 Hz) and 0.90 (3H, t, J=7.4 Hz).

3-Butyl-8-chloro-7-ethylxanthine (12c)

(29) ##STR00235##

(30) Under argon, to a solution of 3-butyl-7-ethylxanthine (1.0 g, 4.2 mmol) in dimethylformamide (30 mL) was added N-chlorosuccinimide (622 mg, 4.6 mmol) by solid fraction. The resulting mixture was stirred at room temperature overnight. The solvent was distillated off under reduced pressure and the residue was dissolved in CH.sub.2Cl.sub.2 (50 mL), then washed with H.sub.2O. The solvent was distillated off under reduced pressure to afford the 3-butyl-8-chloro-7-ethylxanthine (1.1 g, quantitative) as white solid. .sup.1H NMR (250 MHz, MeOD) δ 4.38 (2H, q, J=7.3 Hz), 3.98 (2H, t, J=7.3 Hz), 1.71 (2H, quint, J=7.3 Hz), 1.45-1.32 (5H, m) and 0.97 (3H, t, J=7.3 Hz).

3-Butyl-7-ethylxanthine (6a)

(31) ##STR00236##

(32) To a suspension of 7-ethylxanthine (3.61 g, 10 mmol) and potassium carbonate (3.04 g, 11 mmol) in dimethylformamide (300 mL) was injected butyl iodide (2.28 mL, 10 mmol). The mixture was stirred at room temperature for 5 h, until the complete consumption of substrate. The solvent was distillated off under reduced pressure and the residue was partitioned between CH.sub.2Cl.sub.2 (100 mL) and aqueous solution of 1N HCl. The aqueous layer was extracted with CH.sub.2Cl.sub.2 (3×20 mL) and the combined organic layers were dried over Na.sub.2SO.sub.4. The solvent was distillated off under reduced pressure until a few volume (15 mL) and a mixture Et.sub.2O-petroleum 2:1 (150 mL) was added to allow a precipitation. The solid was collected by filtration affording the 3-butyl-7-ethylxanthine (2.58 g, 73%) as light yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 9.14 (1H, sbroad), 7.60 (1H, s), 4.33 (2H, q, J=7.2 Hz), 4.08 (2H, t, J=7.5 Hz), 1.75 (2H, quint, J=7.5 Hz), 1.53 (3H, t, J=7.2 Hz), 1.90 (2H, sex, J=7.5 Hz) and 0.95 (3H, t, J=7.5 Hz); .sup.13C NMR (100 MHz, CDCl.sub.3) δ 154.7, 150.9, 150.7, 140.7, 107.3, 42.7, 42.4, 30.2, 19.9, 16.4 and 13.7. HRMS (ESI+) for C.sub.11H.sub.17N.sub.4O.sub.2 (M+H) calcd, 237.1346; found, 237.1345.

7-Ethylxanthine (3a)

(33) ##STR00237##

(34) At 50° C., to a solution of 7-ethylguanine (6.2 g, 34.6 mmol) in a mixture of acetic acid (50 mL) and water (5 mL) was added dropwise a solution of sodium nitrite (9.6 g, 0.14 mol) in water (15 mL) over a period of 15 min. After addition the reaction mixture was stirred for 1 h then concentrated under reduced pressure. The residue was poured into cold H.sub.2O (50 mL) and the precipitate collected by filtration was dried to afford 7-ethylxanthine (5.53 g, 89%) as white solid. .sup.1H NMR (250 MHz, DMSO-d6) δ 11.54 (1H, sbroad), 10.86 (1H, sbroad), 7.97 (1H, s), 4.20 (2H, q, J=7.1 Hz) and 1.37 (3H, t, J=7.1 Hz). HRMS (ESI+) for C.sub.7H8N.sub.4O.sub.2(M+H) calcd, 181.0720; found, 181.0720.

7-ethylguanine (1c)

(35) ##STR00238##

(36) Under argon, a suspension of guanosine (10 g, 35.5 mmol) and ethyl iodide (6.8 mL, 85 mmol) in dimethylacetamide (100 mL) was heated at 60° C. overnight. Then reaction mixture was diluted with aqueous 3N HCl (90 mL) and heated at 110° C. for 1 h. The resulting solution was then treated with aqueous 10% NH.sub.3 until pH>8 to allow the precipitation. The solid was collected by filtration and washed with Et.sub.2O to afford 7-ethylguanine (6.16 g, 95%) as light brown powder. .sup.1H NMR (250 MHz, DMSO-d6) δ 7.95 (1H, s), 4.17 (2H, q, J=7.0 Hz) and 1.32 (3H, t, J=7.0 Hz); .sup.13C NMR (63 MHz, DMSO-d6) δ 155.6, 151.5, 149.7, 142.1, 106.2, 41.5 and 16.4.

Example 3: 3-Butyl-8-(5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (28b)

(37) ##STR00239##

(38) To a solution of 5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-thiol (58 mg, 0.32 mmol) in dimethylformamide (2 mL) was injected diisopropylethylamine (55 μL, 0.32 mmol). After 10 min under stirring, a solution of 3-butyl-8-chloromethyl-7-ethylxanthine (43 mg, 0.16 mmol) in dimethylformamide (2 mL) was added dropwise. The mixture was heated at 60° C. for 24 h then allowed to cool at room temperature. Na.sub.2S.sub.2O.sub.4 was added in solid fraction and the resulting mixture was stirred at room temperature for additional 3 h. The solvent was distillated off under reduced pressure and the residue was purified by column chromatography (EtOAc-MeOH 10:0.5) to afford the 3-butyl-8-(5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (28 mg, 43%) as white solid. Rf=0.40. .sup.1H NMR (250 MHz, CDCl.sub.3) δ 8.27 (1H, sbroad), 6.80 (1H, s), 4.53 (2H, q, J=7.1 Hz), 4.08 (2H, t, J=7.4 Hz), 2.72 (3H, s), 2.63 (3H, s), 1.80-1.63 (4H, m), 1.47 (3H, t, J=7.3 Hz), 1.38 (2H, sex, J=7.3 Hz) and 0.92 (3H, t, J=7.4 Hz).

Use of Compounds According to the Invention

Material and Methods

Example 4: HTRF Assay

(39) HTRF assays were performed in white 384 Well Small Volume™ HiBase Polystyrene Microplates (Greiner) with a total working volume of 20 μL. Compounds were dispensed, with 200 nL per well (1% final DMSO), from a concentration stock of 1 mM in 100% DMSO and with serial DMSO dilutions, using a Mosquito Crystal pipetting robot platform (TTP labtech). The IC.sub.50 measurements were performed in triplicates (Table 2 and 3). All HTRF reagents were purchased from CisBio Bioassays and reconstituted according to the supplier protocols. For each assay 14.7 μL of mix 1 (protein+peptide) is added in the assay wells, containing previously dispensed inhibitors, according to the final concentration and buffer described in Table 4, using a Biomek NX MC pipetting robot (Beckman). Then, 5.1 μL of mix 2 (donnor+acceptor) is added. HTRF signals were measured, after a final incubation (overnight at room temperature), using a PHERAstar FS (BMG Labtech) with an excitation filter at 337 nm and fluorescence wavelength measurement at 620 and 665 nm, an integration delay of 60 μs and an integration time of 500 μs. Results were analyzed with a two-wavelengths signal ratio: [intensity (665 nm)/intensity (620 nm)]*10.sup.4. Percentage of inhibition was calculated using the following equation: % inhibition=[(compound signal)−(min signal)]/[(max signal)−(min signal)]*100, where ‘max signal’ is the signal ratio with the compound vehicle alone (DMSO) and ‘min signal’ the signal ratio without peptide. For IC.sub.50 measurements, values were normalized and fitted with Prism (GraphPad software) using the following equation: Y=100/(1+((X/IC.sub.50){circumflex over ( )}Hill slope)).

(40) TABLE-US-00005 TABLE 2 Effect of various compounds of invention on BRD4(BD1) activity.sup.a Cmpd (IC.sub.50 Cmpd (IC.sub.50 Cmpd (IC.sub.50 Cmpd (IC.sub.50 Cmpd (IC.sub.50 Cmpd (IC.sub.50 μM) μM) μM) μM) μM) μM) 1a (NC) 1b (NC) 1c (NC) 2a (NC) 2b (NC) 2c (NC) 3a (NC) 3b (NC) 3c (NC) 4a (NC) 4b (9) 4c (NC) 5a (70.3 ± 5b (52.5 ± 5c (NC) 6a (13.0 ± 6b (11.9 ± 6c (2.6 ± 0.1) 4.9) 7.3) 0.3) 0.6) 7a (2.6 ± 0.1) 7b (2.6 ± 0.1) 7c (17.9 ± 8a (NC) 8b (NC) 8c (10.7 ± 1.3) 0.4) 9a (NC) 10c (NC) 11b (NC) 11c (NC) 12a (NC) 12b (<100) 12c (NC) 14c (<100) 15a (35.1 ± 15b (ND) 15c (100) 16a (NC) 2.9) 17a (NC) 17b (NC) 17c (NC) 18a (NC) 18b (NC) 18c (NC) 19a (93.3 ± 19c (NC) 20a (ND) 20b (ND) 21a (45.6 ± 16.6) 4.9) 21b (89.1 ± 22a (26.4 ± 22b (NC) 23a (NC) 23b (NC) 24a (NC) 9.3) 0.7) 24b (69.6 ± 25a (12.6 ± 25b (46.6 ± 26a (7.9 ± 26b (5.0 ± 27a (100) 5.2) 0.3) 4.6) 0.2) 0.1) 27b (20.4 ± 28a (NC) 28b (NC) 29a (NC) 29b (ND) 30a (18.3 ± 3.6) 1.4) 30b (ND) 31a (46.8 ± 31b (59.6 ± 32a (54.0 ± 7.8) 7.8) 4.8) .sup.aDrug concentration that inhibits protein-protein interaction by 50%. Data are the mean ± standard deviation (SD) of three experiments. NC abbreviation stands for Not Converged (IC.sub.50 > maximal concentration of compound used). ND abbreviation stands for Not Determined.

Example 4 Bis: HTRF Assay

(41) After the efficient synthesis of the focused library in 96 well plates with the Accelerator Synthetizer SLT100, the focused library has been directly transferred to a Labcyte Access/Echo® Laboratory Workstation to assess the compounds for their ability to disrupt bromodmain/histone complexes using the homogeneous time-resolved fluorescence (HTRF®) technology (IC50 μM, table 2bis).

(42) TABLE-US-00006 TABLE 2 bis Effect of various compounds of invention on BRD4(BD1) activity.sup.a HTRF (IC.sub.50, μM) 33 8.5 34 44.5 35 19.2 36 3 37 16.6 38 48.2 39 38.2 40 33 41 6.4 42 45.3 43 33.4 44 21.9 45 11.6 46 20.1 47 30.1 48 35.2 49 35.1 51 30.2 52 33.04 53 4.4 54 7.1 55 14.2 56 13.2 57 7.5 58 2.7 59 6.9 60 8.4 61 4.7 62 8.9 63 8.9 64 5.1 65 32.7 66 16.8 67 8.9 68 35.5 69 8.4 70 6.5 71 17.8 72 4.4 73 7.5 74 5.5 75 3.9 76 12.7 77 26.8 78 15.7 79 4.2 80 10.6 .sup.aDrug concentration that inhibits protein-protein interaction by 50%.

Example 5: Bromodomain Selectivity Profiles

(43) Selectivity profiles of bromodomain inhibitors were performed as described in HTRF screen section. Concentration of histone peptide was optimized to ensure sufficient signal to noise ratio, sufficient sensitivity for detection of weak inhibitors and comparable data from one bromodomain to another. HTRF detection reagents (EPIgeneous™ Binding Domain kits) were purchased from Cisbio Bioassays and used according to supplier's protocol. GST tagged bromodomain proteins were purchased from BPS Bioscience and histone peptide from Anaspec.

(44) TABLE-US-00007 TABLE 3 Selectivity profiles of compounds of invention towards the BET IC.sub.50 (μM).sup.a Bromodomain 4b 6a 7a 22a 25a 26a 26b 27b.sup.c BRD4(BD1) 9 13.0 ± 0.3  2.6 ± 0.1 26.4 ± 0.7 12.6 ± 0.3  7.9 ± 0.2  5.0 ± 0.1 20.4 ± 3.6 BRD3(BD1) 14  4.8 ± 0.3 1.8 ± 1.1 31.7 ± 1.1 40.7 ± 2.2 43.5 ± 7.1 62.0 ± 7.8 NC BRD2(BD1) NC 18.8 ± 0.7  4.7 ± 0.1 30.0 ± 0.9 30.1 ± 1.3 31.9 ± 0.5 59.0 ± 2.9 NC BRDT(BD1) 27  7.9 ± 0.4 2.3 ± 0.2 >100 65.3 ± 8.1 42.5 ± 3.9 >100 NC BRD4(BD2) 5 8.1 ± 0.3 1.9 ± 0.1 NC.sup.d >100 >100 NC NC BRD3(BD2) 4 5.8 ± 0.2 1.8 ± 0.2 NC.sup.d 23.9 ± 1.4  2.1 ± 0.5 NC NC BRD2(BD2) 7 6.6 ± 0.3 1.6 ± 0.2 NC.sup.d NC  9.5 ± 0.5 NC NC ATAD2.sup.b ND NC NC NC >100 56.4 ± 7.9 >100 NC .sup.aDrug concentration that inhibits protein-protein interaction by 50%. Data are the mean ± standard deviation (SD) of three experiments. NC abbreviation stands for Not Converged. ND abbreviation stands for Not Determined. .sup.bATAD2 is used as a non-BET family member bromodomain control. .sup.cData above 25 μM have been excluded due to precipitation and fluorescence interference at 620 nm. .sup.dFluorescence interference at 620 nm with BD2.

(45) TABLE-US-00008 TABLE 4 HTRF selectivity experimental procedures.sup.a MIX 1 MIX 2 Assay Protein name (nM) Peptide name (nM) Donnor name (nM) Acceptor name (nM) buffer.sup.b DMSO GST- 5 H4 KAc 5/8/12/16 25 MAb Anti GST- 0.5 Streptavidin d2 3.125 Buffer A 1% BRD4(BD1) peptide Keu GST- 5 H4 KAc 5/8/12/16 10 MAb Anti GST- 0.5 Streptavidin d2 1.25 Buffer A 1% BRD3(BD1) peptide Keu GST- 5 H4 KAc 5/8/12/16 15 MAb Anti GST- 0.5 Streptavidin d2 1.875 Buffer A 1% BRD2(BD1) peptide Keu GST- 5 H4 KAc 5/8/12/16 50 MAb Anti GST- 0.5 Streptavidin 6.3 Buffer A 1% BRDT(BD1) peptide Keu XL665 GST- 5 H4 KAc 5/8/12/16 75 MAb Anti GST- 0.5 Streptavidin 9.375 Buffer A 1% BRD4(BD2) peptide Keu XL665 GST- 5 H4 KAc 5/8/12/16 150 MAb Anti GST- 0.5 Streptavidin 18.75 Buffer A 1% BRD3(BD2) peptide Keu XL665 GST- 5 H4 KAc 5/8/12/16 25 MAb Anti GST- 0.5 Streptavidin 3.125 Buffer A 1% BRD2(BD2) peptide Keu XL665 GST-ATAD2 5 H4 KAc 5/8/12/16 10 MAb Anti GST- 0.5 Streptavidin 10 Buffer B 1% peptide KTb XL665 .sup.aFor each bromodomain, concentrations of protein, peptide, donor and acceptor have been optimized and are presented in this summary table also indicating the donor and acceptor type as well as the final DMSO concentration and buffer composition. .sup.bBuffer A: 50 mM KPO.sub.4, pH 7, 100 mM KF, BSA 0.1%; Buffer B: 50 mM Hepes, pH 7.5, 150 mM NaCl, BSA 0.1%.

Example 6: Cell-Based Assays

(46) Cells and Cell Culture

(47) The human leukemia cell line Jurkat (ATCC® TIB-152) was maintained in RPMI-1640 medium supplemented with 10% FBS at 37° C. and 5% CO.sub.2. Human osteosarcoma cell line (U2OS, ATCC® HTB-96™) was maintained in DMEM supplemented with 10% FBS at 37° C. and 5% CO.sub.2.

(48) Cytotoxicity Experiments

(49) In antiproliferative assays, compounds were assayed for their growth inhibiting activity towards the described cancer cell lines using the Cell Titer-Glo Luminescent Cell Viability Assay as described by the manufacturer (Promega Corporation). Briefly, 104 cells were plated onto 96 well-plates (white with clear bottom (3610, Corning Costar)) in 100 μL media per well immediately before assay. Compounds were added at different concentrations (ranging from 100 to 0.05 μM) to each well and cell cultures were incubated 37° C. during 72 h. Vehicle (DMSO) was used as control and all compounds were tested in constant percentage of DMSO (1%). After addition of 50 μL Cell Titer-GLO, Luminescence was measured using a Centro luminometer LB960 (Berthold). Dose-response curves were generated and effective dose 50 values (EC.sub.50) were calculated using non-linear regression analysis (Graph Pad Prism).

(50) Western Blot

(51) Non-treated Jurkat cells and Jurkat cells treated during 24 h with compound of the invention 26b, or DMSO were lysed in RIPA lysis buffer (Tris HCl pH=7.5 50 mM, NaCl 150 mM, Triton 1%, SDS 0.1%, sodium deoxycholate 1%) supplemented with protease inhibitor cocktail (P8340, Sigma-Aldrich) and 1 mM PhenylMethylSulfonyl Fluoride (Sigma-Aldrich). 75 μg of protein were loaded onto 10% acrylamide SDS/PAGE and then transferred to nitrocellulose Hybond C-extra membranes, 45 micron (GE Healthcare). The membranes were saturated with 5% (wt/vol) skim milk in TBST [Tris-buffered saline/0.1% (vol/vol) Tween 20] 1 h at room temperature and incubated with anti-myc antibody (clone 9E10, sc40 Santa Cruz) at a 1:500 dilution in 0.5% (wt/vol) skimmed milk in TBST overnight at 4° C. Membranes were then washed with TBST, incubated with an HRP-conjugated anti-mouse secondary antibody (polyclonal goat anti mouse P0447, Pierce) at a 1:20,000 dilution in TBST 1 h at room temperature. Immunoreactive bands were revealed using SuperSignal™ West Pico Extended Duration Substrate (Pierce) detection reagents. Quantification was performed using ImageJ software.

(52) In cell-based assays, 26b reduced Jurkat T cells viability in a dose-dependent manner with an EC.sub.50 of 27 μM. Also, 26b down regulated c-Myc, a pro-oncogene contributing to the pathogenesis of numerous human cancers, in the same range of concentration (FIGS. 1A and 1B).

Example 7: Specific Structural Elements of BET Protein Having a Key Role in the Selectivity

(53) The Inventors follow-up on of a previous mid-throughput screen (MTS) using the 2P213D chemical library, a structurally diverse ‘protein-protein interaction inhibition (2P2I)-oriented’ collection of compounds.sup.18-23 by focusing on a selective BRD4 (BD1) acetylated-mimic xanthine inhibitor. Among the 17 hits that were identified following this screen, one xanthine derivative was found to present a low micromolar IC.sub.50 measured by homogeneous time resolved fluorescence (HTRF) and an unforeseen selectivity profile among the BET family members. A structure-based investigation program around xanthine scaffold containing compounds was the starting point of this observed selectivity with a key role identified for the bromodomains ZA loop.

(54) Results and Discussion

(55) An MTS of the 2P2I.sub.3D chemical library against BRD4 (BD1), identified 17 hits with affinities within the low micromolar range..sup.23 The inventors set-up isothermal titration calorimetry (ITC) as orthogonal assay and were able to validate a direct binding for 7 out of the 17 compounds tested, with ligand efficiency (LE) values ranging from 0.20 up to 0.23. Among them, 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b), a xanthine derivative, presented a Kd of 1.4 μM, driven by a large favorable entropy term (−TΔS of −7.1 Kcal/mol) as demonstrated by ITC. This biophysical result suggested conformational changes and water rearrangement upon binding of the compound. Moreover, an HTRF experiment against seven out of the eight BD1 and BD2 BET domains (BRDT (BD2) was not available from the company Cisbio) pinpointed a 10 fold lower IC.sub.50 for 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) towards BRD4(BD1) (IC.sub.50 of 5 μM) as compared to its relatives BD1 (IC.sub.50's>50 μM), and no detectable inhibition of the BD2 counterparts (Table 3). In cell-based assays, 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) reduced Jurkat T cells viability in a dose-dependent manner with an EC.sub.50 of 27 μM. Also, 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) down regulated c-Myc, a pro-oncogene contributing to the pathogenesis of numerous human cancers, in the same range of concentration. The Inventors next solved the crystal structure of 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) in complex with BRD4(BD1). The co-crystal revealed the globular domain organization for BRD4(BD1),.sup.24 and a well-defined electron density for 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) with the expected binding mode of an acetyl-lysine mimetic.sup.25 forming the canonical hydrogen bond with the conserved asparagine N140 and a water mediated hydrogen bond with Y97 also linking the inhibitor to the conserved water network at the bottom of the binding pocket. Surprisingly, the triazolopyrimidinyl moiety stacked against the ZA channel, occupying the space at the rim of the acetyl-lysine binding site, a binding mode that has also recently been reported for benzimidazolone derivative inhibitors targeting BAZ2B bromodomain..sup.26-28 This triazolo moiety establishes a hydrogen bond interaction with the main chain (NH amido group) of D88 a residue conserved throughout the BET family. This interaction orientates the triazolopyrimidinyl fragment in the ZA channel while its ring system is locked from one side by van der Walls contacts with L92, and from the other side with Q85—the pyrimidine ring sits tightly on this glutamine which is bent by 90° to adopt this complementary orientation. This BD1-conserved amino acid (except in BRDT (BD1)) is replaced by larger residues such as arginine or lysine in the BD2 sub-family. This difference could be responsible for the observed selectivity profile of 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) towards BRD4-BRD3-BRD2-(BD1) vs all the other BET tested.

(56) In order to gain better understanding of this selectivity profile, the Inventors undertook a structure-based program aiming to define the structure/affinity and structure/selectivity relationships by decomposing this model compound.

(57) The xanthine 2b and the corresponding mono- and unsymmetrically dialkylated derivatives 3a, 6a, and 7a were further analyzed by HTRF, ITC, and x-ray crystallography, when available (Table 1′ and table 2). Chemical variations of the xanthine mimicking the reference probe (6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine-6-acetic acid 1,1-dimethylethyl ester (JQ1, PDB accession code 3MXF),.sup.24 such as a substitution of the butyl group (compound 6a) by a chlorobenzyl one (compound 7a) allowed an optimization of the core with an IC.sub.50 of 2.6 μM as measured by HTRF (2 fold increased potency) and a Kd of 1.8 μM as measured by ITC. This optimized core presented a favorable enthalpy change of −6.3 kcal/mol. More importantly, the selectivity profile of this optimized core 7a displayed a pan-BET inhibition profile, similarly to 6a. Analysis of the co-crystal structures confirmed a characteristic recognition of the pocket by this core, as exemplified by the superimposition of 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) with 7a. The core was thus not responsible (as expected) for the observed selectivity profiles of 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b).

(58) The Inventors next questioned whether the observed selectivity profile of 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) was due to the triazolo and/or to the pyrimidinyl moiety of the core extension. To address this purpose, a series of N-3, N-7 disubstituted triazoloxanthine derivatives 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) and 8-(6-Butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-3-(4-chlorobenzyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (27b), 8-(5-Amino-1H-[1,2,4]triazol-3-ylsulfanylmethyl)-3-(4-chlorobenzyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (22a), 8-(5-Amino-1H-[1,2,4]triazol-3-ylsulfanylmethyl)-3-butyl-7-ethyl-3,7-dihydro-purine-2,6-dione (21b) were synthesized.

(59) After evaluation, compound 8-(5-Amino-1H-[1,2,4]triazol-3-ylsulfanylmethyl)-3-(4-chlorobenzyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (22a) revealed an IC.sub.50 of 26.4 μM in HTRF which represents a 10-fold decrease in potency as compared to 7a (Table 2, table 2′ and table 3). Compound 8-(5-Amino-1H-[1,2,4]triazol-3-ylsulfanylmethyl)-3-(4-chlorobenzyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (22a), however, exhibited a shift of selectivity towards BET-BD1 domains (Table 3). The introduction of the triazolo fragment was thus bringing a decrease of potency, yet driving a commencement of selectivity among BET bromodomains (BD1 vs BD2).

(60) A potential hydrogen bond between the triazolo fragment and D88, conserved in all the BET bromodomains, could be stabilized with certain bromodomains and not with others, due to differences in the dynamics of the ZA loop, which has been demonstrated to be crucial in the binding kinetics of several BET-BRDi..sup.24,31,32 Superimposition of BRD4 with or without compound 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) illustrates structural rearrangements with displacement of the ZA loop up to 2.7 Å. These rearrangements are not observed with the ‘pan inhibitor’ xanthine scaffold derivative (compound 7a), thus validating this assumption. The role of this loop and its associated variable dynamics would also explain the IC.sub.50 observed for 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) towards BRD4(BD1) vs BRD3 (BD1) and BRD2(BD1), this hydrogen bond being more stable with BRD4(BD1). Analysis of the protein-ligand(s) interactions including hydrophobic and hydrogen-bonding contacts was carried out using LigPlot+,.sup.33. This weak interaction prevented us from solving a non-ambiguous 3D structure of 8-(5-Amino-1H-[1,2,4]triazol-3-ylsulfanylmethyl)-3-(4-chlorobenzyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (22a) in complex with BRD4(BD1). However, crystals with 50% occupancy of 8-(5-Amino-1H-[1,2,4]triazol-3-ylsulfanylmethyl)-3-(4-chlorobenzyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (22a) highlighted that this compound was still interacting with BRD4(BD1) and that hydrogen bond between the triazolo moiety and the main chain (NH amido group) of D88 was present, with BRD4(BD1). The triazolo was thus explaining only partly the observed selectivity profiles of 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b). An explanation for the selectivity profile of 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) was finally confirmed with the synthesis of 8-(6-Butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-3-(4-chlorobenzyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (27b), the N-chlorobenzyl analogue of 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b). Of note, this compound was found to be less active than the original compound 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) (IC.sub.50 values of 20 μM vs 5 μM, respectively). However, the selectivity profile of this compound was similar to that of 3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b), i.e., selective towards BRD4(BD1). Altogether, these data suggest that the N.sub.3,N.sub.7-dialkylated xanthine core (see 6a and 7a) is responsible for the pan-BET inhibition while condensed with triazolo moiety (see 8-(5-Amino-1H-[1,2,4]triazol-3-ylsulfanylmethyl)-3-(4-chlorobenzyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (22a) it allows the preferential inhibition of BET-BD1 domains forming a hydrogen bond with the conserved D88 residue. Further condensation with pyrimidinyl moiety drives the corresponding triazolopyrimidinyl xanthine derivatives (3-butyl-8-(6-butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-7-ethylxanthine (26b) and 8-(6-Butyl-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylsulfanylmethyl)-3-(4-chlorobenzyl)-7-ethyl-3,7-dihydro-purine-2,6-dione (27b) to be selective towards BRD4(BD1) due to van der Waals interaction with the side chain of Q85. A steric clash prevents this interaction with the R present at the same position in BRDT(BD1) or the K present in the other BRDX(BD2) bromodomains. These findings suggest potential mechanism for the selectivity of BET bromodomains.

CONCLUSION

(61) In this study, the Inventors investigated the key structural feature responsible for the selectivity of a xanthine-based BRD4(BD1) inhibitor identified through MTS.

(62) This compound represents the first low micromolar selective inhibitor targeting BRD4(BD1) with a >10-fold ratio in binding affinity towards any other BET bromodomain tested, yet presenting low but dose-response down regulation of c-Myc levels in cell-based assay. The co-crystal structure revealed an original orientation of the triazolopyrimidinyl moiety, expanding into the ZA channel, setting the basis of a structure-selectivity relationship inside the BET family. Even though it is conserved throughout the BET family, the different dynamic behavior of the ZA loop could be exploited to tune the selectivity of BET inhibitors.

(63) TABLE-US-00009 TABLE 1′ Effect of various substituted xanthine on BRD4(BD1) activity Substituent BRD4(BD1) Compd R.sub.1 R.sub.3 R.sub.7 R.sub.8 IC.sub.50 (μM).sup.a K.sub.D (μM).sup.b LE.sup.c 2b H H H H NA — NA 3a H H Et H NA — NA 6a H Bu Et H 13.0 ± 0.3 — 0.40 7a H 4-ClBn Et H  2.6 ± 0.1 1.8 0.37 .sup.aDrug concentration that inhibits protein-protein interaction by 50%. Data are the mean ± standard deviation (SD) of three experiments. .sup.bKd determined by Isothermal Titration Calorimetry. .sup.CLigand Efficiency (LE) defined as the ratio of the log of the IC.sub.50 to the number (N) of non-hydrogen atoms of the compound LE = 1.4(−log(IC.sub.50)/N).

(64) TABLE-US-00010 TABLE 2 Effect of various triazolo-substituted xanthine derivatives on BRD4(BD1) activity Substituent BRD4(BD1) Cmpd R.sub.1 R.sub.3 R.sub.7 R.sub.8 IC.sub.50 (μM).sup.a K.sub.D (μM).sup.b LE.sup.c 21b H Bu Et 0 >50 — NA 22a H 4-ClBn Et 26.4 ± 0.7  — 0.17 26b H Bu Et 5.0 ± 0.1 1.4 0.22 27b H 4-ClBn Et 20.4 ± 3.6  — 0.15 .sup.aDrug concentration that inhibits protein-protein interaction by 50%. Data are the mean ± standard deviation (SD) of three experiments. .sup.bKd determined by Isothermal Titration Calorimetry. .sup.cLigand Efficiency (LE) defined as the ratio of the IC.sub.50 to the number (N) of non-hydrogen atoms of the compound LE = 1.4(-log IC.sub.50)/N.
Protein Expression and Purification.

(65) For Isothermal Titration Calorimetry (ITC) and crystallogenesis, BRD4(BD1) was produced and purified using a histidine tag affinity chromatography as described by Filipakopoulos et al.9 For these experiments, a pNIC28-BSA4 expression vector containing BRD4(BD1) and a Tobacco Etch Virus (TEV) protease cleavage site have kindly been provided by Stefan Knapp laboratory from the SGC at the University of Oxford. After size exclusion chromatography, the fractions presenting pure BRD4(BD1) after TEV cleavage of the histidine tag were pooled and concentrated to 25 mg/mL for crystallogenesis. For ITC assays, the protein was concentrated up to at 6 mg/mL and the DTT was removed using a buffer exchange column (PD10 from GE healthcare) equilibrated with 10 mM HEPES pH=7.5, 150 mM NaCl. For Homogeneous Time Resolved Fluorimetry (HTRF) experiments, a BRD4(BD1) synthetic gene that includes a TEV cleavage site was purchased from LifeTechnology in a pDONR transport vector before cloning into a pDESTTM15 expression vector for GST affinity purification. Protein production and purification was carried out using similar protocols and buffers as used for the His-BRD4(BD1) system. Purification was carried on GST affinity resin (Thermo Scientific) and reduced glutathione was used for protein release. GST-BRD4(BD1) was further purified by size exclusion chromatography on a Superdex 16/60 Hiload column (GE Healthcare) using 20 mM TRIS pH=8.0, 150 mM NaCl Buffer.

(66) Isothermal Titration Calorimetry.

(67) ITC was used to evaluate the thermodynamics parameters of the binding between BRD4(BD1) and the selected compounds, using ITC conditions previously described by Filippakopoulos et al.8 Purified BRD4(BD1) was extensively dialyzed in the ITC buffer containing 10 mM Hepes pH=7.5 and 150 mM NaCl. Compounds were diluted directly in the last protein dialysate prior to experiments. Titrations were carried out on a MicroCal ITC200 microcalorimeter (GE Healthcare, Piscataway, N.J.). Each experiment was designed using a titrant concentration (protein in the syringe) set 10 to 15 times the analyte concentration (compound in the cell generally between 10 and 35 μM) and using 13 injections at 35° C. A first small injection (generally 0.2 μL) was included in the titration protocol in order to remove air bubbles trapped in the syringe prior titration. Raw data were scaled after setting the zero to the titration saturation heat value. Integrated raw ITC data were fitted to a one site non-linear least squares fit model using the MicroCal Origin plugin as implemented in Origin 7 (Origin Lab). Finally, ΔG and TΔS values were calculated from the fitted ΔH and KA values using the equations ΔG=−R.T. ln KA and ΔG=ΔH−TΔS.

(68) Crystallography.

(69) BRD4(BD1)-Inhibitor co-crystallization was performed at 19° C. (292K) using the hanging drop vapor diffusion method. For the complex with 1 mM of compound 26b, 25 mg/mL of BRD4(BD1) preparation was mixed at a 1:1 ratio with the precipitant solution (200 mM ammonium sulfate, 100 mM Tris pH=8.5, 25% PEG3350) and crystals grew to diffracting quality within 3-5 days. For the complex with 2.5 mM of 7a, 16 mg/mL of BRD4(BD1) preparation was mixed at a 1:1 ratio with the precipitant solution (250 mM ammonium sulfate, 100 mM Tris pH=8.5, 19% PEG3350) and crystals grew to diffracting quality within 15 days. For the complex with 22a, 2.5 mM of compound and 12 mg/mL of BRD4(BD1) preparation was mixed at a 1:1 ratio with the precipitant solution (300 mM Na Formate, 100 mM NaCl, 22% PEG3350, 10% Ethylene glycol). Crystals grew to diffracting quality within 1 month. Crystals were cryo-protected using the precipitant solution supplemented with 10% ethylene glycol for 26b and 7a or with 10% glycerol for 22a and were flash frozen in liquid nitrogen. Data for 26b, 7a and 22a were respectively collected at the AFMB laboratory—Marseilles (Bruker AXS MICROSTAR equipped with a Mar345 detector), and on the ESRF beamlines ID23-2 and ID29. Indexing, integration and scaling were performed using XDS.10 Initial phases were calculated by molecular replacement with Phaser MR (CCP4 suite) 11 using a model of the first domain of BRD4 (extracted from the Protein Data Bank accession code: 2OSS). Initial models for the protein and the ligands were built in COOT.12 The cycles of refinement were carried out with Refmac5 (CCP4 suite). The models and structures factors have been deposited with Protein Data Bank accession code for compound 26b: 5EGU, compound 7a: 5EIS and compound 22a: 5EI4.

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