Targeted ubiquitination degradation BRD4 protein compound, preparation method therefor and application thereof

Abstract

A compound represented by formula (I) or a tautomer, optical isomer, deuterated substance, oxynitride, solvate, pharmaceutically acceptable salt or prodrug thereof can be used in preparing a pharmaceutical composition. The compound or the pharmaceutical composition can be used in the preparation a drug for the prevention and/or treatment of cancer, tumors, viral infections, depression, neurological disorders, trauma, age-related cataracts, organ transplant rejection or autoimmune diseases.

Claims

1. A compound of formula (I) or a tautomer, optical isomer, solvate, or pharmaceutically acceptable salt thereof; ##STR00069## wherein, R is ##STR00070## X is amino or substituted amino, wherein the substituent is C.sub.1-6 alkyl and C.sub.1-6 alkoxy; Y is amino, substituted or unsubstituted saturated 5-7 membered heterocycle, saturated heterospirocycle, 5-7 membered saturated bicyclic hetrocycle or 5-6 membered heteroaryl; the heteroatoms of saturated 5-7 membered heterocycloalkyl is selected from O, N and S, wherein the number of N heteroatom is 1, 2, or 3, and the number of O or S heteroatom is 0, 1, or 2, the substituted saturated 5-7 membered heterocycloalkyl means that the saturated 5-7 membered heterocycloalkyl is independently substituted with one or more substituents selected from halogen, hydroxy, cyano, nitro, amino, carbonyl, C.sub.1-6 alkoxy, C.sub.1-6 alkyl, C.sub.3-8 cycloalkyl, aryl or C.sub.5-7 heteroaryl; the saturated heteromonospirocycloalkyl has heteroatoms selected from O, N and S, wherein the number of N heteroatom is 1, 2 or 3, the number of O or S heteroatoms is 0, 1, or 2, and the saturated heteromonospirocycloalkyl is selected from the group consisting of 3-membered/S-membered, 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, and 5-membered/6-membered ring, the substituted saturated heteromonospirocycloalkyl means that the saturated heteromonospirocycloalkyl is independently substituted with one or more substituents selected from halogen, hydroxy, cyano, nitro, amino, carbonyl, C.sub.1-6 alkoxy, C.sub.1-6 alkyl, C.sub.3-8 cycloalkyl, aryl or C.sub.5-7 heteroaryl; the saturated heterofused cycloalkyl contains one or two heteroatoms independently selected from O, N and S in addition to carbon atoms, and the saturated heterofused cycloalkyl is selected from the group consisting of 5-membered/5-membered and 5-membered/6-membered bicyclic fused heterocyclic group, the substituted saturated heterofused cycloalkyl means that the saturated heterofused cycloalkyl is independently substituted with one or more substituents selected from halogen, hydroxy, cyano, nitro, amino, carbonyl, C.sub.1-4 alkoxy, C.sub.1-4 alkyl, C.sub.3-8 cycloalkyl, aryl and C.sub.5-7 heteroaryl; the heteroaryl group contains one or two heteroatoms independently selected from O, N and S in addition to carbon atoms, the substituted heteroaryl means that the heteroaryl is independently substituted with one or more substituents selected from halogen, hydroxy, cyano, nitro, amino, carbonyl, C.sub.1-6 alkoxy, C.sub.1-6 alkyl, C.sub.3-8 cycloalkyl, aryl and C.sub.5-7 heteroaryl; L is (CH.sub.2).sub.n, CH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.m or CH.sub.2R.sub.1, n is 1, 2, 3, 4, 5 or 6; m is 1, 2, 3 or 4; R.sub.1 is selected from optionally substituted cycloalkyl, heterocycloalkyl, haloheterocycloalkyl, aryl or heteroaryl; the substituent is independently selected from one or more groups selected from halogen, hydroxyl, cyano, nitro, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, haloheterocycloalkyl, aryl and heteroaryl; W is CH.sub.2, NH, O, CONH or COO; Z is CH.sub.2 or CO.

2. The compound of formula (I) or a tautomer, optical isomer, solvate, or pharmaceutically acceptable salt thereof according to claim 1; ##STR00071## wherein, R is ##STR00072## X is amino or substituted amino, and the substituent is selected from C.sub.1-6 alkyl and C.sub.1-6 alkoxy; Y is amino, substituted or unsubstituted saturated 5-7 membered heterocycloalkyl, saturated heteromonospirocycloalkyl, saturated heterofused cycloalkyl or heteroaryl; the substituted or unsubstituted saturated 5-7 membered heterocycloalkyl has heteroatoms selected from O, N and S, wherein the number of N heteroatom is 1, 2 or 3, and the number of O or S heteroatom is 1 or 2, the substituted saturated 5-7 membered heterocycloalkyl is independently substituted with one or more substituents selected from halogen, hydroxy, cyano, nitro, amino, carbonyl, C.sub.1-6 alkoxy, C.sub.1-6 alkyl, C.sub.3-8 cycloalkyl, aryl and C.sub.5-7 heteroaryl; the substituted or unsubstituted saturated heteromonospirocycloalkyl has heteroatoms selected from O, N and S, wherein the number of N heteroatom is 1, 2 or 3, the number of O or S heteroatoms is 1 or 2, and the saturated heteromonospirocycloalkyl is selected from the group consisting of 3-membered/5-membered, 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, and 5-membered/6-membered ring, which is independently substituted with one or more substituents selected from halogen, hydroxy, cyano, nitro, amino, carbonyl, C.sub.1-6 alkoxy, C.sub.1-6 alkyl, C.sub.3-8 cycloalkyl, aryl and C.sub.5-7 heteroaryl; the substituted or unsubstituted saturated heterofused cycloalkyl contains one or two heteroatoms independently selected from O, N and S in addition to carbon atoms, and the saturated heterofused cycloalkyl is selected from the group consisting of 5-membered/5-membered and 5-membered/6-membered bicyclic fused heterocyclic group, which is independently substituted with one or more substituents selected from halogen, hydroxy, cyano, nitro, amino, carbonyl, C.sub.1-4 alkoxy, C.sub.1-4 alkyl, C.sub.3-8 cycloalkyl, aryl and C.sub.5-7 heteroaryl; the heteroaryl group contains one or two heteroatoms independently selected from O, N and S in addition to carbon atoms, the heteroaryl is independently substituted with one or more substituents selected from halogen, hydroxy, cyano, nitro, amino, carbonyl, C.sub.1-6 alkoxy, C.sub.1-6 alkyl, C.sub.3-8 cycloalkyl, aryl and C.sub.5-7 heteroaryl; L is (CH.sub.2).sub.n, CH.sub.2CH.sub.2 (OCH.sub.2CH.sub.2).sub.m or CH.sub.2R.sub.1; n is 1, 2, 3, 4, 5 or 6; m is 1, 2, 3 or 4; R.sub.1 is optionally substituted cycloalkyl, heterocycloalkyl, haloheterocycloalkyl, aryl or heteroaryl; the substituent is independently selected from one or more groups consisting of halogen, hydroxyl, cyano, nitro, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, haloheterocycloalkyl, aryl or heteroaryl; W is CH.sub.2, amino, O, CONH or COO; Z is CH.sub.2 or CO.

3. The compound of formula (I) or a tautomer, optical isomer, solvate, or pharmaceutically acceptable salt thereof according to claim 1, wherein X is amino; Y is amino, or substituted or unsubstituted saturated 5-7 membered heterocycloalkyl.

4. The compound of formula (I) or a tautomer, optical isomer, solvate, or pharmaceutically acceptable salt thereof according to claim 1, wherein X is amino; Y is amino or piperazinyl.

5. The compound of formula (I) or a tautomer, optical isomer, solvate, or pharmaceutically acceptable salt thereof according to claim 1, wherein L is (CH.sub.2).sub.n, CH.sub.2CH.sub.2 (OCH.sub.2CH.sub.2).sub.m, n is 1, 2, 3, 4, 5 or 6; m is 1, 2, 3 or 4.

6. The compound of formula (I) or a tautomer, optical isomer, solvate, or pharmaceutically acceptable salt thereof according to claim 1, wherein the configuration of * in the compound of formula (I) is S type.

7. The compound of formula (I) or a tautomer, optical isomer, solvate, or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the following: ##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078## ##STR00079##

8. A pharmaceutical composition comprising the compound of formula (I) or a tautomer, optical isomer, solvate, or pharmaceutically acceptable salt thereof according to claim 1.

9. A method for preparing the compound of formula (I) or a tautomer, optical isomer, solvate, or pharmaceutically acceptable salt thereof according to claim 1, comprising subjecting the compound of formula (M) and the compound of formula (C) to condensation reaction to afford the compound of formula (I), ##STR00080## wherein, R, L, W and Z are as defined in claim 1.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the basic principle of PROTAC technology.

(2) FIG. 2 shows the BRD4-PROTAC protein degradation mechanism of the compound of the present invention.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

(3) The preparation method of the present invention will be further described in detail below in conjunction with specific examples. It should be understood that the following examples are merely illustrative and explanation of the present invention, and should not be consider to limit the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of the present invention. Unless otherwise specified in the following examples, all temperatures are set in degrees Celsius. Unless otherwise specified, the raw material compounds are synthesized by the method described in present application or are commercially available, and are purchased from the following manufacturers: Bailingwei, Beijing Yinuokai Technology Co., Ltd., Aladdin reagent, Alfa Aesar, Shaoyuan Chemical Technology Co., Ltd. etc.

(4) The abbreviations used in the Preparation Examples, Examples and elsewhere in present application are as follows: DCM dichloromethane DIPEA N,N-diisopropylethylamine DMF N,N-dimethylformamide EtOAc ethyl acetate h hour HATU O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate MeOH methanol TFA trifluoroacetic acid

Preparation Example 1

(6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]thiazolo[4,3-a][1,4]diazepine-6-acetic Acid (Parent Compound)

(5) ##STR00015##

(6) In a 500 ml reaction flask, a solution of 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]diazepine-6-yl)acetate ((+)-JQ1) (2.30 g, 5.00 mmol) dissolved in dichloromethane (100 ml) was provided. Then TFA (20 mL) was added to the solution and stirring continued for 4 h at room temperature, The mixture was evaporated under reduced pressure to afford the title compound (6S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]thiazolo[4,3-a][1,4]diazepine-6-acetic acid (2.00 g), the product was used to the next step without purification.

Preparation Example 2: Preparation of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N-(piperazin-1-yl)acetamide (Compound (M-1))

(7) ##STR00016##

Step 1: Preparation of tert-butyl (s)-4-(2-(4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetamido)piperazine-1-carboxylate

(8) In a 100 ml reaction flask, a solution of the parent compound (1) (0.40 g, 1 mmol) dissolved in DMF (20.0 ml) was provided. Then DIPEA (522 l, 3 mmol), HATU (86.0 mg, 3 mmol) and tert-butyl 4-aminopiperazine-1-carboxylate (0.20 g, 1 mmol) were added to the solution, the mixture was stirred for 2 h at room temperature. After the reaction was completed, ethyl acetate and water were added, the organic layers were separated and evaporated under reduced pressure to afford the crude product tert-butyl (s)-4-(2-(4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetamido)piperazine-1-carboxylate. The product is directly subjected to the next step without purification.

Step 2: Preparation of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N-(piperazin-1-yl)acetamide

(9) In a 100 ml reaction flask, Tert-butyl (s)-4-(2-(4-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl) acetamido)piperazine-1-carboxylate (0.50 g, 0.86 mmol) was dissolved in DCM (100 ml), and TFA (5 ml) was added to the solution. The mixture was stirred for 4 h at room temperature, and then evaporated under reduced pressure to afford 0.40 g of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)-N-(piperazin-1-yl)acetamide (compound (M-1)), the product was used to the next step without purification.

Preparation Example 3: Preparation of (S)-1-(4-aminopiperazin-1-yl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazole[4,3-a][1,4]diaza-6-yl)ethanone (Compound (M-2))

(10) ##STR00017##

(11) (S)-1-(4-aminopiperazine-1-yl)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazole[4,3-a][1,4]diaza-6-yl)ethanone (compound (M-2), 0.40 g) was prepared in a similar to that in Example 2, except that 4-amino piperazine-1-tert-butyl carbamate was replaced with piperazine-1-tert-butyl carboxylate.

Preparation Example 4: Preparation of (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiophene[3,2-f][1,2,4]triazole[4,3-a][1,4]diazapine-6-yl)acethydrazine (Compound (M-3))

(12) ##STR00018##

(13) (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thiophene[3,2-f][1,2,4]triazole[4,3-a][1,4]diazapine-6-yl)acetylhydrazine (compound (M-3), 0.35 g) was prepared in a similar to that in Example 3, except that tert-butyl 4-aminopiperazine-1-carbamate was replaced by tert-butyl carbazate.

Preparation Example 5: Preparation of 4-(2-(2,6-dioxapiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-4-oxobutanoic Acid (Compound (C-1))

(14) ##STR00019##

(15) In a 250 ml reaction flask, a solution of Lenalidomide (1.28 g, 4.9 mmol) and succinic anhydride (0.64 g, 6.4 mmol) were dissolved in toluene (100 ml). The mixture was heated to 125 C. and reacted for 5.0 h, and then cooled to room temperature. The mixture was vacuum filtered and dried to obtain 1.70 g of 4-(2-(2,6-dioxapiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-4-oxobutyric acid (compound (C-1)).

Preparation Example 6: Preparation of 5-(2-(2,6-dioxapiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-5-oxopentanoic Acid (Compound (C-2))

(16) 5-(2-(2,6-dioxapiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-5-oxopentanoic acid (compound (C-2), 1.65 g) was prepared in a similar to that in Example 5, except that succinic anhydride was replaced with glutaric anhydride.

Preparation Example 7: Preparation of 6-(2-(2,6-dioxapiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-6-oxohexanoic Acid (Compound (C-3))

(17) 6-(2-(2,6-dioxapiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-6-oxohexanoic acid (compound of formula (C-3), 1.21 g) was prepared in a similar to that in Example 5, except that succinic anhydride was replaced with adipic anhydride.

Preparation Example 8: Preparation of 2-((2-(2,6-dioxapiperazin-3-yl)-1,3-dioxaindol-4-yl)amino)acetic Acid (Compound (C-4))

(18) ##STR00020##

Step 1: preparation of tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoindolin-4-yl)amino) acetate

(19) In a 100 ml reaction flask, a solution of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (0.30 g, 1.1 mmol) was dissolved in DMF, DIPEA (3841, 2.2 mmol), tert-butyl glycine (0.17 g, 1.3 mmol) was added to the solution, and then the mixture was heated to 90 C. for 2 h. The mixture was cooled to room temperature, water and ethyl acetate were added to the mixture. The organic phase was separated and evaporated under reduced pressure to afford tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino) acetate (0.38 g). The product was used to the next step without purification.

Step 2: preparation of 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)acetic acid (compound (C-4))

(20) In a 100 ml reaction flask, a solution of Tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino) acetate (0.38 g, 0.98 mmol) was dissolved in DCM (100 ml), and TFA (5 ml) was added to the solution. The mixture was stirring for 4 h at room temperature, and then evaporated under reduced pressure to afford of 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)acetic acid (compound (C-4), 0.30 g). The product was used to the next step without purification.

Preparation Example 9: Preparation of 3-((2-(2,6-dioxapiperazin-3-yl)-1,3-dioxaindol-4-yl)amino)propionic Acid (Compound (C-5))

(21) 3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propionic acid (compound (C-5), 0.42 g) was prepared in a similar to that in Example 8, except that tert-butyl glycine was replaced with tert-butyl alanine. The product was used to the next step without purification.

Preparation Example 10: Preparation of 4-((2-(2,6-dioxapiperazin-3-yl)-1,3-dioxaindol-4-yl)amino)butanoic Acid (Compound (C-6))

(22) 4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyric acid (compound of formula (C-6), 0.25 g) was prepared in a similar to that in Example 8, except that tert-butyl glycine was replaced with tert-butyl butyrate, The product was used to the next step without purification.

Preparation Example 11: Preparation of 5-((2-(2,6-dioxapiperazin-3-yl)-1,3-dioxaindol-4-yl)amino)pentanoic Acid (Compound (C-7))

(23) 5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentanoic acid (compound (C-7), 0.12 g) was prepared in a similar to that in Example 8, except that tert-butyl glycine was replaced with tert-butyl pentanoate. The product was used to the next step without purification.

Preparation Example 12: Preparation of 3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propionic Acid (Compound (C-8))

(24) ##STR00021##

(25) 3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)propionic acid (compound (C-8), 0.30 g) was prepared in a similar to that in Example 8, except that tert-butyl glycine was replaced with tert-butyl 3-[2-(2-aminoethoxy)ethoxy]propionate.

Preparation Example 13: Preparation of 3-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxypropionic acid (compound of formula (C-9))

(26) 3-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxypropionic acid (compound (C-9), 0.37 g) was prepared in a similar to that in Example 12, except that tert-butyl 3-[2-(2-aminoethoxy)ethoxy]propionate was replaced with tert-butyl 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propionate.

Preparation Example 14: Preparation of 1-((2-(2,6-dioxapiperazin-3-yl)-1,3-dioxaindol-4-yl)amino)-3,6,9,12-tetraoxapentadecane-15-Acid (Compound (C-10))

(27) 1-((2-(2,6-dioxapiperazin-3-yl)-1,3-dioxa indol-4-yl)amino)-3,6,9,12-tetraoxapentadecane-15-acid (compound of formula (C-10), 0.26 g) was prepared in a similar to that in Example 12, except that tert-butyl 3-[2-(2-aminoethoxy)ethoxy]propionate was replaced with 2-[2-[2-[2-(2-tert-butoxy carbonylethoxy)ethoxy]ethoxy]ethoxy]ethylamine.

Preparation Example 15: Preparation of 2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)acetic Acid (Compound (C-11))

(28) ##STR00022##

Step 1: Preparation of tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino) acetate

(29) A solution of Lenalidomide (259 mg, 1.0 mmol) dissolved in N,N-dimethylformamide (20 ml) was provided, and tert-butyl bromoacetate (234 mg, 1.2 mmol), and potassium carbonate (276 mg, 2.0 mmol), potassium iodide (8 mg, 0.05 mmol) were added to the solution. The mixture was stirred at 80 C. After the reaction was completed, ethyl acetate and water were added, and the organic phase was washed with water for twice. The organic phase was separated, and purified by silica gel chromatography to obtain tert-butyl 2-((2-(2,6-dioxopiperidine-3-yl)-1-oxoisoindol-4-yl)amino) acetate (150 mg).

Step 2: Preparation of 2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)acetic Acid (Compound (C-11))

(30) A solution of Tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino) acetate (0.15 g, 0.40 mmol) dissolved in DCM (20 ml) was provided, and trifluoroacetic acid (7 ml) was added to the solution. The mixture was stirred at room temperature. After the reaction was completed, the mixture was distilled under reduced pressure to obtain 2-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)acetic acid (compound (C-11), 0.10 g, oil). The product was used to the next step without purification.

Preparation Example 16: Preparation of 3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)propionic Acid (Compound (C-12))

(31) 3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)propionic acid (compound (C-12), 0.05 g, oil) was prepared in a similar to that in Example 15, except that tert-butyl bromoacetate was replaced with tert-butyl bromopropionate, the product is directly used to the next step without purification.

Preparation Example 17: Preparation of 4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)butanoic Acid (Compound (C-13))

(32) 4-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)butyric acid (compound (C-13), 0.13 g, oil) was prepared in a similar to that in Example 15, except that tert-butyl bromoacetate was replaced with tert-butyl bromobutyrate. The product was directly used to the next step without purification.

Preparation Example 18: Preparation of 5-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)pentanoic Acid (Compound (C-14))

(33) 5-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl)amino)pentanoic acid (compound of formula (C-14), 0.13 g, oil) was prepared in a similar to that in Example 15, except that tert-butyl bromoacetate was replaced with tert-butyl bromovalerate. The product was directly used to the next step without purification.

Preparation Example 19: Preparation of 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic Acid (compound (C-15))

(34) ##STR00023##

Step 1: Preparation of tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy) acetate

(35) A solution of 2-(2,6-Dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (274 mg, 1.0 mmol) dissolved in N,N-dimethylformamide (20 ml) was provided, then tert-butyl bromoacetate (234 mg, 1.2 mmol), potassium carbonate (276 mg, 2.0 mmol) and potassium iodide (8 mg, 0.05 mmol) were added to the solution and stirred at 80 C. After the reaction was completed, ethyl acetate and water were added, and the organic phase was washed twice with water. The organic phase was separated, and purified by column chromatography to obtain 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy) tert-butyl acetate (150 mg).

Step 2: Preparation of 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic Acid (Compound of Formula (C-6))

(36) A solution of Tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy) acetate (0.15 g, 0.37 mmol) dissolved in dichloromethane (20 ml) was provided, then TFA (7 ml) was added to the solution. The mixture was stirred at room temperature. After the reaction was completed, the mixture was distilled under reduced pressure to obtain 2-((2-(2,6-dioxopiperidine)-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic acid (compound of formula (C-15), 0.10 g, oil), the product was directly used to the next step without purification.

Example 1: Preparation of 4-(4-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetamido)piperazin-1-yl)-N-(2-(2,6-dioxpiperazin-3-yl)-1-oxoisoindolin-4-yl)-4-oxobutanamide

(37) ##STR00024##

(38) In a 100 ml reaction flask, a solution of the compound of formula M-1 (53 mg, 0.11 mmol) dissolved in DMF (10.0 ml) was provided, and then HATU (40 l, 0.23 mmol) and the compound C-1 (39 mg, 0.11 mmol) were added to the solution. The mixture was stirred for 2.0 h at room temperature. TLC showed that raw material was consumed. The reaction solution was poured into water (100.0 ml), extracted with DCM, dried over anhydrous sodium sulfate, evaporated under reduced pressure, and purified and separated by preparative liquid chromatography (dichloromethane:methanol=30:1) to afford 4-(4-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diaza-6-yl)acetamido)piperazin-1-yl)-N-(2-(2,6-dioxopiperazin-3-yl)-1-oxoisoindolin-4-yl)-4-oxobutanamide 30 mg, the yield was 33%.

Example 2-45: Preparation of the Compound of Example 2-45

(39) The products were prepared in a similar to that in Example 1, except that the compound of formula M1 and the compound of formula C1 were replaced with corresponding M-1-M-3 and C-1-C-15 respectively, the detailed are shown below:

(40) TABLE-US-00001 Ex- Com- Com- am- pound pound ple M C Product Compound 2 M-1 C-2 3 M-1 C-3 4 M-1 C-4 5 M-1 C-5 6 M-1 C-6 7 M-1 C-7 0 8 M-1 C-8 9 M-1 C-9 MC-9 10 M-1 C-10 11 M-1 C-11 12 M-1 C-12 13 M C-13 14 M-1 C-14 15 M-1 C-15 16 M-2 C-1 17 M-2 C-2 0 18 M-2 C-3 19 M-2 C-4 20 M-2 C-5 21 M-2 C-6 22 M-2 C-7 23 M-2 C-8 24 M-2 C-9 25 M-2 C-10 26 M-2 C-11 27 M-2 C-12 0 28 M-2 C-13 29 M-2 C-14 30 M-2 C-15 31 M-3 C-1 32 M-3 C-2 33 M-3 C-3 34 M-3 C-4 35 M-3 C-5 36 M-3 C-6 37 M-3 C-7 0 38 M-3 C-8 39 M-3 C-9 40 M-3 C-10 41 M-3 C-11 42 M-3 C-12 43 M-3 C-13 44 M-3 C-14 45 M-3 C-15

(41) Physical characterization of specific compounds were as follows:

(42) TABLE-US-00002 Example .sup.1H NMR(600 MHz, DMSO) MS[M + H].sup.+ 1 .sup.1H NMR (600 MHz, DMSO-d.sub.6) : 1.044-1.067 (m, 1H), 1.625-1.633 (m, 825.3 3H), 2.043-2.052 (m, 1H), 2.301-2.384(m, 1H), 2.412(s, 3H), 2.596-2.749 (m, 10H), 2.823-2.923(m, 1H), 3.085-3.150(m, 1H) 3.170-3.230 (m, 1H), 3.431-3.451(m, 1H), 3.529-3.556 (m, 3H), 4.321-4.388(m, 3H), 4.417- 4.548 (m, 1H), 5.137-5.167 (m, 1H), 7.417-7.454(m, 2H), 7.485-7.512 (m, 4H), , 7.837-7.848(m, 1H), 9.367 (s, 1H) 9.858(s, 1H), 11.028(s, 1H) 4 .sup.1H NMR (600 MHz, CDCl.sub.3) : 0.903-0.941 (m, 1H), 1.266-1.277 (m, 2H), 797.3 1.323-1.359 (m, 1H), 1.444-1.573 (m, 1H), 1.613-1.923 (m, 3H), 2.127(s, 1H), 2.451-2.521(s, 3H) 2.687-2.728 (m, 3H), 2.807-2.921 (m, 3H), 3.089- 3.108 (m, 1H), 3.457-3.864(m, 4H), 4.074-4.235 (m, 2H), 4.651-4.680 (m, 1H), 4.937 (s, 1H), 6.811-6.825 (m, 1H), , 7.027-7.217(m, 2H), 7.344- 7.500 (m, 5H) 8.002-8.164(m, 1H), 8.780-9.197(m, 1H) 8 .sup.1H NMR (600 MHz, CDCl.sub.3) : 0.892-0.923 (m, 1H), 1.244-1.286 (m, 2H), 899.3 1.352-1.386 (m, 1H), 1.444-1.573 (m, 1H), 1.656-1.989 (m, 3H), 2.125(s, 1H), 2.441-2.521(s, 3H) 2.677-2.725 (m, 3H), 2.827-2.931 (m, 3H), 3.089- 3.108 (m, 1H), 3.457-3.864(m, 4H), 4.074-4.235 (m, 2H), 4.281-4.305 (m, 2H), 4.651-4.680 (m, 1H), 4.937 (s, 1H), 6.861-6.885 (m, 1H), , 7.037- 7.214(m, 2H), 7.344-7.500 (m, 5H) 8.012-8.163(m, 1H), 8.770-9.195(m, 1H) 9 .sup.1H NMR (600 MHz, CDCl.sub.3) : 0.812-0.911 (m, 1H), 1.218-1.237 (m, 2H), 943.5 1.325-1.355 (m, 3H), 1.414-1.493 (m, 1H), 1.642-1.943 (m, 3H), 2.337(s, 1H), 2.451-2.521(s, 3H) 2.637-2.768 (m, 3H), 2.827-2.926 (m, 3H), 3.089- 3.138 (m, 1H), 3.257-3.364(m, 4H), 4.251-4.568 (m, 1H), 4.737 (s, 1H), 6.795-6.845 (m, 1H), , 7.127-7.257(m, 2H), 7.374-7.615 (m, 5H) 8.015- 8.189(m, 1H), 8.475-9.082(m, 1H) 11 .sup.1H NMR (600 MHz, CDCl.sub.3) : 0.803-0.941 (m, 1H), 1.216-1.247 (m, 2H), 783.2 1.323-1.359 (m, 3H), 1.434-1.503 (m, 1H), 1.643-1.933 (m, 3H), 2.327(s, 1H), 2.451-2.521(s, 3H) 2.637-2.768 (m, 3H), 2.827-2.926 (m, 3H), 3.089- 3.138 (m, 1H), 3.257-3.364(m, 4H), 3.894-4.235 (m, 2H), 4.251-4.568 (m, 1H), 4.737 (s, 1H), 6.791-6.835 (m, 1H), , 7.127-7.257(m, 2H), 7.384- 7.610 (m, 5H) 8.012-8.174(m, 1H), 8.480-9.097(m, 1H) 15 .sup.1H NMR (600 MHz, CDCl.sub.3) : 0.952-0.974 (m, 1H), 1.236-1.257 (m, 2H), 798.1 1.303-1.338 (m, 1H), 1.432-1.536 (m, 1H), 1.643-1.923 (m, 3H), 2.031(s, 1H), 2.341-2.601(s, 3H) 2.687-2.752 (m, 3H), 2.832-2.921 (m, 3H), 3.189- 3.258 (m, 1H), 3.457-3.654 (m, 4H), 4.174-4.229 (m, 2H), 4.551-4.647 (m, 1H), 4.877 (s, 1H), 6.741-6.830 (m, 1H), 7.127-7.257(m, 2H), 7.484-7.596 (m, 4H) 8.102-8.234(m, 1H), 8.880-9.267(m, 1H) 16 .sup.1H NMR (600 MHz, DMSO-d.sub.6) : 1.154-1.237 (m, 1H), 1.575-1.603 (m, 825.2 3H), 2.043-2.152 (m, 1H), 2.321-2.414(m, 1H), 2.475(s, 3H), 2.536-2.751 (m, 10H), 2.825-2.931(m, 1H), 3.089-3.169(m, 1H) 3.241-3.301 (m, 1H), 3.446-3.473(m, 1H), 3.560-3.654 (m, 3H), 4.171-4.296(m, 3H), 4.452- 4.618 (m, 1H), 5.037-5.567 (m, 1H), 7.467-7.514(m, 2H), 7.585-7.691 (m, 4H), 7.837-7.948(m, 1H), 9.351 (s, 1H) 9.889(s, 1H), 11.008(s, 1H) 19 .sup.1H NMR (600 MHz, CDCl.sub.3) : 0.913-0.953 (m, 1H), 1.281-1.291 (m, 797.1 2H), 1.343-1.369 (m, 1H), 1.464-1.532 (m, 1H), 1.599-1.923 (m, 3H), 2.139(s, 1H), 2.449-2.521(s, 3H) 2.675-2.726 (m, 3H), 2.843-2.964 (m, 3H), 3.034-3.112 (m, 1H), 3.507-3.844(m, 4H), 4.024-4.221 (m, 2H), 4.632- 4.655 (m, 1H), 4.926 (s, 1H), 6.821-6.845 (m, 1H), , 7.104-7.209(m, 2H), 7.384-7.560 (m, 5H) 8.022-8.164(m, 1H), 8.810-9.167(m, 1H) 23 .sup.1H NMR (600 MHz, CDCl.sub.3) : 0.947-0.968 (m, 1H), 1.281-1.291 (m, 2H), 899.5 1.383-1.412 (m, 1H), 1.454-1.539 (m, 1H), 1.588-1.963 (m, 3H), 2.126(s, 1H), 2.449-2.521(s, 3H) 2.675-2.726 (m, 3H), 2.843-2.964 (m, 3H), 3.034- 3.112 (m, 1H), 3.221-3.335 (m, 2H), 3.507-3.844(m, 4H), 4.024-4.221 (m, 2H), 4.228-4.442 (m, 2H), 4.542-4.642 (m, 1H), 4.937 (s, 1H), 6.837-6.853 (m, 1H), , 7.254-7.279(m, 2H), 7.354-7.460 (m, 5H) 8.032-8.154(m, 1H), 8.826-9.187(m, 1H) 24 .sup.1H NMR (600 MHz, CDCl.sub.3) : 0.945-0.997 (m, 1H), 1.301-1.331 (m, 2H), 943.3 1.353-1.369 (m, 1H), 1.464-1.532 (m, 1H), 1.599-1.923 (m, 3H), 2.329(s, 1H), 2.449-2.521(s, 3H) 2.675-2.726 (m, 3H), 2.843-2.964 (m, 3H), 3.034- 3.112 (m, 1H), 3.221-3.335 (m, 2H), 3.507-3.844(m, 4H), 4.024-4.221 (m, 2H), 4.229-4.310 (m, 2H), 4.328-4.452 (m, 2H), 4.510-4.610 (m, 2H), 4.632-4.655 (m, 1H), 4.886 (s, 1H), 6.821-6.845 (m, 1H), , 7.204- 7.269(m, 2H), 7.394-7.540 (m, 5H) 8.055-8.184(m, 1H), 8.890-9.163(m, 1H) 26 .sup.1H NMR (600 MHz, CDCl.sub.3) : 0.899-0.921 (m, 1H), 1.248-1.297 (m, 2H), 783.3 1.330-1.401 (m, 1H), 1.431-1.521 (m, 3H), 1.579-1.873 (m, 3H), 2.089(s, 1H), 2.348-2.497(s, 3H) 2.535-2.716 (m, 3H), 2.823-2.924 (m, 3H), 3.024- 3.212 (m, 1H), 3.497-3.794(m, 4H), 3.978-4.191 (m, 2H), 4.602-4.648 (m, 1H), 4.906 (s, 1H), 6.811-6.865 (m, 1H), 7.104-7.213(m, 2H), 7.352-7.592 (m, 5H) 8.021-8.163(m, 1H), 8.812-9.155(m, 1H) 30 .sup.1H NMR (600 MHz, CDCl.sub.3) : 0.940-0.977 (m, 1H), 1.201-1.242 (m, 2H), 798.2 1.313-1.333 (m, 1H), 1.421-1.537 (m, 1H), 1.655-1.903 (m, 3H), 2.021(s, 1H), 2.348-2.597(s, 3H) 2.656-2.749 (m, 3H), 2.803-2.926 (m, 3H), 3.181- 3.268 (m, 1H), 3.447-3.616 (m, 4H), 4.141-4.222 (m, 2H), 4.534-4.638 (m, 1H), 4.871 (s, 1H), 6.741-6.841 (m, 1H), 7.127-7.327(m, 2H), 7.484-7.656 (m, 4H) 8.112-8.244(m, 1H), 8.892-9.272(m, 1H) 31 .sup.1H NMR (600 MHz, DMSO-d.sub.6) : 1.609-1.628 (m, 3H), 1.988-2.007 (m, 756.5 1H), 2.333-2.407(m, 4H), 2.501-2.598 (m, 5H), 2.647-2.671 (m, 2H), 2.275-2.291 (m, 1H), 3..154-3.172 (m, 2H), 3.323-3.354 (m, 1H), 4.326- 4.394 (m, 2H), 4.501-4.521 (m, 1H), 5.119-5.149 (m, 1H), 7.423-7.452 (m, 6H), 7.830-7.880 (m, 1H), 9.898-9.943(d, 2H), 10.143(s, 1H), 10.995- 11.007(m, 1H). 34 .sup.1H NMR (600 MHz, CDCl.sub.3) : 0.853-0.907 (m, 1H), 1.264 (s, 3H), 1.668- 830.5 1.685 (m, 3H), 2.088 (m, 2H), 2.414 (s, 3H), 2.549-2.569 (m, 2H), 2.682- 2.691 (m, 2H), 2.728-2.741(m, 2H), 3.443-3.506 (m, 2H), 3.572-3.584 (m, 1H), 3.644-3.685 (m, 3H), 3.733-3.789 (m, 3H), 4.612-4.622 (m, 1H), 4.934-4.978 (m, 1H), 6.569 (s, 1H), 6.893-6.907 (m, 1H), 7.098-7.110 (m, 1H), 7.315-7.347 (m, 2H), 7.435-7.503(m, 3H), 9.041-9.241(m, 2H), 9.450-9.650(m, 1H).

Biological Example 1: Effects of Compounds on the Proliferation of RS4; 11 and MM.1S Cells

(43) Cell lines for test: acute leukemia cell line RS4; 11 and multiple myeloma cell line MM.1S

(44) Test method: MTT (thiazole blue) method, which was also called MTT colorimetry assay, was a method used to detect cell survival and growth. The principle of the assay was that the succinate dehydrogenase in mitochondria of living cells could reduce the exogenous MTT to water-insoluble blue-purple crystal formazan and deposit it in the cells, while dead cells have no such function. Dimethyl sulfoxide (DMSO) could dissolve formazan in cells, and its light absorption value was measured at 550 nm wavelength by enzyme-linked immunoassay, which could indirectly reflect the number of living cells. Within a certain range of cell numbers, the amount of MTT crystal formation is proportional to the number of cells. This method has been widely used in the activity detection of some biologically active factors, large-scale anti-tumor drug screening, cytotoxicity test, and tumor radiosensitivity determination.

(45) Experiment Procedure:

(46) Dosage Design

(47) The concentration gradient of the compound was as follows: 0-25.6 pM-128 pM-640 pM-3.2 nm-16 nM-80 nM-400 nM-2 M-10 M; (n=3)

(48) Detection and Calculation

(49) 72 h after drug administration, 20 l per well of MTT working solution (5 mg/ml) was added, and incubated at 37 C. for 4 h, centrifuged in plate centrifuge at 1000 rpm/min for 5 min. 180 l of medium was aspirated and 150 l DMSO was added in RS4; 11 groups; 200 l of medium was aspirated and 150 l DMSO was added in MM.1S group; the mixture was shaken and mixed on microwell shaker, and the bottom of the plate was wiped, and the optical density (OD) was measured at 550 nm by microplate reader. The IC.sub.50 of the half inhibitory concentration was calculated by LOGIT method.

(50) Experimental Results:

(51) TABLE-US-00003 IC.sub.50 (nM) Medicine RS4; 11 MM.1S JQ1 C C dBET1 C C ARV-825 B B The compound in Example 1 A A The compound in Example 4 A A The compound in Example 5 A A The compound in Example 8 B A The compound in Example 9 A B The compound in Example 11 A A The compound in Example 12 A A The compound in Example 15 A A The compound in Example 16 A A The compound in Example 19 A A The compound in Example 20 A A The compound in Example 26 B B The compound in Example 31 B B The compound in Example 34 A A C: IC.sub.50 100 nM, B: 20 nM IC.sub.50 < 100 nM, A: IC.sub.50 < 20 nM.

(52) The experimental results showed that the compound of present invention has good anti-tumor activity. Compared with JQ1, dBET1 and ARV-825, the compound of present invention showed better inhibitory activity, anti-proliferation and apoptosis-inducing ability.

Biological Example 2: Study on the Degradation Mechanism of BRD4-PROTAC Protein

(53) The purpose of the experiment: the influence of BRD4-PROTAC on the degradation of the target protein was observed by MG132 pretreatment to study whether the test compound degrade the target protein through the protease degradation pathway.

(54) Experiment Method:

(55) RS4; 11 and MM.1S cells were cultured in vitro and inoculated in 6-well plate. When the cells were grown to about 80%, MG132 was added to a final concentration of 50 uM. 2 Hours later, the compound of Example 1 and the compound of Example 16 were added to a concentration of 100 nM, and the cells was collected after treated for 4 h. The protein was extracted, and Westing Bloting was performed to detect the BRD4 protein level.

(56) The experimental results were shown in FIG. 2.

(57) The experimental results showed that the compound of the present invention could effectively degrade various tumor cell target protein BRD4; and the protein degradation could be blocked by MG-132, which indicates that the protein degradation of the compound of the present invention was dependent on the ubiquitination-proteasome pathway.

(58) The embodiments of the present invention have been described above. However, the present invention is not limited to the above-mentioned embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.