CN116253730A - A kind of PROTAC compound targeting to degrade HDAC7 protein and its preparation method and application - Google Patents

Abstract

The invention discloses a PROTAC compound targeting to degrade HDAC7 protein, its preparation method and application. The PROTAC compounds targeting the degradation of HDAC7 protein have the potential to degrade HDAC7 on NB4 cells and have significant proliferation inhibitory activity on acute myeloid lymphoblastic leukemia and various diffuse large B lymphocyte lines. Both therapy and as a tool molecule to explore the biological function of HDAC7 have broad application prospects.

Description

A PROTAC compound targeting degradation of HDAC7 protein and its preparation method and application

Technical Field

The present invention relates to the field of medical technology, and in particular to a PROTAC compound that targets the degradation of HDAC7 protein, a preparation method thereof, and an application thereof in the anti-tumor field.

Background Art

HDAC (Histone Deacetylase) is the full name of histone deacetylase, which is an epigenetic regulatory factor. Its main function is to deacetylate lysine residues on histones. Among the 11 classic HDACs that rely on zinc ion domains, they are mainly divided into 4 categories. Class I HDACs include HDAC1, 2, 3, and 8; Class IIa HDACs include HDAC4, HDAC5, HDAC7, and HDAC9; Class IIb HDACs include HDAC6 and HDAC10; and Class IV HDACs include HDAC11. Class IIa HDACs mainly regulate cell and developmental processes through enzymatic and non-enzymatic mechanisms.

Over the past 20 years, HDAC7 protein has been widely studied and has been shown to play a key regulatory role in many physiological and pathological processes. After a series of molecular, cellular, in vivo and disease-related studies, researchers have found that HDAC7 plays a key regulatory role in immune system diseases, renal cancer, non-small cell lung cancer, colon cancer, gastric cancer, multiple myeloma and other diseases through various mechanisms. HDAC7 protein controls development, angiogenesis, immune regulation, inflammation and metabolism by regulating gene expression, cell proliferation, differentiation and survival. In short, HDAC7 is closely related to cancer, inflammation, metabolism and fibrosis. However, the mechanism of action of class IIa HDACs as epigenetic metalloenzymes is not clear. First, histones may not be their catalytic substrates. Literature shows that the deacetylation function of lysine on histones of class IIa HDAC is about 1000 times weaker than that of class I HDAC. Therefore, some researchers have proposed that class IIa HDAC may act as a chaperone protein of class I HDAC, or that it has undiscovered new catalytic substrates. At present, the small molecule inhibitors of class IIa HDAC mainly include compounds such as TMP269, TMP195, CHDI-390576 and NVS-HD1. However, they are unable to selectively inhibit HDAC7 protein. At the same time, their proliferation inhibition effect at the cellular level is generally weak, making it difficult to achieve the ideal therapeutic effect and preventing further drugability research.

The mechanism of action of proteolysis targeting chimera (PROTAC) is different from that of traditional small molecule inhibitors that inhibit the function of target proteins by binding to the active sites of target proteins (occupancy driven). It mainly utilizes the inherent ubiquitin-proteasome system in the body to degrade target proteins (event driven) to achieve the purpose of treatment. PROTAC is a bifunctional molecule composed of three parts, namely, a small molecule ligand of the target protein, an E3 ubiquitin protein ligase ligand, and a connecting chain connecting the two. PROTAC degrades the target protein, making both the kinase and non-kinase functions of the target protein unable to function, which has greater advantages than small molecule inhibitors that can only inhibit kinase function. There are currently no reports of PROTAC molecules directly related to HDAC7. Therefore, it is necessary to develop a PROTAC compound that targets and degrades HDAC7 protein.

Summary of the invention

The purpose of the present invention is to provide a PROTAC compound for targeted degradation of HDAC7 protein and its preparation method and application. The PROTAC compound for targeted degradation of HDAC7 protein has the ability to significantly degrade HDAC7 at a concentration of 1 μM in NB4 cells and has a certain ability to inhibit the proliferation of NB4 cells. It can be developed as a potential drug for acute myeloid lymphocytic leukemia and diffuse large B-cell lymphocytic leukemia and has broad application prospects.

In order to achieve the above object, the present invention adopts the following technical solution:

In a first aspect of the present invention, a PROTAC compound targeting degradation of HDAC7 protein is provided, wherein the PROTAC compound targeting degradation of HDAC7 protein has a structural formula shown in the following formula (I):

or a stereoisomer thereof or a pharmaceutically acceptable salt thereof;

in:

Linker is a connecting group selected from one or more of the following groups: linear or branched alkyl, alkoxy, alkylamino, alkenyl, alkynyl, alkenylene, alkynylene, alkynoxy, alkynamino, cycloalkyl, heterocycloalkyl, spirocycloalkyl, heterospirocycloalkyl, _cycloalkyl , heterocycloalkyl, aromatic ring, heteroaromatic ring, -CO( _CH2 ) _n- , -CO(CH2)nO-, -( _CH2 ) _nNH2- , -( _CH2 )nNR1-, _-CO ( _CH2 )nNH-, -CO( _CH2 ) _nNR1- , -(CH2) _nCONH ( _CH2 ) _n- , -( _CH2 ) _{nCONH ( CH2 ) nNH- , -} ( _{CH2 ) nCONH (} CH2 _{) nNR1-} , _-CO ( _{CH2 )} nNHCO( _CH2 ) _n - , -CO(CH ₂ ) _n NHCO(CH ₂ ) _n NH- , -CO(CH ₂ ) _n NHCO(CH ₂ ) _n NR ₁ -, -CO(CH ₂ OCH ₂ ) _n CH ₂ NHCO(CH ₂ ) _n -, -CO(CH ₂ OCH ₂ ) _n CH ₂ NHCO(CH ₂ ) _n NH-, -CO(CH ₂ OCH ₂ ) _n CH ₂ NHCO(CH ₂ ) _n NR ₁ -, -CH ₂ (CH ₂ OCH ₂ ) _n CH ₂ -, -CH ₂ (CH ₂ OCH ₂ ) _n CH ₂ NH-, -CH ₂ (CH ₂ OCH ₂ ) _n CH ₂ NR ₁ -, -CH ₂ (CH ₂ OCH ₂ ) _n CH ₂ O-, -CH ₂ CONHCH ₂ (CH ₂ OCH ₂ ) _n CH ₂ NH-, -CH ₂ CONHCH ₂ (CH ₂ OCH ₂ ) _n CH ₂ NR ₁ -, -CH ₂ CONHCH ₂ (CH ₂ OCH ₂ ) _n CH ₂ NHCO(CH ₂ ) _n NH-, -CH ₂ CONHCH ₂ (CH ₂ OCH ₂ ) _n CH ₂ NHCO(CH ₂ ) _n NR ₁ -, -CH ₂ CONHCH ₂ (CH ₂ OCH ₂ ) _n CO-, -CH ₂ (CH ₂ OCH ₂ ) _n CH ₂ CO-, or any combination thereof,

n represents a natural number from 1 to 20;

_R1 is H or _C1-10 alkyl.

Linker is a connecting group, selected from one or more of the following groups such as polycyclic halogenated heterocycle, alkenyl, alkynyl, alkynyloxy, alkynylamino, polycyclic alkyl, polycyclic heterocyclic alkyl, polycyclic spiroalkyl, polycyclic heterospiroalkyl, polycyclic cycloalkyl, polycyclic heterocycloalkyl, aromatic ring, halogenated aromatic ring or any combination thereof, wherein the ring system combination is any combination of 1-4 ring systems, the ring system size ranges from four-membered ring to six-membered ring, and the ring system types include bridged ring, spiro ring, and cyclocyclic ring. These include but are not limited to the following structures:

E3 ligand is an E3 ligase ligand selected from one of the following ligands:

Furthermore, the HDAC7-PROTAC compound provided by the present invention is a compound as shown below or its stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug, and the specific structure is shown in Table 1.

Table 1: Overview of PROTAC compounds targeting degradation of HDAC7 protein

In the second aspect of the present invention, a pharmaceutical composition for anti-acute myeloid lymphocytic leukemia and diffuse large B-cell lymphoma is provided, comprising the PROTAC compound targeting the degradation of HDAC7 protein or its pharmacologically or physiologically acceptable stereoisomer, geometric isomer, tautomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition also includes a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or a combination thereof. The dosage form of the pharmaceutical composition is mainly an injection, an oral agent, and a mucosal administration agent.

The pharmaceutical composition further comprises other drugs having the effect of treating or preventing acute myeloid lymphocytic leukemia or diffuse large B-cell lymphoma.

In a third aspect of the present invention, a method for preparing the PROTAC compound targeting the degradation of HDAC7 protein is provided, the method comprising:

The PROTAC compounds targeting the degradation of HDAC7 mutant proteins are divided into four categories: A, B, C, and D. Among them,

1. Class A PROTAC compounds are compounds represented by general formula (I), wherein the linker is a piperidine carboxylic acid derivative of different lengths and sizes, and the E3 ligand is a 4-substituted amine derivative.

2. Class B PROTAC compounds are compounds represented by general formula (I), wherein the linker is a benzoic acid derivative of different lengths and sizes, and the E3 ligand is a 4-substituted amine derivative.

3. Class C PROTAC compounds are compounds represented by general formula (I), wherein the linker is a nitrogen-containing heterocyclic derivative of different lengths and sizes, and the E3 ligand is a 4-substituted amine derivative.

4. Class D PROTAC compounds are compounds represented by general formula (I), wherein the linker is a PEG chain of varying degrees and sizes, and the E3 ligand is a 3-substituted amine derivative or a VHL ligand.

The preparation route of class A PROTAC compounds is as follows:

Compound 8 was dissolved in dichloromethane, trifluoroacetic acid was added to remove the Boc protecting group, and then condensed with 1-Boc-4-piperidinic acid under HATU and DIPEA to obtain compound 9. Compound 9 was de-Boced under trifluoroacetic acid to obtain compound 10. Compound 11 was subjected to nucleophilic reaction with various nitrogen-containing heterocycles under DIPEA to obtain 12a-d, and then oxidized by DMP to obtain 13a-d. Compound 10 and compound 13a-d were subjected to reductive amination under sodium cyanoborohydride and glacial acetic acid to obtain four compounds A1-A4.

The preparation route of class B PROTAC compounds is as follows:

Compound 8 was dissolved in dichloromethane, trifluoroacetic acid was added to remove the Boc protecting group, and then condensed with 1-Boc-4-(4-carboxyphenyl)piperidine or 1-Boc-4-(4-carboxyphenyl)piperazine under HATU and DIPEA to obtain compounds 14a-b. Compounds 14a-b were deprotected from Boc under trifluoroacetic acid to obtain compounds 15a-b. Compounds 15a-b and compounds 13a-d were reductively aminated under sodium cyanoborohydride and glacial acetic acid to obtain four compounds B1-B4.

The preparation route of class C PROTAC compounds is as follows:

Compound 8 was dissolved in dichloromethane, trifluoroacetic acid was added to remove the Boc protecting group, and then the compound 16a-d was reductively aminated with the aldehyde derivative under the conditions of sodium cyanoborohydride and glacial acetic acid to obtain compounds 16a-d, and the Boc of compound 16a-d was removed under trifluoroacetic acid to obtain compounds 17a-d. Compounds 17a-d and compounds 13a-d were reductively aminated under the conditions of sodium cyanoborohydride and glacial acetic acid to obtain five compounds C1-5; compound 11 was reacted with tert-butyl ester derivatives under alkaline conditions to obtain compounds 18a-b, and the Boc protecting group was removed under trifluoroacetic acid to obtain 19a-b. Compounds 17a-d and compounds 19a-b were subjected to acid-amine condensation reaction under the conditions of HATU and DIPEA to obtain six compounds C6-C11. The preparation route of class D PROTAC compounds is as follows:

After removing the Boc protecting group from compounds 20a-b and 21a-b, respectively, acid-amine condensation was carried out under HATU and DIPEA conditions to obtain compound D1-2. After removing the Boc protecting group from compounds 21c and 22, respectively, acid-amine condensation was carried out under HATU and DIPEA conditions to obtain compound D3.

Beneficial effects of the present invention:

The present invention synthesizes and screens a series of compounds, which are novel HDAC7-PROTAC degraders. The inventors confirmed through western blot experiments that the novel HDAC7-PROTAC degraders provided by the present invention have a significant degradation effect on HDAC7 in NB4 cells, and further proved through cell proliferation inhibition experiments that the series of compounds effectively inhibit the cell proliferation of NB4, DHL4, DHL2, and Jeko-2, while the positive compound TMP269 has weak or no obvious inhibition of proliferation on these cells, further expanding the scope of indications, so that the novel HDAC7-PROTAC has the potential to treat acute myeloid lymphocytic leukemia and diffuse large B-lymphocytic leukemia.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Proliferation inhibition of the compounds of the present invention on SU-DHL4 cells;

Figure 2: Proliferation inhibition of the compounds of the present invention on SU-DHL2 cells;

Figure 3: Preliminary pharmacokinetics of the compounds of the present invention in ICR mice.

DETAILED DESCRIPTION

The following is a detailed description of some embodiments of the present invention. All the following embodiments are only examples for the purpose of illustrating the present invention in detail so as to deepen the understanding of the present invention and should not be construed as limiting the present invention.

In the description of the entire embodiment, the raw materials, methods and professional terms used are generally recognized in the field. Unless otherwise specified, if there is any ambiguity, the description in the present invention shall prevail. The various raw materials, reagents, instruments and equipment used in the present invention, unless otherwise specified, can be purchased from the market or can be obtained by existing methods.

The following will explain in detail a novel PROTAC compound for targeting the degradation of HDAC7 protein, its preparation method, and its application in acute myeloid lymphocytic leukemia and diffuse large B-cell lymphocytic leukemia through the specific synthesis method and relevant biological evaluation data of the embodiment.

Example 1

Synthesis of POI target head molecule 1

Step 1: Preparation of 3-(N-hydroxyurea)benzoic acid (Compound 2)

3-Cyanobenzoic acid (Compound 1, 20.0 g, 0.1 mol) was dissolved in 1000 mL of ethanol solution, and an aqueous solution (150 mL) of hydroxylamine hydrochloride (19.0 g, 0.3 mol), an aqueous solution (150 mL) of sodium carbonate (24.0 g, 0.2 mol), and 8-hydroxyquinoline (100.0 mg, 0.7 mmol) were added in sequence. The mixture was refluxed at 80°C with stirring for 4 h, the solvent was dried by spin drying, the mixture was diluted with water, and 2N HCl was added to acidify the mixture to a pH of about 3. A white solid was precipitated, which was washed with water and acetone in sequence. After drying under reduced pressure, 18.2 g of a white solid was obtained. The yield was 76%; ESI-MS: 179.1 [MH] ⁺ ; ¹ H NMR (500MHz, DMSO-d6) δ9.77 (s, 1H), 8.28 (t, J = 1.8 Hz, 1H), 7.95 (dt, J = 7.7, 1.5 Hz, 1H), 7.90 (dt, J = 7.8, 1.5 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 5.94 (s, 2H).

Step 2: Preparation of 3-(5-trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzoic acid (Compound 3)

3-(N-Hydroxyureayl)benzoic acid (compound 2, 5.0 g, 27.8 mmol) was dissolved in 100 mL of anhydrous pyridine solution, cooled to 0°C, and trifluoromethylacetic anhydride (11.6 mL, 83.4 mmol) was slowly added dropwise. After the addition, the temperature was raised to 50°C for reaction for 3 h. 2N HCl was added to acidify to pH 3-4 under ice bath conditions, and water was added to dilute. The mixture was extracted with ethyl acetate for multiple times. The organic layers were combined, mixed with silica gel, the solvent was dried by spin drying, and purified by column chromatography with PE:EA=3:1 to obtain 3.5 g of a white solid, with a yield of 49%; ESI-MS: 257.1 [MH] ^- ; ¹ H NMR (500MHz, DMSO-d6) δ13.46(s,1H),8.57(q,J＝1.8Hz,1H),8.35–8.28(m,1H),8.22(dq,J＝7.8,1.5Hz,1H),7.78(m,1H).

Step 3: Preparation of 2-(4-phenylthiazol-2-yl)acetonitrile (Compound 5)

2-Cyanothioacetamide (compound 4, 10.1 g, 0.1 mol) and 2-bromoacetophenone (20.0 g, 0.1 mol) were dissolved in 400 mL of anhydrous ethanol, refluxed at 80°C for 4 h, added with saturated sodium bicarbonate aqueous solution, adjusted the pH to alkaline, extracted with EA for multiple times, combined the organic layers, mixed with silica gel, spin-dried the solvent, and purified by column with PE:EA=10:1 to obtain 18.9 g of a yellow solid, yield: 95%; ESI-MS: 201.1 [M+H] ⁺ ; ¹ H NMR (500 MHz, DMSO-d6) δ8.14 (s, 1H), 8.03–7.93 (m, 2H), 7.49–7.43 (m, 2H), 7.41–7.33 (m, 1H), 4.65 (s, 2H).

Step 4: Preparation of tert-butyl 4-cyano-4-(4-phenylthiazol-2-yl)piperidine-1-carboxylate (Compound 6)

2-(4-phenylthiazol-2-yl)acetonitrile (compound 5, 5.0 g, 25.0 mmol) was dissolved in anhydrous DMF, sodium hydride (1.8 g, 75.0 mmol) was added under ice bath condition, the reaction was carried out at room temperature for 1 h, tert-butyl bis(2-chloroethyl)carbamate (9.0 g, 37.5 mmol) was added, the reaction was carried out at 80°C for 3 h, the mixture was diluted with saturated ammonium chloride solution, EA was extracted several times, the organic layers were combined, the sample was mixed with silica gel, the solvent was dried by spin drying, and the mixture was purified by column with PE:EA=5:1 to obtain 3.8 g of light yellow solid, yield: 41%; ESI-MS: 370.2 [M+H] ⁺ ; ¹ H NMR(500MHz,Chloroform-d)δ7.79–7.74(m,2H),7.34(s,1H),7.29(dd,J＝8.3,6.8Hz,2H),7.24–7.18(m,1H),4.12(d,J＝26.0Hz,2H),3.13(s,2H),2.21(dq,J＝13 .6,2.9Hz,2H),2.08(m,2H),1.38(s,9H).

Step 5: Preparation of tert-butyl 4-aminomethyl-4-(4-phenylthiazol-2-yl)piperidine-1-carboxylate (Compound 7)

Tert-butyl 4-cyano-4-(4-phenylthiazol-2-yl)piperidine-1-carboxylate (Compound 6, 5.0 g, 13.6 mmol) was dissolved in 60 mL of dry tetrahydrofuran solution, and lithium aluminum hydride (1.0 g, 26.3 mmol) was slowly added. The reaction was carried out at room temperature for 3 h. Water was slowly added dropwise under an ice bath until no more bubbles were generated. The mixture was diluted with water, extracted with DCM for several times, the organic layers were combined, the samples were mixed with silica gel, the solvent was dried by spin drying, and purified by column chromatography with dichloromethane: methanol = 30:1 to obtain 2.8 g of a light yellow liquid, with a yield of 55%; ESI-MS: 374.2 [M+H] ⁺ ; ¹ H NMR (400MHz, DMSO-d6) δ8.02(s,1H),7.93(d,J＝7.9Hz,2H),7.42(t,J＝7.6Hz,2H),7.34(t,J＝7.2Hz,1H),3.73(d,J＝13.4Hz,2H),3.01(s,2H),2.80(s,2H),2 .12(d,J＝13.9Hz,2H),1.84–1.72(m,2H),1.40(s,9H).

Step 6: Preparation of tert-butyl 4-(4-phenylthiazol-2-yl)-4-(3-(5-trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidine-1-carboxylate (Compound 8)

3-(5-Trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzoic acid (Compound 3, 3.0 g, 11.6 mmol) and tert-butyl 4-aminomethyl-4-(4-phenylthiazol-2-yl)piperidine-1-carboxylate (Compound 7, 5.6 g, 15.0 mmol) were dissolved in DCM, and HOBt (2.6 g, 17.4 mmol), EDCI (3.3 g, 17.4 mmol), and DIPEA (6.2 mL, 34.8 mmol) were added. The mixture was reacted at room temperature for 3 h, diluted with water, extracted with DCM for several times, the organic layers were combined, dried over anhydrous Na ₂ SO ₄ , mixed with silica gel, the solvent was spin-dried, and purified by column chromatography with PE:EA=5:1 to obtain 5.1 g of a white solid, with a yield of 71%; ESI-MS: 614.2 [M+H] ⁺ ; ¹ H NMR (500MHz, DMSO-d6) δ8.79(t,J＝6.5Hz,1H),8.45(p,J＝1.8Hz,1H),8.18(dq,J＝7.9,1.6Hz,1H),8.12–8.03(m,2H),7.94(dt,J＝8.2,1.6Hz,2H),7.68(td,J =7.8,2.5Hz ,1H),7.38(t,J＝7.6Hz,2H),7.29(td,J＝7.3,1.5Hz,1H),3.84(d,J＝13.3Hz,2H),3.65–3.54(m,2H),2.97(d,J＝12.6Hz,2H),2.36–2.21(m,2H),1.88( m,2H),1.38(s,9H).

Example 2

Synthesis of HDAC7-PROTAC molecule A1

Step 1: Preparation of tert-butyl 4-(4-phenylthiazol-2-yl)-4-(3-(5-trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidine-1-carbonyl)piperidine-1-carboxylate (Compound 9)

Tert-butyl 4-(4-phenylthiazol-2-yl)-4-(3-(5-trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidine-1-carboxylate (Compound 8, 250.0 mg, 0.4 mmol) was dissolved in 20 mL of DCM, 2 mL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 1 h. The solvent was dried by spin drying, 20 mL of DMF solution was added, 1-(tert-butyloxycarbonyl)piperidine-4-carboxylic acid (121.0 mg, 0.5 mmol), HATU (230.0 mg, 0.6 mmol), DIPEA (0.2 mL, 1.2 mmol) were added, and the mixture was stirred at room temperature for 2 h. Water was added to dilute the mixture, and EA was extracted several times. The organic layers were combined and anhydrous Na ₂ SO ₄ Dry, mix with silica gel, spin dry the solvent, and purify by column with dichloromethane:methanol=40:1 to obtain 120 mg of white solid, yield: 41%; ESI-MS: 725.3 [M+H] ⁺ . ¹ H NMR (500MHz, DMSO-d6) δ8.83 (t, J=6.3 Hz, 1H), 8.48 (t, J=1.8 Hz, 1H), 8.23–8.17 (m, 1H), 8.12–8.06 (m, 2H), 8.00–7.94 (m, 2H), 7.69 (t, J=7.8 Hz, 1H), 7.40 (dd, J=8.3, 7.1 Hz, 2H), 7.35–7.28 (m, 1H), 4.22 (d, J ＝13.1Hz,1H),4.09–3.82(m,3H),3.72–3.54(m,2H),3.41(s,2H),3.24(t,J＝12.0Hz,1H),2.93–2.74(m,4H),2.40(d,J＝13.5Hz,1H),2.29(d,J＝13.5 Hz,1H),2.02–1.84(m,2H),1.66–1.53(m,2H),1.40(s,9H).

Step 2: Preparation of N-((4-(4-phenylthiazol-2-yl)-1-(piperidine-4-carbonyl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (Compound 10)

Tert-butyl 4-(4-phenylthiazol-2-yl)-4-(3-(5-trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidine-1-carbonyl)piperidine-1-carboxylate (Compound 9, 250.0 mg, 0.3 mmol) was dissolved in 20 mL of DCM, 2 mL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 1 h. The solvent was dried by spin drying to obtain 215 mg of an oily liquid, which was used directly in the next step without purification. Yield: 100%; ESI-MS: 625.3 [M+H] ⁺ .

Step 3: Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-(4-hydroxypiperidin-1-yl)isoindoline-1,3-dione (Compound 12a)

2-(2,6-Dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (Compound 11, 276.0 mg, 1.0 mmol) was dissolved in 20 mL of DMSO, 4-hydroxypiperidine hydrochloride (206.0 mg, 1.5 mmol) and DIPEA (0.5 ml, 3.0 mmol) were added, and the mixture was reacted at 90°C for 4 h, and water was added for dilution. EA was extracted several times, the organic liquid was combined, washed with water several times, dried over anhydrous Na ₂ SO ₄ , mixed with silica gel, dried by spin drying, and purified by column chromatography with dichloromethane:methanol=40:1 to obtain 200 mg of a yellow solid, yield: 56%; ESI-MS: 358.1 [M+H] ⁺ ; ¹ H NMR (500MHz, DMSO-d6) δ11.09(s,1H),7.65(d,J＝8.5Hz,1H),7.32(d,J＝2.4Hz,1H),7.24(dd,J＝8.7,2.4Hz,1H),5.07(dd,J＝12.8,5.4Hz,1H),4.78(d,J＝4.1Hz, 1H), 3.82(m,2H),3.75(m,1H),3.25–3.18(m,2H),2.94–2.83(m,1H),2.63–2.52(m,2H),2.28(t,J＝7.4Hz,1H),2.07–1.97(m,1H),1.81(m,2H),1.50(q ,J=7.2,6.7Hz,1H).

Step 4: Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-(4-oxopiperidin-1-yl)isoindoline-1,3-dione (Compound 13a)

2-(2,6-dioxopiperidin-3-yl)-5-(4-hydroxypiperidin-1-yl)isoindoline-1,3-dione (Compound 12a, 357.0 mg, 1.0 mmol) was dissolved in 20 mL of DCM, and DMP (848.0 mg, 2.0 mmol) was added, stirred at room temperature for 2 h, filtered through celite, washed with DCM several times, dried over anhydrous Na ₂ SO ₄ , and the solvent was dried by spin drying to obtain 355 mg of a yellow liquid, which was directly used in the next step. Yield: 100%; ESI-MS: 356.1 [M+H] ⁺ .

Step 5: Preparation of N-((1-(1'-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-[1,4'-bipiperidinyl]-4-carbonyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-trifluoromethyl-1,2,4-oxadiazol-3-yl)benzamide (Compound A1)

N-((4-(4-phenylthiazol-2-yl)-1-(piperidine-4-carbonyl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (Compound 10, 321.0 mg, 0.5 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-(4-oxopiperidin-1-yl)isoindoline-1,3-dione (Compound 13a, 284.0 mg, 0.8 mmol) were dissolved in 20 mL of DCM, sodium cyanoborohydride (94.0 mg, 1.5 mmol) was added, glacial acetic acid was added dropwise to make the pH value of the system reach 3-5, the mixture was stirred at room temperature for 5 h, diluted with water, extracted with EA several times, the organic layers were combined, and anhydrous Na ₂ SO ₄ Dry, mix with silica gel, spin dry the solvent, and purify with dichloromethane:methanol=35:1 to obtain 150 mg of yellow solid, yield: 30%; ESI-MS: 964.3 [M+H] ⁺ . ¹ H NMR (500MHz, DMSO-d ₆ )δ11.08(s,1H),8.78(t,J＝6.4Hz,1H),8.43(d,J＝1.9Hz,1H),8.19(dt,J＝7.8,1.4Hz,1H),8.09(s,1H),8.04(dt,J＝7.9,1.5Hz,1H),7.96–7.90(m,2H) ,7.72–7.63(m,2H),7.39(t,J＝7.6Hz,2H),7.34–7.28(m,2H),7.24(dd,J＝8.7,2.3Hz,1H),5.06(dd,J＝1 2.8,5.4Hz,1H),4.18(d,J＝13.4Hz,1H),4.07(d,J＝12.8Hz,2H),3.91(d,J＝13.5Hz,1H),3.58(t,J＝7.1Hz,2H),3.19(t,J＝12.2Hz,1H),3.03–2.76(m,7H ),2.62–2.52(m,3H),2.31(dd,J＝52.8,13.8Hz,4H),2.00(m,2H),1.94–1.75(m,5H),1.67–1.52(m,4H).

Example 3

The synthesis process of HDAC7-PROTAC molecule A2-4 is the same as that of compound A1. Compound 11 is used as the raw material, and reacts with 4-piperidinemethanol hydrochloride, 4-piperidineethanol, and 3-methylhydroxyazetidine hydrochloride under alkaline conditions to obtain intermediates 12b-d (12b: 2-(2,6-dioxopiperidin-3-yl)-5-(4-(hydroxymethyl)piperidin-1-yl)isoindoline-1,3-dione; 12c: 2-(2,6-dioxopiperidin-3-yl)-5-(4-(hydroxyethyl)piperidin-1-yl)isoindoline-1,3-dione; 12d: 2-(2,6-dioxopiperidin-3-yl)-5-(3- (hydroxymethyl)azetidine-1-yl)isoindolin-1,3-dione), and then oxidized by DMP to obtain intermediates 13b-d (13b: 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-diketoisoindolin-5-yl)piperidine-4-carboxaldehyde; 13c: 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-diketoisoindolin-5-yl)piperidin-4-yl)acetaldehyde; 13d: 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-diketoisoindolin-5-yl)azetidine-3-carboxaldehyde), and then reacted with compound 10 for acid amine condensation to obtain compounds A2-A4. The structural formulas of this series of compounds are shown in the following table.

Example 4

Synthesis of HDAC7-PROTAC molecule B1

Step 1: Preparation of tert-butyl 4-(4-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide)methyl)piperidine-1-carbonyl)phenyl)piperidine-1-carboxylate (Compound 14a)

Tert-butyl 4-(4-phenylthiazol-2-yl)-4-(3-(5-trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidine-1-carboxylate (Compound 8, 250.0 mg, 0.4 mmol) was dissolved in DCM, 2 mL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 1 h. The solvent was dried by spin drying, 20 mL of DMF solution was added, 4-(1-(tert-butyloxycarbonyl)piperidin-4-yl)benzoic acid (162.0 mg, 0.5 mmol), HATU (230.0 mg, 0.6 mmol), and DIPEA (0.2 mL, 1.2 mmol) were added, and the mixture was stirred at room temperature for 2 h. Water was added to dilute the mixture, and EA was extracted several times. The organic layers were combined and anhydrous Na ₂ SO ₄ Dry, mix with silica gel, spin dry the solvent, and purify by column with dichloromethane:methanol=35:1 to obtain 180 mg of white solid, yield: 56%; ESI-MS: 801.3 [M+H] ⁺ . ¹ H NMR (500MHz, DMSO-d6) δ8.81 (t, J=6.4 Hz, 1H), 8.46 (t, J=1.8 Hz, 1H), 8.19 (m, 1H), 8.12–8.05 (m, 2H), 7.98–7.91 (m, 2H), 7.69 (t, J=7.8 Hz, 1H), 7.39 (dd, J=8.3, 7.0 Hz, 2H), 7.34–7.27 (m, 5H), 4.31 (s ,1H),4.09(s,2H),3.62(d,J＝5.6Hz,3H),3.26–3.02(m,2H),2.74(d,J＝3.5Hz,2H),2.70(h,J＝3.4Hz,1H),2.33(d,J＝43.5Hz,2H),1.97(d,J＝38.0Hz,2 H),1.80–1.70(m,2H),1.51(m,2H),1.42(s,9H).

Step 2: Preparation of N-((4-(4-phenylthiazol-2-yl)-1-(4-(piperidin-4-yl)benzoyl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (Compound 15a)

Tert-butyl 4-(4-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide)methyl)piperidine-1-carbonyl ) phenyl)piperidine-1-carboxylate (Compound 14a, 400.0 mg, 0.5 mmol) was dissolved in 30 mL of DCM, 2 mL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 1 h. The solvent was dried by spin drying to obtain 350 mg of an oily liquid, which was used directly in the next step without purification. Yield: 100%; ESI-MS: 701.3 [M+H] ⁺ .

Step 3: Preparation of N-((1-(4-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperidin-4-yl)benzoyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (Compound B1)

N-((4-(4-phenylthiazol-2-yl)-1-(4-(piperidin-4-yl)benzoyl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (Compound 15a, 350.0 mg, 0.5 mmol) and 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidine-4-carbaldehyde (Compound 13b, 295.0 mg, 0.8 mmol) were dissolved in 30 mL of DCM, sodium cyanoborohydride (94.0 mg, 1.5 mmol) was added, glacial acetic acid was added dropwise to adjust the pH value of the system to 3-5, the mixture was stirred at room temperature for 5 h, diluted with water, extracted with EA several times, the organic layers were combined, and anhydrous Na ₂ SO ₄ Dry, mix with silica gel, spin dry the solvent, and purify with dichloromethane:methanol=35:1 to obtain 180 mg of yellow solid, yield: 34%; ESI-MS: 1054.4 [M+H] ⁺ . ¹ H NMR (500MHz, DMSO-d ₆ )δ11.09(s,1H),8.78(t,J＝6.4Hz,1H),8.44(d,J＝1.8Hz,1H),8.19(dt,J＝7.7,1.5Hz,1H),8.11–8.03(m,2H),7.98–7.91(m,2H),7.72–7.63(m,2H),7.38(dd,J＝8.3,7.0Hz,2H),7.34–7.27(m,6H),7.22(dd,J＝8.8 ,2.3Hz,1H),5.08(dd,J＝12.9,5.4Hz,1H),4.30(s,1H),4.04(d,J＝12.8Hz,2H),3.62(d,J＝6.3Hz,3H),3.09(s,2H),3.01–2.85(m,5H),2.64–2.52(m,3 H),2.40–2.15(m,4H),2.06–1.73(m,11H),1.71–1.60(m,2H). ¹³ C NMR(126MHz,DMSO-d ₆ )δ173.93,173.25,170.56,169.58,168.57,168.12,167.43,166.29,155.48,154.23,136.19,134.78,134.52,134.35,131.76,130.35,130.07,1 29.07,1 28.27,127.46,127.13,126.81,126.49,125.46,125.03,118.04,117.85,114.98,108.19,54.62,49.75,49.23,47.73,45.70,33.17,31.47,30. 14,22.69.

Example 5

Synthesis of HDAC7-PROTAC molecules B2-B4

The operation process is the same as that of the synthesis of compound B1. Compound 15a is used as the raw material, and is subjected to reductive amination with compound 13d to obtain compound B2; compound 8 is subjected to acid-amine condensation with 4-(4-(tert-butyloxycarbonyl)piperazine-1-yl)benzoic acid under HATU and DIPEA conditions after the Boc protecting group is removed. Compound 14b (4-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl) ... The Boc protecting group was removed under trifluoroacetic acid conditions to obtain compound 15b (N-((4-(4-phenylthiazol-2-yl)-1-(4-(piperazin-1-yl)benzoyl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide). Compound 15b and compound 13b were subjected to reductive amination to obtain compound B3; compound 15b and compound 13d were subjected to reductive amination to obtain compound B4. The structural formulas of this series of compounds are shown in the following table.

Example 6

Synthesis of HDAC7-PROTAC molecule C1

Step 1: Preparation of tert-butyl 4-(2-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)ethyl)piperidine-1-carboxylate (Compound 16a)

4-(4-phenylthiazol-2-yl)-4-(3-(5-trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidine-1-carboxylic acid tert-butyl ester (Compound 8, 250.0 mg, 0.4 mmol) was dissolved in DCM, 2 mL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 1 h. The solvent was dried by spin drying, 20 mL of DCM solution was added, 4-(2-oxoethyl)piperidine-1-carboxylic acid tert-butyl ester (120.0 mg, 0.5 mmol) and sodium cyanoborohydride (78.0 mg, 1.2 mmol) were added, glacial acetic acid was added dropwise to adjust the pH value of the system to 3-5, the mixture was stirred at room temperature for 2 h, water was added to dilute, EA was extracted several times, the organic layers were combined, and anhydrous Na ₂ SO ₄ Dry, mix with silica gel, spin dry the solvent, and purify by column with dichloromethane:methanol=40:1 to obtain 175 mg of white solid, yield: 60%; ESI-MS: 725.3 [M+H] ⁺ . ¹ HNMR(500MHz,Chloroform-d)δ8.49(d,J＝1.8Hz,1H),8.22(d,J＝7.8Hz,1H),7.99(dt,J＝7.9,1.5Hz,1H),7.88–7.84(m,2H),7.60–7.55(m,2H),7.42–7.32(m,4H) ,4.21–3.82(m,5H),3.66–3.39(m,3H),3.03(s,2H),2.64(d,J＝15.3Hz,4H),2.45(s,2H),1.97(s,2H),1.76–1.59(m,5H),1.44(s,9H).

Step 2: Preparation of N-((4-(4-phenylthiazol-2-yl)-1-(2-(piperidin-4-yl)ethyl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (Compound 17a)

Tert-butyl 4-(2-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)ethyl)piperidine-1-carboxylate (Compound 16a, 362.0 mg, 0.5 mmol) was dissolved in 30 mL of DCM, 2 mL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 1 h. The solvent was dried by spin drying to obtain 312 mg of an oily liquid, which was used directly in the next step without purification. Yield: 100%; ESI-MS: 625.3 [M+H] ⁺ .

Step 3: Preparation of N-((1-(2-(1'-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-[1,4'-bipiperidinyl]-4-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (Compound C1)

N-((4-(4-phenylthiazol-2-yl)-1-(2-(piperidin-4-yl)ethyl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (Compound 17a, 312.0 mg, 0.5 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-(4-oxopiperidin-1-yl)isoindoline-1,3-dione (Compound 13a, 284.0 mg, 0.8 mmol) were dissolved in 30 mL of DCM, sodium cyanoborohydride (94.0 mg, 1.5 mmol) was added, glacial acetic acid was added dropwise to adjust the pH value of the system to 3-5, the mixture was stirred at room temperature for 5 h, diluted with water, extracted with EA several times, the organic layers were combined, and anhydrous Na ₂ SO ₄ Dry, mix with silica gel, spin dry the solvent, purify with dichloromethane:methanol=35:1, and obtain 200 mg of yellow solid, yield: 41%; ESI-MS: 964.4 [M+H] ⁺ . ¹ HNMR (500MHz, DMSO-d ₆ )δ11.08(s,1H),8.85–8.72(m,1H),8.44(s,1H),8.22–8.17(m,1H),8.12–8.02(m,2H),7.96–7.91(m,2H),7.68(dt,J＝10.4,8.0Hz,2H),7.39(t,J＝7.7Hz,2H),7.31(dd,J＝16.6,9 .3Hz,2H),7.25(d,J＝9.0Hz,1H),5.07(dd,J＝12.7,5.5Hz,1H),4.12(s,3H),3.78–3.38(m,4H),3.21–2.68(m,9H),2.65–2.52(m,3H),2.46–1.82(m,14 H),1.66(d,J＝28.2Hz,3H).

Example 7

Synthesis of HDAC7-PROTAC molecules C2-C5

The operation process is the same as the synthesis of compound C1. Compound 17a is used as the raw material and is reductively aminized with compound 13b and compound 13c to obtain compound C2 and compound C3 respectively; compound 8 is subjected to reductive amination with 3-(2-oxoethyl)azetidine-1-carboxylic acid tert-butyl ester and 3-formylazetidine-1-carboxylic acid tert-butyl ester under sodium cyanoborohydride and glacial acetic acid to obtain compounds 16c-d (16c: 3-(2-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)ethyl)azetidine-1-carboxylic acid tert-butyl ester; 16d: 3-((4-(4-phenylthiazol-2-yl)-4- tert-butyl ((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide)methyl)piperidin-1-yl)methyl)azetidine-1-carboxylate); Compound 16c-d was deprotected by removal of the Boc protecting group under trifluoroacetic acid conditions to give compounds 17c-d (17c: N-((1-(2-(azetidin-3-yl)ethyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide; 17d: N-((1-(2-(azetidin-3-yl)methyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide). Compound 17c and compound 13d undergo reductive amination to obtain compound C4; compound 17d and compound 13d undergo reductive amination to obtain compound C4. The structural formulas of this series of compounds are shown in the following table.

Example 8

Synthesis of HDAC7-PROTAC molecule C6

Step 1: Preparation of tert-butyl 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidine-4-carboxylate (Compound 18a)

2-(2,6-Dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (Compound 11, 276.0 mg, 1.0 mmol) was dissolved in 20 mL of DMSO solution, tert-butyl 4-piperidinylcarboxylate (130.0 mg, 0.7 mmol) and DIPEA (0.3 mL, 1.5 mmol) were added, and the mixture was reacted at 90°C for 3 h, diluted with water, extracted with EA for several times, the organic layers were combined, dried over anhydrous Na ₂ SO ₄ , mixed with silica gel, the solvent was dried, and purified by column chromatography with dichloromethane:methanol=80:1 to obtain 200 mg of a yellow solid, with a yield of 45%; ESI-MS: 442.2 [M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d6) δ11,10(s,1H),7,61(d,J＝8.5Hz,1H),7.35(s,1H),7.21(d,J＝8.6Hz,1H),5.05(dd,J＝12.9,5.3Hz,1H),3.92(d,J＝13.3Hz,2H),3.03(t ,J＝11.4Hz,2H),2.95-2.80(m,1H),2.54(d,J＝15.6Hz,2H),2.00(d,J＝5.4Hz,1H),1.82(d,J＝10.9Hz,2H).1.53(d,J＝10.2Hz,2H).1.42(s,9H).

Step 2: Preparation of 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidine-4-carboxylic acid (Compound 19a)

Tert-butyl 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidine-4-carboxylate (Compound 18a, 441.0 mg, 1.0 mmol) was dissolved in 20 mL of DCM, and 2 mL of trifluoroacetic acid was added. The mixture was reacted at room temperature for 1 h, and the solvent was dried by spin drying to obtain 385 mg of a yellow liquid, which was directly used in the next step without purification. Yield: 100%; ESI-MS: 386.2 [M+H] ⁺ .

Step 3: Preparation of N-((1-(2-(1-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidine-4-carbonyl)piperidin-4-yl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (Compound C6)

N-((4-(4-phenylthiazol-2-yl)-1-(2-(piperidin-4-yl)ethyl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (Compound 17a, 312.0 mg, 0.5 mmol) and 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidine-4-carboxylic acid (Compound 19a, 308.0 mg, 0.8 mmol) were dissolved in 30 mL of DMF solution, HATU (285.0 mg, 0.8 mmol) and DIPEA (0.3 mL, 1.5 mmol) were added, and the mixture was reacted at room temperature for 2 h, diluted with water, extracted with EA for several times, and the organic layers were combined and anhydrous Na ₂ SO ₄ Dry, mix with silica gel, spin dry the solvent, and purify by column with dichloromethane:methanol=40:1 to obtain 120 mg of yellow solid, yield: 24%; ESI-MS: 992.4 [M+H] ⁺ . ¹ H NMR (500MHz, DMSO-d6) δ11.09(s,1H),8.75(t,J＝6.5Hz,1H),8.45(d,J＝1.8Hz,1H),8.19(d,J＝7.7Hz,1H),8.09–8.03(m,2H),7.96–7.91(m,2H),7.72–7.63 (m,2H),7.39(t,J＝7.6Hz,2H),7.32–7.28(m,2H),7.22(dd,J＝8.8,2.3Hz,1H),5.08(dd,J＝10.8,5.4Hz, 1H),4.33(d,J＝12.6Hz,1H),4.07–4.00(m,2H),3.96(d,J＝13.1Hz,1H),3.56(d,J＝6.2Hz,2H),3.12–2.74(m,8H),2.64–2.53(m,2H),2.46(d,J＝12.9Hz ¹³ C NMR (126MHz, DMSO-d6) δ173.26,172.26,170.55,168.58,168.07,167.42,166.13,155.28,154.21,136.24,134.86,134.51,131.73,130.31,130.07,12 9.07,128.22, 126.80,126.44,125.46,125.03,118.05,114.72,108.23,55.80,50.18,49.22,47.18,45.30(d,J=24.5Hz),41.84,37.34,34.24,33.37,32.17,3 1.46,27.97,22.67.

Example 9

Synthesis of HDAC7-PROTAC molecules C7-C11

The operation process is the same as the synthesis of compound C6. Compound 11 reacts with tert-butyl azetidine-3-carboxylate hydrochloride under DMSO and DIPEA to obtain 18b (tert-butyl 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)azetidine-4-carboxylate), and de-Boc is carried out under trifluoroacetic acid to obtain 19b (1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)azetidine-3-carboxylic acid). Compound 17d is condensed with compound 19b to obtain C7; compound 17c is condensed with compound 19b to obtain C8; compound 17b is condensed with compound 19a to obtain compound C9; compound 17b is condensed with compound 19b to obtain compound C10; compound 17a is condensed with compound 19b to obtain compound C11. The structural formula of this series of compounds is shown in the following table.

Example 10

Synthesis of HDAC7-PROTAC molecule D1

Step 1: Preparation of tert-butyl (2-(2-(2-(4-(4-phenylthiazol-2-yl)-4-(3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)carbamate (Compound 21a)

Tert-butyl 4-(4-phenylthiazol-2-yl)-4-(3-(5-trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidine-1-carboxylate (Compound 8, 250.0 mg, 0.4 mmol) was dissolved in DCM, 2 mL of trifluoroacetic acid was added, the reaction was carried out at room temperature for 1 h, the solvent was dried by spin drying, 20 mL of DMF solution was added, potassium carbonate (69.0 mg, 0.5 mmol) was added, the reaction was carried out at room temperature for 2 h, water was added for dilution, EA was extracted several times, the organic layers were combined, dried over anhydrous Na ₂ SO ₄ , mixed with silica gel, the solvent was dried by spin drying, and purified by column chromatography with dichloromethane:methanol=45:1 to obtain 180 mg of a white solid, Yield: 57%; ESI-MS: 789.3[M+H] ⁺ . ¹ H NMR (500MHz, DMSO-d6) δ8.47(s,1H),8.25–8.01(m,4H),7.94(d,J＝7.7Hz,2H),7.70(t,J＝7.9Hz,1H),7.40(t,J＝7.6Hz,2H),7.31(t,J＝7.3Hz,1H),5.12(td, J＝10.7,5.3Hz,1H),3.49(d,J＝20.8Hz,22H),2.00(dd,J＝13.2,4.1Hz,2H),1.61(t,J＝12.0Hz,2H),1.36(s,9H).

Step 2: Preparation of N-((1-(15-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-13-oxo-3,6,9-trioxa-12-azapentadecanyl)-4-(4-phenylthiazol-2-yl)piperidin-4-yl)methyl)-3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide (Compound D1)

Compound 20b (464.0 mg, 1.2 mmol) and compound 21a (788.0 mg, 1.0 mmol) were dissolved in 30 mL of DCM solution, 2 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2 h. After the solvent was dried by spin drying, DMF solution was added respectively. The reaction mixtures were combined, HATU (570.0 mg, 1.5 mmol) and DIPEA (0.5 mL, 3 mmol) were added, and the mixture was stirred at room temperature for 2 h. Water was added for dilution, and EA was extracted several times. The organic layers were combined, dried over anhydrous Na ₂ SO ₄ , mixed with silica gel, the solvent was dried by spin drying, and purified by column chromatography using dichloromethane:methanol=30:1 to obtain 457 mg of a yellow-green solid, with a yield of 45%; ESI-MS: 1016.3 [M+H] ⁺ . ¹ H NMR (500 MHz, DMSO-d ₆ )δ11.10(s,1H),8.78(s,1H),8.45(d,J＝2.0Hz,1H),8.21–8.16(m,1H),8.07(q,J＝4.7,3.5Hz,3H),7.96–7.91(m,2H),7.69(t,J＝7.8Hz,1H),7.58(dd, J＝8.6,7.0Hz,1H),7.38(t,J＝7.6Hz,2H),7.29(t,J＝7.3Hz,1H),7.12(d,J ＝8.7Hz,1H),7.03(d,J＝7.1Hz,1H),6.71(t,J＝6.0Hz,1H),5.05(dd,J＝12.8,5.5Hz,1H),3.57–3.44(m,17H),3.20(q,J＝5.8Hz,3H),2.89(m,2H),2.62–2 .52(m,3H),2.42(t,J＝6.5Hz,3H),2.37–2.30(m,3H),2.06–1.97(m,3H). ¹³ CNMR(126MHz,DMSO-d ₆ )δ173.22,171.01,170.47,169.26,168.59,167.75,166.17,154.25,146.64,136.70,136.21,134.83,132.65,131.73,130.33,130.06,129.06,128.24,126.80,126.46,125.03,117.62,114.82,111.02,109.78,70.10(d,J＝9.8Hz),69.55,50.29,49.04,35.22,31.46,22.64.

Embodiment 11

Synthesis of HDAC7-PROTAC molecules D2-D3

The operation process is the same as that of the synthesis of compound D1. Compound 20a ((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)glycine tert-butyl ester) and compound 21b ((2-(2-(2-(2-oxo-2-(4-(4-phenylthiazol-2-yl)-4-((3-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamide)methyl)piperidin-1-yl)ethoxy)ethoxy)ethoxy)ethyl)carbamic acid tert-butyl ester) are respectively de-Bocated and then subjected to acid-amine condensation reaction under HATU and DIPEA conditions to obtain compound D2; Compound 21c (1-(4-phenylthiazol-2-yl)-4-(3-(5-trifluoromethyl)-1,2,4-oxadiazol-3-yl)benzamido)methyl)piperidin-1-yl)-3,6,9,12-tetraoxapentadecan-15-acid tert-butyl ester) and compound 22 ((S)-1-((2S,4R)-4-hydroxy-2-(4-(4-methylthiazol-5-yl)benzylcarbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutane-2-yl)carbamic acid tert-butyl ester) were subjected to acid amine condensation under HATU and DIPEA conditions to obtain compound D3. The structural formulas of this series of compounds are shown in the following table.

Example 12

In vitro enzyme activity of the compounds of the present invention

1. Experimental Methods

Instrument: TECAN SPARK microplate reader (TECAN, Switzerland)

Materials: HDAC7 protein, purified from SF9 cells; substrate Ac-Leu-Lys(TFAc)-AMC

Sample processing: The sample was dissolved in DMSO, stored at low temperature, and diluted in a gradient manner, and the concentration of DMSO in the final system was controlled within a range that did not affect the enzyme activity detection. The positive compound used in the experiment was TMP269.

2. Experimental process: 100nM HDAC7 protein and 50μM substrate were dissolved in kinase reaction buffer (500mMNaCl, 50mM Tris, PH8.0). 0.3μM and 3μM compounds were added to the reaction system (100μL), and the drug-free group, positive compound (TMP269, 0.3μM and 3μM) and blank control group were set up at the same time. Two sub-wells were set for each concentration of each sample. After sufficient dissolution, the system was transferred to a 96-well plate, and 100μl 10mg/ml trypsin was added and incubated at 37°C. The fluorescence value (absorption light 460nm, excitation light 390nm) was detected by an enzyme reader to indicate the release of AMC. The sample enzyme activity inhibition rate was calculated by the sample reading, and the calculation formula was: (RFU without drug-RFU compound)/RFU without drug x100%. The results are shown in Table 1.

3. Experimental results:

The experimental results show that the compounds of the present invention modified with TMP269 as the target head have weak binding ability with the HDAC7 protein, indicating that the compounds of the present invention have the potential to exert the PROTAC degradation mechanism and do not rely on strong inhibition of the target head to exert their efficacy.

Table 1 Enzyme activity inhibition rate of the compounds of the present invention at a concentration of 1 μM

"A" represents >75%; "B" represents 50%-75%; "C" represents 15%-50%; "D" represents <15% Example 13

Degradation of HDAC7 in NB4 cells at 1 μM and 5 μM concentrations of the compounds of the present invention

1. Experimental methods:

Cell culture and administration: NB4 cells were cultured in a 37°C incubator containing 5% CO ₂ , and the culture conditions were RPMI-1640 + 10% Gibco serum. Anti-mycoplasma drugs were given three generations before recovery. The cell suspension was added to a 15mL centrifuge tube, centrifuged at 1500rpm for 4min, the supernatant was discarded, and the cells were resuspended in 2mL of culture medium and counted. The cell suspension was seeded in a 6-well plate, 500,000 cells per well, and the test compound was added at an appropriate concentration. Samples were collected after 12-24h.

Sample preparation: At the end of the action time, the cells were collected and washed with PBS; according to the cell amount, the corresponding volume of 4% SDS was added to lyse the cells, and the cells were sonicated until they were no longer viscous; centrifuged at 12,000g for 30 minutes at room temperature; the supernatant was transferred to a new EP tube for protein quantification.

Take 2mg/mL BSA standard and dilute it by half to the concentration used for the standard curve, which is 2mg/mL, 1mg/mL, 0.5mg/mL, 0.25mg/mL, and 0.0625mg/mL in sequence; according to the calculation that each sample requires 200μL of solution A and 4μL of solution B in the BCA quantitative kit, take the corresponding volume of solution A and solution B (volume ratio is 50:1) and mix them evenly; take 10μL of BSA of different concentrations and samples and add them to a 96-well plate respectively, then add 200μL of mixed solution A and solution B, gently tap and place at 37℃ in the dark for 30min; after the reaction, measure the absorbance value at 562nm, calculate the sample protein concentration using the standard curve, take 20μg of protein and add a certain amount of 6×Loading buffer according to the volume to make the final concentration 1×Loading buffer; heat at 95℃ for denaturation for 10min, centrifuge and mix after cooling for Western Blot experiment, and freeze the remaining samples at -80℃. The degradation of HDAC7 in NB4 cells by the compounds at concentrations of 1 μM and 5 μM was tested. The results are shown in Table 2.

2. Experimental results

The experimental results show that the compounds of the present invention can degrade HDAC7 in NB4 cells, and series C and series D can significantly degrade HDAC7.

Table 2 Degradation screening test of the compounds of the present invention in NB4 cells

"A" stands for >75%; "B" stands for 50%-75%; "C" stands for 15%-50%; "D" stands for <15%

Embodiment 14

Proliferation inhibition of the compounds of the present invention on NB4 cells

1. Experimental Methods

The CCK8 method was used to test the proliferation inhibitory effects of different compounds on NB4 cells with TMP269 as a positive control. The IC ₅₀ values were calculated using Graphpad Prism V9.3.0 software. The results are shown in Table 3.

2. Experimental results

The experimental results show that the proliferation inhibition activity of the compound of the present invention in NB4 cells is significantly better than that of the positive molecule TMP269.

Table 3 Proliferation inhibition of the compounds of the present invention on NB4 cells

No. TMP269 C6 C7 C8 C10 D3 IC ₅₀ (μM) D A C B C A

"A" represents 0-1μM; "B" represents 1-5μM; "C" represents 5-15μM; "D" represents >15μM

Embodiment 15

Proliferation inhibition of the compounds of the present invention on various DLBCL cells

1. Experimental Methods

The CCK8 method was used to test the proliferation inhibitory effects of different compounds on various DLBCL cells with TMP269 as a positive control. The IC ₅₀ values were calculated using Graphpad Prism V9.3.0 software. The results are shown in Table 4.

2. Experimental results

The experimental results show that the compound of the present invention has significantly better proliferation inhibition activity than the positive molecule TMP269 in various DLBCL cell lines.

Table 4 Proliferation inhibition of the compounds of the present invention on multifocal lymphoma cells

"++++" represents 0-1.5μM; "+++" represents 1.5-3μM; "++" represents 3-13μM; "+" represents >13μM

Example 16

Preliminary pharmacokinetics of the compounds of the present invention in ICR mice

1. Experimental Methods

ICR mice were given oral gavage at a dose of 30 mg/kg, and 150 μL to 200 μL of mouse blood was collected by orbital blood sampling at eight time points: 0, 0.25, 0.5, 1, 2, 4, 8, and 24 h. The blood sample was centrifuged at 4 ° C and 5000 rpm for 15 min, and the supernatant was aspirated to EP tube for storage. 50 μL of the supernatant of the sample to be tested was aspirated to EP tube, 200 μL of chromatographic acetonitrile was added, and centrifuged at 4 ° C and 13500 rpm for 10 min, and the supernatant was aspirated to the sample bottle. The compound concentration was determined using a Waters Xevo TQ high performance liquid-mass spectrometer, and loratadine was used as an internal standard compound. Pharmacokinetic parameters such as C _max , T _max , T _1/2 and AUC were calculated, and graphed using Graphpad 9.3.0. The results are shown in Table 5.

2. Experimental results

Table 5 Preliminary pharmacokinetic parameters of compounds C6 and C7

The experimental results show that in ICR mice, compound C6 and compound C7 have certain oral absorption activity under the condition of oral gavage at 30 mg/kg.

Claims (9)

1. A compound, characterized in that it has a structure represented by general formula (I):

or its stereoisomers, geometric isomers, tautomers, N-oxides, hydrates, solvates, metabolites, or pharmaceutically acceptable salts thereof; in: Linker is a connecting group, and is selected from one or more of the following groups: linear or branched alkyl, alkoxy, alkylamino, halogenated heterocycle, alkenyl, alkynyl, alkenylene, alkynylene, alkynoxy, alkynamino, cycloalkyl, heterocycloalkyl, spirocycloalkyl, heterospirocycloalkyl, cycloalkyl, heterocycloalkyl, aromatic ring, _halogenated aromatic ring, heteroaromatic ring, _-CO ( _CH2 ) _n- , -CO ₍ CH2) _nO- , -(CH2) _nNH2- , -(CH2)nNR1-, -CO(CH2)nNH-, -CO _{( CH2 ) nNR1- , -} ( _CH2 ) _nCONH ( _CH2 ) _{n- ,} -( _CH2 ) _nCONH (CH2) _{nNH- ,} - _{( CH2 )} nCONH( _CH2 ) _nNR1- , _-CO ( _CH2 ) _n NHCO(CH ₂ ) _n -, -CO(CH ₂ ) _n NHCO(CH ₂ ) _n NH-, -CO(CH ₂ ) _n NHCO(CH ₂ ) _n NR ₁ -, -CO(CH ₂ OCH ₂ ) _n CH ₂ NHCO(CH ₂ ) _n -, -CO(CH ₂ OCH ₂ ) _n CH ₂ NHCO(CH ₂ ) _n NH-, -CO(CH ₂ OCH ₂ ) _n CH ₂ NHCO(CH ₂ ) _n NR ₁ -, -CH ₂ (CH ₂ OCH ₂ ) _n CH ₂ -, -CH ₂ (CH ₂ OCH ₂ ) _n CH ₂ NH-, -CH ₂ (CH ₂ OCH ₂ ) _n CH ₂ NR ₁ -, -CH ₂ (CH ₂ OCH ₂ ) _n CH ₂ O-, -CH ₂ CONHCH ₂ (CH ₂ OCH ₂ ) _n CH ₂ NH-, -CH ₂ CONHCH ₂ (CH ₂ OCH ₂ ) _n CH ₂ NR ₁ -, -CH ₂ CONHCH ₂ (CH ₂ OCH ₂ ) _n CH ₂ NHCO(CH ₂ ) _n NH-, -CH ₂ CONHCH ₂ (CH ₂ OCH ₂ ) _n CH ₂ NHCO(CH ₂ ) _n NR ₁ -, -CH ₂ CONHCH ₂ (CH ₂ OCH ₂ ) _n CO-, -CH ₂ (CH ₂ OCH ₂ ) _n CH ₂ CO-, or any combination thereof; n represents a natural number from 1 to 20; _R1 is H or _C1-10 alkyl; E3 ligand is the E3 ligase ligand of CRBN or VHL. 2. The compound according to claim 1, characterized in that: Linker is a connecting group, wherein the ring system combination is any combination of 1-4 ring systems, the size of the ring system is a four-membered ring to a six-membered ring, and the types of ring systems include bridged rings, spiro rings, and fused rings; including but not limited to the following structures:

3. The compound according to claim 1, characterized in that: E3 ligand is an E3 ligase ligand selected from one of the following ligands:

4. The compound according to claim 1, characterized in that it is selected from the following compounds or their stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs:

5. A pharmaceutical composition, characterized in that it comprises one or more compounds according to any one of claims 1 to 4. 6. A pharmaceutical preparation, characterized in that it comprises one or more of the compounds or compositions described in any one of claims 1 to 5. 7. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating a disease that benefits from HDAC7 protein degradation. 8. The use according to claim 7, characterized in that the disease is selected from: one or more of acute myeloid lymphocytic leukemia, diffuse large B-cell lymphoma, multiple myeloma, chronic lymphocytic leukemia, chronic myeloid leukemia, mantle cell lymphoma, non-small cell lung cancer, esophageal cancer, gastric cancer, colon cancer, breast cancer, kidney cancer, and liver cancer. 9. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating AML and DLBCL.

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