CN116836179A - Bromodomain protein BRD4 inhibitors with tumor targeting - Google Patents

Abstract

The invention relates to a compound formed by a bromodomain protein BRD4 inhibitor and a heat shock protein HSP90 inhibitor, which has certain anti-tumor activity and can overcome the shortcomings of the BRD4 inhibitor's poor selectivity for tumor tissue and high toxicity.

Description

Tumor-targeted bromodomain protein BRD4 inhibitors

Technical Field

The present invention relates to a compound formed by a BRD4 protein inhibitor and an HSP90 inhibitor, which has certain anti-tumor activity and can overcome the problem of tumor tissue selectivity of general BRD4 inhibitors.

Background Art

The bromodomain and extra-terminal (BET) protein family is involved in the occurrence and development of many human diseases and plays an important role in biological processes such as DNA damage repair, transcriptional expression, and chromosome remodeling. The family mainly includes four subtypes: BRD2, BRD3, BRD4, and BRDT. Bromodomain protein 4 (BRD4) in the BET family is the most important member of the family. It can regulate DNA repair and control gene expression through histone acetylation, thereby affecting the cell cycle. BRD4 has two bromodomains, BD1 and BD2, and its mechanism of action is to bind to the hyperacetylated histone region on chromatin, accumulate during transcription, and promote gene transcription in the initiation and elongation steps.

BRD4 can promote the development and metastasis of tumors and is overexpressed in many tumors such as melanoma, myeloma and breast cancer. The specific mechanisms of BRD4 are: promoting the transcription of c-MYC gene and B lymphocytic neoplasm 2 gene; upregulating the expression of PD-L1 in tumor cells and promoting tumor cell proliferation; aggravating immunosuppression; repairing damaged DNA; and promoting telomere extension. The important role of BRD4 in tumor tissue makes this target a focus of drug development.

Currently, the strategies adopted include inhibiting BRD4 or directly using degraders to degrade BRD4 overexpressed in tumor cells. Studies have shown that a variety of BRD4 inhibitors and degraders have good anti-tumor effects, including the earliest inhibitor (+)-JQ-1. Many of them are currently in the clinical research stage and show good application prospects. This chapter will select (+)-JQ-1, which has the most thorough research on drug structure-activity relationship and biological activity, as the target protein ligand of BRD4.

Although BRD4 inhibitors have shown good efficacy for a variety of disease types, especially hematological tumors, they still have some application limitations. Since these drugs are based on the pharmacological principle of occupancy-driven reversible binding to target proteins, high doses are often required to achieve therapeutic effects. Clinical trials show dose-limiting toxicity such as thrombocytopenia, fatigue, diarrhea, vomiting, anemia, and hyperbilirubinemia. Some inhibitors, such as OTX-015 and IBET-151, were forced to terminate due to their limited therapeutic effects on solid tumors. Pan-BET inhibitors, due to their lack of selectivity for multiple bromodomains, can lead to off-target effects and serious safety issues. In recent years, more and more selective BRD4 inhibitors have been developed. These inhibitors only interfere with one of the two domains on BRD4, which improves the selectivity of the drug, but the potency needs to be further improved.

The continuous development of PROTAC technology has provided new ideas for the application of BRD4 inhibitors. BRD4 ligand, E3 ubiquitin ligase ligand and intermediate linker constitute the basic structure of BRD4 degraders based on PROTAC technology. In recent years, researchers have designed and synthesized a series of BRD4 degraders, which can be divided into two categories according to the type of E3 ubiquitin ligase: CRBN-based BRD4 degraders, such as dBET-1, BETd-260, ARV-825; and VHL-based BRD4 degraders, such as MZ1, MZP-54, ARV-771. Taking BETd-260 as an example, the degrader can effectively induce the degradation of the target protein at a concentration of DC50=30pM in hematological tumors, and the in vitro anti-tumor effect is also one order of magnitude higher than that of the corresponding inhibitor. BRD4 degraders can effectively induce BRD4 degradation, and their ability to inhibit tumor cell growth and promote cell apoptosis is far better than that of the corresponding inhibitors. It is a very promising tumor treatment strategy against BRD4.

Although BRD4 degraders based on the mainstream E3 ubiquitin ligase ligands VHL and CRBN have good efficacy, there are also some problems. For example, both are non-tissue-specific E3 ligases, which may cause systemic off-target and toxic side effects; both are non-essential proteins for tumor growth, and PROTAC small molecule drugs based on them are prone to drug resistance.

In order to overcome the shortcomings of BRD4 inhibitors and PROTACs, improve the anti-tumor activity of drugs and reduce toxic side effects, on the one hand, WEE1 inhibitors with high selectivity and low toxicity can be developed; on the other hand, the activity of drugs can be improved by exploring the combination of WEE1 inhibitors with other drugs or making them into compounds.

As a chaperone protein, HSP90 is one of the proteins with the highest intracellular content, accounting for about 1-3% of the total cellular protein, and is also a known tumor target. Since HSP90 is widely distributed and usually highly expressed in tumor tissues, inhibitors targeting this target have strong tumor tissue selectivity, and HSP90 has gradually become a hot spot in anti-tumor drug research in recent years. Clinical experimental results show that HSP90 inhibitors often require high doses to be effective. Because of this, HSP90 exhibits more side effects in clinical practice, such as visual toxicity, gastrointestinal toxicity, liver toxicity, neurotoxicity, etc., which greatly limits its application, and no HSP90 inhibitor has been approved for marketing so far. In addition, there are reports in the art that HSP90 inhibitors are used in combination with other anticancer substances, and there are also reports that HSP90 inhibitors are made into compounds with BRD4 inhibitors, but specific activity data are not disclosed. Therefore, the present invention will design a series of HSP90-BRD4 compound molecules for the BRD4 inhibitor (+)-JQ-1.

Summary of the invention

The present invention has found in experiments that due to the continuous secretion of HSP90 in tumor cells, when the BRD4 inhibitor forms a compound with the HSP90 inhibitor, the HSP90 inhibitor can be used to target tumor cells, thereby significantly improving the effect of the BRD4 inhibitor in inhibiting the target protein in tumor cells, thereby solving the shortcomings of the existing BRD4 inhibitors in terms of selectivity and large toxic side effects. In addition, the present invention has also found that HSP90, as a chaperone protein, can mediate the folding and maturation of more than 400 client proteins. BRD4, as one of its client proteins, may be selectively degraded in the compounds of the present invention through the action of the HSP90 inhibitor.

Therefore, the present invention relates to a compound comprising a BRD4 inhibitory portion and a HSP90 inhibitory portion, which are connected by a connecting chain; or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.

In one embodiment, the aforementioned connecting chain refers to a bond or connecting group that forms a covalent bond between the HSP90 inhibitory portion and the BRD4 inhibitory portion, and these connecting chains include: chemical bonds (including carbon-carbon bonds, carbon-nitrogen bonds, carbon-sulfur bonds, carbon-phosphorus bonds, nitrogen-nitrogen bonds), ester bonds (including carboxylate bonds, sulfonate bonds, carbamate bonds), carbonyl groups (-C(＝O)-), C _1-6 alkylene groups, amide bonds, ether bonds, disulfide bonds, etc. and combinations thereof.

In a preferred embodiment of the compound, the HSP90 inhibitory moiety includes but is not limited to the following structures, and structures or groups derived from these structures that can form various covalent bonds with the connecting chain:

or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.

In a preferred embodiment of the compound, the BRD4 inhibitory portion includes but is not limited to the following structures, and structures or groups derived from these structures that can form various types of covalent bonds with the connecting chain:

or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.

In a preferred embodiment of the compound, the HSP90 inhibitory portion includes the structure of the above-mentioned NVP-AUY922, AT13387 or STA9090 analogs, or a structure derived from NVP-AUY922, AT13387 or STA9090 analogs; the BRD4 inhibitory portion includes the structure of the above-mentioned (+)-JQ-1, or a structure derived from (+)-JQ-1; the connecting chain is selected from a chemical bond, an amide bond, a carbonyl group, a C _1-6 alkylene group and a combination thereof,

or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.

In a more preferred embodiment of said compound,

The HSP90 inhibitory moiety comprises a monovalent group obtained by removing a hydrogen atom from the following structure:

wherein R ₁ represents -COOH, R ₂ represents -NHC(=O)CH ₂ CH ₂ COOH, and R ₃ represents -CH ₂ COOH;

The BRD4 inhibitory moiety comprises a monovalent group obtained by removing a hydrogen atom from the following structure:

The connecting chain is selected from the group consisting of chemical bonds: -NH-(CH ₂ )n-NH-, -NH-(CH ₂ CH ₂ O)n-CH ₂ CH ₂ -NH-; or pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, racemates or prodrugs thereof.

In a further preferred embodiment, the compounds of the present invention are HTT-1 to HTT-18 as shown below,

or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof.

In one embodiment, the present invention relates to a pharmaceutical composition comprising a compound of the present invention as described above or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrug thereof, and one or more pharmaceutically acceptable carriers.

In one embodiment, the pharmaceutical composition of the present invention further comprises other therapeutic drugs. In another embodiment, the pharmaceutical composition of the present invention is used in combination with other therapeutic drugs. The other therapeutic drugs are, for example, tumor chemotherapy drugs, tumor targeted drugs, tumor immunotherapy drugs, tumor drug conjugates (such as antibody drug conjugates, small molecule drug compounds and micro drug compounds).

Another embodiment relates to the use of the compound or pharmaceutical composition of the present invention for preparing a medicament for preventing or treating tumors.

The compound of the present invention has good BRD4 inhibitory activity and HSP90 inhibitory activity, and in particular, the BRD4 inhibitory activity is enhanced due to the targeting effect brought about by the HSP90 inhibitory activity, which solves the shortcomings of poor targeting and large toxic side effects of BRD4 inhibitors to a certain extent, and provides a promising option for the clinical treatment of cancer. The compound of the present invention has achieved a certain cancer cell inhibitory effect in a cancer cell line (MM1S).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Western analysis of tumor cells MM1S treated with compound HTT-4 for 24 hours.

DETAILED DESCRIPTION

The term "modification" used in this specification refers to the structural modification of a compound for the purpose of converting a substance with certain activity (such as BRD4 inhibitors and HSP90 inhibitors) into a compound, but the modified part still contains the main structure or active structure of the original substance and retains (and may even improve after forming the compound) the pharmacological activity of the original inhibitor.

The term "inhibitory moiety" (such as BRD4 inhibitory moiety and HSP90 inhibitory moiety) used in this specification refers to the part obtained by transforming the corresponding inhibitor (such as BRD4 inhibitor and HSP90 inhibitor) as described above, which can be a monovalent or higher-valent group and is connected to a connecting chain.

The term "connector chain" as used in this specification refers to a chemical part that connects two or more inhibitory parts, which can be a chemical bond or a chemical group as defined above. The connecting chain (or part thereof) can exist in the active substance molecule before modification or in the inhibitory part after modification, or it can be a newly added connecting bond or group.

The term "compound" used in the present specification refers to a compound obtained by linking at least two of the above-mentioned inhibitory moieties via a linker.

The term "prodrug" as used in this specification is a substance that is administered in an inactive or incompletely active form and subsequently converted into an active substance (e.g., a compound of the present invention) through a metabolic process. Prodrugs can be used to improve the absorption, distribution, metabolism, and/or excretion of active substances, and can also be used to improve the selectivity of active substances for specific cell or process interactions, thereby, for example, reducing adverse reactions or unintended effects of active substances.

The term "about" used in this specification indicates a range of 10% above and below the corresponding numerical value. For example, if the concentration of a certain component is about 5mM, it indicates that its concentration is 4.5-5.5mM; if the concentration range of a certain component is about 5-10mM, it indicates that its concentration range is 4.5-11mM.

In addition, other terms used in this specification have common meanings in the art.

In order to obtain the compounds of the present invention, the active substances need to be first modified, and then the modified substances are connected through a connecting chain. The following uses the BRD4 inhibitor (+)-JQ-1 and the HSP90 inhibitors NVP-AUY922, AT13387 and STA9090 analogs as examples to illustrate the general reaction route.

First, as shown in Reaction Scheme 1, the BRD4 inhibitor (+)-JQ-1 (A1) can be transformed into Compound 2 containing a carboxyl group according to conventional organic chemical reaction synthesis methods.

Next, as shown in Reaction Scheme 2, HSP90 inhibitors NVP-AUY922, AT13387 and STA9090 can be transformed into compounds 3, 5, 6 containing a carboxyl group, and then connected to the aforementioned compound 2 via a linker.

The specific method for synthesizing the compound will be further described in detail in the Examples section below.

The compounds of the present invention can be used to prepare drugs for treating tumors. Specifically, the tumors include, but are not limited to, pancreatic cancer, colon cancer, colorectal cancer, ovarian cancer, cervical cancer, cardia cancer, testicular cancer, prostate cancer, liver cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, head and neck cell cancer, bladder cancer, gastric cancer, kidney cancer, bile duct cancer, brain cancer, breast cancer, epithelial cell cancer, skin cancer, esophageal cancer, lymphoma, glioma, melanoma, multiple myeloma and leukemia, including metastatic lesions in other tissues or organs far away from the primary site of the tumor.

The compounds of the present invention can be administered to a subject, such as a human patient, using any convenient means that can produce the desired result. For example, the compounds can be formulated into pharmaceutical compositions as described above, and/or formulated into known or newly developed dosage forms (such as tablets, capsules, injections, etc.).

In addition, the compounds of the present invention can be used in combination with other therapeutic drugs. The other therapeutic drugs include tumor chemotherapy drugs, tumor targeting drugs, tumor immunotherapy drugs, tumor drug conjugates (such as antibody drug conjugates, small molecule drug compounds and micro drug compounds). The compounds of the present invention can be made into the same dosage form with other therapeutic drugs, or they can be made into separate dosage forms.

More specific embodiments of the present invention will be illustratively explained through the following examples in conjunction with the accompanying drawings, but it should be appreciated that these examples are not intended to limit the scope of the present invention.

Example

The synthetic raw materials, test substances, etc. used in the examples of this specification are all known to those skilled in the art and can be obtained by commercially available or literature methods. The test or characterization methods used are also methods known to those skilled in the art.

In the intermediate examples below, the sources of A1 to A4 as starting materials are respectively referred to the following documents: J. Med. Chem. 2020, 63, 5421-5441; Expert Opin. Ther. Patents 2014, 24(2), 185-199; J. Med. Chem. 2014, 57, 2258-2274; Mol Cancer Ther 2020 19(8): 1613–1622.

In the following pharmacological examples, the compounds HTT-1 to HTT-18 obtained in product examples 1 to 6 are referred to as "compounds of the present invention." In addition, the cell lines used in the pharmacological examples were derived from: Shanghai Institute of Materia Medica, University of Chinese Academy of Sciences (SIMM).

Intermediate Example 1

Compound A1 (2.0 g, 1.7 mmol) was dissolved in 10 mL of 50% TFA/DCM solution and reacted for 3 h at room temperature. After the reaction was completed, the solvent was removed under reduced pressure to obtain the target product 2 as a yellow solid (1.5 g) with a yield of 86%.

Intermediate Example 2

Compound A2 (2.0 g, 3.2 mmol) was dissolved in 20 mL of methanol, 10% palladium carbon (200 mg) was added, nitrogen and hydrogen were replaced, and the reaction was carried out at room temperature for 8 h. After the reaction was complete, diatomaceous earth was filtered and the solvent was removed under reduced pressure to obtain a crude product. The crude product was separated by silica gel column chromatography to obtain the target product 3, 1.35 g of off-white solid, with a yield of 85%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ10.62(s,2H),9.78(s,1H),9.68(s,1H),7.69(s,2H),7.37–7.16(m,5H),6.80(s,2H),6.74(s,1H),3.47(s,4H),3.19(m,1 H), 2.40 (s, 4H), 1.17 (d, J = 6.9Hz, 6H). ¹³ C NMR (100MHz, DMSO-d ₆ )δ173.2,161.9,158.3,151.9,132.46,132.1,130.9,129.1,122.2,114.5,100.8,97.8,66.0,62.3,53.0,26.2,22.4.

Intermediate Example 3

Under nitrogen, compound A3 (5.0 g, 9.58 mmol) was dissolved in 50 mL of methanol, 1.5 g of 10% Pd/C was added, hydrogen was replaced, and the reaction was carried out for 4 h at room temperature. After the reaction was complete, diatomaceous earth was used for suction filtration to remove palladium carbon. The filtrate was removed under reduced pressure to obtain a crude product. The crude product was slurried with DCM and methanol (10:1) to obtain compound 4, 2.54 g of yellow solid, with a yield of 85%. ¹ HNMR (400MHz, DMSO-d ₆ ) δ9.89(s,1H),9.77(s,1H),7.65(d,J＝0.6Hz,1H),7.27(d,J＝7.5Hz,1H),6.82–6.66(m,2H),6.52(s,1H),4.66(dd,J＝35.0,5.3 Hz, 2H), 4.21 (t, J＝7.1Hz, 2H), 3.31–2.97 (m, 3H), 1.22 (d, J＝6.8Hz, 6H). ¹³ C NMR (100MHz, DMSO-d ₆ )δ168.12,161.88,157.58,145.00,135.15,131.62,128.07,127.30,119.12,115.05,113.96,112.23,101.86,48.07,27.13,26.65,22.38.

Intermediate Example 4

Compound 4 (2.0 g, 6.41 mmol) and succinic anhydride (774 mg, 7.69 mmol) were dissolved in 20 mL of toluene, heated to reflux, and reacted for 12 h. After the reaction was complete, the solvent was removed under reduced pressure to obtain a crude product. The crude product was chromatographed on a silica gel column to obtain compound 5 as a light yellow solid (2.24 g) with a yield of 85%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ9.89 (s, 1H), 9.72 (s, 1H), 9.19 (s, 1H), 7.65 (d, J = 0.6Hz, 1H), 7.56 (dd, J = 7.5, 1.5Hz, 1H), 7.34 (q, J = 1.1Hz, 1H), 7.30 (d, J = 7.5Hz ,1H),6.53(s,1H),4.30–4.05(m,2H),3.16(dtd,J＝18.9,7.0,0.9Hz,3H),2.68–2.59(m,2H),2.59–2.48(m,2H),1.22(d,J＝6.8Hz,6H). ¹³ C NMR (100MHz ,DMSO-d ₆ )δ175.04,170.94,167.33,161.76,158.59,138.62,137.57,130.15,127.67,126.80,119.68,117.32,116.56,113.66,101.77,47.57,31.77,28. 84,27.00,22.43.

Intermediate Example 5

Compound A4 (1.0 g, 1.73 mmol) was dissolved in 20 mL of 20% TFA/DCM solution and reacted for 2 h at room temperature. After the reaction was completed, the solvent was removed under reduced pressure to obtain the target product 6 as a yellow solid (750 mg) with a yield of 83%.

Intermediate Example 6: General Synthesis Route of 7a-7d

Compound 2 (1 eq), 2a-2d (1 eq) and HATU (1.2 eq) were added to a two-necked bottle in sequence, and anhydrous DMF solution was added by injection under nitrogen protection. After stirring and dissolving, DIPEA (3 eq) was added dropwise. After the addition was completed, the reaction system was stirred at room temperature overnight. After the reaction was detected to be complete, water was added to quench, and the crude product was extracted with ethyl acetate. After drying, it was separated and purified by silica gel column chromatography to obtain the corresponding products 7a-7d.

Synthesis of compound 7a

Compound 7a was obtained using compounds 2 and 2a as raw materials according to the general synthesis method of intermediate Example 6. The light yellow solid was 150 mg with a yield of 90%. 1H NMR (400 MHz, DMSO) δ8.23 (t, J = 5.1 Hz, 1H), 7.49 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 6.83-6.77 (m, 1H), 4.50 (t, J = 7.0 Hz, 1H), 3.17-3.09 (m, 4H), 3.05-3.01 (m, 2H), 2.60 (s, 3H), 2.41 (s, 3H), 1.63 (s, 3H), 1.38 (s, 9H).13C NMR (101MHz, DMSO) δ170.20,169.74,163.49,161.71,156.08,155.58,150.30,137.23,135.67,132.75,131.17,130.63,130.31,130.04,128.95,78.15 ,54.24,54.07,28.69,23.09,18.55,17.20,14.54,13.15,12.96,11.77.

Synthesis of compound 7b

Compound 7b was obtained by using compound 2 and 2b as raw materials according to the general synthesis method of intermediate Example 6, as a light yellow solid (155 mg), with a yield of 89%. 1H NMR (400MHz, DMSO) δ8.20 (t, J = 5.5 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 6.78 (s, 1H), 4.50 (dd, J = 8.3, 5.8 Hz, 1H), 3.21-2.94 (m, 6H), 2.60 (s, 3H), 2.41 (s, 3H), 1.61 (d, J = 8.3 Hz, 3H), 1.37 (s, 9H), 1.25(d,J＝5.6Hz,2H).13CNMR(101MHz,DMSO)δ169.99,163.51,156.03,155.58,150.29,137.24,135.65,132.75,131.16,130.63,130.30,130.04,128.9 3,77.93,54.35,38.13,36.74,28.72,14.53,13.15,11.78.

Synthesis of compound 7c

Compound 7c was obtained by using compound 2 and 2c as raw materials according to the general synthesis method of intermediate Example 6. The light yellow solid was 140 mg and the yield was 90%. 1H NMR (400MHz, DMSO) δ8.18 (t, J = 5.4 Hz, 1H), 7.50 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 5.8 Hz, 1H), 4.50 (dd, J = 8.2, 5.9 Hz, 1H), 3.20-3.02 (m, 4H), 2.89 (s, 2H), 2.59 (s, 3H), 2.41 (s, 3H), 1.62 (s, 3H), 1.42 (s, 4H), 1.37 (s, 9H).13C NMR (101MHz, DMSO) δ169.81,169.35,163.47,161.35,156.08,155.60,150.27,137.24,135.68,132.74,131.17,130.58,130.29,130.04,128.96,77.80 ,55.39,54.35,38.74,28.74,27.43,27.06,23.08,14.53,13.15,11.78.

Synthesis of compound 7d

Compound 7d was obtained by using compound 2 and 2d as raw materials according to the general synthesis method of intermediate Example 6. The light yellow solid was 140 mg and the yield was 88%. 1H NMR (400MHz, DMSO) δ8.16 (t, J = 5.4 Hz, 1H), 7.49 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 6.77 (s, 1H), 4.50 (t, J = 7.0 Hz, 1H), 3.16 (m, J = 18.9, 12.3, 5.5 Hz, 4H), 2.91-2.87 (m, 2H), 2.59 (s, 3H), 2.41 (s, 3H), 1.62 (s, 3H), 1.37 (s, 9H), 1.28-1.24 (m, 8H).13C NMR (101MHz, DMSO) δ169.77,163.46,156.05,155.60,150.26,137.24,135.71,132.74,131.17,130.57,130.29,130.06,128.93,77.74,54.39,54.08,3 8.89,38.13,29.94,29.69,28.74,26.55,26.49,18.56,17.21,14.52,13.15,11.76.

Intermediate Example 7: General Synthesis Method of Compounds 8a to 8d

Compounds 7a-7d (1 eq) were added to a mixed solution of trifluoroacetic acid and dichloromethane (1:1 ratio), stirred at room temperature for 3 hours, and concentrated under reduced pressure after the reaction was complete to obtain yellow solid compounds 8a-8d.

Intermediate Example 8

Compounds 2 (1 eq), 2e; 2f (1 eq) and HATU (1.2 eq) were added to a two-necked bottle in sequence, and anhydrous DMF solution was added by injection under nitrogen protection. After stirring and dissolving, DIPEA (3 eq) was added dropwise. After the addition was completed, the reaction system was stirred at room temperature overnight. After the reaction was detected to be complete, water was added to quench, and the crude product was extracted with ethyl acetate. After drying, it was separated and purified by silica gel column chromatography to obtain the corresponding products 9e; 9f.

Intermediate Example 9

Compound 9e; 9f (1 eq) was added to a mixed solution of trifluoroacetic acid and dichloromethane (1:1 ratio), and stirred at room temperature for 3 hours. After the reaction was detected to be complete, it was concentrated under reduced pressure to obtain yellow solid compound 10e; 10f.

Product Example 1: General Synthesis Method of Compounds HTT-1 to HTT-4

Compounds 8a-8d (1 eq), 5 (1 eq) and HATU (1.2 eq) were added to a two-necked bottle in sequence, and anhydrous DMF solution was added by injection under nitrogen protection. After stirring and dissolving, DIPEA (3 eq) was added dropwise. After the addition was completed, the reaction system was stirred at room temperature overnight. After the reaction was detected to be complete, water was added to quench, and the crude product was extracted with ethyl acetate. After drying, it was separated and purified by silica gel column chromatography to obtain the corresponding products HTT-1-HTT-4.

Synthesis of Compound HTT-1

Using compounds 8a and 5 as raw materials, HTT-1 was obtained according to the general synthesis method of compounds HTT-1 to HTT-4, with a white solid of 45 mg and a yield of 65%. 1H NMR (400MHz, DMSO) δ9.90 (s, 1H), 9.74 (s, 1H), 9.61 (s, 1H), 8.25 (s, 1H), 7.91 (s, 1H), 7.57 (s, 1H), 7.49 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 8.2 Hz, 3H), 7.26 (s, 1H), 6.97 (s, 1H), 6.40 (s, 1H ),4.51(t,J＝6.8Hz,1H),3.95(t,J＝7.8Hz,2H),3.25(d,J＝6.6Hz,2H),3.13(d,J＝20.9Hz,6H),3.02(t,J＝7.7Hz,3H),2.59(s,3H),2.41(s,5H),1.62(s,3 H),1.12(d,J＝6.7Hz,6H).13C NMR (101MHz, DMSO) δ171.94,170.55,170.22,167.72,163.53,157.13,155.59,153.47,150.35,138.66,137.21,135.74,135.51,133.40,132.73,131.1 7,130.63,130.3 2,130.05,128.96,126.00,117.75,116.36,115.67,102.86,54.26,49.28,38.95,38.82,38.12,32.07,30.92,27.93,26.30,23.14,14.54,13.1 5,11.78.HRMS(ESI):m/z calcd for(M+H)+:837.3952; found:837.3635.

Synthesis of Compound HTT-2

Using compounds 8b and 5 as raw materials, HTT-2 was obtained according to the general synthesis method of compounds HTT-1 to HTT-4, with a white solid of 40 mg and a yield of 75%. 1H NMR (400MHz, DMSO) δ9.87 (s, 1H), 9.74 (s, 1H), 9.61 (s, 1H), 8.19 (s, 1H), 7.87 (s, 1H), 7.56 (s, 1H), 7.49 (d, J = 8.5 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 7.2 Hz, 1H ),6.97(s,1H),6.40(s,1H),4.51(t,J＝7.0Hz,1H),3.94(t,J＝8.1Hz,2H),3.27–2.97(m,11H),2.59(s,4H),2.40(s,5H),1.64–1.55(m,5H),1.12(d, J＝6.8Hz,6H).13C NMR (101MHz, DMSO) δ171.65,170.55,169.94,163.53,157.14,155.59,153.51,150.30,137.24,135.68,133.43,132.73,131.16,130.61,130.32,130.0 5,128.96,126.01,117.7 6,116.39,102.90,100.00,54.34,40.63,40.42,40.21,40.00,39.80,39.59,39.38,38.13,36.95,32.15,30.90,29.79,26.31,23.13,14.52,13 .15,11.78.HRMS(ESI):m/zcalcd for(M+H)+:851.4631; found:851.4611.

Synthesis of Compound HTT-3

Using compounds 8c and 5 as raw materials, HTT-3 was obtained according to the general synthesis method of compounds HTT-1 to HTT-4, 36 mg of white solid, with a yield of 75%. 1H NMR(400MHz,DMSO)δ9.89(s,1H),9.77(s,1H),9.65(s,1H),8.21(s,1H),7.87(s,1H),7.56(s,1H),7.48(s,2H),7.43(s,2H),7.27(s,1H),6.97(s,1 H),6.41(s,1H),4.48(s,1H),3.95(s,2H),3.02(s,10H),2.55(s,3H),2.41(s,4H),1.62(s,3H),1.44(s,4H),1.24(s,3H),1.12(d,J＝4.8Hz,6H).13 C NMR (101MHz, DMSO) δ171.54,170.57,169.84,163.49,157.15,155.59,153.48,150.30,143.32,137.22,135.70,135.59,133.44,132.73,131.17,130.5 9,130.30,130.04,128.95,12 5.99,124.18,119.20,117.73,117.70,116.36,115.64,110.63,102.88,54.35,49.25,38.71,38.09,32.21,30.92,27.15,27.08,26.30,23.13, 14.53,13.15,11.78.HRMS(ESI):m/z calcd for(M+H)+:865.4862; found:865.4811.

Synthesis of Compound HTT-4

Compounds 8d and 5 were used as raw materials and HTT-4 was obtained according to the general synthesis method of compounds HTT-1 to HTT-4. The white solid was 50 mg and the yield was 70%. 1H NMR (400MHz, DMSO) δ9.88 (s, 1H), 9.75 (s, 1H), 9.62 (s, 1H), 8.18 (t, J = 5.5 Hz, 1H), 7.85 (t, J = 5.4 Hz, 1H), 7.56 (s, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.42 (d, J = 8.5 Hz, 2H), 7.26 (s, 1H), 6.98 (s, 1H), 6.40 (s, 1 H),4.51(t,J＝7.1Hz,1H),3.95(t,J＝8.3Hz,2H),3.26–3.20(m,2H),3.13–3.00(m,8H),2.59(s,3H),2.40(d,J＝8.6Hz,5H),1.62(s,3H),1.48–1.34(m ,5H),1.31–1.25(m,5H),1.12(d,J＝6.9Hz,6H).13C NMR (101MHz, DMSO) δ171.47,170.57,169.82,167.74,163.48,157.14,155.60,153.49,150.28,138.64,137.22,135.72,135.58,133.43,132.72,131.1 8,130.57,130.29,130.04, 128.93,126.01,117.73,116.35,115.65,102.88,54.38,49.30,38.90,38.12,32.22,30.92,29.65,29.60,28.00,26.57,26.55,26.31,23.12,1 4.52,13.14,11.76.HRMS(ESI):m/z calcd for(M+H)+:893.5532; found:893.5012.

Product Example 2: General Synthesis Method of Compounds HTT-5 to HTT-8

Compounds 8a-8d (1 eq), 3 (1 eq) and HATU (1.2 eq) were added to a two-necked bottle in sequence, and anhydrous DMF solution was added by injection under nitrogen protection. After stirring and dissolving, DIPEA (3 eq) was added dropwise. After the addition was completed, the reaction system was stirred at room temperature overnight. After the reaction was detected to be complete, water was added to quench, and the crude product was extracted with ethyl acetate. After drying, it was separated and purified by silica gel column chromatography to obtain the corresponding products HTT-5-HTT-8.

Synthesis of Compound HTT-5

Using compounds 8a and 3 as raw materials, the general synthesis method of compounds HTT-5 to HTT-8 gave HTT-5, 40 mg of white solid, with a yield of 60%. 1H NMR (400MHz, DMSO) δ9.90 (s, 1H), 9.74 (s, 1H), 9.61 (s, 1H), 8.25 (s, 1H), 7.91 (s, 1H), 7.57 (s, 1H), 7.49 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 8.2 Hz, 3H), 7.26 (s, 1H), 6.97 (s, 1H), 6.40 (s, 1H ),4.51(t,J＝6.8Hz,1H),3.95(t,J＝7.8Hz,2H),3.25(d,J＝6.6Hz,2H),3.13(d,J＝20.9Hz,6H),3.02(t,J＝7.7Hz,3H),2.59(s,3H),2.41(s,5H),1.62(s,3 H),1.12(d,J＝6.7Hz,6H).13C NMR (101MHz, DMSO) δ171.94,170.55,170.22,167.72,163.53,157.13,155.59,153.47,150.35,138.66,137.21,135.74,135.51,133.40,132.71,131.1 7,130.63,130.3 2,130.05,128.96,126.00,117.75,116.36,115.67,102.86,54.26,49.28,38.95,38.82,38.12,32.07,30.92,27.93,26.30,23.14,14.54,13.1 5,11.78.HRMS(ESI):m/z calcd for(M+H)+:865.4862; found:865.4811.

Synthesis of Compound HTT-6

Using compounds 8b and 3 as raw materials, HTT-6 was obtained according to the general synthesis method of compounds HTT-5 to HTT-8, with a white solid of 35 mg and a yield of 55%. 1H NMR (400MHz, DMSO) δ9.95 (s, 1H), 9.69 (s, 1H), 9.60 (s, 1H), 8.25 (s, 1H), 7.91 (s, 1H), 7.57 (s, 1H), 7.49 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 8.2 Hz, 3H), 7.26 (s, 1H), 6.97 (s, 1H), 6.45 (s, 1H ),4.51(t,J＝6.8Hz,1H),3.95(t,J＝7.8Hz,2H),3.25(d,J＝6.6Hz,2H),3.13(d,J＝20.9Hz,6H),3.02(t,J＝7.7Hz,3H),2.59(s,3H),2.41(s,5H),1.62(s,3 H),1.12(d,J＝6.7Hz,6H).13C NMR (101MHz, DMSO) δ171.94,170.55,170.22,167.72,163.53,157.13,155.59,153.47,150.35,138.66,137.21,135.74,135.51,133.40,132.73,131.1 7,130.63,130.3 2,130.05,128.96,126.00,117.75,116.35,115.67,102.86,54.20,49.28,38.95,38.82,38.12,32.07,30.92,27.93,26.30,23.11,14.54,13.1 5,11.71.HRMS(ESI):m/z calcd for(M+H)+:877.4604; found:877.4591.

Synthesis of Compound HTT-7

Using compounds 8c and 3 as raw materials, HTT-7 was obtained according to the general synthesis method of compounds HTT-5 to HTT-8, 45 mg of white solid, with a yield of 60%. 1H NMR (400MHz, MeOD) δ7.41(dd,J＝17.4,8.0Hz,4H),7.30–7.20(m,4H),6.78(s,1H),6.35(s,1H),4.67–4.60(m,1H),3.64(s,4H),3.49–3.33(m,7H),3. 28(s,1H),3.10–3.01(m,1H),2.68(s,3H),2.43(s,6H),1.66(d,J＝17.9Hz,7H),1.29(s,1H),0.95(d,J＝6.8Hz,6H).13C NMR (101MHz, DMSO) δ170.13,169.58,163.51,157.78,156.63,156.52,155.59,154.90,150.28,148.20,140.10,137.22,135.70,134.44,132.75,131.1 7,130.62,130.28,130.04,129. 52,128.96,127.75,126.33,125.91,103.06,102.96,61.91,61.84,54.39,53.41,53.02,38.18,36.52,36.22,34.02,30.07,25.71,22.78,14.9 6,14.55,13.13,11.78.HRMS(ESI):m/z calcd for(M+H)+:891.5102; found:891.5032.

Synthesis of Compound HTT-8

Using compounds 8d and 3 as raw materials, HTT-8 was obtained according to the general synthetic method of compounds HTT-5 to HTT-8, 45 mg of white solid, with a yield of 70%. 1H NMR (400MHz, MeOD) δ7.38(dd,J＝17.4,8.0Hz,4H),7.30–7.25(m,4H),6.77(s,1H),6.35(s,1H),4.67–4.60(m,1H),3.64(s,4H),3.49–3.33(m,7H),3. 28(s,1H),3.11–3.01(m,1H),2.68(s,3H),2.43(s,6H),1.66(m,J＝17.9Hz,11H),1.29(s,1H),0.93(d,J＝6.8Hz,6H).13C NMR (101MHz, DMSO) δ170.13,169.58,163.51,157.78,156.63,156.52,155.59,154.90,150.28,148.20,140.10,137.22,135.70,134.44,132.75,131.1 7,130.62,130.28,130.04,129. 52,128.96,127.75,126.33,125.91,103.06,102.96,61.91,61.84,54.39,53.41,53.02,38.18,36.52,36.22,34.02,30.07,25.75,22.78,14.9 6,14.55,13.13,11.78.HRMS(ESI):m/z calcd for(M+H)+:919.5536; found:919.5499.

Product Example 3: General Synthesis Method of Compounds HTT-9 to HTT-12

Compounds 8a-8d (1 eq), 6 (1 eq) and HATU (1.2 eq) were added to a two-necked bottle in sequence, and anhydrous DMF solution was added by injection under nitrogen protection. After stirring and dissolving, DIPEA (3 eq) was added dropwise. After the addition was completed, the reaction system was stirred at room temperature overnight. After the reaction was detected to be complete, water was added to quench, and the crude product was extracted with ethyl acetate. After drying, it was separated and purified by silica gel column chromatography to obtain the corresponding products HTT-9-HTT-12.

Synthesis of Compound HTT-9

Using compounds 8a and 6 as raw materials, HTT-9 was obtained according to the general synthesis method of compounds HTT-9 to HTT-12, with a white solid of 35 mg and a yield of 55%. 1H NMR (400MHz, DMSO) δ10.59 (s, 1H), 9.76 (s, 1H), 8.95 (t, J = 5.6 Hz, 1H), 8.28 (s, 1H), 7.79 (s, 1H), 7.48 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.3 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 8.1 Hz, 2H), 6.57 (s, 1H),6.35(s,1H),4.52(t,J＝6.9Hz,1H),3.44(s,2H),3.28–3.15(m,9H),2.88(d,J＝10.1Hz,3H),2.59(s,3H),2.40(d,J＝17.1Hz,10H),1.62(s,3H), 1.03(t,J＝7.1Hz,3H),0.79(d,J＝6.7Hz,6H).13C NMR (101MHz, DMSO) δ170.33,169.94,163.53,157.75,156.62,156.53,155.57,154.88,150.31,148.21,140.09,137.24,135.69,134.43,132.74,131.1 7,130.63,130.31,130.0 2,129.58,128.95,127.74,126.32,125.94,103.04,102.97,61.94,61.75,54.23,53.40,53.03,38.87,38.13,34.02,25.70,22.77,14.95,14.5 4,13.13,11.77.HRMS(ESI):m/z calcd for(M+H)+:947.5911; found:947.5801.

Synthesis of Compound HTT-10

Using compounds 8b and 6 as raw materials, HTT-10 was obtained according to the general synthesis method of compounds HTT-9 to HTT-12, with a white solid of 39 mg and a yield of 60%. 1H NMR (400MHz, DMSO) δ10.60 (s, 1H), 9.78 (s, 1H), 8.96 (t, J = 5.8 Hz, 1H), 8.26 (t, J = 5.5 Hz, 1H), 7.79 (t, J = 5.8 Hz, 1H), 7.49 (d, J = 8.6 Hz, 2H), 7.43 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.3 Hz, 2H), 7.29 (d, J = 8.3 Hz, 2H), 6.57 (s, 1H), 6.3 6(s,1H),4.52(dd,J＝8.2,5.9Hz,1H),3.46(s,1H),3.39–3.29(m,6H),3.17(dd,J＝13.4,6.9Hz,6H),2.89(d,J＝9.9Hz,3H),2.59(s,3H),2.41(d,J＝17. 4Hz,10H),1.61(s,3H),1.03(t,J＝7.2Hz,3H),0.79(d,J＝6.9Hz,6H).13C NMR (101MHz, DMSO) δ170.13,169.58,163.51,157.78,156.63,156.52,155.59,154.90,150.28,148.20,140.10,137.22,135.70,134.44,132.75,131.1 7,130.62,130.28,130.04,129. 52,128.96,127.75,126.33,125.91,103.06,102.96,61.91,61.84,54.39,53.41,53.02,38.18,36.52,36.22,34.02,30.07,25.71,22.78,14.9 6,14.55,13.13,11.78.HRMS(ESI):m/z calcd for(M+H)+:961.6113; found:961.5819.

Synthesis of compound HTT-11

Using compounds 8c and 6 as raw materials, HTT-11 was obtained according to the general synthesis method of compounds HTT-9 to HTT-12, with a white solid of 45 mg and a yield of 78%. 1H NMR (400MHz, DMSO) δ10.59 (s, 1H), 9.70 (s, 1H), 8.95 (t, J = 5.7 Hz, 1H), 8.20 (t, J = 5.2 Hz, 1H), 7.59 (t, J = 5.6 Hz, 1H), 7.50 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.2 Hz, 2H), 7.29 (d, J = 8 .2Hz,2H),6.57(s,1H),6.34(s,1H),4.54–4.49(m,1H),3.48(s,1H),3.30–3.08(m,10H),2.90(s,3H),2.59(s,3H),2.42(d,J＝14.0Hz,10H),1.62( s,3H),1.44(s,4H),1.03(t,J＝7.1Hz,3H),0.80(d ,J＝6.8Hz,6H).13CNMR(101MHz,DMSO)δ169.83,169.47,163.48,157.74,156.62,156.52,155.59,154.89,150.28,148.20,140.08,137.24,135.69,134. 44,132.74,131.17,130.58,130.29,130.04 ,129.56,128.96,127.75,126.36,125.92,103.04,102.99,61.90,61.83,54.35,53.40,53.02,38.71,38.37,38.12,34.02,27.23,27.14,25.71 ,22.79,14.95,14.54,13.14,11.77.HRMS(ESI):m/z calcd for(M+H)+:975.6813; found:975.6599.

Synthesis of Compound HTT-12

Compounds 8d and 6 were used as raw materials and HTT-12 was obtained according to the general synthesis method of compounds HTT-9 to HTT-12. The white solid was 35 mg and the yield was 55%. 1H NMR (400MHz, DMSO) δ10.60 (s, 1H), 9.73 (s, 1H), 8.95 (t, J = 5.8 Hz, 1H), 8.17 (t, J = 5.4 Hz, 1H), 7.65 (t, J = 5.8 Hz, 1H), 7.48 (d, J = 8.5 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 6.55 (s, 1H), 6.34 (s, 1H),4.55–4.48(m,1H),3.49(s,2H),3.27–3.03(m,9H),2.93–2.84(m,3H),2.59(s,3H),2.42(d,J＝12.0Hz,10H),1.62(s,3H),1.41(dd,J＝13.7,6. 8Hz,4H),1.26(d,J＝15.9Hz,4H),1.03(t,J＝7.2Hz,3H),0.79(d,J＝6.9Hz,6H).13C NMR (101MHz, DMSO) δ169.80,169.36,163.47,157.74,156.62,156.54,155.60,154.88,150.27,148.21,140.09,137.23,135.71,134.44,132.73,131.1 8,130.57,130.28,130.05,129.57,128.93,127.75,126.33,125 .91,103.04,102.99,61.89,61.82,54.39,53.37,53.03,40.63,40.42,40.21,40.00,39.79,39.58,39.37,38.82,38.53,38.13,34.02,29.66,2 6.52,26.47,25.71,22.79,14.95,14.52,13.15,11.76.HRMS(ESI):m/z calcd for(M+H)+:1003.6880; found:1003.6723.

Product Example 4: General Synthesis Method of Compounds HTT-13 and HTT-14

Compounds 10e; 10f (1 eq), 5 (1 eq) and HATU (1.2 eq) were added to a two-necked bottle in sequence, and anhydrous DMF solution was added by injection under nitrogen protection. After stirring and dissolving, DIPEA (3 eq) was added dropwise. After the addition was completed, the reaction system was stirred at room temperature overnight. After the reaction was detected to be complete, water was added to quench, and the crude product was extracted with ethyl acetate. After drying, it was separated and purified by silica gel column chromatography to obtain the corresponding products HTT-13; HTT-14.

Synthesis of compound HTT-13

Using compounds 10e and 5 as raw materials, HTT-13 was obtained according to the general synthesis method of compounds HTT-13 and HTT-14, with a white solid of 35 mg and a yield of 75%. ¹ H NMR (400MHz, DMSO) δ9.87 (s, 1H), 9.73 (s, 1H), 9.60 (s, 1H), 8.28 (t, J = 5.3 Hz, 1H), 7.95 (t, J = 5.2 Hz, 1H), 7.55 (s, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.40 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 4.6 Hz, 1H), 6.97 (s, 1H), 6.39 (s, 1H), 4. 51(t,J＝7.0Hz,1H),3.94(t,J＝8.2Hz,2H),3.44(dd,J＝12.2,5.9Hz,5H),3.25(dt,J＝14.7,6.0Hz,7H),3.11–3.06(m,1H),3.02(t,J＝8.2Hz,2H),2.59(s, 3H), 2.42 (d, J = 8.2Hz, 6H), 1.61 (s, 3H), 1.11 (d, J = 6.9Hz, 6H). ¹³ C NMR (101MHz, DMSO) δ171.84,170.53,170.19,157.12,155.57,153.47,150.30,137.19,135.72,132.71,131.10,130.62,130.36,130.03,128.94, 125.97,69.44,69.38,54.32,39.02,30.74,26.26,23.13,14.53,13.13,11.74.HRMS(ESI):m/z calcd for(M+H) ⁺ :881.4580; found:881.4329.

Synthesis of Compound HTT-14

Compounds 10f and 5 were used as raw materials and HTT-14 was obtained according to the general synthesis method of compounds HTT-13 and HTT-14. The white solid was 40 mg and the yield was 70%. 1H NMR (400MHz, DMSO) δ9.87 (s, 1H), 9.73 (s, 1H), 9.61 (s, 1H), 8.30 (t, J = 5.5 Hz, 1H), 7.95 (t, J = 5.5 Hz, 1H), 7.55 (s, 1H), 7.47 (s, 2H), 7.42 (d, J = 8.5 Hz, 2H), 7.25 (s, 1H), 6.97 (s, 1H), 6.40 (s, 1H), 4.50 (d, J = 7 .7Hz,1H),3.95(t,J＝8.3Hz,2H),3.53(s,5H),3.46(t,J＝5.8Hz,3H),3.41(t,J＝5.9Hz,4H),3.29–3.19(m,7H),3.05(dt,J＝16.5,7.6Hz,4H),2.59(s,3H ),2.41(s,5H),1.62(s,3H),1.12(d,J＝6.9Hz,6H).13C NMR (101MHz, DMSO) δ171.82,170.54,170.18,163.49,157.13,155.57,153.48,150.30,137.23,135.69,133.43,132.74,131.17,130.62,130.30,130.0 2,128.9 3,126.01,117.73,116.35,115.67,102.87,70.05,69.68,69.62,54.30,39.11,39.05,37.97,32.07,30.75,26.30,23.13,14.53,13.15,11.77. HRMS(ESI):m/z calcd for(M+H)+:925.5233; found:925.4905.

Product Example 5: General Synthesis Method of Compounds HTT-15 and HTT-16

Compounds 10e; 10f (1 eq), 3 (1 eq) and HATU (1.2 eq) were added to a two-necked bottle in sequence, and anhydrous DMF solution was added by injection under nitrogen protection. After stirring and dissolving, DIPEA (3 eq) was added dropwise. After the addition was completed, the reaction system was stirred at room temperature overnight. After the reaction was detected to be complete, water was added to quench, and the crude product was extracted with ethyl acetate. After drying, it was separated and purified by silica gel column chromatography to obtain the corresponding products HTT-15; HTT-16.

Synthesis of Compound HTT-15

Using compounds 10e and 3 as raw materials, HTT-15 was obtained according to the general synthesis method of compounds HTT-15 and HTT-16, with a white solid of 35 mg and a yield of 79%. 1H NMR (400MHz, DMSO) δ10.09 (s, 1H), 9.73 (s, 1H), 8.96 (t, J = 5.8 Hz, 1H), 8.28 (t, J = 5.4 Hz, 1H), 7.72 (t, J = 5.6 Hz, 1H), 7.48 (d, J = 8.6 Hz, 2H), 7.46 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 8.3 Hz, 2H), 6. 1 .61(s,3H),1.03(t,J＝7.2Hz,3H),0.79(d,J＝6.9Hz,6H).13C NMR (101MHz, DMSO) δ170.17,169.76,163.48,157.74,156.61,156.52,155.57,154.88,150.27,148.20,140.10,137.24,135.70,134.43,132.75,131.1 6,130.61,130.30,130.02,129. 55,128.93,127.73,126.33,125.92,103.03,101.98,69.34,61.86,61.69,54.33,53.34,53.06,39.10,38.51,38.02,34.01,25.70,22.78,14.9 6,14.53,13.14,11.76.HRMS(ESI):m/z calcd for(M+H)+:907.5312; found:907.4849.

Synthesis of Compound HTT-16

Compounds 10f and 3 were used as raw materials and HTT-16 was obtained according to the general synthesis method of compounds HTT-15 and HTT-16. HTT-16 was obtained as a white solid (45 mg) with a yield of 75%. 1H NMR (400MHz, DMSO) δ9.87 (s, 1H), 9.73 (s, 1H), 9.60 (s, 1H), 8.28 (t, J = 5.3 Hz, 1H), 7.95 (t, J = 5.2 Hz, 1H), 7.55 (s, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 4.6 Hz, 1H), 6.97 (s, 1H), 6.39 (s, 1H), 4.51 (t,J＝7.0Hz,1H),3.94(t,J＝8.2Hz,2H),3.44(dd,J＝12.2,5.9Hz,6H),3.25(dt,J＝14.7,6.0Hz,10H),3.11–3.06(m,1H),3.02(t,J＝8.2Hz,2H),2.59(s,3 H),2.42(d,J＝8.2Hz,6H),1.61(s,3H),1.18(d,J＝6.9Hz,6H).13C NMR (101MHz, DMSO) δ171.84,170.53,170.19,157.12,155.57,153.47,150.30,137.19,135.72,132.71,131.10,130.62,130.36,130.03,128.98,125.9 7,69.44,69.38,54.52,39.02,30.74,26.26,23.13,14.53,13.13,11.74.HRMS(ESI):m/z calcd for(M+H)+:951.5466; found:951.5378.

Product Example 6: General Synthesis Method of Compounds HTT-17 and HTT-18

Compounds 10e; 10f (1 eq), 6 (1 eq) and HATU (1.2 eq) were added to a two-necked bottle in sequence, and anhydrous DMF solution was added by injection under nitrogen protection. After stirring and dissolving, DIPEA (3 eq) was added dropwise. After the addition was completed, the reaction system was stirred at room temperature overnight. After the reaction was detected to be complete, water was added to quench, and the crude product was extracted with ethyl acetate. After drying, it was separated and purified by silica gel column chromatography to obtain the corresponding products HTT-17; HTT-18.

Synthesis of compound HTT-17

Using compounds 10e and 6 as raw materials, HTT-17 was obtained according to the general synthesis method of compounds HTT-17 and HTT-18, with a white solid content of 25 mg and a yield of 70%. 1H NMR (400MHz, DMSO) δ10.61 (s, 1H), 9.76 (s, 1H), 8.96 (t, J = 5.8 Hz, 1H), 8.28 (t, J = 5.4 Hz, 1H), 7.72 (t, J = 5.6 Hz, 1H), 7.46 (d, J = 8.6 Hz, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 6. 1 .61(s,3H),1.03(t,J＝7.2Hz,3H),0.79(d,J＝6.9Hz,6H).13C NMR (101MHz, DMSO) δ170.17,169.74,163.48,157.74,156.61,156.52,155.57,154.88,150.27,148.20,140.10,137.24,135.70,134.43,132.75,131.1 6,130.61,130.30,130.02,129. 55,128.93,127.73,126.33,125.92,103.03,102.98,69.34,61.86,61.69,54.33,53.34,53.06,39.10,38.51,38.02,34.01,25.70,22.78,14.9 6,14.53,13.14,11.76.HRMS(ESI):m/z calcd for(M+H)+:991.6533; found:991.6250.

Synthesis of compound HTT-18

Using compounds 10f and 6 as raw materials, according to the general synthesis method of compounds HTT-17 and HTT-18, white solid HTT-18 was obtained, 55 mg of white solid, with a yield of 75%. 1H NMR (400MHz, DMSO) δ10.61 (s, 1H), 9.76 (s, 1H), 8.96 (t, J = 5.8Hz, 1H), 8.28 (t, J = 5.4Hz, 1H), 7.72 (t, J = 5.6Hz, 1H), 7.46 (d, J = 8.6Hz, 2H), 7.41 (d, J = 8. 5Hz,2H),7.33(d,J＝8.3Hz,2H),7.29(d,J＝8.3Hz,2H),6.57(s,1H),6.34(s,1H),4.55–4.50(m,1H),3.46(d,J＝5.7 Hz,8H),3.32–3.13(m,11H),2.91(d, J＝10.1 Hz,3H),2.59(s,3H),2.41(d,J＝14.2 Hz,10H),1.61(s,3H),1.03(t,J＝7.2 Hz,3H),0.79(d,J＝6.9 Hz,6H).13CNMR(101 MHz,DMSO)δ170.17,169.74,163.48 ,157.74,156.61,156.52,155.58,154.88,150.27,148.20,140.10,137.24,135.70,134.43,132.75,131.16,130.65,130.30,130.02,129.55, 128.93,127.73,126.33,125.95,103.03,102.98,69.34,61.86,61.69,54.33,53.34,53.06,39.10,38.51,38.02,34.01,25.70,22.78,14.96,14.50,13.14,11.66. HRMS (ESI): m/z calcd for (M+H)+: 1035.6833; found: 1035.6523. Pharmacological Example 1: HSP90 enzyme inhibition activity experiment of compounds HTT-1 to HTT-18

To evaluate the effect of the compound on HSP90 enzyme activity, the HSP90 enzyme activity was tested by fluorescence polarization. A series of dilutions of the test compound were prepared with a 10% DMSO buffer solution, and 10 μL of the dilution was added to a 100 μL reaction, and the reaction was carried out at room temperature. A 100 μL mixture of 5nM FITC-labeled geldanamycin and the test compound was reacted for 3 hours. The fluorescence intensity was measured using a Tecan Infinite M1000 microplate reader at 485nm and 530nm excitation, and the fluorescence intensity was converted to fluorescence polarization using Tecan Magellan 6 software. The activity percentage of the compound was calculated according to the following formula: Activity % = (FP-FPb) / (FPt-FPb) × 100%, where FP = fluorescence polarization in the presence of the compound.

Table 1. HSP90 enzyme activity of compounds

^a Data is the mean value of two independent determinations.

^b Positive control.

As shown in Table 1, the inhibitory activity of 18 target compounds on HSP90 enzyme was tested with HSP90 inhibitor STA9090 as positive control. Among them, STA9090 showed extremely strong inhibitory activity against HSP90 (IC ₅₀ = 15nM) kinase. In general, these HSP90-based tumor-targeted BRD4 inhibitors have high inhibition rates on enzymes at 100nM, indicating that the modification and design of HSP90 inhibitors basically do not affect their target activity. Among them, the single-concentration inhibition rates of AT13387-based compounds HTT-1; HTT-3; HTT-4; HTT-13 and the bifunctional molecules HTT-10; HTT-11; HTT-12 based on STA9090 on HSP90 are above 90%. Except for HTT-13 (100nM, Inhibition rate = 93%), the linkers of the other six compounds are all alkyl chains, and the enzyme inhibition activity is best when butane and hexane are used as linkers. The experimental results show that when the linker is an alkyl chain and is longer, the compound has better HSP90 target binding activity.

Pharmacological Example 2: Experimental study on the proliferation inhibition activity of compounds HTT-1, HTT-2, HTT-3, HTT-4, HTT-13 and HTT-14 on tumor cell lines

The CCK8 method was used to detect the inhibition of tumor cell growth by compounds. The specific steps are as follows: cells in the logarithmic growth phase are inoculated into 96-well culture plates at an appropriate density, and 100 μL of complete medium is added to each well for overnight culture. A series of concentrations of compounds are added, and three replicates are set for each concentration, and positive control wells without compound action and negative control wells without cells are set. The cells were cultured at 37°C for 72 hours. After the drug effect ended, 10 μL of CCK-8 reagent was added to each well, placed in a 37°C incubator for 3 hours, and the optical density (OD value) at a wavelength of 450 nm was measured using the full-wavelength microplate reader SpectraMax 190.

Table 2. Inhibitory activity of compounds against MM1S tumor cells in vitro

^a Data is the mean±SD value of three independent determinations.

^b Positive control.

(+)-JQ-1 and HSP90 inhibitor AUY922 were used as positive compounds. As shown in Table 2, on tumor cells MM1S, compared with HSP90 inhibitor AUY922 (IC ₅₀ = 27nM) and BRD4 inhibitor (+)-JQ-1 (IC ₅₀ = 26.5nM), the IC50 of the compounds were all at the micromolar level, among which compound HTT-4 (linker length of 6 carbon atoms, IC ₅₀ = 0.6μM) had a relatively stronger anti-proliferative effect on MM1S cells than other compounds.

Pharmacological Example 3: Western blotting experiment of compound HTT-4

Western experiments were used to further explore the inhibitory ability of compound HTT-4 on tumor cells and the effect on BRD4 protein. Cells were seeded in six-well plates, cultured overnight, and treated with different concentrations of compounds for 24 hours before being collected. Wash once with pre-cooled PBS, and add 1×SDS loading buffer to lyse the cells. Collect the cell lysate, heat it in a boiling water bath for 10 minutes, and centrifuge it at 4℃12000rpm for 5 minutes. Take the supernatant for SDS-PAGE electrophoresis. After the electrophoresis is completed, transfer it. After the transfer is completed, stain it with Ponceau red to determine the transfer situation and the position of the protein band on the nitrocellulose membrane. After marking, the target band is blocked with blocking solution on a shaker at room temperature for 1 hour. Then, the membrane is placed in the primary antibody and incubated overnight at 4 degrees Celsius. Wash it three times with TBST washing solution at room temperature for 10 minutes each time. Add the secondary antibody labeled with horseradish peroxidase and incubate it on a shaker at room temperature for 1 hour. Wash it three times with TBST for 10 minutes each time, develop the color, and expose it.

As shown in Figure 1, after the compound acted on MM1S cells for 24 hours, HTT-4 at a concentration of 0.2 μM could downregulate the expression of BRD4 and c-MYC.

The present invention uses alkyl and PEG linkers to connect HSP90 inhibitors and BRD4 inhibitor modified ligands to design and synthesize a series of tumor-targeted BRD4 inhibitors. In the HSP90 enzyme inhibition experiment, a series of compounds showed good enzyme activity. In the cytotoxicity experiment and Western experiment, the compound HTT-4 showed certain anti-tumor activity. The compounds of the present invention are expected to be used for the treatment of tumors with overexpression of BRD4.

Claims (10)

1. A compound, which includes a BRD4 inhibitory part and an HSP90 inhibitory part, both of which are connected by a connecting chain; or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate thereof body or prodrug; 2. The compound according to claim 1, wherein the connecting chain comprises a bond or connecting group that forms a covalent bond between the HSP90 inhibitory moiety and the BRD4 inhibitory moiety, and the connecting chain is selected from the group consisting of: chemical bonds, ester bonds, Carbonyl group, C _1-6 alkylene group, amide bond, ether bond, disulfide bond and combinations thereof; or its pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, racemates or prodrugs. 3. The compound according to claim 1 or 2, wherein the HSP90 inhibitory moiety is selected from the following structures, and structures or groups derived from these structures that can form various types of covalent bonds with the connecting chain: or its pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, racemates or prodrugs. 4. The compound according to claim 1, wherein the BRD4 inhibitory moiety is selected from the following structures, and structures or groups derived from these structures that can form various types of covalent bonds with the connecting chain: or its pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, racemates or prodrugs. 5. The compound according to claim 3 or 4, wherein the HSP90 inhibitory moiety includes the structure of the NVP-AUY922, AT13387 or STA9090 analog, or a structure derived from the NVP-AUY922, AT13387 or STA9090 analog; BRD4 The inhibitory moiety includes the structure of (+)-JQ-1, or a structure derived from (+)-JQ-1; the connecting chain is selected from chemical bonds, amide bonds, carbonyl groups, C _1-6 alkylene groups and combinations thereof, or its pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, racemates or prodrugs. 6. The compound of claim 3, 4 or 5, wherein, The HSP90 inhibitory moiety consists of a monovalent group obtained by removing one hydrogen atom from the following structure: Among them, R ₁ represents -COOH, R ₂ represents -NHC(=O)CH ₂ CH ₂ COOH, and R ₃ represents -CH ₂ COOH; The BRD4 inhibitory moiety consists of a monovalent group obtained by removing one hydrogen atom from the following structure: The connecting chain is selected from chemical bonds: -NH-(CH ₂ )n-NH-, -NH-(CH ₂ CH ₂ O)n-CH ₂ CH ₂ -NH-; or its pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, racemates or prodrugs. 7. The compound according to any one of the preceding claims, which is HTT-1 to HTT-18 as shown below, or its pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, racemates or prodrugs. 8. A pharmaceutical composition comprising a compound according to any preceding claim or a pharmaceutically acceptable salt, solvate, tautomer, stereoisomer, racemate or prodrugs, and one or more pharmaceutical carriers. 9. The pharmaceutical composition according to claim 8, which further comprises other therapeutic drugs, and the other therapeutic drugs are tumor chemotherapy drugs, tumor targeted drugs, tumor immunotherapy drugs and tumor drug conjugates. 10. Use of a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to any one of claims 8 to 9 for the preparation of a drug for preventing or treating tumors.