A brd9 inhibitor, preparation method and application thereof

Abstract

The embodiment of the application provides a BRD9 inhibitor, a preparation method and application thereof. The BRD9 inhibitor comprises a compound shown in the following formula 1 and / or a salt of the compound shown in the formula 1: formula 1 in the formula 1, L is selected from a cyclic group containing a heteroatom, and the number of carbon atoms of the cyclic group is 1-14, and the heteroatom comprises one or more of O, N and S. The L structure of the BRD9 inhibitor can be flexibly adapted to the active binding site of BRD9, can be efficiently and specifically combined with the BRD9 target and inhibit the biological activity, greatly improves the targeted combination efficiency and inhibition specificity compared with traditional inhibitors; meanwhile, the optimized L structure significantly enhances the in-vivo stability and drugability of the inhibitor, effectively reduces the off-target toxicity, and exhibits more precise targeted treatment efficiency for BRD9-dependent tumors, and the modifiability of the L structure also provides sufficient space for subsequent structure optimization, activity improvement for different tumor subtypes.

Description

Technical Field

[0001] This invention relates to the field of drug synthesis and application technology, and in particular to a BRD9 inhibitor, its preparation method and its application. Background Technology

[0002] Epigenetic regulation is one of the core mechanisms for maintaining normal cellular physiological functions and pathological processes. Among them, the bromodomain, as a "reader" module that specifically recognizes histone acetylated lysine residues, participates in gene transcription regulation by mediating protein-protein interactions and plays an irreplaceable role in key life activities such as cell proliferation, differentiation, and apoptosis. It has become an important research hotspot and drug development target in the field of epigenetics. BRD9 (Bromodomain Containing 9), as a specific subunit of the BAF (BRG1 / BRM-associated factor) chromatin remodeling complex, recruits the BAF complex to specific genomic sites through the specific binding of its bromodomain to histone acetylation markers. With the help of the complex's ATPase activity, it remodels chromatin conformation, thereby regulating the transcriptional activation or inhibition of downstream key genes, and constitutes a core node in the gene expression regulatory network.

[0003] In recent years, the oncogenic mechanism and drug targeting potential of BRD9 have been gradually revealed, promoting the research progress of related inhibitors. Studies have confirmed that BRD9 plays an oncogene role in various malignant tumors: in diseases such as synovial sarcoma and malignant rhabdoid tumor, the loss or mutation of the core subunits of the BAF complex (such as SMARCB1) leads to the dysfunction of the complex, and the survival of tumor cells is highly dependent on the BRD9-stable atypical BAF (ncBAF) complex. This "synthetic lethality" effect provides a unique therapeutic window for precision therapy targeting BRD9; in addition, BRD9 is abnormally highly expressed or hyperfunctional in tumors such as acute myeloid leukemia (AML) and pancreatic cancer, directly driving the proliferation and survival of tumor cells. In existing technologies, designing bifunctional chimeric molecules (i.e., PROTACs, protein degradation-targeting chimeras) to achieve precise degradation of BRD9 has become a new direction for overcoming the aforementioned bottlenecks. These molecules can specifically bind to BRD9 at one end, while the other end targets and recruits naturally occurring E3 ubiquitin ligases in cells. By leveraging the specific ubiquitination labeling function of E3 enzymes on target proteins, BRD9 is guided to be recognized and degraded by the proteasome, achieving an upgrade in target regulation from "activity inhibition" to "complete protein clearance." However, existing BRD9-targeting PROTACs still face key technical challenges: poor adaptability of E3 ubiquitin ligases to binding ligands leads to low synergistic binding efficiency between the dual-targeting molecules and BRD9 and E3; unreasonable linker structure design makes it difficult to balance the binding affinity and degradation kinetics at both ends; and in terms of activity intensity, targeting selectivity, druggability (such as solubility and metabolic stability), and tumor subtype adaptability, they still do not fully meet the needs of clinical translation, indicating significant room for development of novel and highly effective BRD9 inhibitors.

[0004] Therefore, in the research and clinical translation of BRD9 inhibitors, how to design novel target molecules to efficiently inhibit BRD9 activity and synergistically improve targeting selectivity and druggability, while effectively solving the problems of insufficient activity intensity, poor targeting, short druggability and limited tumor subtype adaptability of existing candidate compounds, and further enhancing the precision and safety of treatment for BRD9-dependent tumors, is a technical challenge that urgently needs to be overcome in this field. Summary of the Invention

[0005] This invention provides a BRD9 inhibitor, its preparation method, and its application. The BRD9 inhibitor has excellent target adaptability and target selectivity, which helps to improve the pharmacokinetic stability and drugability of the compound, while also possessing good potential for structural modification and further drug optimization.

[0006] This invention provides a BRD9 inhibitor comprising a compound of Formula 1 and / or a salt of the compound of Formula 1:

[0007] .

[0008] In Formula 1, L is selected from cyclic groups containing heteroatoms, and the number of carbon atoms in the cyclic group is 1 to 14. The heteroatoms include one or more of O, N, and S.

[0009] According to one embodiment of the present invention, the cyclic group is selected from at least one of cycloalkyl, heterocycloalkyl, and aryl groups.

[0010] According to one embodiment of the present invention, the structure of L is selected from one of the following formulas 2-1 to 2-15:

[0011]

[0012] In Equation 2-1, n is 0 or 1; m is an integer from 0 to 4;

[0013] In Formula 2-13, n1 is 0 or 1; n2 is an integer from 0 to 4; R7 is selected from substituted or unsubstituted alkyl groups and substituted or unsubstituted cycloalkyl groups; M is N or C;

[0014] In Equation 2-14, n3 and n4 are independently 0 or 1;

[0015] In Equation 2-15, n5, n6, and n7 are independently 0 or 1; Q is N or C.

[0016] According to one embodiment of the present invention, the structure of the BRD9 inhibitor is selected from one of the following formulas 3-1 to 3-8:

[0017] .

[0018] According to one embodiment of the present invention, the salt of the compound shown in Formula 1 includes a salt formed by the compound shown in Formula 1 and an acid, wherein the acid includes one or more of hydrochloric acid, phosphoric acid, citric acid, and tartaric acid.

[0019] In another aspect, the present invention provides a method for preparing a BRD9 inhibitor, comprising the following steps: mixing compound 5, a bicyclic alkyl carboxylic acid compound, and a urea condensing agent, and performing a condensation reaction to obtain the BRD9 inhibitor; wherein the structural formula of compound 5 is shown in Formula 4:

[0020] Formula 4.

[0021] According to one embodiment of the present invention, the bicyclic alkyl carboxylic acid compound includes bicyclic [1.1.0]butane-1-carboxylic acid.

[0022] According to one embodiment of the present invention, the urea condensing agent comprises 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.

[0023] According to one embodiment of the present invention, the condensation reaction is carried out at a temperature of 25°C to 45°C for a time of 1 h to 3 h.

[0024] According to one embodiment of the present invention, the preparation process of compound 5 includes: mixing compound 4 containing a Boc protecting group with an acidic deprotecting agent to carry out a deprotection reaction of the Boc protecting group to obtain compound 5; wherein, the structural formula of compound 4 containing a Boc protecting group is shown in Formula 5:

[0025] Formula 5.

[0026] According to one embodiment of the present invention, the acidic deprotecting agent comprises a solution of 1,4-dioxane hydrogen chloride.

[0027] According to one embodiment of the present invention, the temperature of the deprotection reaction is 18°C ​​to 25°C, and the time is 1 h to 3 h.

[0028] According to one embodiment of the present invention, the preparation process of compound 4 containing a Boc protecting group includes: mixing compound 3 and a heteroatom-containing cyclic group compound containing an N-Boc protecting group, and carrying out a reduction reaction under the action of a reducing agent to obtain compound 4 containing a Boc protecting group; wherein, the heteroatom-containing cyclic group containing an N-Boc protecting group has the structure of L as described in any one of claims 1-3; the structural formula of compound 3 is shown in Formula 6:

[0029] Formula 6.

[0030] According to one embodiment of the present invention, the reducing agent comprises sodium triacetoxyborohydride.

[0031] According to one embodiment of the present invention, the reduction reaction takes 6 h to 12 h and the temperature is 0 °C to 5 °C.

[0032] According to one embodiment of the present invention, the preparation process of compound 3 includes: mixing a halonaridinone compound, an alkali metal hydride and an alkyl halide, and performing an alkylation reaction to obtain compound 2; mixing compound 2 with a borate ester compound and a haloalkoxy aromatic aldehyde, and performing a Suzuki coupling reaction under the action of a catalyst to obtain compound 3.

[0033] In another aspect, the present invention provides a pharmaceutical composition comprising the BRD9 inhibitor described herein, or a BRD9 inhibitor prepared by the method described herein.

[0034] In another aspect, the present invention provides the use of a BRD9 inhibitor in the preparation of a medicament for the treatment of at least one of leukemia, lymphoma, breast cancer, kidney cancer, colorectal cancer, liver cancer, small cell lung cancer, human bone marrow rhabdomyosarcoma, pancreatic cancer, and malignant glioblastoma.

[0035] The implementation of this invention has at least the following beneficial effects: The BRD9 inhibitor provided by this invention has excellent target adaptation ability and target selectivity, which helps to improve the pharmacokinetic stability and drugability of the compound, while having good potential for structural modification and further drug optimization. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. The specific embodiments listed below are merely descriptions of the principles and features of this invention, and the examples given are only for explaining this invention and are not intended to limit the scope of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0037] This invention provides a BRD9 inhibitor, comprising a compound of Formula 1 and / or a salt of the compound of Formula 1:

[0038] Formula 1;

[0039] In Formula 1, L is selected from cyclic groups containing heteroatoms, and the number of carbon atoms in the cyclic groups is 1 to 14. The heteroatoms include one or more of O, N, and S.

[0040] According to the inventors' research, the aforementioned BRD9 inhibitor contains an L-structure. The heteroatoms and cyclic groups in the L-structure work synergistically to improve the specific binding efficiency and binding strength between the inhibitor and the BRD9 target. Specifically, the L-structure, as the core linker arm of the BRD9 binding backbone, contains O, N, and S heteroatoms with unique electronic properties. These heteroatoms can form specific hydrogen bonds or electrostatic interactions with amino acid residues on the BRD9 bromine domain. Furthermore, the spatially optimized skeleton configuration of the L-structure complements the hydrophobic pocket spatial conformation of the BRD9 bromine domain, effectively reducing steric hindrance and energy loss during the binding of the inhibitor to the BRD9 target. This solves the problems of poor ligand compatibility and low binding rates of traditional inhibitors to the BRD9 target.

[0041] Furthermore, the design of the cyclic group with 1-14 carbon atoms allows the L-structure to extend into the deep region of the BRD9 active site, enhancing the binding strength to the target through hydrophobic interactions. Simultaneously, the spatial rigidity of the cyclic framework promotes the stability of the inhibitor's binding to the BRD9 target, avoiding insufficient binding depth due to excessively short carbon chains or steric hindrance caused by excessively long carbon chains. At the same time, the introduction of heteroatoms (O / N / S) can adjust the electron cloud distribution and chemical bond flexibility of the L-structure, enabling it to maintain a certain rigidity to preserve spatial complementarity during binding, while also possessing appropriate flexibility to dynamically adapt to the conformational fluctuations of BRD9. It is this "balanced adjustment of rigidity and flexibility" that allows the inhibitor to significantly enhance its binding affinity to the BRD9 target and optimize its binding kinetics. The structural diversity and modifiability of the L-structure provide ample space for subsequent structural iterations to optimize the drug-likeness of inhibitors targeting different tumor subtypes.

[0042] In this invention, the number of carbon atoms in the cyclic group is 1 to 14. Here, the number of carbon atoms refers to the total number of carbon atoms contained in the complete L-linking group, not the number of carbon atoms in the cyclic parent ring skeleton. The number of carbon atoms of 1 and 2 corresponds only to the short alkyl linking segments such as methylene and methyl connected to both ends of the cyclic parent ring. The L group is composed of the cyclic parent ring + side / linking short carbon chains, and the total number of carbon atoms of the two is in the range of 1 to 14.

[0043] Specifically, the salts of the compounds shown in Formula 1 above include pharmaceutically acceptable salts.

[0044] In some embodiments, the cyclic group is selected from at least one of cycloalkyl, heterocycloalkyl, and aryl groups. By adjusting the flexibility, rigidity, and electronic properties of the L-structure, the cyclic group enhances the adaptability and stability of the inhibitor's binding to the BRD9 target, optimizes drug development, and simultaneously reserves space for tumor subtype adaptation and structural iteration.

[0045] Specifically, cycloalkyl refers to a saturated cyclic group whose cyclic skeleton consists only of carbon atoms, with heteroatoms attached to the carbon atoms of the cycloalkyl ring as substituents; heterocycloalkyl is a saturated cyclic group whose cyclic skeleton consists of carbon atoms and O, N, and S heteroatoms; aryl includes benzene rings or pyridine rings, with heteroatoms attached to the aromatic ring as substituents.

[0046] In some embodiments, the structure of L described above is selected from one of the following formulas 2-1 to 2-15:

[0047]

[0048] In Equation 2-1, n is 0 or 1; m is an integer from 0 to 4;

[0049] In Formula 2-13, n1 is 0 or 1; n2 is an integer from 0 to 4; R7 is selected from substituted or unsubstituted alkyl groups and substituted or unsubstituted cycloalkyl groups; M is N or C;

[0050] In Equation 2-14, n3 and n4 are independently 0 or 1;

[0051] In Equation 2-15, n5, n6, and n7 are independently 0 or 1; Q is N or C.

[0052] The diverse L structures and substituents of Formulas 2-1 to 2-15, along with the adjustment of the numerical range, can be adapted to the structural characteristics of different BRD9 mutants, enhance the binding complementarity and binding stability between the inhibitor and the BRD9 target, efficiently optimize the binding affinity and druggability of the inhibitor to the BRD9 target, and improve the adaptability and target selectivity of the inhibitor to different tumor subtypes, leaving ample room for subsequent structural optimization.

[0053] When m=n=1 in Equation 2-1, the above cyclic group can also be called a piperazine ring.

[0054] Specifically, the substituents mentioned above include one or more of the following: deuterium, halogen, carbonyl, carboxyl, hydroxyl, amino, nitro, cyano, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 alkoxycarbonyl, C1-C6 amide, C2-C12 ester, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted five- or six-membered heteroaryl, 3-12-membered heterocyclic, and 3-12-membered cycloalkyl.

[0055] In some embodiments, the structure of the above-mentioned BRD9 inhibitor is selected from one of the following formulas 3-1 to 3-8:

[0056] .

[0057] In the structures of the aforementioned BRD9 inhibitors, the L structure is selected from Formulas 2-1 to 2-15. The diversity of the L structure helps to enhance target selectivity to reduce off-target effects, while adapting to the needs of different tumor subtypes and optimizing drug efficacy.

[0058] In some embodiments, the salt of the compound shown in Formula 1 above includes a salt formed by the compound shown in Formula 1 and an acid. The presence of the above-mentioned BRD9 inhibitors in salt form helps to improve the water solubility of the BRD9 inhibitors, helps to meet the requirements of oral formulations, enhances chemical stability and in vivo bioavailability, reduces administration irritation, and is suitable for multiple routes of administration such as oral and injectable, further improving the clinical applicability of BRD9 inhibitors.

[0059] Specifically, the aforementioned acids are pharmaceutically acceptable acids, which may include pharmaceutically acceptable inorganic acids and / or organic acids; the aforementioned inorganic acids include hydrochloric acid and / or phosphoric acid, and the aforementioned organic acids include citric acid and / or tartaric acid.

[0060] In summary, the L structure of the BRD9 inhibitor provided by this invention can flexibly adapt to the active binding site of BRD9, effectively inhibiting the biological activity of BRD9 protein; at the same time, it improves in vivo stability and drug-likeness, enhances target selectivity, reduces off-target toxicity, and has more precise therapeutic efficacy for BRD9-dependent tumors. The modifiability of its L structure also provides ample space for subsequent structural optimization.

[0061] This invention also provides a method for preparing the above-mentioned BRD9 inhibitor, comprising the following steps: mixing compound 5, a bicyclic alkyl carboxylic acid compound, and a urea condensing agent, and performing a condensation reaction to obtain the BRD9 inhibitor; wherein, the structural formula of compound 5 is shown in Formula 4:

[0062] Formula 4.

[0063] This invention utilizes a condensation reaction between compound 5, a bicyclic alkyl carboxylic acid compound, and a urea-based condensing agent to efficiently and gently assemble the molecular backbone, precisely constructing the characteristic structure of the target BRD9 inhibitor. This reaction method exhibits high specificity, stably achieving effective connection between the core structure and the rigid bicyclic structure, reducing byproduct formation, ensuring precise product structure and stable activity, and significantly improving the preparation efficiency and purity of the target product.

[0064] In some embodiments, the above-mentioned bicyclic alkyl carboxylic acid compounds include bicyclic [1.1.0]butane-1-carboxylic acid. The rigid bicyclic structure can fill the spatial gaps of the BRD9 active site, enhancing the binding with BRD9; at the same time, its spatial configuration can synergize with the L structure to optimize the binding angle between the BRD9 inhibitor and the BRD9 target, thereby helping to solve the problems of insufficient inhibitor activity and poor adaptability to the BRD9 target.

[0065] Specifically, the molar ratio of compound 5 to the bicyclic alkyl carboxylic acid compound can be (1~1.5):1, for example 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1 or any range between the above two.

[0066] In some embodiments, the urea condensing agent described above includes 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU). Urea condensing agents facilitate the activation of carboxyl groups to form reactive ester intermediates, accelerating the condensation reaction rate. Simultaneously, they exhibit high reaction selectivity, do not undergo side reactions with heteroatoms of the L-structure, and reduce the formation of byproducts.

[0067] Specifically, the molar ratio of compound 5 to the urea condensing agent can be 1:(1~2), for example 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2 or any two of the above.

[0068] During the above condensation reaction, alkaline substances can be added to neutralize the acid produced by the condensation reaction, which helps to maintain the stability of the reaction environment.

[0069] Specifically, the alkaline substances mentioned above include diisopropylethylamine (DIEA).

[0070] Specifically, the molar ratio of the above-mentioned compound 5 to the above-mentioned alkaline substance can be 1:(3~6), for example 1:3, 1:4, 1:4.5, 1:5, 1:5.5, 1:6 or any two of the above.

[0071] In some embodiments, the temperature of the condensation reaction can be 25°C to 45°C, for example, 25°C, 30°C, 35°C, 40°C, 45°C, or any two of the above; the time can be 1 h to 3 h, for example, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, or any two of the above. Controlling the temperature and time of the condensation reaction within the above ranges helps the condensation reaction to proceed fully, improving the reaction conversion rate and yield, while avoiding over-reaction that leads to impurity formation and product decomposition, thus balancing reaction efficiency and product purity.

[0072] Specifically, the above-mentioned bicycloalkyl carboxylic acid compounds and urea condensing agents were dissolved in an absolutely anhydrous N,N-dimethylformamide (DMF) solution and mixed to obtain a second reaction system; compound 5 and an alkaline substance were added to the second reaction system to carry out a condensation reaction; after the reaction was carried out for 1 h to 3 h, water was added to quench the reaction; the organic phase was extracted, washed, dried, filtered, concentrated and separated by chromatography using ethyl acetate to obtain the above-mentioned BRD9 inhibitor.

[0073] In some embodiments, the preparation process of compound 5 includes: mixing compound 4 containing a Boc protecting group with an acidic deprotecting agent to carry out a deprotection reaction of the Boc protecting group to obtain compound 5; wherein, the structural formula of compound 4 containing a Boc protecting group is shown in Formula 5:

[0074] Formula 5.

[0075] By deprotecting intermediates containing Boc protecting groups, the amino protecting groups can be precisely removed, exposing the active amino sites on the cyclic groups and providing effective reaction sites for subsequent condensation reactions. This deprotection process is mild and thorough, without damaging the overall molecular skeleton structure, and can stably obtain structurally intact and highly pure intermediate compound 5, ensuring the smooth progress of subsequent reactions.

[0076] In some embodiments, the acidic deprotecting agent described above comprises a 1,4-dioxane solution (HCl / 1,4-Dioxane). This acidic deprotecting agent, under mild conditions at room temperature, facilitates the removal of the Boc protecting group from compound 4, exposing the amino active site on the L structure and providing a highly active substrate for subsequent condensation reactions.

[0077] Specifically, the molar concentration of the above-mentioned 1,4-dioxane hydrogen chloride solution can be 2 mol / L to 4 mol / L, for example, 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L or any two of the above.

[0078] Specifically, the molar ratio of the compound 4 containing the Boc protecting group to the acidic deprotecting agent can be 1:(10~20), for example 1:10, 1:12, 1:14, 1:16, 1:18, 1:20 or any range between the above.

[0079] In some embodiments, the temperature of the deprotection reaction can be 18°C ​​to 25°C, for example, 18°C, 20°C, 22°C, 24°C, 25°C, or any two of the above; the time can be 1 h to 3 h, for example, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, or any two of the above. Controlling the temperature and time of the deprotection reaction within the above ranges helps to completely remove the Boc protecting group, improve the yield and purity of the intermediate, and avoids excessive protonation of the amino group and degradation of the product due to excessive reaction time, thus providing a structurally stable and sufficiently active intermediate for subsequent condensation reactions.

[0080] In some embodiments, compound 3 is mixed with a heteroatom-containing cyclic group compound containing an N-Boc protecting group, and a reduction reaction is carried out under the action of a reducing agent to obtain compound 4 containing a Boc protecting group; wherein, the heteroatom-containing cyclic group containing an N-Boc protecting group has the structure of L according to any one of claims 1-3; the structural formula of compound 3 is shown in Formula 6:

[0081] Formula 6.

[0082] By reducing compound 3 with a heteroatom-containing cyclic group containing an N-Boc protecting group in the presence of a reducing agent, a stable and efficient connection between the aromatic aldehyde site and the heterocyclic group can be achieved, precisely constructing a key intermediate framework with a protecting group. This reaction site exhibits high selectivity, stably introducing heteroatom-containing cyclic groups with excellent target adaptability, laying the structural foundation for the final product's excellent BRD9 inhibitory activity, while effectively improving the structural regularity and preparation stability of the intermediate.

[0083] Specifically, the molar ratio of the above-mentioned compound 3 to the above-mentioned compound containing a heteroatom-containing cyclic group with an N-Boc protecting group can be 1:(1~1.5), for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5 or any range between the above two.

[0084] In some embodiments, the reducing agent includes sodium triacetoxyborohydride. Sodium triacetoxyborohydride has mild reducing properties, which helps to improve the selectivity of the reduction reaction, increase product purity, and reduce subsequent purification costs; the mild reducing properties can protect the functional groups in compound 3 from being reduced, which is beneficial to maintaining the structural integrity of the raw materials and products.

[0085] Specifically, the molar ratio of the above-mentioned compound 3 to the above-mentioned reducing agent can be 1:(2.5~3.5), for example 1:2.5, 1:2.7, 1:2.9, 1:3.3, 1:3.5 or any range between the above two.

[0086] Specifically, the reducing agent is added in 2 to 3 batches to the reaction system containing compound 3 and a heteroatom-containing cyclic group containing an N-Boc protecting group in order to control the reaction rate.

[0087] In some embodiments, the reduction reaction time can be 6 h to 12 h, for example, 6 h, 8 h, 10 h, 11 h, 12 h, or any two of the above; the temperature can be 0 °C to 5 °C, for example, 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, or any two of the above. The reduction reaction can also be carried out under ice bath conditions. Carrying the reduction reaction at low temperatures helps suppress the formation of side reactions and improve product purity; a time of not less than 6 h helps ensure the reaction proceeds fully, avoiding waste of raw materials due to incomplete reaction; a time of not more than 12 h helps reduce energy consumption and production cycle costs.

[0088] Specifically, the process of mixing compound 3 with a heteroatom-containing cyclic group compound containing an N-Boc protecting group and carrying out a reduction reaction under the action of a reducing agent includes: dissolving compound 3 and the heteroatom-containing cyclic group compound containing an N-Boc protecting group in an absolutely anhydrous dichloromethane solvent and mixing them to obtain a first reaction system; adding a reducing agent in batches to the first reaction system under ice bath conditions to carry out a reduction reaction; quenching the reaction with water after 1 h to 3 h; and using dichloromethane to extract, wash, dry, filter, concentrate, and separate the organic phase by chromatography to obtain the above-mentioned compound 4 containing a Boc protecting group.

[0089] According to the inventors' research, this preparation method constructs the core structure of the BRD9 inhibitor through a three-step synergistic process of "reduction → deprotection → condensation," enhancing the binding compatibility and binding strength between the BRD9 inhibitor and the BRD9 target. The product purity and yield are stable, making it suitable for large-scale production. Specifically, through a reduction reaction, an L-structure containing an N-Boc protecting group is introduced into compound 3. The introduction of the N-Boc protecting group helps avoid side reactions of the heteroatoms in the L-structure during the reaction, preserving an intact active site for subsequent binding to the BRD9 target. It also helps enhance molecular flexibility to adapt to the binding conformation of the BRD9 target protein. Under the action of an acidic deprotecting agent, the Boc protecting group is removed, exposing the amino group of the L-structure, providing a highly active substrate for the subsequent condensation reaction. A urea-based condensing agent activates the carboxyl group of the bicyclic alkyl carboxylic acid compound, promoting the condensation reaction between the amino group of compound 5 and the bicyclic alkyl carboxylic acid compound. The rigid structure of the bicyclic alkyl carboxylic acid compound enhances the spatial complementarity with the BRD9 active site, helping to solve the problem of insufficient inhibitor activity against the BRD9 target.

[0090] In some embodiments, compound 3 is prepared by a process including the following steps: mixing a halonaridinone compound, an alkali metal hydride, and an alkyl halide, and performing an alkylation reaction to obtain compound 2; mixing compound 2 with a borate ester compound and a haloalkoxy aromatic aldehyde, and performing a Suzuki coupling reaction under the action of a catalyst to obtain compound 3.

[0091] This invention employs a stepwise synergistic approach combining alkylation and Suzuki coupling reactions. First, alkylation modifies the parent alkyl group of a halonaphthidium compound; then, Suzuki coupling is used to achieve precise directional assembly of the aryl fragment, efficiently yielding compound 3. This process exhibits excellent reaction selectivity, helping to reduce side reactions and impurity formation, and improving the structural accuracy and purity of compound 3.

[0092] Specifically, the aforementioned halonaphthidone compounds include 4-iodo-2,7-naphthid-1(2H)-one. Halonaphthidone compounds are readily activated by alkali metal hydrides in alkylation reactions, which helps improve the reaction efficiency; simultaneously, they are compatible with the catalytic system of subsequent Suzuki coupling reactions, enhancing the conversion rate of aryl fragment splicing. This substrate exhibits structural stability and strong reaction compatibility, effectively reducing side reactions and providing reliable assurance for the precise synthesis of subsequent intermediates and the pharmacodynamic stability of the final product.

[0093] The structural formula of the above-mentioned 4-iodo-2,7-naphthidine-1(2H)-one is shown in Formula 7:

[0094] Formula 7.

[0095] In some embodiments, the alkali metal hydride includes NaH. As a strongly basic alkali metal hydride, NaH facilitates the formation of a nitrogen anion intermediate with excellent stability, significantly enhancing the nucleophilic reactivity of the substrate and contributing to the improved synthesis conversion rate of compound 2.

[0096] Specifically, the molar ratio of the above-mentioned halonaridinone compounds to the above-mentioned alkali metal hydrides can be 1:(1~3), for example 1:1, 1:1.5, 1:2, 1:2.5, 1:3 or any two of the above.

[0097] In some embodiments, the alkyl halide includes CH3I. The addition of the alkyl halide facilitates the N-alkylation conversion of halonaphthidium ketones, improves the conversion and selectivity of the alkylation reaction, reduces the formation of byproducts, and helps to increase the structural purity and yield of compound 2.

[0098] Specifically, the molar ratio of the above-mentioned halonaridinone compounds to the above-mentioned alkyl halides can be 1:(1~2.5), for example 1:1, 1:1.5, 1:2, 1:2.5 or any range between the above two.

[0099] Specifically, the temperature of the alkylation reaction is maintained below 0°C; the alkylation reaction time can be 3 h to 7 h, for example, 3 h, 4 h, 5 h, 6 h, 7 h or any two of the above.

[0100] In some embodiments, the above-mentioned haloalkoxy aromatic aldehydes include 4-bromo-2,6-dimethoxybenzaldehyde. As a haloalkoxy aromatic aldehyde, the bromine atom on its benzene ring is a highly reactive electrophilic reaction site, which can undergo a palladium-catalyzed Suzuki coupling reaction to achieve efficient coupling with compound 2.

[0101] Specifically, the structural formula of the above-mentioned 4-bromo-2,6-dimethoxybenzaldehyde is shown in Formula 8:

[0102] Formula 8.

[0103] Specifically, the molar ratio of compound 2 to the haloalkoxy aromatic aldehyde can be 1:(1~1.5), for example 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5 or any range between the above.

[0104] In some embodiments, the above-mentioned borate esters include bispinacol borate. As a borate ester, bispinacol borate exhibits moderate reactivity and excellent stability, enabling it to react efficiently with haloalkoxy aromatic aldehydes under catalytic conditions to directionally generate arylborate intermediates. The compound possesses suitable steric hindrance, effectively suppressing side reactions and improving the synthesis efficiency and purity of the intermediate. This provides a structurally stable and reactivity-matched coupling substrate for the subsequent Suzuki coupling reaction with compound 2.

[0105] Specifically, the molar ratio of compound 2 to the borate ester compound can be 1:(1~1.5), for example 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5 or any range between the above.

[0106] In some embodiments, the catalyst described above comprises Pd(dppf)Cl2. This catalyst exhibits high catalytic activity and outstanding selectivity, and can efficiently catalyze the Suzuki coupling reaction between the arylboronic ester intermediate and compound 2, thereby helping to lower the activation energy and accelerate the reaction rate.

[0107] Specifically, the molar ratio of compound 2 to the catalyst can be 1:(0.05~0.2), for example 1:0.05, 1:0.1, 1:0.14, 1:0.18, 1:0.2 or any two of the above.

[0108] In the above condensation reaction, alkali metal carboxylates and / or inorganic bases can be added to neutralize the acid produced by the Suzuki coupling reaction, helping to maintain the stability of the reaction environment. Simultaneously, this helps to improve the catalytic efficiency of the catalyst and facilitates the stable formation of the arylboronic ester intermediate. Specifically, the alkali metal carboxylates include KOAc; the inorganic bases include K₂CO₃.

[0109] Specifically, the molar ratio of the above-mentioned compound 2 to the above-mentioned alkali metal carboxylate can be 1:(2~4), for example, 1:2, 1:2.5, 1:3, 1:3.5, 1:4 or any two of the above; the molar ratio of the above-mentioned compound 2 to the above-mentioned inorganic base can be 1:(2~3), for example, 1:2, 1:2.5, 1:3 or any two of the above.

[0110] Specifically, a halonaridinone compound was dissolved in an absolutely anhydrous N,N-dimethylformamide (DMF) solution, and an alkali metal hydride was added in batches at 0℃~3℃, with stirring for 20 min~40 min to obtain a third reaction system. An alkyl halide was then added dropwise to the third reaction system, and an alkylation reaction was carried out at 0℃~3℃ for 3 h~7 h. After filtration and drying, compound 2 was obtained. Under an inert atmosphere, a haloalkoxy aromatic aldehyde, a borate ester compound, an alkali metal carboxylate, and a catalyst were dissolved in an anhydrous 1,4-dioxane solution and mixed to obtain a fourth reaction system. The fourth reaction system was heated at 95℃~105℃ for 6 h~24 h. Compound 2, an inorganic base, deionized water, and a catalyst were then added to this reaction system, and a Suzuki coupling reaction was carried out at 95℃~105℃ for 6 h~24 h. After the reaction, the reaction product was filtered, washed, dried, concentrated, and separated by chromatography to obtain compound 3.

[0111] Specifically, the molar concentration of the absolutely anhydrous N,N-dimethylformamide solution can be 0.05 mol / L to 0.35 mol / L, for example, 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.35 mol / L, or any two of the above; the molar concentration of the anhydrous 1,4-dioxane solution can be 0.05 mol / L to 0.2 mol / L, for example, 0.05 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, or any two of the above.

[0112] Specifically, in the step of filtering the reaction products, diatomaceous earth (Celite) can be used as the filter medium; in the step of washing the reaction products, dichloromethane (DCM) can be used as the washing agent.

[0113] Specifically, the preparation process of the BRD9 inhibitor of the present invention is carried out in an inert gas atmosphere; the inert gas includes nitrogen.

[0114] Specifically, in the preparation process of the BRD9 inhibitor of the present invention, the drying steps of the reaction products involved all use anhydrous sodium sulfate as a drying agent.

[0115] In the specific implementation, under an inert gas atmosphere, halonaridinone compounds are dissolved in an absolutely anhydrous N,N-dimethylformamide (DMF) solution. Alkali metal hydrides are added in batches at 0°C to 3°C, and the mixture is stirred for 20 to 40 minutes to obtain a third reaction system. Alkyl halides are then added dropwise to the third reaction system, and an alkylation reaction is carried out at 0°C to 3°C for 3 to 7 hours. Deionized water is added to form a precipitate. The precipitate is filtered and dried to obtain compound 2. Haloalkoxy aromatic aldehydes, borate esters, alkali metal carboxylates, and catalysts are dissolved in anhydrous 1,4-dioxane solution and mixed to obtain a fourth reaction system. The fourth reaction system is heated at 95°C to 105°C for 6 to 24 hours. The temperature of the reaction system is cooled to room temperature, and then compound 2, an inorganic base, deionized water, and a catalyst are added to the reaction system. A Suzuki coupling reaction is carried out at 95°C to 105°C for 6 to 24 hours. h; After the coupling reaction is completed, the reaction product is filtered, washed, dried, concentrated, and separated by chromatography using DCM to obtain compound 3; Compound 3 and a heteroatom-containing cyclic compound containing an N-Boc protecting group are dissolved in anhydrous dichloromethane solvent and mixed to obtain the first reaction system; Under ice bath conditions, a reducing agent is added in batches to the first reaction system to carry out a reduction reaction, and after 1 h to 3 h, water is added to quench the reaction; The organic phase of the reaction is separated, and the organic phase is extracted with dichloromethane, washed with saturated NaCl solution, dried, filtered, concentrated, and separated by chromatography to obtain compound 4 containing a Boc protecting group; Compound 4 containing a Boc protecting group is mixed with an acidic deprotecting agent at room temperature to carry out a deprotection reaction of the Boc protecting group, and after 1 h to 4 h, the solvent in the reaction system is dried by rotation to obtain compound 5; The above bicyclic alkyl carboxylic acid compound and urea condensing agent are dissolved in anhydrous N,N-dimethylformamide (DMF) solution and mixed at room temperature for 5 h. The second reaction system was obtained after 15 min; compound 5 and an alkaline substance were added to the second reaction system to carry out a condensation reaction; after 1 h to 3 h, water was added to quench the reaction; the organic phase was extracted, washed, dried, filtered, concentrated and separated by chromatography using ethyl acetate to obtain the above BRD9 inhibitor.

[0116] This invention also provides a pharmaceutical composition comprising the above-described BRD9 inhibitor or the BRD9 inhibitor prepared by the above-described preparation method.

[0117] The pharmaceutical composition provided by this invention contains a high-purity, structurally precise, and activity-stable BRD9 inhibitor prepared by the method of this invention. It can specifically target the BRD9 protein, efficiently block its binding to acetylated histones, inhibit the abnormal activation of downstream oncogenic signaling pathways, and thereby regulate the expression of genes related to tumor cell proliferation, apoptosis, and invasion, thus exerting significant anti-tumor activity. At the same time, the composition has good drug-like properties, excellent biocompatibility and formulation stability, which helps to achieve effective delivery and in vivo action of the inhibitor, providing a safe and effective treatment option for BRD9-target-related malignant tumors and inflammatory diseases. Moreover, the preparation process ensures the uniformity of inhibitor quality, resulting in good batch-to-batch stability of the pharmaceutical composition, and it has good potential for clinical translation and large-scale application.

[0118] This invention also provides an application of the above-mentioned BRD9 inhibitor in the preparation of a drug, which is used to treat at least one of leukemia, lymphoma, breast cancer, kidney cancer, colorectal cancer, liver cancer, small cell lung cancer, human bone marrow rhabdomyosarcoma, pancreatic cancer, and malignant glioblastoma.

[0119] The BRD9 inhibitor provided by this invention can help regulate tumor-related signaling pathways by targeting and inhibiting the function of the BRD9 protein. It also has good target selectivity and can reduce the toxic side effects on normal tissues while exerting strong anti-tumor activity. It provides a new targeted therapy candidate drug and clinical treatment strategy for the above-mentioned malignant tumors, and has important clinical application value and translational prospects.

[0120] The present invention will be further described below through specific embodiments.

[0121] Example 1

[0122]

[0123] S1. As shown in synthetic route 1, in a 25 mL round-bottom flask under a N2 atmosphere, 4-iodo-2,7-naphthidine-1(2H)-one 1 (544 mg, 2 mmol, 1.0 eq) was dissolved in anhydrous DMF (10 mL, 0.2 mol / L). NaH (4 mmol, 2.0 eq) was added in portions at 0 °C, and the mixture was stirred for 30 min to obtain the third reaction system. CH3I (3 mmol, 1.5 eq) was then added dropwise to the third reaction system, and an alkylation reaction was carried out at 0 °C for 5 h. Deionized water was added to form a precipitate. The precipitate was filtered and dried to obtain compound 2 (300 mg, 52%). Electrospray ionization mass spectrometry (ESI-MS) m / z 287.1 [M + H] + ;

[0124] S2. In a 25 mL round-bottom flask under a N2 atmosphere, 4-bromo-2,6-dimethoxybenzaldehyde (490 mg, 2 mmol, 1.0 eq) was dissolved in anhydrous 1,4-dioxane (10 mL, 0.1 mol / L). Pd(dppf)Cl2 (146 mg, 0.2 mmol, 0.1 eq), bis(pinacol)boronic acid ester (610 mg, 2.4 mmol, 1.2 eq), and KOAc (588 mg, 6 mmol, 3.0 eq) were added and mixed to obtain the fourth reaction system. The fourth reaction system was heated at 100 °C for 12 h. The temperature of the reaction system was cooled to room temperature, and then compound 2 (572 mg, 2 mmol, 1.0 eq), K2CO3 (504 mg, 4 mmol, 2.0 eq), deionized water (1 mL), and Pd(dppf)Cl2 (146 mg, 0.2 mmol, 0.1 eq) were added to the reaction system. (mg, 0.2 mmol, 0.1 eq) were subjected to a Suzuki coupling reaction at 100 °C for 12 h. After the coupling reaction was completed, the reaction product was filtered with diatomaceous earth, washed with DCM, dried and concentrated with anhydrous sodium sulfate. Finally, the concentrate was separated by silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain the above compound 3.

[0125] S3. In a 25 mL round-bottom flask, compound 3 (504 mg, 1.5 mmol, 1.0 eq) and N-Boc piperazine (335 mg, 1.8 mmol, 1.2 eq) were dissolved in absolutely anhydrous dichloromethane (15 mL) and mixed to obtain the first reaction system. Under ice bath conditions, sodium triacetoxyborohydride (954 mg, 4.5 mmol, 3.0 eq) was added in portions to the first reaction system to carry out the reduction reaction. After 6 h of reaction, water (10 mL) was added to quench the reaction. The organic phase of the reaction was separated and extracted three times with dichloromethane (10 mL), washed three times with saturated NaCl solution, and then dried with anhydrous sodium sulfate. The mixture was filtered and concentrated. Finally, the concentrate was separated by silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain compound 4 (PC01-1) containing the Boc protecting group.

[0126] S4. In a 10 mL round-bottom flask, PC01-1 (99 mg, 0.2 mmol) and HCl / 1,4-Dioxane (4 mol / L, 2 mL) were mixed at room temperature (25 °C) to carry out the deprotection reaction of the Boc protecting group. The reaction was carried out for 2 h, and the solvent in the reaction system was dried by rotating to obtain compound 5 (PC01-2, 69 mg, yield 80%).

[0127] S5. In a 10 mL round-bottom flask, bicyclo[1.1.0]butane-1-carboxylic acid (9.8 mg, 0.1 mmol, 1.0 eq) and HATU (57 mg, 0.15 mmol, 1.5 eq) were dissolved in anhydrous DMF (2 mL) solution and mixed at room temperature (25 °C) for 10 min to obtain a second reaction system. PC01-2 (60 mg, 0.12 mmol, 1.0 eq) and DIEA (34.5 mg, 0.5 mmol, 5.0 eq) were added to the second reaction system to carry out a condensation reaction. After 2 h of reaction, water (2 mL) was added to quench the reaction. The organic phase was extracted three times with ethyl acetate (3 mL), and the organic phases were combined and then extracted successively with water (3 mL) and saturated brine (3 mL). The sample was washed three times with anhydrous sodium sulfate, then dried, filtered, and concentrated. Finally, the concentrate was subjected to silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain the BRD9 inhibitor, named PC01 (41 mg, yield 86%, ESI-MS: m / z 457.2 [M + H]). + ).

[0128] PC01: 1 H NMR (500 MHz, CDCl3) δ 9.22 (d, J = 1.4 Hz, 1H), 8.66 (dd, J= 4.6, 1.5 Hz, 1H), 7.88 (q, J = 1.0 Hz, 1H), 7.29 (d, J = 4.6 Hz, 1H), 6.83(s, 2H), 3.89 (d, J = 6.2 Hz, 8H), 3.60 (d, J = 1.1 Hz, 3H), 3.56 – 3.41 (m,4H), 2.71 – 2.64 (m, 4H), 2.03 (dd, J = 6.1, 4.1 Hz, 2H), 1.78 (dd, J = 6.1,4.1 Hz, 2H), 1.70 (t, J = 4.0 Hz, 4H), 1.63 (q, J = 4.2 Hz, 1H), 1.59 – 1.53(m, 4H).

[0129] The electrospray ionization mass spectrometry (ESI-MS) and proton nuclear magnetic resonance spectra of the BRD9 inhibitors prepared in Example 1, the following examples, and the comparative examples were determined using the following methods:

[0130] 1. ESI-MS Determination Method: Weigh the BRD9 inhibitor, dissolve and dilute it with chromatographically pure methanol to prepare a 1 mg / mL test solution. Filter the solution through a 0.22 μm organic phase filter membrane and perform the determination using an electrospray ionization mass spectrometer (Agilent 1200LC / G6110A, XBridge column, 4.6 mm × 50 mm, 3.5 μm, column temperature 50°C, flow rate 1.8 mL / min). The instrument is set to positive ion ionization mode (ESI). + The drying gas temperature was set to 325℃, the drying gas flow rate to 8 L / min, the nebulizing gas pressure to 25 psi, the capillary voltage to 3.5 kV, and the mass scan range to 100~800 m / z. Data was acquired in full scan mode, the quasi-molecular ion peak was recorded, and the molecular ion mass-to-charge ratio of the compound was confirmed.

[0131] 2. 1 The method for H NMR determination: Weigh 8 mg of BRD9 inhibitor and place it in an NMR sample tube. Add 0.6 mL of deuterated chloroform (CDCl3) and sonicate until completely dissolved. Use tetramethylsilane (TMS) as an internal standard (chemical shift δ = 0.00 ppm). Measure the NMR using a 500 MHz NMR spectrometer (Bruker-500 MHz NMR spectrometer) with 16 scans. After acquisition, Fourier transform, phase correction, baseline correction, and integration are performed using the instrument's software. Record the chemical shift (δ), coupling constant (J), and integration area of ​​each hydrogen atom to resolve the hydrogen nucleus structure information of the compound.

[0132] Example 2

[0133]

[0134] Steps S1 to S2 are the same as in Example 1;

[0135] S3. As shown in synthetic route 2, in a 25 mL round-bottom flask, compound 3 (504 mg, 1.5 mmol, 1.0 eq) and 3-Boc-3,9-diazaspiro[5.5]undecane (457 mg, 1.8 mmol, 1.2 eq) were dissolved in absolutely anhydrous dichloromethane (15 mL) and mixed to obtain the first reaction system. Under ice bath conditions, sodium triacetoxyborohydride (954 mg, 4.5 mmol, 3.0 eq) was added in portions to the first reaction system to carry out the reduction reaction. After 6 h of reaction, water (10 mL) was added to quench the reaction. The organic phase of the reaction was separated using dichloromethane (10 mL). The organic phase was extracted three times with anhydrous sodium sulfate (mL), washed three times with saturated NaCl solution, dried with anhydrous sodium sulfate, filtered, and concentrated. Finally, the concentrate was separated by silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain compound 4 (PC02-1) containing a Boc protecting group.

[0136] S4. In a 10 mL round-bottom flask, PC02-1 (113 mg, 0.2 mmol) and HCl / 1,4-Dioxane (4 mol / L, 2 mL) were mixed at room temperature (25 °C) to carry out the deprotection reaction of the Boc protecting group. The reaction was carried out for 2 h, and the solvent in the reaction system was dried by rotation to obtain compound 5 (PC02-2, 91 mg, yield 91%).

[0137] S5. In a 10 mL round-bottom flask, bicyclo[1.1.0]butane-1-carboxylic acid (9.8 mg, 0.1 mmol, 1.0 eq) and HATU (57 mg, 0.15 mmol, 1.5 eq) were dissolved in anhydrous DMF (2 mL) solution and mixed at room temperature (25 °C) for 10 min to obtain a second reaction system. PC02-2 (60 mg, 0.12 mmol, 1.2 eq) and DIEA (34.5 mg, 0.5 mmol, 5.0 eq) were added to the second reaction system to carry out a condensation reaction. After 2 h of reaction, water (2 mL) was added to quench the reaction. The organic phase was extracted three times with ethyl acetate (3 mL), and the organic phases were combined and then extracted successively with water (3 mL) and saturated brine (3 mL). The sample was washed three times with anhydrous sodium sulfate, then dried, filtered, and concentrated. Finally, the concentrate was subjected to silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain the BRD9 inhibitor, named PC02 (41 mg, yield 76%, ESI-MS: m / z 543.3 [M + H]). + ).

[0138] PC02:1 H NMR (500 MHz, CDCl3) δ 9.22 (d, J = 1.4 Hz, 1H), 8.66 (dd, J= 4.6, 1.5 Hz, 1H), 7.88 (q, J = 1.0 Hz, 1H), 7.29 (d, J = 4.6 Hz, 1H), 6.83(s, 2H), 3.90 (d, J = 4.8 Hz, 8H), 3.60 (d, J = 1.1 Hz, 3H), 3.53 – 3.48 (m,4H), 2.73 – 2.68 (m, 4H), 2.03 (dd, J = 6.1, 4.1 Hz, 2H), 1.78 (dd, J = 6.1,4.1 Hz, 2H), 1.62 (p, J = 4.1 Hz, 1H).

[0139] Example 3

[0140]

[0141] Steps S1 to S2 are the same as in Example 1;

[0142] S3. As shown in synthetic route 3, in a 25 mL round-bottom flask, compound 3 (504 mg, 1.5 mmol, 1.0 eq) and 8-Boc-2,8-diazaspiro[4.5]decane (432 mg, 1.8 mmol, 1.2 eq) were dissolved in absolutely anhydrous dichloromethane (15 mL) and mixed to obtain the first reaction system. Under ice bath conditions, sodium triacetoxyborohydride (954 mg, 4.5 mmol, 3.0 eq) was added in portions to the first reaction system to carry out the reduction reaction. After 6 h of reaction, water (10 mL) was added to quench the reaction. The organic phase of the reaction was separated using dichloromethane (10 mL). The organic phase was extracted three times with a solution of saturated NaCl solution, washed three times, dried with anhydrous sodium sulfate, filtered, and concentrated. Finally, the concentrate was separated by silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain compound 4 (PC03-1) containing a Boc protecting group.

[0143] S4. In a 10 mL round-bottom flask, PC03-1 (110 mg, 0.2 mmol) and HCl / 1,4-Dioxane (4 mol / L, 2 mL) were mixed at room temperature (25 °C) to carry out the deprotection reaction of the Boc protecting group. The reaction was carried out for 2 h, and the solvent in the reaction system was dried by rotating to obtain compound 5 (PC03-2, 83 mg, yield 86%).

[0144] S5. In a 10 mL round-bottom flask, bicyclo[1.1.0]butane-1-carboxylic acid (9.8 mg, 0.1 mmol, 1.0 eq) and HATU (57 mg, 0.15 mmol, 1.5 eq) were dissolved in anhydrous DMF (2 mL) solution and mixed at room temperature (25 °C) for 10 min to obtain a second reaction system. PC03-2 (58 mg, 0.12 mmol, 1.2 eq) and DIEA (34.5 mg, 0.5 mmol, 5.0 eq) were added to the second reaction system to carry out a condensation reaction. After 2 h of reaction, water (2 mL) was added to quench the reaction. The organic phase was extracted three times with ethyl acetate (3 mL), and the organic phases were combined and then extracted successively with water (3 mL) and saturated brine (3 mL). The sample was washed three times with anhydrous sodium sulfate, then dried, filtered, and concentrated. Finally, the concentrate was subjected to silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain the BRD9 inhibitor, named PC03 (43 mg, yield 81%, ESI-MS: m / z 529.3 [M + H]). + ).

[0145] PC03: 1 H NMR (500 MHz, CDCl3) δ 9.22 (d, J = 1.4 Hz, 1H), 8.66 (dd, J= 4.6, 1.5 Hz, 1H), 7.88 (q, J = 1.0 Hz, 1H), 7.29 (d, J = 4.6 Hz, 1H), 6.83(s, 2H), 3.91 (d, J = 12.8 Hz, 8H), 3.70 – 3.59 (m, 5H), 3.31 (ddd, J = 12.3,5.3, 2.7 Hz, 2H), 2.70 (dd, J = 4.1, 3.0 Hz, 2H), 2.60 (d, J = 0.9 Hz, 2H),2.03 (dd, J = 6.1, 4.1 Hz, 2H), 1.86 – 1.75 (m, 4H), 1.69 – 1.52 (m, 5H).

[0146] Example 4

[0147]

[0148] Steps S1 to S2 are the same as in Example 1;

[0149] S3. As shown in synthetic route 4, in a 25 mL round-bottom flask, compound 3 (504 mg, 1.5 mmol, 1.0 eq) and 7-Boc-2,7-diazaspiro[3.5]nonane (407 mg, 1.8 mmol, 1.2 eq) were dissolved in absolutely anhydrous dichloromethane (15 mL) and mixed to obtain the first reaction system. Under ice bath conditions, sodium triacetoxyborohydride (954 mg, 4.5 mmol, 3.0 eq) was added in portions to the first reaction system to carry out the reduction reaction. After 6 h of reaction, water (10 mL) was added to quench the reaction. The organic phase of the reaction was separated using dichloromethane (10 mL). The organic phase was extracted three times with anhydrous sodium sulfate (mL), washed three times with saturated NaCl solution, dried with anhydrous sodium sulfate, filtered, and concentrated. Finally, the concentrate was separated by silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain compound 4 (PC04-1) containing a Boc protecting group.

[0150] S4. In a 10 mL round-bottom flask, PC04-1 (107 mg, 0.2 mmol) was mixed with HCl / 1,4-Dioxane (4 mol / L, 2 mL) at room temperature to carry out the deprotection reaction of the Boc protecting group. The reaction was carried out for 2 h, and the solvent in the reaction system was dried by rotation to obtain compound 5 (PC04-2, 75 mg, yield 80%).

[0151] S5. In a 10 mL round-bottom flask, bicyclo[1.1.0]butane-1-carboxylic acid (9.8 mg, 0.1 mmol, 1.0 eq) and HATU (57 mg, 0.15 mmol, 1.5 eq) were dissolved in anhydrous DMF (2 mL) solution and mixed at room temperature (25 °C) for 10 min to obtain a second reaction system. PC04-2 (56 mg, 0.12 mmol, 1.2 eq) and DIEA (34.5 mg, 0.5 mmol, 5.0 eq) were added to the second reaction system to carry out a condensation reaction. After 2 h of reaction, water (2 mL) was added to quench the reaction. The organic phase was extracted three times with ethyl acetate (3 mL), and the organic phases were combined and then extracted successively with water (3 mL) and saturated brine (3 mL). The sample was washed three times with anhydrous sodium sulfate, then dried, filtered, and concentrated. Finally, the concentrate was subjected to silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain the BRD9 inhibitor, named PC04 (41 mg, yield 79%, ESI-MS: m / z 515.3 [M + H]). + ).

[0152] PC04:1 H NMR (500 MHz, CDCl3) δ 9.22 (d, J = 1.4 Hz, 1H), 8.66 (dd, J= 4.6, 1.5 Hz, 1H), 7.88 (q, J = 1.0 Hz, 1H), 7.29 (d, J = 4.6 Hz, 1H), 6.83(s, 1.78 (dd, J = 6.0, 4.1Hz, 2H), 1.74 (t, J = 3.3 Hz, 4H), 1.62 (p, J = 4.1 Hz, 1H).

[0153] Example 5

[0154]

[0155] Steps S1 to S2 are the same as in Example 1;

[0156] S3. As shown in synthetic route 5, in a 25 mL round-bottom flask, compound 3 (504 mg, 1.5 mmol, 1.0 eq) and tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (356 mg, 1.8 mmol, 1.2 eq) were dissolved in absolutely anhydrous dichloromethane (15 mL) and mixed to obtain the first reaction system. Under ice bath conditions, sodium triacetoxyborohydride (954 mg, 4.5 mmol, 3.0 eq) was added in portions to the first reaction system to carry out the reduction reaction. After 6 h of reaction, water (10 mL) was added to quench the reaction. The organic phase of the reaction was separated using dichloromethane (10 mL). The organic phase was extracted three times with anhydrous sodium sulfate (mL), washed three times with saturated NaCl solution, dried with anhydrous sodium sulfate, filtered, and concentrated. Finally, the concentrate was separated by silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain compound 4 (PC05-1) containing a Boc protecting group.

[0157] S4. In a 10 mL round-bottom flask, PC05-1 (101 mg, 0.2 mmol) and HCl / 1,4-Dioxane (4 mol / L, 2 mL) were mixed at room temperature (25 °C) to carry out the deprotection reaction of the Boc protecting group. The reaction was carried out for 2 h, and the solvent in the reaction system was dried by rotating to obtain compound 5 (PC05-2, 69 mg, yield 78%).

[0158] S5. In a 10 mL round-bottom flask, bicyclo[1.1.0]butane-1-carboxylic acid (9.8 mg, 0.1 mmol, 1.0 eq) and HATU (57 mg, 0.15 mmol, 1.5 eq) were dissolved in anhydrous DMF (2 mL) solution and mixed at room temperature (25 °C) for 10 min to obtain a second reaction system. PC05-2 (53 mg, 0.12 mmol, 1.2 eq) and DIEA (34.5 mg, 0.5 mmol, 5.0 eq) were added to the second reaction system to carry out a condensation reaction. After 2 h of reaction, water (2 mL) was added to quench the reaction. The organic phase was extracted three times with ethyl acetate (3 mL), and the organic phases were combined and then extracted successively with water (3 mL) and saturated brine (3 mL). The sample was washed three times with anhydrous sodium sulfate, then dried, filtered, and concentrated. Finally, the concentrate was subjected to silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain the BRD9 inhibitor, named PC05 (41 mg, yield 84%, ESI-MS: m / z 487.1 [M + H]). + ).

[0159] PC05: 1 H NMR (500 MHz, CDCl3) δ 9.43 (s, 1H), 8.44 (d, J = 7.5 Hz,1H), 8.15 (s, 1H), 7.54 (d, J = 7.4 Hz, 1H), 6.76 (s, 2H), 3.81 (s, 6H), 3.72(s, 4H), 3.66 (s, 2H), 3.64 (s, 3H), 3.22 (s, 4H), 1.98 (p, J = 10.6 Hz, 1H), 1.29 (dd, J = 12.3, 10.6 Hz, 2H), 1.04 (dd, J = 12.4, 10.6 Hz, 2H).

[0160] Example 6

[0161]

[0162] Steps S1 to S2 are the same as in Example 1;

[0163] S3. As shown in synthetic route 6, in a 25 mL round-bottom flask, compound 3 (504 mg, 1.5 mmol, 1.0 eq) and 6-Boc-2,6-diazaspiro[3.4]octane (382 mg, 1.8 mmol, 1.2 eq) were dissolved in absolutely anhydrous dichloromethane (15 mL) and mixed to obtain the first reaction system. Under ice bath conditions, sodium triacetoxyborohydride (954 mg, 4.5 mmol, 3.0 eq) was added in portions to the first reaction system to carry out the reduction reaction. After 6 h of reaction, water (10 mL) was added to quench the reaction. The organic phase of the reaction was separated using dichloromethane (10 mL). The organic phase was extracted three times with anhydrous sodium sulfate (mL), washed three times with saturated NaCl solution, dried with anhydrous sodium sulfate, filtered, and concentrated. Finally, the concentrate was separated by silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain compound 4 (PC06-1) containing a Boc protecting group.

[0164] S4. In a 10 mL round-bottom flask, PC06-1 (104 mg, 0.2 mmol) was mixed with HCl / 1,4-Dioxane (4 mol / L, 2 mL) at room temperature (25 °C) to carry out the deprotection reaction of the Boc protecting group. The reaction was carried out for 2 h, and the solvent in the reaction system was dried by rotating to obtain compound 5 (PC06-2, 83 mg, yield 91%).

[0165] S5. In a 10 mL round-bottom flask, bicyclo[1.1.0]butane-1-carboxylic acid (9.8 mg, 0.1 mmol, 1.0 eq) and HATU (57 mg, 0.15 mmol, 1.5 eq) were dissolved in anhydrous DMF (2 mL) solution and mixed at room temperature for 10 min to obtain a second reaction system. PC06-2 (55 mg, 0.12 mmol, 1.2 eq) and DIEA (34.5 mg, 0.5 mmol, 5.0 eq) were added to the second reaction system to carry out a condensation reaction. After 2 h of reaction, water (2 mL) was added to quench the reaction. The organic phase was extracted three times with ethyl acetate (3 mL), and the organic phases were combined and then extracted successively with water (3 mL) and saturated brine (3 mL). The sample was washed three times with anhydrous sodium sulfate, then dried, filtered, and concentrated. Finally, the concentrate was subjected to silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain the BRD9 inhibitor, named PC06 (42 mg, yield 83%, ESI-MS: m / z 501.4 [M + H]). + ).

[0166] PC06: 1 H NMR (500 MHz, CDCl3) δ 9.22 (d, J = 1.4 Hz, 1H), 8.66 (dd, J= 4.6, 1.5 Hz, 1H), 7.88 (q, J = 1.0 Hz, 1H), 7.29 (d, J = 4.6 Hz, 1H), 6.83(s, 2H), 3.93 (s, 2H), 3.89 (s, 6H), 3.60 (d, J = 1.1 Hz, 3H), 3.50 – 3.45(m, 4H), 3.06 (s, 1H), 3.04 (s, 1H), 2.81 (s, 1H), 2.79 (s, 1H), 2.03 (dd, J= 6.1, 4.1 Hz, 2H), 1.81 – 1.76 (m, 4H), 1.62 (p, J = 4.1 Hz, 1H).

[0167] Example 7

[0168]

[0169] Steps S1 to S2 are the same as in Example 1;

[0170] S3. As shown in synthetic route 7, in a 25 mL round-bottom flask, compound 3 (504 mg, 1.5 mmol, 1.0 eq) and 2-Boc-2,5-diazabicyclo[2.2.1]heptane (356 mg, 1.8 mmol, 1.2 eq) were dissolved in absolutely anhydrous dichloromethane (15 mL) and mixed to obtain the first reaction system. Under ice bath conditions, sodium triacetoxyborohydride (954 mg, 4.5 mmol, 3.0 eq) was added in portions to the first reaction system to carry out the reduction reaction. After 6 h of reaction, water (10 mL) was added to quench the reaction. The organic phase of the reaction was separated using dichloromethane (10 mL). The organic phase was extracted three times with anhydrous sodium sulfate (mL), washed three times with saturated NaCl solution, dried with anhydrous sodium sulfate, filtered, and concentrated. Finally, the concentrate was separated by silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain compound 4 (PC07-1) containing a Boc protecting group.

[0171] S4. In a 10 mL round-bottom flask, PC07-1 (107 mg, 0.2 mmol) was mixed with HCl / 1,4-Dioxane (4 mol / L, 2 mL) at room temperature to carry out the deprotection reaction of the Boc protecting group. The reaction was carried out for 2 h, and the solvent in the reaction system was dried by rotating to obtain compound 5 (PC07-2, 80 mg, yield 85%).

[0172] S5. In a 10 mL round-bottom flask, bicyclo[1.1.0]butane-1-carboxylic acid (9.8 mg, 0.1 mmol, 1.0 eq) and HATU (57 mg, 0.15 mmol, 1.5 eq) were dissolved in anhydrous DMF (2 mL) solution and mixed at room temperature for 10 min to obtain a second reaction system. PC07-2 (56.5 mg, 0.12 mmol, 1.2 eq) and DIEA (34.5 mg, 0.5 mmol, 5.0 eq) were added to the second reaction system to carry out a condensation reaction. After 2 h of reaction, water (2 mL) was added to quench the reaction. The organic phase was extracted three times with ethyl acetate (3 mL), and the organic phases were combined and then extracted successively with water (3 mL) and saturated brine (3 mL). The organic phase was washed three times with anhydrous sodium sulfate, then dried, filtered, and concentrated. Finally, the concentrate was subjected to silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain the BRD9 inhibitor, named PC07 (39 mg, yield 76%, ESI-MS: m / z 515.1 [M + H]). + ).

[0173] PC07:1 H NMR (500 MHz, CDCl3) δ 9.22 (d, J = 1.4 Hz, 1H), 8.66 (dd, J= 4.6, 1.5 Hz, 1H), 7.88 (q, J = 1.0 Hz, 1H), 7.29 (d, J = 4.6 Hz, 1H), 6.83(s, 2H), 4.13 (dqd, J = 6.1, 3.6, 2.6 Hz, 1H), 3.89 (s, 6H), 3.77 (s, 2H), 3.69 (ddd, J = 11.9, 7.9, 5.3 Hz, 1H), 3.60 (d, J = 1.1 Hz, 3H), 3.42 (ddd, J= 11.9, 7.9, 5.3 Hz, 1H), 2.91 (dqd, J = 6.1, 3.6, 2.6 Hz, 1H), 2.78 (ddd, J= 11.9, 7.9, 5.3 Hz, 1H), 2.62 (ddd, J = 11.9, 7.9, 5.3 Hz, 1H), 2.06 (dd, J= 6.0, 4.1 Hz, 2H), 1.96 – 1.78 (m, 7H), 1.68 – 1.57 (m, 2H).

[0174] Example 8

[0175]

[0176] Steps S1 to S2 are the same as in Example 1;

[0177] S3. As shown in synthetic route 8, in a 25 mL round-bottom flask, compound 3 (504 mg, 1.5 mmol, 1.0 eq) and 2-Boc-decahydro-2,6-naphthidine (432 mg, 1.8 mmol, 1.2 eq) were dissolved in absolutely anhydrous dichloromethane solvent (15 mL) and mixed to obtain the first reaction system. Under ice bath conditions, sodium triacetoxyborohydride (954 mg, 4.5 mmol, 3.0 eq) was added in portions to the first reaction system to carry out the reduction reaction. After 6 h of reaction, water (10 mL) was added to quench the reaction. The organic phase of the reaction was separated and extracted three times with dichloromethane (10 mL), washed three times with saturated NaCl solution, and then dried with anhydrous sodium sulfate. The mixture was filtered and concentrated. Finally, the concentrate was separated by silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain compound 4 (PC08-1) containing the Boc protecting group.

[0178] S4. In a 10 mL round-bottom flask, PC08-1 (110 mg, 0.2 mmol) was mixed with HCl / 1,4-Dioxane (4 mol / L, 2 mL) at room temperature to carry out the deprotection reaction of the Boc protecting group. The reaction was carried out for 2 h, and the solvent in the reaction system was dried by rotation to obtain compound 5 (PC08-2, 81 mg, yield 83%).

[0179] S5. In a 10 mL round-bottom flask, bicyclo[1.1.0]butane-1-carboxylic acid (9.8 mg, 0.1 mmol, 1.0 eq) and HATU (57 mg, 0.15 mmol, 1.5 eq) were dissolved in anhydrous DMF (2 mL) solution and mixed at room temperature for 10 min to obtain a second reaction system. PC08-2 (58 mg, 0.12 mmol, 1.2 eq) and DIEA (34.5 mg, 0.5 mmol, 5.0 eq) were added to the second reaction system to carry out a condensation reaction. After 2 h of reaction, water (2 mL) was added to quench the reaction. The organic phase was extracted three times with ethyl acetate (3 mL), and the organic phases were combined and then extracted successively with water (3 mL) and saturated brine (3 mL). The sample was washed three times with anhydrous sodium sulfate, then dried, filtered, and concentrated. Finally, the concentrate was subjected to silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain the BRD9 inhibitor, named PC08 (46 mg, yield 87%, ESI-MS: m / z 529.12 [M + H]). + ).

[0180] PC08: 1H NMR (500 MHz, CDCl3) δ 9.22 (d, J = 1.4 Hz, 1H), 8.66 (dd, J= 4.6, 1.5 Hz, 1H), 7.88 (q, J = 1.0 Hz, 1H), 7.29 (d, J = 4.6 Hz, 1H), 6.83(s, 2H), 3.89 (s, 6H), 3.85 – 3.70 (m, 2H), 3.66 (dd, J = 11.2, 3.1 Hz, 1H), 3.60 (d, J = 1.1 Hz, 3H), 3.58 – 3.51 (m, 2H), 3.41 (dd, J = 11.4, 3.1 Hz,1H), 2.79 (dd, J = 11.2, 3.6 Hz, 1H), 2.75 – 2.61 (m, 2H), 2.50 (dd, J =11.2, 3.6 Hz, 1H), 2.07 – 1.96 (m, 3H), 1.95 – 1.80 (m, 3H), 1.78 (dd, J =6.1, 4.1 Hz, 2H), 1.67 – 1.51 (m, 3H).

[0181] Comparative Example 1

[0182]

[0183] Steps S1 to S2 are the same as in Example 1;

[0184] S3. In a 25 mL round-bottom flask, compound 3 (504 mg, 1.5 mmol, 1.0 eq) and N-Boc-1,6-hexanediamine (389 mg, 1.8 mmol, 1.2 eq) were dissolved in absolutely anhydrous dichloromethane (15 mL) and mixed to obtain the first reaction system. Under ice bath conditions, sodium triacetoxyborohydride (954 mg, 4.5 mmol, 3.0 eq) was added in portions to the first reaction system to carry out the reduction reaction. After 6 h of reaction, water (10 mL) was added to quench the reaction. The organic phase of the reaction was separated and extracted three times with dichloromethane (10 mL), washed three times with saturated NaCl solution, and then dried with anhydrous sodium sulfate. The mixture was filtered and concentrated. Finally, the concentrate was separated by silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain compound 4 (PC09-1) containing a Boc protecting group.

[0185] S4. In a 10 mL round-bottom flask, PC09-1 (105 mg, 0.2 mmol) was mixed with HCl / 1,4-Dioxane (4 mol / L, 2 mL) at room temperature to carry out the deprotection reaction of the Boc protecting group. The reaction was carried out for 2 h, and the solvent in the reaction system was dried by rotating to obtain compound 5 (PC09-2, 76 mg, yield 82%).

[0186] S5. In a 10 mL round-bottom flask, bicyclo[1.1.0]butane-1-carboxylic acid (9.8 mg, 0.1 mmol, 1.0 eq) and HATU (57 mg, 0.15 mmol, 1.5 eq) were dissolved in anhydrous DMF (2 mL) solution and mixed at room temperature for 10 min to obtain a second reaction system. PC09-2 (55 mg, 0.12 mmol, 1.2 eq) and DIEA (34.5 mg, 0.5 mmol, 5.0 eq) were added to the second reaction system to carry out a condensation reaction. After 2 h of reaction, water (2 mL) was added to quench the reaction. The organic phase was extracted three times with ethyl acetate (3 mL), and the organic phases were combined and then extracted successively with water (3 mL) and saturated brine (3 mL). The sample was washed three times with anhydrous sodium sulfate, then dried, filtered, and concentrated. Finally, the concentrate was subjected to silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain the BRD9 inhibitor, named PC09 (39 mg, yield 77%, ESI-MS: m / z 505.30 [M + H]). + .).

[0187] PC09: 1 H NMR (500 MHz, CDCl3) δ 9.25 (s, 1H), 8.36 (s, 1H), 8.12 (s,1H), 7.58 (s, 1H), 7.29 (s, 1H), 6.78 – 6.58 (m, 2H), 4.01 (s, 1H), 3.94 (s,1H), 3.81 (s, 6H), 3.64 (s, 3H), 3.23 (s, 1H), 3.12 (d, J = 5.2 Hz, 2H), 2.61(d, J = 10.6 Hz, 2H), 1.98 (s, 1H), 1.47 (d, J = 3.4 Hz, 4H), 1.44 – 1.31 (m,4H), 1.33 – 1.21 (m, 2H), 1.14 – 0.98 (m, 2H).

[0188] Comparative Example 2

[0189]

[0190] Steps S1 to S2 are the same as in Example 1;

[0191] S3. In a 25 mL round-bottom flask, compound 3 (504 mg, 1.5 mmol, 1.0 eq) and N-Boc-1,14-tetradecanediamine (590.4 mg, 1.8 mmol, 1.2 eq) were dissolved in absolutely anhydrous dichloromethane (15 mL) and mixed to obtain the first reaction system. Under ice bath conditions, sodium triacetoxyborohydride (954 mg, 4.5 mmol, 3.0 eq) was added in portions to the first reaction system to carry out the reduction reaction. After 6 h of reaction, water (10 mL) was added to quench the reaction. The organic phase of the reaction was separated and extracted three times with dichloromethane (10 mL), washed three times with saturated NaCl solution, and then dried with anhydrous sodium sulfate. The mixture was filtered and concentrated. Finally, the concentrate was separated by silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain compound 4 (PC10-1) containing a Boc protecting group.

[0192] S4. In a 10 mL round-bottom flask, PC10-1 (127 mg, 0.2 mmol) was mixed with HCl / 1,4-Dioxane (4 mol / L, 2 mL) at room temperature (25 °C) to carry out the deprotection reaction of the Boc protecting group. The reaction was carried out for 2 h, and the solvent in the reaction system was dried by rotation to obtain compound 5 (PC10-2, 93 mg, yield 81%).

[0193] S5. In a 10 mL round-bottom flask, bicyclo[1.1.0]butane-1-carboxylic acid (9.8 mg, 0.1 mmol, 1.0 eq) and HATU (57 mg, 0.15 mmol, 1.5 eq) were dissolved in anhydrous DMF (2 mL) solution and mixed at room temperature (25 °C) for 10 min to obtain a second reaction system. PC10-2 (69 mg, 0.12 mmol, 1.2 eq) and DIEA (34.5 mg, 0.5 mmol, 5.0 eq) were added to the second reaction system to carry out a condensation reaction. After 2 h of reaction, water (2 mL) was added to quench the reaction. The organic phase was extracted three times with ethyl acetate (3 mL), and the organic phases were combined and then extracted successively with water (3 mL) and saturated brine (3 mL). The sample was washed three times with anhydrous sodium sulfate, then dried, filtered, and concentrated. Finally, the concentrate was subjected to silica gel column chromatography (Greagent brand, Shanghai Titan Technology Co., Ltd.) to obtain the BRD9 inhibitor, named PC10 (48 mg, yield 78%, ESI-MS: m / z 617.40 [M + H]). + .).

[0194] PC10: 1 H NMR (500 MHz, CDCl3) δ 9.42 (s, 1H), 8.44 (s, 1H), 8.11 (s,1H), 7.56 (s, 1H), 7.30 (s, 1H), 6.79 – 6.73 (m, 2H), 4.04 (s, 1H), 3.97 (s,1H), 3.81 (s, 6H), 3.64 (s, 3H), 3.14 (s, 1H), 3.11 (d, J = 31.9 Hz, 2H), 2.65 (d, J = 14.7 Hz, 2H), 1.98 (s, 1H), 1.84 (s, 1H), 1.46 (s, 2H), 1.44 –1.21 (m, 23H), 1.15 – 0.94 (m, 2H).

[0195] The effects of the BRD9 inhibitors prepared in the above examples and comparative examples on cell viability were determined using the following assay methods:

[0196] Assay of the antiproliferative activity of BRD9 inhibitor against acute myeloid leukemia cell line NB4: NB4 cell line was cultured and seeded at a density of 5000 cells per well in 96-well plates and cultured to the logarithmic growth phase; 10 μM (1×10⁻⁶) BRD9 inhibitor was added to each well. -5 mol / L), 40 μM (4×10-5 The cells were cultured for 72 hours with a BRD9 inhibitor (mol / L), and then incubated for 4 hours with 10 μL of MTT (Meilun Biotechnology) cell proliferation assay solution. The absorbance at 490 nm was measured. 10 mL of dimethyl sulfoxide (DMSO) solution was added to the adherent cells and incubated for 4 hours. The absorbance at 490 nm was measured as a control group, and the inhibition rate was calculated.

[0197] Table 1

[0198]

[0199] Note: ++ indicates an inhibition rate of 50-70%; +++ indicates an inhibition rate of 70-90%.

[0200] As shown in Table 1, the BRD9 inhibitors prepared in Examples 1 to 8 of this invention all exhibited significant anti-proliferative activity against NB4 cells, with inhibition rates reaching 50%–70% at a concentration of 10 μM and mostly reaching 70%–90% at a concentration of 40 μM. In contrast, the compounds in Comparative Examples 1 and 2, which used chain-like diamines as the L-structure, showed inhibition rates of less than 20% at the same concentration, indicating almost no significant activity. This result fully demonstrates that the core technical feature of this invention is the use of a defined cyclic group containing heteroatoms as the L-structure. The spatial rigidity of the cyclic skeleton and the synergistic effect of the heteroatoms embedded within the ring can significantly improve the binding efficiency of the inhibitor to the BRD9 target, while simply adjusting the carbon chain length cannot achieve effective inhibition.

[0201] Further analysis of the internal differences in the examples revealed that compounds employing different heterocyclic structures (piperazine ring, spirocyclic, bicyclic, decahydronaphthidine, etc.) all exhibited good activity, demonstrating the rationality and universality of the heterocyclic structure limitation range. Specifically, PC01 containing structural formula 2-1, PC02 containing structural formula 2-12, PC04 containing structural formula 2-4, PC05 containing structural formula 2-5, PC06 containing structural formula 2-8, and PC07 containing structural formula 2-3 achieved inhibition rates of 70%–90% at a concentration of 40 μM. These L-structures showed a higher degree of matching with BRD9, and the rigidity and spatial orientation of the ring system were more conducive to the formation of a stable target-inhibitor complex. In contrast, PC08 and PC03 containing structural formula 2-2 achieved inhibition rates of 50%–70% at a concentration of 40 μM, indicating that these specific heterocyclic skeletons possessed superior spatial complementarity and binding affinity.

[0202] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A BRD9 inhibitor, characterized in that it comprises a compound of Formula 1 and / or a salt of the compound of Formula 1: Formula 1 In Formula 1, L is selected from cyclic groups containing heteroatoms, and the number of carbon atoms in the cyclic group is 1 to 14. The heteroatoms include one or more of O, N, and S.

2. The BRD9 inhibitor according to claim 1, characterized in that, The cyclic group is selected from at least one of cycloalkyl, heterocycloalkyl, and aryl groups.

3. The BRD9 inhibitor according to claim 1, characterized in that, The structure of L is selected from one of the following formulas 2-1 to 2-15: In Equation 2-1, n is 0 or 1; m is an integer from 0 to 4; In Formula 2-13, n1 is 0 or 1; n2 is an integer from 0 to 4; R7 is selected from substituted or unsubstituted alkyl groups and substituted or unsubstituted cycloalkyl groups; M is N or C; In Equation 2-14, n3 and n4 are independently 0 or 1; In Equation 2-15, n5, n6, and n7 are independently 0 or 1; Q is N or C.

4. The BRD9 inhibitor according to claim 3, characterized in that, The structure of the BRD9 inhibitor is selected from one of the following formulas 3-1 to 3-8: 。 5. The BRD9 inhibitor according to claim 1 or 4, characterized in that, Salts of the compound shown in Formula 1 include salts formed by the compound shown in Formula 1 and an acid, wherein the acid includes one or more of hydrochloric acid, phosphoric acid, citric acid, and tartaric acid.

6. A method for preparing the BRD9 inhibitor according to any one of claims 1-5, characterized in that, Includes the following steps: Compound 5, a bicycloalkylcarboxylic acid compound, and a urea condensing agent were mixed and subjected to a condensation reaction to obtain the BRD9 inhibitor; wherein, the structural formula of compound 5 is shown in Formula 4: Formula 4.

7. The method for preparing the BRD9 inhibitor according to claim 6, characterized in that, The bicyclic alkyl carboxylic acid compounds include bicyclic [1.1.0]butane-1-carboxylic acid; And / or, the urea condensing agent comprises 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; And / or, the condensation reaction is carried out at a temperature of 25°C to 45°C for a time of 1 h to 3 h.

8. The method for preparing the BRD9 inhibitor according to claim 6 or 7, characterized in that, The preparation process of compound 5 includes: mixing compound 4 containing a Boc protecting group with an acidic deprotecting agent to carry out a deprotection reaction of the Boc protecting group, thereby obtaining compound 5; wherein, the structural formula of compound 4 containing a Boc protecting group is shown in Formula 5: Formula 5.

9. The method for preparing the BRD9 inhibitor according to claim 8, characterized in that, The acidic deprotecting agent includes a solution of 1,4-dioxane hydrogen chloride; And / or, the deprotection reaction is carried out at a temperature of 18°C ​​to 25°C for a time of 1 h to 3 h.

10. The method for preparing the BRD9 inhibitor according to claim 8 or 9, characterized in that, The preparation process of compound 4 containing the Boc protecting group includes: mixing compound 3 and a heteroatom-containing cyclic group compound containing the N-Boc protecting group, and carrying out a reduction reaction under the action of a reducing agent to obtain compound 4 containing the Boc protecting group; wherein, the heteroatom-containing cyclic group containing the N-Boc protecting group has the structure of L as described in any one of claims 1-3; the structural formula of compound 3 is shown in Formula 6: Formula 6.

11. The method for preparing the BRD9 inhibitor according to claim 10, characterized in that, The reducing agent includes sodium triacetoxyborohydride; And / or, the reduction reaction takes 6 h to 12 h and is carried out at a temperature of 0 °C to 5 °C.

12. The method for preparing the BRD9 inhibitor according to claim 10 or 11, characterized in that, The preparation process of compound 3 includes: A mixture of halonaridinone compounds, alkali metal hydrides, and alkyl halides was subjected to an alkylation reaction to yield compound 2. Compound 2 was mixed with borate esters and haloalkoxy aromatic aldehydes, and then subjected to a Suzuki coupling reaction in the presence of a catalyst to obtain compound 3.

13. A pharmaceutical composition, characterized in that, Includes the BRD9 inhibitor as described in any one of claims 1-5, or the BRD9 inhibitor prepared by the preparation method described in any one of claims 6-12.

14. The use of a BRD9 inhibitor as claimed in any one of claims 1-5 in the preparation of a medicament, characterized in that, The drug is used to treat at least one of the following: leukemia, lymphoma, breast cancer, kidney cancer, colorectal cancer, liver cancer, small cell lung cancer, human bone marrow rhabdomyosarcoma, pancreatic cancer, and malignant glioblastoma of the brain.

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