A cbp / p300 inhibitor, methods of making, pharmaceutical compositions, and uses thereof

Abstract

The present application relates to a CBP / P300 inhibitor, a preparation method thereof, a pharmaceutical composition and uses thereof. Specifically, the present application provides a class of DELs shown in formula I, based on the DELs, the present application provides a compound having a structure shown in formula II, wherein the definitions of each group and substituent are described in the specification; the present application also discloses a preparation method of the compound of formula II and uses thereof in preventing and / or treating diseases related to abnormal activity of CBP / P300.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more specifically to a CBP / P300 inhibitor, its preparation method, pharmaceutical composition, and its uses. Background Technology

[0002] CBP / P300 is an important macromolecular protein among histone acetyltransferases (HATs). Due to their high structural homology, they are considered to have similar functions. CBP / P300 has multiple functions, promoting transcription. First, also known as KAT3a, it is an acetyltransferase that catalyzes the acetylation of histones and other proteins. Second, it contains a bromodomain, enabling it to perform acetylation recognition functions similar to proteins such as BRD2 / 3 / 4, recruiting transcription initiation complexes to begin transcription. Third, CBP / P300 also functions as a protein ubiquitin ligase, potentially playing a role in protein ubiquitination and degradation. Increasing research confirms that CBP / P300 dysfunction is associated with various human diseases, such as cancer, diabetes, inflammation, and heart disease, particularly cancer. For example, literature has reported that abnormal or mutated CBP / P300 expression is associated with many cancers, including colitis, lung cancer, pancreatic pain, and acute myeloid leukemia. Inhibiting the activity of CBP / P300 histone acetyltransferase or inhibiting CBP's activity as a transcriptional coactivator has been found to suppress the growth of neuroblastoma, pancreatic cancer, and acute myeloid leukemia in vitro and in vivo. In lung cancer, CBP has been found to be highly expressed in different lung cancer cell lines and is positively correlated with hTERT expression. Patients with CBP-positive expression have significantly lower overall survival and disease-free survival than those with CBP-negative expression. CBP / P300 has long been considered an important target for treating diseases including cancer. However, due to the significant similarity between the bromodomain (BRD) of CBP / P300 and targets such as BRD4, developing highly selective inhibitors of CBP / P300 is challenging. Although various structural types of CBP / P300 small molecule inhibitors have been developed, only CellCentric's CCS1477 has entered Phase 2b clinical trials for the treatment of advanced drug-resistant prostate cancer. Therefore, developing small molecule inhibitors for tumors associated with CBP / P300 mutations remains of great significance.

[0003] Currently, the commonly used technique for discovering lead compounds in small molecule drug development is high-throughput screening of large compound libraries. High-throughput screening requires a large compound library (1-2 million compounds), which is costly to maintain and has a long screening cycle. The cost of reagents for developing the assay and the overall screening cycle limit the number of targets that can be screened within a given set of resources.

[0004] Furthermore, with the rapid development of computational chemistry, structure-based computer-aided drug design virtual screening has become an important technique for discovering small molecule lead compounds. Despite significant advancements in virtual screening, the small molecules obtained still present challenges such as insufficient activity and difficulties in further optimization.

[0005] Furthermore, fragment-based drug discovery is a recently developed lead compound discovery technology. After more than a decade of development, drugs based on this technology have already been approved for the market. In fact, fragment-based drug discovery is closely linked to structure-based drug design. Both research methods begin with the discovery of high-quality molecular fragments targeting specific sites of action. However, screening molecular fragments is a resource-intensive and time-consuming process. While commonly used fragment screening techniques such as X-ray and NMR have good success rates, the slow pace of the entire process severely limits the number of targets that can be screened and the speed of project progress. Therefore, lead compound discovery still requires the development of cheaper and faster technologies to accelerate the screening process for new targets, thereby speeding up the development of innovative drugs.

[0006] The synthesis and screening of DNA-encoded libraries (DELs) was proposed in the early 1990s by Professors Sydney Breener and Richard Lerner of the Scripps Research Institute. It combines the advantages of combinatorial chemistry (efficiently synthesizing compound libraries) with molecular biology and chemical bioinformatics, and has been greatly promoted by the development of nucleic acid-compatible chemical reactions and high-throughput sequencing technologies. The success of target screening largely depends on the structural diversity and spatial quantity of the screening compound library. DEL synthesis employs a combinatorial chemistry split-pool strategy, utilizing multiple nucleic acid-compatible chemical reactions, a vast building block (BB) reagent library, and a unique compound encoding method. This allows for the synthesis of millions or even tens of billions of compounds with DNA sequences in a short time, effectively meeting the structural diversity and spatial quantity requirements for screening. DEL screening is primarily based on affinity. Because this screening involves screening the target protein and the entire compound library, rather than screening individual compounds one by one, it significantly improves screening efficiency, shortens drug development cycles, and reduces development costs. Furthermore, structure-activity relationship (SAR) data can be analyzed from screening data, providing a reference for the rational design of drug molecules and accelerating the hit-to-lead process. Currently, DEL synthesis and screening have become one of the most cutting-edge technologies in the field of new drug discovery, and have been widely used in the synthesis and screening of lead compounds in new drug development, with several candidate drugs already in clinical trials. Summary of the Invention

[0007] One object of the present invention is to provide a DEL with benzopyrazolinone as the main structural backbone as shown in general formula (I) and a method for preparing the same.

[0008] Another object of the present invention is to provide compounds of general formula (II), their tautomers, enantiomers, diastereomers, racemates, metabolic precursors, pharmaceutically acceptable salts, esters, prodrugs or hydrates thereof.

[0009] Another objective of this invention is to provide an important intermediate for this type of compound and a method for its preparation.

[0010] Another object of the present invention is to provide the use of such compounds in the preparation of medicaments for the prevention and treatment of diseases associated with abnormal activity of CBP / P300, including gastrointestinal cancer, cervical cancer, bladder cancer, laryngeal cancer, liver cancer, breast cancer, lung cancer, oral cancer, ovarian cancer, diabetes, inflammation, and other diseases.

[0011] In a first aspect, the present invention provides a nucleic acid-encoded compound library DEL represented by Formula I.

[0012]

[0013] The DEL is prepared as follows:

[0014] 1) The reaction of AOP headpiece-primer, DEL cycle1-encoding and compound BB-1 yields DEL-Cycle1;

[0015] 2) DEL-Cycle1 DEL cycle2-encoding reacts with compound BB-2 to give DEL-Cycle2;

[0016] in,

[0017] The AOP headpiece has the following structure:

[0018]

[0019] The primer has the following structure: AAATCGATGTG11 pairs of deoxyribonucleotides;

[0020] DEL cycle1-encoding is composed of 9 pairs of deoxyribonucleotides and two deoxynucleotides, as shown in cccctatgtCA.

[0021] Compound BB-1 is selected from the compounds listed in Table 1;

[0022] The structure of DEL-Cycle1 is as follows:

[0023] DEL cycle2-encoding consists of 11 pairs of deoxyribonucleotides and two deoxynucleotides, as shown in aatgtcgtcCT.

[0024] Compound BB-2 is selected from the compounds listed in Table 2;

[0025] The structure of DEL-Cycle2 is as follows:

[0026]

[0027] In another preferred embodiment, the DEL is prepared as follows:

[0028]

[0029] in,

[0030] Compound BB-1 is selected from the following group of compounds: Furthermore, compound BB-1 is selected from the compounds listed in Table 1;

[0031] Compound BB-2 is Furthermore, compound BB-2 is selected from the compounds listed in Table 2.

[0032] In a second aspect, the present invention provides a compound of Formula II, or a tautomer, enantiomer, diastereomer, racemate, metabolic precursor, pharmaceutically acceptable salt, ester, prodrug, or hydrate thereof.

[0033]

[0034] in,

[0035] A is selected from the following group: -O-, -NH-, -NH-S(O)2-, -NH-CO-NH-, -NH-CO-O-, -NHSO2-;

[0036] R1 is selected from the following group: C1-C3 alkyl, C5-C8 cycloalkyl, halo-C1-C3 alkyl, hydroxy-substituted C1-C3 alkyl;

[0037] B is selected from the following group: C1-C3 alkyl, C5-C8 cycloalkyl, C5-C12 aryl, 5-8 membered heterocyclic or 5-8 membered heteroaryl containing 1-3 heteroatoms selected from N, O, and S, C5-C8 cycloalkyl and C6-C12 aryl, 5-8 membered heterocyclic or 5-8 membered heteroaryl and C6-C12 aryl containing 1-3 heteroatoms selected from N, O, and S;

[0038] R2 is selected from the following group: C6-C12 aryl, 5-8 membered heterocyclic group or 5-8 membered heteroaryl containing 1-3 heteroatoms selected from N, O, and S;

[0039] C is selected from the following group: C1-C3 alkyl, C5-C8 cycloalkyl, C5-C12 aryl, 5-8 membered heterocyclic or 5-8 membered heteroaryl containing 1-3 heteroatoms selected from N, O, and S, C5-C8 cycloalkyl and C6-C12 aryl, 5-8 membered heterocyclic or 5-8 membered heteroaryl and C6-C12 aryl containing 1-3 heteroatoms selected from N, O, and S;

[0040] R3 is selected from the group consisting of: C6-C12 aryl, 5-8 membered heterocyclic group or 5-8 membered heteroaryl containing 1-3 heteroatoms selected from N, O, S; wherein the aryl, heterocyclic or heteroaryl group is optionally substituted by 1-3 substituents from the group consisting of: halogen, C1-C6 alkyl, C1-C6 alkoxy, OH.

[0041] In another preferred embodiment,

[0042] A is -NH-;

[0043] R1 is a C1-C3 alkyl group;

[0044] B is a C1-C3 alkyl group;

[0045] R2 is a C6-C12 aryl group;

[0046] C is a C1-C3 alkyl group;

[0047] R3 is a halogenated C6-C12 aryl group.

[0048] In another preferred embodiment, the compound is

[0049] In a third aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a safe and effective amount of the compound described in the second aspect of the present invention, or a tautomer, enantiomer, diastereomer, racemate, metabolic precursor, pharmaceutically acceptable salt, ester, prodrug, or hydrate thereof.

[0050] In a fourth aspect, the present invention provides the use of the compound described in the second aspect of the present invention, or its tautomers, enantiomers, diastereomers, racemates, metabolic precursors, pharmaceutically acceptable salts, esters, prodrugs, or hydrates thereof, for the preparation of a drug targeting CBP / P300.

[0051] In a fifth aspect, the present invention provides the use of the compound described in the second aspect of the present invention, or its tautomers, enantiomers, diastereomers, racemates, metabolic precursors, pharmaceutically acceptable salts, esters, prodrugs, or hydrates thereof, for the preparation of a medicament for the prevention and / or treatment of diseases related to abnormal CBP / P300 activity.

[0052] In another preferred embodiment, the diseases associated with abnormal CBP / P300 activity are selected from the group consisting of: gastrointestinal cancer, cervical cancer, bladder cancer, laryngeal cancer, liver cancer, breast cancer, lung cancer, oral cancer, ovarian cancer, diabetes, inflammation, pulmonary dysfunction, pain (including but not limited to complex regional pain syndrome), macular degeneration and related functional disorders, skin diseases, immunodeficiency diseases, and damage and functional disorders of the central nervous system.

[0053] In another preferred embodiment, the diseases associated with abnormal CBP / P300 activity include, but are not limited to, diseases selected from the group consisting of: skin cancer (such as melanoma), lymphatic system cancers, breast cancer, cervical cancer, uterine cancer, digestive tract cancers, lung cancer, ovarian cancer, prostate cancer, colon cancer, rectal cancer, oral cancer, brain tumors, head and neck cancer, pharyngeal cancer, testicular cancer, kidney cancer, pancreatic cancer, spleen cancer, liver cancer, bladder cancer, laryngeal cancer, and cancers associated with AIDS.

[0054] In another preferred embodiment, the diseases associated with abnormal CBP / P300 activity include, but are not limited to, diseases selected from the group consisting of hematologic malignancies and myeloma.

[0055] In another preferred embodiment, the hematologic malignancy is acute or chronic leukemia.

[0056] In another preferred embodiment, the myeloma is a multiple myeloma.

[0057] In another preferred embodiment, the compound is used to prevent or treat primary and metastatic tumors.

[0058] In a sixth aspect, the present invention provides a drug screening method, which uses the DEL described in claim 1 for drug screening.

[0059] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0060] Figure 1 This is the result of DEL cycle 1-encoding mass spectrometry detection (taken from well 1).

[0061] Figure 2 This is the result of DEL cycle 1-encoding electrophoresis (taken from well 1).

[0062] Figure 3 These are the results of DEL-Cycle1 mass spectrometry analysis (taken from well 1).

[0063] Figure 4 These are the mass spectrometry results from DEL-Cycle1. (A) The mass spectrometry results show the 208 amino acids pooled together after the reaction with DELcycle1-encoding; (B) The mass spectrometry results after the protecting group was removed.

[0064] Figure 5 The results are from DEL-Cycle1 mass spectrometry (475 components). Figure (A) shows the mass spectrometry results after the reaction with 2-nitrobenzyl alcohol. Figure (B) shows the mass spectrometry results after the reaction and pooling together.

[0065] Figure 6 This is the result of DEL cycle2-encoding mass spectrometry detection (taken from well 1).

[0066] Figure 7 This is the result of DEL cycle 2-encoding electrophoresis.

[0067] Figure 8The results of DEL-Cycle2 mass spectrometry (taken from well 1) are shown in Figure (A). Figure (B) shows the mass spectrometry results of the photoreaction products.

[0068] Figure 9 These are the mass spectrometry results of DEL-Cycle2 pooled together.

[0069] Figure 10 This is the hydrogen NMR spectrum of DNA-6a.

[0070] Figure 11 This is the carbon NMR spectrum of DNA-6a. Detailed Implementation

[0071] Through long-term and in-depth research, the inventors have prepared and provided a DEL (nucleic acid-encoded compound library) with benzopyrazolinone as the main structural backbone. Based on the DEL, a class of CBP / P300 inhibitors were screened. These inhibitors can effectively inhibit CBP / P300 activity or inhibit the activity of CBP as a transcriptional co-activator, and have high selective inhibitory activity against CBP / P300, providing a new option for the prevention and / or treatment of diseases related to abnormal CBP / P300 activity. Based on this, the inventors completed this invention.

[0072] the term

[0073] In this invention, unless otherwise specified, the terms used have the general meanings known to those skilled in the art.

[0074] In this invention, the term "halogen" refers to F, Cl, Br, or I.

[0075] In this invention, "C1-C6 alkyl" refers to a straight-chain or branched alkyl group comprising 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, neopentyl, pterpentyl, or similar groups.

[0076] In this invention, the term "C2-C6 alkenyl" refers to a straight-chain or branched alkenyl group having 2-6 carbon atoms and containing a double bond, and includes, without limitation, vinyl, propenyl, butenyl, isobutenyl, pentenyl, and hexenyl groups.

[0077] In this invention, the term "C2-C6 ynyl" refers to a straight-chain or branched ynyl group having 2-6 carbon atoms and containing a triple bond, and includes, without limitation, ethynyl, propynyl, butynyl, isobutynyl, pentylyl, and hexynyl.

[0078] In this invention, the term "C3-C8 cycloalkyl" refers to a cyclic alkyl group having 3-8 carbon atoms on a ring, and includes, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.

[0079] In this invention, the term "C1-C6 alkoxy" refers to a straight-chain or branched alkoxy group having 1-6 carbon atoms, and includes, without limitation, methoxy, ethoxy, propoxy, isopropoxy, and butoxy. Preferably, it is a C1-C4 alkoxy group.

[0080] In this invention, the term "heterocyclic group" refers to a 4-8 membered heterocyclic group containing 1, 2 or 3 heteroatoms selected from N, O, and S, including (but not limited to) the following groups: propylene oxide, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, piperazineyl, morpholinyl, and thiomorpholinyl.

[0081] In this invention, the terms "aromatic ring" or "aryl" have the same meaning, and are preferably "C6-C10 aryl". The term "C6-C10 aryl" refers to an aromatic cyclic group with 6-10 carbon atoms that does not contain heteroatoms on the ring, such as phenyl, naphthyl, etc.

[0082] In this invention, the terms "aromatic heterocycle" or "heteroaryl" have the same meaning, referring to a heteroaromatic group containing one or more heteroatoms. For example, "C3-C10 heteroaryl" refers to an aromatic heterocycle containing 1 to 4 heteroatoms selected from oxygen, sulfur, and nitrogen, and 3 to 10 carbon atoms. Non-limiting examples include: furanyl, thiophene, pyridinyl, pyrazolyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted.

[0083] In this invention, the term "halogenated" refers to being replaced by a halogen.

[0084] In this invention, the term "substitution" refers to the substitution of one or more hydrogen atoms on a specific group by a specific substituent. The specific substituent is the substituent described accordingly above, or the substituent appearing in the various embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substituted site of that group, and the substituents may be the same or different at each position. Those skilled in the art will understand that the combinations of substituents contemplated in this invention are stable or chemically feasible combinations. Such substituents include, but are not limited to: halogens, hydroxyl groups, carboxyl groups (-COOH), C1-C6 alkyl groups, C2-C6 alkenyl groups, C2-C6 alkynyl groups, C3-C8 cycloalkyl groups, 3- to 12-membered heterocyclic groups, aryl groups, heteroaryl groups, C1-C8 aldehyde groups, C2-C10 acyl groups, C2-C10 ester groups, amino groups, C1-C6 alkoxy groups, C1-C10 sulfonyl groups, etc.

[0085] In this invention, the term 1-6 refers to 1, 2, 3, 4, 5, or 6. Other similar terms each have a similar meaning independently.

[0086] The term "tautomer" refers to structural isomers that readily transform into each other through a chemical reaction in which they are typically isomers, resulting in the movement of hydrogen atoms or protons accompanied by the transformation of single bonds and adjacent double bonds.

[0087] The term "enantiomer" refers to a stereoisomer that is a mirror image of another and cannot be superimposed.

[0088] The term "diastere" refers to a stereoisomer that has two or more chiral neutrals and is not a mirror image of another.

[0089] A racemic mixture is a stereoisomer that is a mirror image of another, with opposite optical rotations that can cancel each other out.

[0090] "Metabolic precursors" refer to a class of compounds that are inactive or have low activity in vitro, but release active drugs and exert their medicinal effects in vivo through metabolic pathways.

[0091] "Medicinal salts" refer to the salts formed by drug molecules with corresponding organic acids, inorganic acids, or organic bases, such as hydrochloric acid, formic acid, trifluoroacetic acid, succinic acid, and methanesulfonates.

[0092] "Prodrugs" are compounds that are inactive or have low activity in vitro, but release active drugs in vivo through enzymatic or non-enzymatic transformation to exert their medicinal effects.

[0093] "Hydrate" refers to a compound that contains water.

[0094] It should be understood that when a group exists simultaneously at multiple different positions in a compound, its definition at each position is independent and can be the same or different. That is, the term "selected from the following group:" and the term "each independently selected from the following group:" have the same meaning.

[0095] DEL and its preparation

[0096] The DEL represented by general formula (I) of this invention is prepared as follows:

[0097]

[0098] The structure of the AOP headpiece, as reported in the literature, is as follows:

[0099]

[0100] The structures of the compound BB used in Cycle1 are shown in Table 1, which contains a total of 473 compounds.

[0101] Table 1: The structure of BB in Cycle1.

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] The BBs used in Cycle2 are shown in Table 2, comprising a total of 294 compounds.

[0119] Table 2: The structure of BB in Cycle2.

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131] CBP / P300 inhibitors

[0132] This invention provides compounds of Formula II, or tautomers, enantiomers, diastereomers, racemates, metabolic precursors, pharmaceutically acceptable salts, esters, prodrugs, or hydrates thereof.

[0133]

[0134] The groups are as defined above.

[0135] In another preferred embodiment, each group in the compound of formula II is independently the corresponding group in the specific compound.

[0136] As used herein, the terms "pharmaceutically acceptable salt" or "medicinal salt" refer to salts formed by the compounds of the present invention with acids or bases that are suitable for use as medicines. Pharmaceutically acceptable salts include both inorganic and organic salts. A preferred class of salts are those formed by the compounds of the present invention with acids. Suitable acids for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid.

[0137] Another preferred class of salts are salts formed by the compounds of the present invention with a base, such as alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (such as lower alkanol ammonium salts and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0138] The term "solvate" refers to a complex formed by the coordination of the compound of the present invention with solvent molecules in a specific ratio. "Hydrate" refers to a complex formed by the coordination of the compound of the present invention with water.

[0139] Furthermore, the compounds of the present invention also include prodrugs of the compounds. The term "prodrug" includes substances that are themselves biologically active or inactive, and which, when taken by an appropriate method, are metabolized or chemically reacted in the human body to form the compounds, or salts or solutions of the compounds. The prodrugs include (but are not limited to) carboxylic acid esters, carbonates, phosphate esters, nitrate esters, sulfate esters, sulfone esters, sulfoxide esters, amino compounds, carbamates, azo compounds, phosphoramides, glucosides, ethers, acetals, etc., of the compounds.

[0140] This invention also includes any of the novel intermediates disclosed herein.

[0141] Pharmaceutical Compositions and Administration

[0142] The present invention also provides pharmaceutical compositions comprising a compound of formula II, or a tautomer thereof, an enantiomer, a diastereomer, a racemic mixture, a metabolic precursor, a pharmaceutically acceptable salt, an ester, a prodrug, or a hydrate thereof.

[0143] Because the compounds of the present invention have excellent antitumor activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate tumor-related diseases.

[0144] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 10-1000 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0145] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0146] The pharmaceutical composition is an injection, capsule, tablet, pill, powder, or granule.

[0147] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

[0148] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0149] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0150] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0151] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0152] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0153] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0154] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0155] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds (such as antitumor drugs).

[0156] The treatment method of the present invention can be used alone or in combination with other treatment methods or drugs.

[0157] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 50–1000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0158] Compared with the prior art, the present invention has the following main advantages:

[0159] (1) The DEL provided in this application contains approximately 140,125 compounds, which are very suitable for drug screening;

[0160] (2) The compound of formula II described in this application has excellent CBP / P300 inhibitory activity (such as excellent selective inhibition) and can be used to prevent and / or treat diseases related to abnormal CBP / P300 activity;

[0161] (3) The DEL synthesis method used in this application can synthesize a large number of compounds with DNA sequences, ranging from millions to tens of billions, in a short period of time, which can better meet the structural diversity and spatial quantity required for screening.

[0162] (4) DEL is mainly based on affinity screening. Since this screening involves screening the target protein and the entire compound library, rather than screening individual compounds one by one, it can significantly improve screening efficiency, shorten the drug development cycle, and reduce development costs.

[0163] (5) The structure-activity relationship (SAR) of the screened Hit can be analyzed from the screening data, providing a reference for the rational design of drug molecules, thereby accelerating the Hit to Lead process.

[0164] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0165] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0166] In all embodiments, the AOP headpiece and primer were purchased from Biosearch Technologies, Novato, CA.

[0167] DNA tags were purchased from General Bio;

[0168] The nucleic acid products were determined by HPLC / ESI-MS and gel electrophoresis. The HPLC model was Agilent Technologies 1260 Infinity II, and the reversed-phase column was OPTI-LYNX. TM Trap Columns, model: C18AQ-40μm; ESI-MS model: G6230B; horizontal electrophoresis tank, model: DYY-6C, purchased from Beijing Lu'an; gel imaging system, model: AZURE biosystems 200.

[0169] 1¹H NMR was recorded using a Bruker Avance Ⅲ-500 NMR spectrometer, with chemical shifts expressed as δ (ppm). Mass spectrometry was performed using UPLC-MS (ESI) with a Waters HPLC-CLASS UPLC and a Waters SQDetector2 MS (ESI). Solvents used in column chromatography, such as petroleum ether, ethyl acetate, and dichloromethane, were purchased from Sinopharm Chemical Reagent Co., Ltd. Thin-layer chromatography silica gel plates (HSGF254) used in reaction detection were from Sinopharm Chemical Reagent Co., Ltd. 200-300 mesh silica gel from Sinopharm Chemical Reagent Co., Ltd. was used for compound separation. 96-well or 384-well plates for the reaction were purchased from Sangon Biotech (Shanghai) Co., Ltd. Reagents, including chiral amino acids, aromatic acids, 2-nitrobenzyl alcohol, agarose, staining agents, and reagents for preparing buffer solutions, were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0170] The ligation buffer composition is 10X Buffer Composition, 400mM Tris-HCl, 100mM MgCl2, 100mM DTT, and 5mM ATP.

[0171] The DEL cycle 1-encoding uses a total of 475 characters, and the structure shown is cccctatgtCA, consisting of 9 pairs of deoxyribonucleotides and two deoxynucleotides, not all of which are listed.

[0172] The DEL cycle 2-encoding uses a total of 295 characters, and the structure shown as aatgtcgtcCT consists of 9 pairs of deoxyribonucleotides and two deoxynucleotides, which are not fully listed.

[0173] The structure of Fmoc is

[0174] The structure of Boc is

[0175] AOP header piece connects to the primer method:

[0176] (1) Dissolve 20 μmol of AOP headpiece in 20 mL of ultrapure water, add 13.34 mL of Primer (1.8 mM, water-soluble), then add 36.66 mL of ultrapure water and 8 mL of 10X ligation T4 buffer, and finally add 2 mL of T4 DNA ligase. Shake and react overnight at 18 °C.

[0177] (2) Add sodium chloride solution (5M) to the reaction solution, the volume of which is 10% of the total volume of the reaction solution (1), and then add frozen ethanol, the volume of which is 2.5 times the total volume after adding sodium chloride. After adding, place it at -80℃ for half an hour.

[0178] (3) Centrifuge the frozen reaction mixture (4℃, 10000 rpm, 30 min), remove the supernatant after centrifugation, cool the precipitate with liquid nitrogen, and freeze-dry to obtain powdered AOP headpiece-primer for later use.

[0179] DEL Construction Method

[0180] Example 1

[0181]

[0182] (1) 2695 μL of 1.15 mM AOP headpiece-primer was added in equal volumes to 475 wells. 2.6 μL of 10x ligation buffer, 0.65 μL of T4 DNA ligase, and 13.1 μL of water were added to each well. Subsequently, 475 different Tag1 solutions (3.97 μL of 1.8 mM aqueous solution) were aliquoted into each well, and the mixture was incubated overnight at 18°C ​​with shaking. The reaction products were analyzed by HPLC / ESI-MS (e.g., [missing information]). Figure 1 (taken from one well) and native-PAGE (4%, TAE, 115v, 45min, SYBR gold staining) detection (e.g.) Figure 2 After confirming complete ligation, add 10% (v / v) 5M NaCl solution and 2.5 times (v / v) cold ethanol (ethanol stored at -80°C) to the reaction mixture, then incubate the mixture at -80°C for 30 minutes. Centrifuge the mixture (4°C, 10000 rpm) for 30 minutes, remove the supernatant, cool the solid precipitate in liquid nitrogen for 30 minutes, lyophilize to obtain DEL cycle1-encoding, and dissolve it in 6.5 μL of pH 9.4 250 mM sodium borate buffer.

[0183] (2) Prepare 200 mM DMA solutions of 208 Fmoc protected amino acids and 64 aromatic acids respectively, and premix them with equal volumes of HATU and DIPEA (both 200 mM DMA solutions). Add the premixed reactants one by one into the 272 wells in (1) for acylation reaction. After overnight at room temperature, pool the 208 Fmoc protected amino acids and 64 aromatic acids separately, post-process them according to the method in step (1), and obtain solids, such as... Figure 3 and Figure 4 As shown in A ( Figure 3For test results taken from a single well, Figure 4 A represents the detection results of the product pooled together after the reaction. The solid product of Fmoc-protected amino acids was dissolved in 1352 μL of an aqueous solution containing 10% piperidine (v / v) and reacted for 2 hours to remove the protecting Fmoc group. The product was then post-processed and purified according to step (1). The OD value of the product was 996 nmol. The results are as follows: Figure 4 As shown in B.

[0184] (3) Add 160 equivalents of PNP (400mM / CH3CN) to the remaining 201 wells in (1) and premix for 5 min. Then add 6.5 μL of 201 diamines or Boc-protected diamines (200mM, dissolved in DMA) and react at room temperature for 3 h. Pool the Boc-protected reaction mixture and process it according to step (1). Remove the Boc protecting group from the obtained solid and mix it with the remaining diamine reactants and purify it to obtain 710 nmol of product.

[0185] (4) Following the acylation method in step (2), the pooled DEL containing 473 BB1 molecules was reacted with 3-nitro-4-(hydroxymethyl)benzoic acid. After post-treatment, a solid was obtained, yielding 1475 nmol. The results are as follows: Figure 5 As shown.

[0186] (5) Dissolve the solid obtained in step (4) in 1092 μL of water and distribute it evenly into 295 reaction wells. Connect tag2 using the tag1 ligation method described in step (1) (2 μL of 10X ligation buffer, 0.5 μL of T4 DNA ligase, 10.75 μL of water, and 3.05 μL of DNA tag (1.8 mM solution) per well). After complete ligation, post-process and purify to obtain DELcycle2-encoding. The detection results are as follows: Figure 6 As shown.

[0187] (6) Add 400 equivalents of amine (10 μL, 200 mM DMA solution) to each of the 294 wells, irradiate the reaction under UV light at 365 nm for 20 min, pool the reaction mixture and purify it to obtain a solid of 962 nmol.

[0188] The BB used to prepare DEL-Cycle1 in the above reaction has a structure selected from Table 1, and the BB used to prepare DEL-Cycle2 in the above reaction has a structure selected from Table 2.

[0189] Specifically, this embodiment uses the following specific substances as an example:

[0190] The tag1 information used in step (1) for the sampling well is: gaatccctgAG.

[0191] The tag2 information used in step (5) for the sampling well is: gacgacattGT.

[0192] The BB-1 molecule used in the sampling well in step (1) is as follows:

[0193] The BB-2 molecular structure used in the sampling well in step (5) is:

[0194] Figure 1 This is the result of DEL cycle 1-encoding mass spectrometry detection (taken from one well).

[0195] from Figure 1 It can be seen that 18885.8052 is the molecular weight of DELcycle1-encoding, indicating that tag1 was successfully linked.

[0196] Figure 2 This is the result of DEL cycle 1-encoding electrophoresis. The electrophoresis image shows a significant change in displacement after tag 1 was connected, indicating successful tag 1 connection. The result is consistent with... Figure 1 Consistent.

[0197] Figure 3 These are the results of DEL-Cycle1 mass spectrometry analysis (taken from well 1).

[0198] from Figure 3 It can be seen that the mass spectrometry shows that the molecular weight peak is the molecular weight peak after the reaction of DEL cycle 1-encoding with small molecules, indicating that the reaction was successful.

[0199] Figure 4 These are the mass spectrometry results from DEL-Cycle1. (A) The mass spectrometry results show the 208 amino acids pooled together after the reaction with DELcycle1-encoding; (B) The mass spectrometry results after the protecting group was removed.

[0200] from Figure 4 It can be seen that the spectral peaks have a significant leftward shift, indicating that the overall molecular weight has decreased and the protecting group has been removed.

[0201] Figure 3 and Figure 4 The differences are: Figure 3 From mass spectrometry results obtained from a single well, the specific molecular weight peak can be determined. Figure 4 Mass spectrometry results pooled together only show the overall mass spectrometry results and cannot be matched one-to-one with specific target molecular weight peaks.

[0202] Figure 5These are the mass spectrometry results from DEL-Cycle1 (475 components). (A) The mass spectrometry results after the reaction with 2-nitrobenzyl alcohol. (B) The mass spectrometry results after the reaction are pooled together.

[0203] from Figure 5 It can be seen that: with Figure 4 The results showed a significant shift in the mass spectrometry peaks, indicating that the reaction was successful.

[0204] Figure 6 This is the result of DEL cycle2-encoding mass spectrometry detection (taken from well 1).

[0205] from Figure 6 It can be concluded that tag2 was successfully connected.

[0206] Figure 7 This is the result of DEL cycle 2-encoding electrophoresis.

[0207] from Figure 7 It can be seen that the electrophoretic displacement changed significantly after tag2 was connected, indicating that tag2 was successfully connected.

[0208] Figure 8 The results of DEL-Cycle2 mass spectrometry (taken from well 1) are shown in Figure (A). Figure (B) shows the mass spectrometry results of the photoreaction products.

[0209] from Figure 8 It can be seen that the molecular weight peak of the photoreaction product is correct, indicating that the pore reaction was successful.

[0210] Figure 9 These are the mass spectrometry results of DEL-Cycle2 pooled together.

[0211] from Figure 9 It can be seen that the mass spectrometry peaks pooled together show a significant shift, indicating that the reaction was successful.

[0212] Example 2

[0213]

[0214] 2-Nitrobenzyl alcohol (6.10 mg, 0.02 mM) and 3-amino-2-(4-chlorobenzyl)propionic acid (17.01 mg, 0.08 mM) were dissolved in 100 mM PBS / MeOH (V / V = 1:1, pH = 8.0). The mixture was irradiated with UV light at 365 nm for 1 h and then incubated at room temperature for 0.5 h. The resulting reaction mixture was extracted three times with ethyl acetate, dried over Na₂SO₄, and filtered. The filtrate was concentrated under vacuum and subjected to column chromatography to give 6.0 mg of the product, with a yield of 92%.

[0215] Example 3

[0216]

[0217] Fmoc-(S)-3-amino-4-phenylbutyric acid (100 mg, 0.23 mmol) was dissolved in DMF (1 mL), and HATU (262 mg, 0.69 mmol) and methylamine hydrochloride (47 mg, 0.69 mmol) were added separately. Then, DIPEA (205 μL, 1.15 mmol) was slowly added dropwise. After the addition was complete, the mixture was reacted at room temperature for 1 hour. The solvent DMF was removed, and the resulting oily mixture was not further separated and proceeded directly to the next reaction step.

[0218] The oily mixture was dissolved in a 1:1 (2 mL) THF / MeOH solution, and 2 mL of 3 M NaOH aqueous solution was added. The mixture was stirred at room temperature until it became clear. After removing the solvent by rotary evaporation, the mixture was dissolved in ethyl acetate and washed three times with saturated sodium chloride aqueous solution. It was dried over anhydrous Na₂SO₄ and filtered. The filtrate was concentrated under vacuum, and the crude product was used directly in the next step without separation. The obtained crude product was dissolved in DMF (1 mL). 4-(hydroxymethyl)-3-nitrobenzoic acid (54 mg, 0.28 mmol) and HATU (261 mg, 0.69 mmol) were added, followed by dropwise addition of DIPEA (205 μL, 1.15 mmol). After reacting at room temperature for 2 hours, the solvent DMF was removed, and the resulting oily mixture was purified by silica gel chromatography using DCM / MeOH ratios of 50:1 to 30:1 as the eluent to give 58 mg of white solid. The three-step yield was 62.5%. 1H NMR (500MHz, DMSO) δ8.58(d,J=8.4Hz,1H),8.40(d,J=1.8Hz,1H),8.11(dd,J=8.1,1.8Hz,1H),7.91(d,J=8.1Hz,1H),7.81(q,J=4.1Hz,1H),7.29(d,J =8.5Hz,2H),7.22(d,J=8.5Hz,2H),5.64(s,1H),4.86(s,2H),4.54–4.45( m,1H),2.90–2.80(m,2H),2.56(d,J=4.6Hz,3H),2.40(d,J=6.9Hz,2H).13C NMR (126MHz, DMSO) δ170.14,163.46,146.53,141.27,137.80,134.02,132.05,131.00,13 0.77,128.46,128.03,122.98,59.88,48.69,40.09,38.70,25.48.LRMS(ESI):406.25(M+H + ).

[0219] The compound containing DNA-5a (2 μM) and 3-amino-2-(4-chlorobenzyl)propionic acid (8 μM) in 100 mM PBS / MeOH (1:1, pH 8.0) was treated with 365 nm UV light for 1 h and incubated at room temperature for 0.5 h. The resulting aqueous solution was extracted three times with ethyl acetate, dried over Na₂SO₄, and filtered. The filtrate was concentrated under vacuum. The crude product was purified by high performance liquid chromatography to obtain the DNA-6a compound in 95% yield. 1H NMR(500MHz,DMSO)δ12.45(s,1H),10.41(s,1H),8.42(d,J=8.4Hz,1H),7.81 (q,J=4.1Hz,1H),7.68(d,J=8.2Hz,1H),7.55(s,1H),7.42(dd,J=8.2,0.8Hz, 1H),7.34–7.29(m,4H),7.26–7.21(m,4H),4.51–4.43(m,1H),4.02(t,J=7.1 Hz,1H),3.97(ddd,J=14.2,6.8,2.8Hz,1H),3.15–3.11(m,1H),2.87–2.80(m, 3H), 2.76 (dd, J=13.9, 5.6Hz, 1H), 2.56 (d, J=4.6Hz, 3H), 2.40–2.36 (m, 2H). 13CNMR(126MHz,DMSO)δ173.75,170.28,165.36,160.25,145.54,137.91,137 .78,137.57,130.99,130.72,128.16,128.02,122.96,119.64,118.76,111. 20,48.59,45.65,44.84,40.43,38.70,34.33,25.49.LRMS(ESI):583.27(M+H + ).

[0220] Figure 10 This is the 1H NMR spectrum of compound dna-6a.

[0221] from Figure 10 It can be seen that the number and shift of hydrogen atoms shown in the 1H NMR spectrum correspond one-to-one with the hydrogen atoms in the compound dna-6a, and the obtained compound is the target compound.

[0222] Figure 11 This is the carbon NMR spectrum of compound dna-6a.

[0223] from Figure 11 It can be seen that the shifts in the carbon NMR spectrum correspond one-to-one with the carbon atoms in DNA-6a, and the obtained compound is the target compound.

[0224] The activity of compound DNA-6a was determined by Shanghai ChemPartner Co., Ltd. using IC50. 50 The value is 1822 nM, and the detection method is as follows:

[0225] (1) Prepare 1* analytical buffer (modified Tris buffer);

[0226] (2) The test compound was diluted to different concentrations, and the final fraction of dimethyl sulfoxide was 1%.

[0227] (3) Prepare enzyme solution in 1* analytical buffer;

[0228] (4) Add the peptide and [ to the buffer solution] 3 Preparation of substrate solution with H]-Ac-CoA;

[0229] (5) Incubate at room temperature for 15 minutes;

[0230] (6) Add 10 μL of substrate solution to each well to start the reaction. Incubate at room temperature for 60 minutes.

[0231] (7) Add cold Ac CoA to the 1x analytical buffer to stop the reaction;

[0232] (8) Transfer 25 μL of liquid from each well from the analytical plate to a flash plate and incubate at room temperature for 1 hour;

[0233] (9) Clean the flash plate three times with ddH2O + 0.1% Tween-20;

[0234] (10) Read the value using a liquid scintillation / luminescence counter;

[0235] (11) Use formula (1) to fit the data in Excel to obtain the suppression value;

[0236] (12) Formula (1): Equation (1): inh%=(Max-Signal) / (Max-Min)*100;

[0237] (13) Use formula (2) to perform XL fitting on the data to obtain the IC50 value;

[0238] (14)Y=Bottom+(Top-Bottom) / (1+(IC50 / X)*HillSlope);

[0239] (15)Y is% inhibition and X is compound concentration.

[0240] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound of formula II, or a pharmaceutically acceptable salt thereof, II in, A is -NH-; R1 is a C1-C3 alkyl group; B is a C1-C3 alkyl group; R2 is a C6-C12 aryl group; C is a C1-C3 alkyl group; R3 is a halogenated C6-C12 aryl group.

2. The compound as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound is .

3. A pharmaceutical composition, characterized in that, The compound of claim 1, or a pharmaceutically acceptable salt thereof, comprises a pharmaceutically acceptable carrier and a safe and effective amount thereof.

4. Use of the compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, Used to prepare drugs targeting CBP / P300.

5. Use of the compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, For the preparation of drugs for the prevention and / or treatment of diseases related to abnormal CBP / P300 activity; The diseases associated with abnormal CBP / P300 activity are selected from the following group: gastrointestinal cancer, cervical cancer, bladder cancer, laryngeal cancer, liver cancer, breast cancer, lung cancer, oral cancer, ovarian cancer, diabetes, inflammation, and pain.

6. The use as described in claim 5, characterized in that, The pain is a complex regional pain syndrome.

Images