WO2020177657A1 - Chemical compound having btk-degrading activity - Google Patents

Abstract

Provided in the present invention is a chemical compound having Btk-degrading activity. Specifically, provided in the present invention is a chemical compound as shown in formula I; definitions of each group are as set forth in the description. The chemical compound of the present invention has good Btk inhibitory activity and can degrade Btk; it may be used to prepare a drug for treatment of disease related to Btk activity. (I)

Description

A class of compounds with Btk degradation activity Technical field

The invention belongs to the field of medicine, and specifically relates to a class of compounds with the activity of degrading tyrosine protein kinase Btk, and preparation and application thereof.

Background technique

Btk (Bruton's tyrosine kinase) is a member of the Tec family of non-receptor tyrosine kinases. It is a gene necessary for cell differentiation and proliferation. It is also used in B-cell lymphoma and acute lymphoblastic leukemia (ALL ) And plasmacytoma. Btk is a key component of the B cell receptor (BCR) signaling pathway, and is a good site for targeted treatment of diseases such as B cell lymphoma.

Btk is a key regulator of B cell development, activation, signal transduction and survival, and is involved in the regulation of angiogenesis, cell proliferation and apoptosis, and cell movement. In addition, Btk is also involved in many other hematopoietic cell signal pathways, for example, involved in the signal pathway mediated by Toll-like receptors and cytokine receptors in macrophages, and involved in the signal transduction of IgE receptors in mast cells.

Recent studies have shown that the Btk signaling pathway is the current non-Hodgkin lymphoma (NHL), especially chronic lymphocytic leukemia (CLL), B cell lymphoma and autoimmune diseases (rheumatoid arthritis, psoriasis, etc.) New hot spots in clinical treatment research (Deng Rong, Zhao Lizhi. Research progress of Btk inhibitors. Pharmaceutical Research, 2014, 33(6): 359-372.).

Therefore, those skilled in the art devote themselves to developing compounds that can degrade Btk activity.

Summary of the invention

The purpose of the present invention is to provide a compound capable of inhibiting and degrading Btk, and its preparation and application.

In the first aspect of the present invention, there is provided a compound represented by the following formula I, or a pharmaceutically acceptable salt thereof:

among them:

A, Z, W ₂ are independently missing or selected from: O, NR ₁ , NR ₁ -CO, CO-NR ₁ , SO, SO ₂ , NR ₁ -SO ₂ , SO ₂ -NR ¹ , C(=O )-O, O-(C=O), C(=O), CH=CH, C≡C, O(CH ₂ ) _h , NH(CH ₂ ) _h , (CR ² ₂ ) _h , (CH ₂ CH ₂ O) _h , (OCH ₂ CH ₂ ) _h , NR ¹ -CO-(CH ₂ ) _h , CO-NR ¹ -(CH ₂ ) _h , CO-NR ¹ -(CH ₂ ) _h -O, 3 ~12-membered substituted or unsubstituted saturated or unsaturated cyclic hydrocarbon or heterocyclic hydrocarbon, substituted or unsubstituted aromatic ring or heteroaromatic ring; wherein R ^{1 is} selected from H, C _1-6 alkyl (straight chain or Branched chain, can be randomly substituted by 1 or more halogen, hydroxyl, cyano, amino or C _1-6 alkoxy), C _1-6 acyl (straight or branched chain, can be randomly substituted by 1 or more Halogen, hydroxy, cyano, amino or C _1-6 alkoxy substituted); wherein RW _{2 is} selected from H, hydroxy, amino, cyano, carboxy, ester, halogen, substituted or unsubstituted C _1-6 alkane Group, alkoxy group, acyl group, 3-12 membered substituted or unsubstituted cyclic hydrocarbon group or heterocyclic hydrocarbon group, substituted or unsubstituted aryl group or heteroaryl group; wherein h is an integer between 0 and 30;

W _{1 is} missing or selected from: C(=O), (CH ₂ )g, O(CH ₂ )g, (OCH ₂ CH ₂ )g; wherein g is preferably an integer between 0 and 10;

B is missing or selected from: O, C, CH, CH ₂ , C=O, S, NR ³ , NR ³ -C(=O), C(=O)-NR ³ , C(=O)-O, OC(=O)-O, NR ³ -C(=O)-O, OC(=O)-NR ³ , NR ³ -C(=O)-NR ⁴ , C with or without substituents _1-12 hydrocarbon group, _3-12 membered substituted or unsubstituted cyclic or heterocyclic hydrocarbon, substituted or unsubstituted aromatic ring or heteroaromatic ring; wherein R ³ and R ⁴ are each independently selected from H, with or C _1-8 hydrocarbon groups without substituents, C _3-12 cyclic hydrocarbon groups, heterocyclic hydrocarbon groups, aryl groups, heteroaryl groups;

X is selected from: C(R ⁵ ) ₂ , C(=O), S(=O), SO ₂ ; wherein R ^{5 is} each independently selected from: H, C _1-6 with or without substituents Hydrocarbyl; or two R ⁵ are connected to each other and form a 3-12-membered saturated or unsaturated carbocyclic or heterocarbocyclic ring (containing 1-3 heteroatoms) together with the connected carbon atoms;

a is an integer between 0-30.

In another preferred example, A is missing, or A is O(CH ₂ ) _h , where h is an integer between 0 and 5, such as h is 1, 2, 3, 4, or 5.

In another preferred example, W _{1 is} missing, or W ₁ is C(=O).

In another preferred example, Z is missing, or Z is O(CH ₂ ) _h , where h is an integer between 0 and 5, such as h is 1, 2, 3, 4, or 5.

In another preferred example, W _{2 is} missing, or W ₂ is NH.

In another preferred example, B is O.

In another preferred example, a is an integer between 2 and 30; preferably, a is an integer between 4 and 20; more preferably, a is an integer between 5 and 10; most preferably, a is An integer between 6 and 8; for example, a is 3, 4, 5, 6, 7, 8, 9, or 10.

In another preferred example, the X is CH ₂ .

In another preferred embodiment, A, Z, W ₂ are each independently missing or selected from: O, NH, C(=O)-O, O-(C=O), C(=O), (CH ₂ )h, CH ₂ -O, O(CH ₂ )h, NH(CH ₂ )h, (CH ₂ -CH ₂ -O)h, (OCH ₂ CH ₂ )h, NH-CO-(CH ₂ )h , CO-NH-(CH ₂ )h, CO-NH-(CH ₂ )hO, substituted or unsubstituted benzene ring, pyridine ring, piperazine ring, homopiperazine ring, piperidine ring, pyrrolidine ring; h is preferably an integer between 0 and 10; W _{1 is} missing or selected from: C(=O), (CH ₂ )g, O(CH ₂ )g, (OCH ₂ CH ₂ )g; wherein g is preferably 0 Integer between to 10; B is missing or selected from: O, C=O, NH, OC(=O), NH-C(=O), C(=O)-NH, C(=O)-O A is an integer between 0 and 30; X is preferably: C(R ⁵ )2, C(=O); wherein R ^{5 is} each independently selected from: H, C1～5 hydrocarbon group; or two R ⁵ mutually Connected and connected to the carbon atom together to form a homo-carbon disubstituted cycloalkane or heterocyclic hydrocarbon group;

In another preferred embodiment, A, Z, W ₂ are each independently missing or selected from: O, NH, C(=O)-O, O-(C=O), C(=O), (CH ₂ )h, CH ₂ -O, O(CH ₂ )h, NH(CH ₂ )h, (CH ₂ -CH ₂ -O)h, (OCH ₂ CH ₂ )h, NH-CO-(CH ₂ )h , CO-NH-(CH ₂ )h, CO-NH-(CH ₂ )hO, substituted or unsubstituted benzene ring, pyridine ring, piperazine ring, homopiperazine ring, piperidine ring, pyrrolidine ring; h is preferably an integer between 0 and 10; W _{1 is} missing or selected from: C(=O), (CH ₂ )g, O(CH ₂ )g, (OCH ₂ CH ₂ )g; wherein g is preferably 0 Integer between to 10; B is missing or selected from: O, C=O, NH, OC(=O), NH-C(=O), C(=O)-NH, C(=O)-O ; A is an integer between 0 and 10; X is preferably C(=O), CH ₂ .

In another preferred example, A, Z and W ₂ are each independently missing or selected from: O, NH, C(=O)-O, O-(C=O), C(=O), (CH ₂ )h, O(CH ₂ )h, NH(CH ₂ )h, (CH ₂ -CH ₂ -O)h, (OCH ₂ CH ₂ )h, NH-CO-(CH ₂ )h, CO-NH- (CH ₂ )h, CO-NH-(CH ₂ )hO; wherein h is preferably an integer between 0 and 10; W _{1 is} missing or selected from: C(=O), (CH ₂ )g, O(CH ₂ ) g, (OCH ₂ CH ₂ )g; wherein g is preferably an integer between 0 and 10; B is preferably: O; a is preferably an integer between 0 and 10; X is preferably C(=O), CH ₂ .

In another preferred example, the compound of the present invention does not include the specific compound disclosed in patent document CN109422752A and/or WO2019042445A1.

In another preferred embodiment, the compound is selected from the following group:

The second aspect of the present invention provides a pharmaceutical composition and a method of administration thereof. The composition contains the compound described in the first aspect, its isomers, prodrugs, pharmaceutically acceptable salts, and pharmaceutically acceptable salts. Acceptable carrier.

In another preferred embodiment, the pharmaceutical composition also contains another one or more anti-tumor agents.

In another preferred embodiment, the pharmaceutical composition is used to inhibit the activity of Bruton's tyrosine protein kinase (Btk) or reduce the level of Bruton's tyrosine protein kinase (Btk).

In another preferred embodiment, the pharmaceutical composition is used to treat diseases related to Bruton's tyrosine protein kinase (Btk) activity or expression.

The third aspect of the present invention provides a use of the compound according to the first aspect of the present invention for:

(a) Preparation of drugs for the treatment of diseases related to the activity or expression of Bruton's tyrosine protein kinase (Btk);

(b) Preparation of Bruton's tyrosine protein kinase (Btk) targeted inhibitors or degradation agents;

(c) Non-therapeutic inhibition or degradation of Bruton's tyrosine protein kinase (Btk) activity in vitro;

(d) Non-therapeutic inhibition of tumor cell proliferation in vitro; and/or

(e) Treatment of diseases related to the activity or expression of Bruton's tyrosine protein kinase (Btk).

In another preferred example, the diseases include tumors and autoimmune diseases; preferably, the tumors include non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), B-cell lymphoma, etc.; The autoimmune diseases include rheumatoid arthritis, psoriasis and the like.

The fourth aspect of the present invention provides a method for preparing the compound of formula I as described in the first aspect of the present invention, including the steps:

(a) In an inert solvent, a compound of formula III is reacted with a compound of formula II to obtain a compound of formula I, where M ₁ is a leaving group such as a hydroxyl group, a halogen, or a sulfonate or W ₁ -M1 is an aldehyde or ketone; In the formula, the definition of each group is as described above.

In another preferred embodiment, the method further includes the steps:

(b) In an inert solvent, a compound of formula IV is reacted with a compound of formula V to obtain a compound of formula II, where M ₂ is a leaving group such as a hydroxyl, halogen, or sulfonate, or Z-M1 is an aldehyde or ketone.

detailed description

After extensive and in-depth research, the inventor prepared a class of compounds with the structure shown in Formula I, and found that it has Bruton's tyrosine protein kinase (Btk) inhibitory and degradation activity. In addition, the compound has an inhibitory effect on Bruton's tyrosine protein kinase (Btk) at a very low concentration, and the inhibitory activity is quite excellent, so it can be used for treatment and Bruton's tyrosine protein kinase (Btk). Sex or expression related diseases such as tumors. The present invention has been completed on this basis.

The present invention discloses a new class of compounds and their use to inhibit and degrade Bruton's tyrosine protein kinase (Btk). These compounds can inhibit and degrade Btk, and can be used to treat tumors or autoimmune diseases.

the term

In the present invention, the term "C _1-8 hydrocarbon group" refers to a functional group containing only two types of atoms, carbon and hydrogen, in which the number of carbon atoms is 1-8. Hydrocarbyl can be regarded as the free radical left after the corresponding hydrocarbon loses one hydrogen atom, which can be alkyl, cycloalkyl, alkenyl or alkynyl, etc.; its structure can be linear, branched or cyclic; It is aliphatic or aromatic.

The term "C _1-6 alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl , Tert-butyl, or similar groups.

The term "alkoxy" as used herein includes O-alkyl, where "alkyl" is as defined above.

The term "halo" as used in the present invention includes fluoro, chloro, bromo or iodo unless otherwise indicated.

The compounds of the present invention may contain double bonds. When containing such double bonds, the compounds of the present invention exist in cis, trans or mixtures thereof.

The halogen described herein includes fluorine, chlorine, bromine and iodine.

Unless otherwise indicated, the alkyl group and the alkyl portion of the alkoxy group referred to herein can be linear, branched or cyclic.

In the present invention, the term "cycloalkyl" refers to a functional group containing both carbon and hydrogen atoms. Including cycloalkyl, cycloalkenyl (containing at least one carbon-carbon double bond) and aryl. They can be single ring, double ring and multiple ring. They can be spiro rings or fused rings.

In the present invention, the term "heterocyclic hydrocarbon group" refers to a functional group containing carbon, hydrogen and at least one heteroatom other than carbon and hydrogen. Including heterocycloalkyl, heterocycloalkenyl (containing at least one carbon-carbon double bond) and heteroaryl. One or more ring-forming atoms in the ring are heteroatoms. The heteroatoms can be O, N, and S atoms, and various combinations thereof. They can be single ring, double ring and multiple ring. They can be spiro rings or fused rings.

In the present invention, the term "substituent" includes but is not limited to fluorine, chlorine, bromine, cyano, hydroxyl, amino, C _1-6 hydrocarbyloxy, C _1-6 halogenated hydrocarbyl, C _1-6 acyl, C _{1 -6} Sulfonyl.

The term "hydrocarbyloxy" as used herein refers to an O-hydrocarbyl group, where the "hydrocarbyl group" is as defined above.

The term "hydrocarbyloxycarbonyl" as used herein refers to a C(=O)O-hydrocarbyl group, where the "hydrocarbyl group" is as defined above.

The term "amino group" as used herein refers to N (H or hydrocarbyl 1) (H or hydrocarbyl 2), where "hydrocarbyl" is as defined above.

The term "aminocarbonyl" as used herein refers to a C(=O)-amine group, where the "amino group" is as defined above.

The term "amide group" as used herein refers to N(H or hydrocarbyl)-C(=O)-hydrocarbyl, where "hydrocarbyl" is as defined above.

In the present invention, the term "comprising", "comprising" or "including" means that various ingredients can be used together in the mixture or composition of the present invention. Therefore, the terms "mainly consisting of" and "consisting of" are included in the term "containing".

In the present invention, the term "pharmaceutically acceptable" ingredients refers to substances that are suitable for humans and/or animals without excessive side effects (such as toxicity, irritation, and allergic reactions), that is, substances that have a reasonable benefit/risk ratio.

In the present invention, the term "effective amount" refers to the amount of a therapeutic agent that treats, alleviates, or prevents the target disease or condition, or shows a detectable therapeutic or preventive effect. The precise effective amount for a subject depends on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Therefore, it is useless to specify an accurate effective amount in advance. However, for a given condition, routine experiments can be used to determine the effective amount, which can be judged by clinicians.

In this article, unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on the group are substituted by a substituent selected from the following group: halogen, unsubstituted or halogenated C _1-6 alkyl, Unsubstituted or halogenated C _2-6 acyl, unsubstituted or halogenated C _1-6 alkyl-hydroxy.

Unless otherwise specified, in the present invention, all compounds appearing are intended to include all possible optical isomers, such as a single chiral compound, or a mixture of various chiral compounds (ie, racemates). In all the compounds of the present invention, each chiral carbon atom may optionally be R configuration or S configuration, or a mixture of R configuration and S configuration.

As used herein, the term "compound of the invention" refers to a compound represented by Formula I. The term also includes various crystal forms, pharmaceutically acceptable salts, hydrates or solvates of the compound of formula I.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention and an acid or base suitable for use as a medicine. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts are the salts of the compounds of this invention with acids. Acids suitable for salt formation include but are not limited to: hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid and other inorganic acids, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, Organic acids such as maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, toluenesulfonic acid, and benzenesulfonic acid; and acidic amino acids such as aspartic acid and glutamic acid.

Compound and pharmaceutically acceptable salt thereof

The present invention relates to a compound of the following formula I or a pharmaceutically acceptable salt thereof;

among them:

A, Z, W ₂ are independently missing or selected from: O, NR ¹ , NR ¹ -CO, CO-NR ¹ , SO, SO ₂ , NR ¹ -SO ₂ , SO ₂ -NR ¹ , C(=O )-O, O-(C=O), C(=O), CH=CH, C≡C, O(CH ₂ ) _h , NH(CH ₂ ) _h , (CR ² ₂ ) _h , (CH ₂ CH ₂ O) _h , (OCH ₂ CH ₂ ) _h , NR ¹ -CO-(CH ₂ ) _h , CO-NR ¹ -(CH ₂ ) _h , CO-NR ¹ -(CH ₂ ) _h -O, 3 ~12-membered substituted or unsubstituted saturated or unsaturated cyclic hydrocarbon or heterocyclic hydrocarbon, substituted or unsubstituted aromatic ring or heteroaromatic ring; wherein R ^{1 is} selected from H, C _1-6 alkyl (straight chain or Branched chain, can be randomly substituted by 1 or more halogen, hydroxyl, cyano, amino or C _1-6 alkoxy), C _1-6 acyl (straight or branched chain, can be randomly substituted by 1 or more Halogen, hydroxy, cyano, amino or C _1-6 alkoxy substituted); wherein RW _{2 is} selected from H, hydroxy, amino, cyano, carboxy, ester, halogen, substituted or unsubstituted C _1-6 alkane Group, alkoxy group, acyl group, 3-12 membered substituted or unsubstituted cyclic hydrocarbon group or heterocyclic hydrocarbon group, substituted or unsubstituted aryl group or heteroaryl group; wherein h is an integer between 0 and 30;

W _{1 is} missing or selected from: C(=O), (CH ₂ )g, O(CH ₂ )g, (OCH ₂ CH ₂ )g; wherein g is preferably an integer between 0 and 10;

B is missing or selected from: O, C, CH, CH ₂ , C=O, S, NR ³ , NR ³ -C(=O), C(=O)-NR ³ , C(=O)-O, OC(=O)-O, NR ³ -C(=O)-O, OC(=O)-NR ³ , NR ³ -C(=O)-NR ⁴ , C with or without substituents _1-12 hydrocarbon group, _3-12 membered substituted or unsubstituted cyclic or heterocyclic hydrocarbon, substituted or unsubstituted aromatic ring or heteroaromatic ring; wherein R ³ and R ⁴ are each independently selected from H, with or C _1-8 hydrocarbon groups without substituents, C _3-12 cyclic hydrocarbon groups, heterocyclic hydrocarbon groups, aryl groups, heteroaryl groups;

X is selected from: C(R ⁵ ) ₂ , C(=O), S(=O), SO ₂ ; wherein R ^{5 is} each independently selected from: H, C _1-6 with or without substituents Hydrocarbyl; or two R ⁵ are connected to each other and form a 3-12-membered saturated or unsaturated carbocyclic or heterocarbocyclic ring (containing 1-3 heteroatoms) together with the connected carbon atoms;

a is an integer between 0-30.

对化合物的活性具有显著的影响。本发明通过大量研究，对化合物中的linker连接方式和结构进行了优化，结果表明linker的连接位置和结构对化合物活性影响极大，比如linker连接哌啶环的两个结构单元处于对位时不具有Btk降解活性，而处于间位时具有Btk降解活性。另外，研究发现，当linker结构中，a为5或以上时，本发明的化合物不仅抗肿瘤活性显著提高，而且对依鲁替尼耐药的肿瘤细胞表现出了显著地抑制作用，取得了预料不到的技术效果。 The inventor found in the research that in the compound of the present invention, the structure of the linker

It has a significant impact on the activity of the compound. The present invention has optimized the linker connection mode and structure in the compound through a large number of studies. The result shows that the linker’s connection position and structure have a great influence on the activity of the compound. It has Btk degradation activity, and has Btk degradation activity when in meta position. In addition, studies have found that when a in the linker structure is 5 or more, the compound of the present invention not only significantly improves anti-tumor activity, but also exhibits a significant inhibitory effect on ibrutinib-resistant tumor cells, which is expected Less technical effect.

The compound package of the present invention can form pharmaceutically acceptable salts with inorganic acids, organic acids or bases. The inorganic acid includes but is not limited to hydrochloric acid, hydrobromic acid, nitric acid, perchloric acid, sulfuric acid or phosphoric acid; the organic acid includes but not limited to methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, benzene Sulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, acetic acid, maleic acid, ascorbic acid, lactic acid, tartaric acid, malonic acid, glycolic acid, succinic acid and propionic acid, etc.; the alkali includes but is not limited to inorganic Salts and amines.

The term pharmaceutically acceptable salts refers to those salts suitable for contact with tissues of humans and mammals without excessive toxicity, irritation, allergic reactions, etc. according to medical judgment. Pharmaceutically acceptable salts are well known in the art.

The present invention also encompasses pharmaceutical compositions containing prodrugs of compounds of formula I. Prodrugs include compounds in which the precursor molecule is covalently bound to the free carboxyl group of the compound of formula I through a carbonate bond, a carbamate bond, an amide bond, an alkyl ester bond, a phosphate bond, and a phosphoramidate bond. On the hydroxyl, amino or amine group.

Compound preparation

Preparation

The preparation method of the compound of formula I of the present invention is described in more detail below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention can also be conveniently prepared by combining various synthetic methods described in this specification or known in the art, and such combinations can be easily performed by those skilled in the art to which the present invention belongs.

The following reaction scheme illustrates the preparation of the compounds of the invention. Unless otherwise indicated, the groups in the reaction scheme and subsequent discussions are as defined above.

Generally speaking, the following scheme can be adopted to obtain the compound of formula I:

The preparation method of the compound of formula I includes the steps:

(a) In an inert solvent, a compound of formula III is reacted with a compound of formula II to obtain a compound of formula I, where M ₁ is a leaving group such as a hydroxyl group, a halogen, or a sulfonate or W ₁ -M1 is an aldehyde or ketone; In the formula, the definition of each group is as described above.

In another preferred embodiment, the method further includes the steps:

(b) In an inert solvent, a compound of formula IV is reacted with a compound of formula V to obtain a compound of formula II, where M ₂ is a leaving group such as a hydroxyl, halogen, or sulfonate, or Z-M1 is an aldehyde or ketone.

Application of compound of formula I

The compound of formula I can be used for one or more of the following purposes:

(a) Preparation of drugs for the treatment of diseases related to the activity or expression of Bruton's tyrosine protein kinase (Btk);

(b) Preparation of Bruton's tyrosine protein kinase (Btk) targeted inhibitors or degradation agents;

(c) Non-therapeutic inhibition or degradation of Bruton's tyrosine protein kinase (Btk) activity in vitro;

(d) Non-therapeutic inhibition of tumor cell proliferation in vitro; and/or

(e) Treatment of diseases related to the activity or expression of Bruton's tyrosine protein kinase (Btk).

In another preferred embodiment, the disease related to the activity or expression of Bruton's tyrosine protein kinase (Btk) is a tumor, preferably a tumor selected from the group consisting of non-small cell lung cancer, inflammatory muscle fiber Cell tumor and so on.

The compound of formula I of the present invention can be used to prepare a pharmaceutical composition, which comprises: (i) an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable Carrier.

In another preferred embodiment, the effective amount refers to a therapeutically effective amount or an inhibitory effective amount.

The compound of formula I of the present invention can also be used in a method for inhibiting or degrading Bruton's tyrosine protein kinase (Btk). The inhibition is non-therapeutic in vitro or can be therapeutic.

In another preferred embodiment, when an inhibitory effective amount of the compound of formula I of the present invention or a pharmaceutically acceptable salt thereof is administered to the inhibitory object, the inhibitory effective amount is 0.001-500 nmol/L, preferably 0.01 -200nmol/L.

In particular, the present invention also provides a method for treating diseases related to the activity or expression of Bruton's tyrosine protein kinase (Btk), the method comprising: administering a therapeutically effective amount of a compound of formula I to a subject, Or the pharmaceutical composition containing the compound of formula I as an active ingredient.

Pharmaceutical composition and method of administration

Since the compound of the present invention has excellent inhibitory activity on Bruton's tyrosine protein kinase (Btk), the compound of the present invention and various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates , And the pharmaceutical composition containing the compound of the present invention as the main active ingredient can be used to treat, prevent, and alleviate diseases related to Btk activity or expression. According to the prior art, the compounds of the present invention can be used to treat diseases including tumors.

The pharmaceutical composition of the present invention contains the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier within a safe and effective amount. The "safe and effective amount" refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Generally, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention/agent, more preferably, 5-200 mg of the compound of the present invention/agent. Preferably, the "one dose" is a capsule or tablet.

)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。 "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which 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 blended with the compound of the present invention and between them without significantly reducing the efficacy of the compound. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and 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 lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The method of administration of the compound or pharmaceutical composition of the present invention is not particularly limited. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration .

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 mixed with the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectant, For example, glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators, such as quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, hard Calcium fatty acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the active compound or the release of the compound in such a composition may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microcapsules with one or more of the above-mentioned excipients.

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

In addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.

In addition to the active compound, the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

The composition for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous 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.

The dosage form of the compound of the present invention for topical administration includes ointment, powder, patch, spray and inhalant. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.

The compound of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage is the pharmaceutically effective dosage considered to be administered. For a 60kg body weight, the daily The administered dose is usually 1 to 2000 mg, preferably 5 to 500 mg. Of course, the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skill range of a skilled physician.

The main advantages of the present invention include:

1. A compound of formula I is provided.

2. A Btk inhibitor with a novel structure and its preparation and application are provided. The inhibitor can inhibit the activity of Btk at a very low concentration.

3. A pharmaceutical composition for treating diseases related to Btk enzyme activity is provided.

4. Through a lot of research, the present invention optimizes the linker connection mode and structure in the compound. It was unexpectedly found that the linker's connection position and structure have a great influence on the activity of the compound. For example, the two structural units connecting the piperidine ring are in the right It has no Btk degradation activity when it is in the position, but it has Btk degradation activity when it is in the meta position. In addition, studies have found that when a is 5 or more in the linker structure, the compound of the present invention not only significantly improves anti-tumor activity, but also exhibits a significant inhibitory effect on ibrutinib-resistant tumor cells, which is expected Less technical effect.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples usually follow the conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.

Example 1 Preparation of Compound 1

first step:

Dissolve 100 mg of 4-hydroxythalidomide, 120 mg of pentaethylene glycol monobenzyl ether, and 100 mg of triphenylphosphine in 10 ml of anhydrous tetrahydrofuran, add 95 mg of diisopropyl azodicarboxylate dropwise, and react at room temperature for 2 hours. THF was removed under reduced pressure and purified by column chromatography to obtain 112 mg of compound 1-1. MS(ESI): 585[M+H] ⁺ .

The second step:

100 mg of compound 1-1 and 50 mg of 10% palladium on carbon were added to 10 ml of methanol and hydrogenated at room temperature overnight. After filtration, the filtrate was concentrated, and the residue was purified by column chromatography to obtain compound 1-2 63mg. MS(ESI): 495[M+H] ⁺ .

third step:

Dissolve 50mg of compound 1-2 in 5ml of dichloromethane, add 47mg of Dess-Martin oxidant, and react at room temperature for 2h. A saturated aqueous solution of sodium bicarbonate and a saturated aqueous solution of sodium thiosulfate were added, stirred for 5 min, and the organic layer was separated, dried over anhydrous sodium sulfate, and concentrated to dryness to obtain compound 1-3 directly for the next reaction.

After dissolving compound 1-3 in 5ml of dichloromethane, add 50mg of ibrutinib intermediate, 1ml of glacial acetic acid and 50mg of sodium triacetoxyborohydride, react at room temperature overnight, distilled off dichloromethane under reduced pressure, and column chromatography After purification, compound 1, 61 mg was obtained. MS(ESI): 863[M+H] ⁺ . ¹ H NMR(400MHz,CDCl3)δ9.78-10.02(br,1H), 8.37(d,J=7.6Hz,1H), 7.59-7.66(m,3H),7.43(d,J=7.2Hz,1H ), 7.36 (m, 2H), 7.22 (m, 1H), 7.05-7.18 (m, 4H), 5.69 (br, 2H), 4.90-5.03 (m, 2H), 4.30 (t, J = 4.8Hz, 2H), 3.60-3.95 (m, 16H), 3.20 (br, 1H), 2.95 (br, 1H), 2.62-2.94 (m, 6H), 2.06-2.1 (m, 4H), 1.83 (m, 2H) .

Example 2 Preparation of Compound 34

first step:

Dissolve 12.01 g of triethylene glycol monobenzyl ether in 150 ml of anhydrous tetrahydrofuran, add 2.00 g of 60% sodium hydride at room temperature, and react under reflux for 1 h. Cool the reaction solution to room temperature, add 6.41 g of tert-butyl acrylate, and react at room temperature overnight. The solvent was removed under reduced pressure, and 6.21 g of compound 34-1 was obtained after purification by column chromatography. MS (ESI): 369 [M+Na] ⁺ .

The second step:

Add 6.00 g of compound 34-1 and 1.50 g of 10% palladium on carbon to 60 ml of methanol, and hydrogenate at room temperature overnight. The reaction solution was filtered with celite, the filter cake was washed with methanol 20ml*3, the filtrate and the washing solution were combined, and concentrated under reduced pressure to obtain compound 34-24.54g. MS(ESI): 279[M+H] ⁺ .

third step:

Dissolve 2.78 g of compound 34-2 in 50 ml of dichloromethane, add 5.00 g of Dess-Martin oxidant, and react at room temperature for 2 hours. Add a saturated aqueous solution of sodium bicarbonate and a saturated aqueous solution of sodium thiosulfate, stir for 10 min, separate the organic layer, concentrate to dryness, and use the crude product directly in the next step.

After the crude product was dissolved in 100ml of dichloroethane, 2.73g of pomalidomide and 5ml of glacial acetic acid were added, and 6.36g of sodium triacetoxyborohydride was added in batches, reacted at room temperature overnight, concentrated under reduced pressure, and purified by column chromatography. Compound 34-3675mg. MS(ESI): 534 [M+H] ⁺ .

the fourth step:

500mg of compound 34-3 was dissolved in 20ml of dichloromethane, 5ml of 2M hydrogen chloride ether solution was added under ice-water bath, and stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to obtain 455 mg of compound 34-4. MS(ESI): 476[MH] ^- .

the fifth step:

Dissolve 50mg of compound 34-4 and 50mg of pomalidomide intermediate in 5ml of dichloromethane, add 20mg of 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodioxide 21 mg of imine hydrochloride, reacted overnight at room temperature. Dichloromethane was distilled off under reduced pressure and purified by column chromatography to obtain compound 34, 45 mg. MS (ESI): 846 [M+H] ⁺ . ¹ H NMR(400MHz,CDCl3)δ9.95(br,1H), 8.38(d,J=7.6Hz,1H), 7.65(m,2H),7.47(m,1H),7.04-7.38(m,8H ), 6.85 (d, J = 8.8 Hz, 1H), 6.47 (m, 1H), 5.83 (br, 2H), 4.90-4, 98 (m, 2H), 3.98 (m, 1H), 3.15 to 3.74 ( m, 19H), 2.10-2.85 (m, 6H), 1.68-2.00 (m, 4H).

Example 3 Synthesis of Compound 2

Step 1: Synthesis of compound 2-1

Dissolve hexaethylene glycol (28.2 g, 100 mmol) in dichloromethane (500 mL), add tert-butyldimethylchlorosilane (15.0 g, 100 mmol) and triethylamine (12.1 g, 120 mmol), and react at room temperature for 16 hours. The reaction solution was washed with 10% potassium dihydrogen sulfate aqueous solution, concentrated and evaporated to dryness, and purified by silica gel column chromatography to obtain colorless oily product 2-1 (17.1 g, yield 43%).

Step 2: Synthesis of compound 2-2

Compound 2-1 (17.0g, 43mmol) was dissolved in anhydrous tetrahydrofuran (400mL), triphenylphosphine (13.5g, 52mmol) and diisopropyl azodicarboxylate (11.3g, 120mmol) were added, and nitrogen atmosphere React at room temperature for 16h. The reaction solution was concentrated and evaporated to dryness, and purified by silica gel column chromatography to obtain a colorless oily product 2-2 (10.1 g, yield 36%).

Step 3: Synthesis of compound 2-3

Compound 2-2 (10.0 g, 15 mmol) was dissolved in 0.1 M hydrochloric acid methanol solution (100 mL), and reacted at room temperature for 4 hours. The reaction solution was concentrated and evaporated to dryness, and purified by silica gel column chromatography to obtain a pale yellow oily product 2-3 (6.6 g, yield 81%).

Step 4: Synthesis of compound 2

Compound 2-3 (6.5 g, 12 mmol) was dissolved in 1,2-dichloroethane (100 mL), and Dess-Martin reagent (10.4 g, 24 mmol) was added in three batches, and reacted at room temperature for 2 hours. The reaction solution was washed successively with 10% sodium thiosulfate solution and 5% sodium carbonate solution and then separated. The organic phase was added with Ibrutinib intermediate (3.1g, 8mmol) and acetic acid (5mL), and boron acetate was added in three batches Sodium hydride (7.6g, 36mmol) was reacted at room temperature for 16h. The reaction solution was filtered, concentrated and evaporated to dryness, and purified by silica gel column chromatography to obtain a yellow solid product 2 (0.99 g, yield 9%).

MS(ESI,m/z):907.2[M+H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ11.11(s,1H), 8.24(s,1H), 7.80(t,J=8.0Hz,1H), 7.69-7.61(m,2H), 7.52( d,J=8.4Hz,1H),7.48–7.39(m,5H),7.23–7.09(m,5H),5.08(dd,J=5.6Hz,1H),4.76(m,1H),4.38–4.28 (m,2H),3.79(t,J=4.8Hz,2H),3.67–3.59(m,2H),3.55–3.39(m,17H),3.11–3.00(m,1H),2.96–2.81(m ,2H), 2.68-2.52(m,4H), 2.10-1.91(m,4H), 1.84-1.59(m,2H).

Example 4 Synthesis of Compound 5

Step 1: Synthesis of compound 5-1

Dissolve pentaethylene glycol (23.8g, 100mmol) in dichloromethane (500mL), add tert-butyldimethylchlorosilane (15.0g, 100mmol) and triethylamine (12.1g, 120mmol), and react at room temperature for 16h. The reaction solution was washed with a 10% potassium dihydrogen sulfate aqueous solution, concentrated and evaporated to dryness, and purified by silica gel column chromatography to obtain a colorless oily product 5-1 (15.8 g, yield 45%).

Step 2: Synthesis of compound 5-2

Compound 5-1 (15.1 g, 43 mmol) was dissolved in dichloromethane (300 mL), p-toluenesulfonyl chloride (9.0 g, 47 mmol) and triethylamine (5.2 g, 51 mmol) were added, and reacted at room temperature for 16 h. The reaction solution was washed with saturated sodium carbonate aqueous solution, concentrated and evaporated to dryness, and purified by silica gel column chromatography to obtain a colorless oily product 5-2 (16.7 g, yield 77%).

The third step: Synthesis of compound 5-3

Compound 5-2 (16.2g, 32mmol) was dissolved in acetonitrile (300mL), phthalimide (5.6g, 38mmol) and potassium carbonate (6.6g, 48mmol) were added, and the reaction was refluxed for 16h. The reaction solution was extracted with ethyl acetate, concentrated and evaporated to dryness, and purified by silica gel column chromatography to obtain a colorless oily product 5-3 (12.3 g, yield 80%).

Step 4: Synthesis of compound 5-4

Compound 5-3 (12.0 g, 25 mmol) was dissolved in 0.1 M hydrochloric acid methanol solution (120 mL) and reacted at room temperature for 4 h. The reaction solution was concentrated and evaporated to dryness, and purified by silica gel column chromatography to obtain a pale yellow oily product 5-4 (8.1 g, yield 90%).

Step 5: Synthesis of compound 5-5

Compound 5-4 (8.0 g, 21 mmol) was dissolved in dichloromethane (200 mL), p-toluenesulfonyl chloride (4.5 g, 24 mmol) and triethylamine (2.7 g, 27 mmol) were added, and reacted at room temperature for 16 h. The reaction solution was washed with saturated sodium carbonate aqueous solution, concentrated and evaporated to dryness, and purified by silica gel column chromatography to obtain a pale yellow oily product 5-5 (8.3 g, yield 73%).

Step 6: Synthesis of compound 5-6

Compound 5-5 (8.0g, 15mmol) was dissolved in tetrahydrofuran (200mL), Ibrutinib intermediate (5.9g, 15mmol) and diisopropylethylamine (2.7g, 21mmol) were added and reacted at 60°C 24h. The reaction solution was washed with a 10% potassium dihydrogen sulfate aqueous solution, concentrated and evaporated to dryness, and purified by silica gel column chromatography to obtain a pale yellow oily product 5-6 (5.3 g, yield 47%).

Step 7: Synthesis of compound 5-7

Compound 5-6 (5.0 g, 6.8 mmol) was dissolved in ethanol (200 mL), hydrazine hydrate (3.4 g, 68 mmol) was added, and reacted at room temperature for 24 hours. After the reaction solution was filtered, the filtrate was concentrated and evaporated to dryness. After purification by silica gel column chromatography, a yellow oily product 5-7 (2.3g, yield 56%) was obtained.

Step 8: Synthesis of compound 5

Compound 5-7 (2.0g, 3.3mmol) was dissolved in tetrahydrofuran (100mL), and 4-floxalidomide (1.09g, 3.9mmol) and diisopropylethylamine (0.64g, 5.0mmol) were added , Reaction at 60℃ for 24h. The reaction solution was concentrated and evaporated to dryness, and purified by silica gel column chromatography to obtain a yellow solid product 5 (0.48 g, yield 17%).

MS(ESI,m/z):862.3[M+H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ11.11(s,1H), 8.27(s,1H), 7.65(d,J=8.4Hz,2H), 7.57(dd,J=7.2Hz,1H) ,7.49–7.40(m,4H),7.25–7.09(m,6H),7.03(d,J=7.2Hz,1H), 6.59(t,J=6.0Hz,1H),5.05(dd,5.6Hz, 1H), 4.76(m,1H), 3.91–3.34(m,23H), 3.17(d,J=5.2Hz,1H), 2.94–2.83(m,1H), 2.64–2.51(m,3H), 2.50 –2.45(m,2H),2.17–1.92(m,4H).

Example 5 Synthesis of Compound 6

Compound 6 was prepared by referring to the synthesis method of compound 5 in Example 4.

MS(ESI,m/z):906.3[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ 11.11 (s, 1H), 8.24 (s, 1H), 7.67 - 7.63 (m, 2H), 7.48 - 7.42 (m, 3H), 7.22 - 7.09 (m ,6H),7.01(dd,J=7.6Hz,2H),6.52(s,2H),5.12(dd,J=5.6Hz,1H),4.76(m,1H),3.89–3.75(m,2H) ,3.54–3.41(m,22H),3.11–2.85(m,3H),2.80–2.70(m,1H),2.12–1.94(m,4H),1.84–1.74(m,1H),1.65(m, 1H), 1.31-1.20 (m, 2H).

Example 6 Synthesis of Compound 7

Compound 7 was prepared by referring to the synthesis method of compound 5 in Example 4.

MS(ESI,m/z): 952.3[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ 11.11 (s, 1H), 8.24 (s, 1H), 7.70-7.62 (m, 2H), 7.53-7.39 (m, 3H), 7.24-7.09 (m ,6H),7.01(t,J=7.6Hz,2H),6.53(s,2H),5.12(dd,J=5.6Hz,1H),4.76(m,1H),3.92-3.72(m,2H) ,3.59–3.37(m,24H),3.33–3.29(m,2H),3.08–2.88(m,2H),2.80–2.64(m,1H),2.48–2.30(m,2H),2.11–1.91( m, 4H), 1.85-1.72 (m, 1H), 1.65 (m, 1H), 1.29-1.18 (m, 1H).

Example 7 Synthesis of Compound 8

Compound 8 was prepared by referring to the synthesis method of compound 5 in Example 4.

MS(ESI,m/z):996.3[M+H] ⁺ .

¹ H NMR(400MHz,DMSO-d6)δ11.11(s,1H), 8.24(s,1H), 7.68-7.62(m,2H), 7.50-7.39(m,3H), 7.24-7.09(m, 6H), 7.01 (t, J = 7.6 Hz, 2H), 6.53 (s, 2H), 5.12 (dd, J = 5.6 Hz, 1H), 4.76 (m, 1H), 3.90–3.72 (m, 2H), 3.59–3.29(m,30H), 3.08–2.88(m,2H), 2.80–2.67(m,1H), 2.48–2.30(m,2H), 2.11–1.91(m,4H), 1.85–1.72(m ,1H),1.65(m,1H),1.29-1.20(m,1H).

In the same way, referring to the methods in the above examples, the compounds in the following table can be obtained:

Example 8 Western blot detection of the activity of the compound to degrade Btk protein

Cell line: Ramos cell line was cultured in an incubator at 37°C, 5% CO ₂ and saturated humidity with RPMI1640 medium containing 10% serum.

Set up a DMSO control group and test compound (0.5 μM). After 24 hours of treatment, cells are collected and pre-chilled cell lysate is added. Placed on ice for 10 minutes, total cell protein is extracted. Protein concentration is determined and quantified by BCA method. Routine gel preparation, sample loading, electrophoresis, transfer to membrane, and block. Dilute the primary antibody in the blocking solution at a ratio of 1:500-5000, immerse the membrane in the diluted primary antibody and incubate overnight at 4°C. After rinsing, dilute the secondary antibody with the blocking solution 1:10000-20000, immerse the membrane in the diluted secondary antibody, and incubate at room temperature for 45 minutes. After rinsing, check the test results on ODYSSEY (Li-COR). GAPDH was used as an internal reference control.

Use Image J software to perform grayscale analysis on each band, and calculate the degradation rate of the compound to degrade Btk protein.

The results showed that the compound of the present invention (0.5μM) has significant degradation activity on Btk protein in Ramos cells, and the degradation rate of compound 1, 2, 5, 6, 8, 10, 11, 20, 21, 25, 26 reached 90 %the above.

Compound BTK degradation rate Compound BTK degradation rate 1 ++++ 11 ++++ 2 ++++ 20 ++++ 5 ++++ twenty one ++++ 6 ++++ 25 ++++ 8 ++++ 26 ++++ 10 ++++ Control Compound I -

Note: In the above table: "++" means the degradation rate is 10%-30%; "+++" means the degradation rate is 30%-90%; "++++" means the degradation rate is >90%; -" represents no degradation activity.

The structure of the control compound I is as follows:

Example 9 CTG method to detect the inhibitory effect of compounds on the proliferation of DOHH2 cells (human B-cell lymphoma cells)

The inhibitory effect of the compound on the proliferation of DOHH2 cells (available from ATCC) was tested in vitro by the CTG method. The cells were cultured in RPMI1640 medium with 10% serum. Inoculate a 96-well plate with 1×10 ⁴ cells/well, and place it in a 37°C, 5% CO ₂ incubator. The incubation time after adding the test compound (10 μM) was 72 hours. Then add an appropriate amount of CTG reagent, measure the luminescence value and calculate the inhibition rate.

Experimental results show that the compounds of the present invention have significant anti-DOHH2 cell proliferation activity, and compounds 1, 2, 3, 5, and 6 have more excellent anti-DOHH2 cell proliferation activity.

Compound Inhibition rate Compound Inhibition rate 1 ++++ 3 ++++ 2 ++++ 5 ++++ 6 ++++ To To

Note: In the above table: "++" means the inhibition rate is 10%-30%; "+++" means the inhibition rate is 30%-90%; "++++" means the inhibition rate is >90%.

Example 10 CTG method to detect the inhibitory effect of the compound on the proliferation of REC-1 and other tumor cells

In vitro CTG method was used to test the compound's proliferation inhibitory effects on REC-1 (human mantle cell tumor cells, available from ATCC) and TMD-8 (human diffuse large B lymphoma cells, available from ATCC). The cells were cultured in RPMI1640 medium with 10% fetal bovine serum. Inoculate a 96-well plate with 1×10 ⁴ cells/well, and place it in a 37°C, 5% CO ₂ incubator. The incubation time after adding the test compound (10 μM) was 72 hours. Then add an appropriate amount of CTG reagent, measure the luminescence value, and calculate the inhibition rate.

Experimental results show that the anti-TMD-8 cell proliferation activity of compounds 1, 2, 5, and 6 is greater than 90%.

Note: In the above table: "++" means the inhibition rate is 10%-30%; "+++" means the inhibition rate is 30%-90%; "++++" means the inhibition rate is >90%.

Example 11 Anti-tumor effect of compound in mouse TMD-8 tumor model

TMD-8 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum FBS. The cells were placed in a 5% CO ₂ incubator at 37°C.

Establish tumor SCID mouse subcutaneous transplantation model by cell inoculation method: Collect tumor cells in logarithmic growth phase, resuspend them in RPMI-1640 medium after counting, add Matrigel 1:1, and adjust the cell suspension concentration to 4×10 ⁷ cells Cells/ml. A 1ml syringe was used to inoculate tumor cells subcutaneously on the right back of SCID mice, 4×10 ⁶ cells/0.1ml/mouse, a total of 35 mice were inoculated.

When the average tumor volume of the animal is about 140 mm ³ , the animals that are too large, too small, or have irregular tumor shapes are eliminated, 30 tumor-bearing mice with a tumor volume of 101.34 ~ 209.86 mm ³ are selected, and the animals are divided into 3 groups by random block method The groups are the model group, the control compound 1 group, and the compound 6 group, with 10 mice in each group. The day of grouping is recorded as Day 0, and the administration is started according to the animal body weight, and the dosage is 30 mg/kg.

After 12 days of administration, the tumor volume was measured and the tumor inhibition rate was calculated. The calculation formula is as follows:

Tumor inhibition rate=(1-administration group tumor volume/model group tumor volume)*100%

The experimental results are shown in the following table, which shows that compound 6 has significant anti-tumor efficacy in tumor models, and its anti-tumor effect is significantly better than the control compound.

Compound Tumor inhibition rate% Control compound 1 44% 6 64%

The structure of control compound 1 is as follows:

Example 12 Western blot detection of the p-Btk inhibitory activity of the compound in the Btk-C481S mutant Hela cell line

Cell line: pre-built Btk-C481S mutation and stable transformation of Hela cell line (reference Chen JG, Liu X, Munshi M, et al. BTK(Cys481Ser) drives ibrutinib resistance via ERK1/2 and protects BTK(wild-type) MYD88 -mutated cells by a paracrine mechanism.Blood.2018;131(18):2047-2059.), cultured in an incubator at 37°C, 5% CO ₂ , and saturated humidity with DMEM high glucose medium containing 10% fetal bovine serum .

Set up DMSO control group, ibrutinib (100nM) group, compound 2 or 6 (100nM) administration group, collect cells after treatment for 24 hours, add pre-cooled cell lysate, place on ice for 10 minutes, extract total cell protein, BCA Method to determine protein concentration and quantify. Routine gel preparation, sample loading, electrophoresis, transfer to membrane, and block. Dilute the primary antibody (Btk or P-Btk) in the blocking solution at a ratio of 1:500-5000, immerse the membrane in the diluted primary antibody and incubate overnight at 4°C. After rinsing, dilute the secondary antibody with the blocking solution 1:10000-20000, immerse the membrane in the diluted secondary antibody, and incubate at room temperature for 45 minutes. After rinsing, check the test results on the gel imager. GAPDH was used as an internal reference control.

Use Image J software to perform grayscale analysis on each band, and calculate the inhibition rate of compound phosphorylation Btk (p-Btk).

The results showed that compound 6 of the present invention has significant degradation activity on Btk-C481S mutant protein in Hela cells, and can significantly inhibit p-Btk, while ibrutinib cannot degrade Btk-C481S mutant protein, and can hardly inhibit p-Btk. Btk. It shows that the compound of the present invention can overcome ibrutinib resistance.

Compound p-Btk inhibition rate Ibrutinib + 2 ++++ 6 ++++

Note: In the above table: "+" means the inhibition rate is 0%-10%; "++++" means the inhibition rate is >90%;

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (14)

A compound represented by the following formula I, or a pharmaceutically acceptable salt thereof:

Among them, A, Z, W ₂ are each independently missing or selected from: O, NR ¹ , NR ¹ -CO, CO-NR ¹ , SO, SO ₂ , NR ¹ -SO ₂ , SO ₂ -NR ¹ , C( =O)-O, O-(C=O), C(=O), CH=CH, C≡C, O(CH ₂ ) _h , NH(CH ₂ ) _h , (CR ² ₂ ) _h , ( CH ₂ CH ₂ O) _h , (OCH ₂ CH ₂ ) _h , NR ¹ -CO-(CH ₂ ) _h , CO-NR ¹ -(CH ₂ ) _h , CO-NR ¹ -(CH ₂ ) _h -O , 3-12-membered substituted or unsubstituted saturated or unsaturated cyclic or heterocyclic hydrocarbon, substituted or unsubstituted aromatic or heteroaromatic ring; wherein R ^{1 is} selected from H, C _1-6 alkyl, C _1-6 acyl; wherein R ^{2 is} selected from H, hydroxy, amino, cyano, carboxy, ester, halogen, substituted or unsubstituted C _1-6 alkyl, alkoxy, acyl, 3-12 membered substituted or Unsubstituted cyclic hydrocarbon group or heterocyclic hydrocarbon group, substituted or unsubstituted aryl group or heteroaryl group; wherein h is an integer between 0 and 30; W _{1 is} missing or selected from: C(=O), (CH ₂ )g, O(CH ₂ )g, (OCH ₂ CH ₂ )g; wherein g is preferably an integer between 0 and 10; B is missing or selected from: O, C, CH, CH ₂ , C=O, S, NR ³ , NR ³ -C(=O), C(=O)-NR ³ , C(=O)-O, OC(=O)-O, NR ³ -C(=O)-O, OC(=O)-NR ³ , NR ³ -C(=O)-NR ⁴ , C with or without substituents _1-12 hydrocarbon group, _3-12 membered substituted or unsubstituted cyclic or heterocyclic hydrocarbon, substituted or unsubstituted aromatic ring or heteroaromatic ring; wherein R ³ and R ⁴ are each independently selected from H, with or C _1-8 hydrocarbon groups without substituents, C _3-12 cyclic hydrocarbon groups, heterocyclic hydrocarbon groups, aryl groups, heteroaryl groups; X is selected from: C(R ⁵ ) ₂ , C(=O), S(=O), SO ₂ ; wherein R ^{5 is} each independently selected from: H, C _1-6 with or without substituents Hydrocarbyl; or two R ⁵ are connected to each other and form a 3-12-membered saturated or unsaturated carbocyclic or heterocarbocyclic ring; a is an integer between 0-30. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: In the formula I, A, Z, and W ₂ are each independently missing or selected from: O, and NH; W _{1 is} missing or C(=O); B is O; a is an integer between 4 and 20; X is: C(R ⁵ ) ₂ , or C(=O), where R ⁵ is H; And, the compound is not compound 16 disclosed in patent document CN109422752A; or the compound is not specific compound 16 disclosed in patent document WO2019042445A1. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: In the formula I, A and Z are each independently missing, W ₂ is NH; W _{1 is} missing; a is an integer between 5-10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: A is missing, or A is O(CH ₂ ) _h , where h is an integer between 0 and 5, such as h is 1, 2, 3, 4, or 5; W _{1 is} missing, or W ₁ is C (=O); Z is missing, or Z is O(CH ₂ ) _h , where h is an integer between 0 and 5, such as h is 1, 2, 3, 4, or 5; W _{2 is} missing, or W ₂ is NH; B is O; and a is an integer between 3 and 30; And the compound is not compound 7, compound 15, or compound 16 disclosed in patent document WO2019042445A1. The compound according to claim 4, or a pharmaceutically acceptable salt thereof, wherein a is an integer between 4 and 20. The compound of claim 5, or a pharmaceutically acceptable salt thereof, wherein a is an integer between 5 and 10. The compound of claim 4, or a pharmaceutically acceptable salt thereof, characterized in that: A is missing, Z is missing, W ₂ is NH, and a is an integer between 5 and 10. The compound of claim 4, or a pharmaceutically acceptable salt thereof, characterized in that: A is missing, Z is missing, W _{1 is} missing, W ₂ is NH, and a is an integer between 5 and 10. The compound according to claim 4, or a pharmaceutically acceptable salt thereof, characterized in that: A is missing, Z is missing, W _{1 is} missing, W _{2 is} missing, and a is an integer between 6 and 8. The compound of claim 4, or a pharmaceutically acceptable salt thereof, wherein: X is CH ₂ or C(=O). The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following group:

A pharmaceutical composition, characterized in that the composition contains the compound of claim 1 or a pharmaceutically acceptable salt, prodrug thereof, and a pharmaceutically acceptable carrier. A use of the compound of claim 1 or a pharmaceutically acceptable salt thereof for: (a) Preparation of drugs for the treatment of diseases related to the activity or expression of Bruton's tyrosine protein kinase (Btk); (b) Preparation of Bruton's tyrosine protein kinase (Btk) targeted inhibitors or degradation agents; (c) Non-therapeutic inhibition or degradation of Bruton's tyrosine protein kinase (Btk) activity in vitro; (d) Non-therapeutic inhibition of tumor cell proliferation in vitro; and/or (e) Treatment of diseases related to the activity or expression of Bruton's tyrosine protein kinase (Btk). The use according to claim 13, wherein the diseases include tumors, autoimmune diseases; preferably, the tumors include non-Hodgkin’s lymphoma (NHL), chronic lymphocytic leukemia (CLL), B Cell lymphoma, etc.; the autoimmune diseases include rheumatoid arthritis, psoriasis and the like.