Small molecule compound serving as blimp1 inhibitor and use thereof

Abstract

The present invention relates to the technical field of organic synthetic drugs, and provides a small molecule compound serving as a Blimp1 inhibitor and a use thereof. The small molecule compound of the present invention is a compound represented by formula I, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. The compound of the present invention can effectively inhibit Blimp1 activity, thereby significantly delaying CAR-T cell exhaustion. Therefore, the compound of the present invention can enhance the anti-tumor effect of CAR-T therapy; moreover, the compound of the present invention can also inhibit the progression of hemophagocytic lymphohistiocytosis. The compound of the present invention is a druggable compound suitable for clinical application, and has good application prospects.

Description

Small molecule compounds as Blimp1 inhibitors and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of organic synthetic drugs, and particularly relates to a small molecule compound as a Blimp1 inhibitor and uses thereof. BACKGROUND

[0002] Chimeric antigen receptor T cell therapy (CAR-T therapy) is a new type of tumor immunotherapy developed in recent years, that is, through genetic engineering of patient's own T cells, so that it can recognize and attack tumor cells. At present, CAR-T cell therapy has achieved good therapeutic effect in hematological tumors, and the effect in solid tumors is poor, but even in hematological tumors, the complete remission rate is only about 50%. There are many reasons for the poor effect of CAR-T cell therapy, and CAR-T cell exhaustion is one of the main reasons. Studies have shown that when T cell exhaustion is inhibited or delayed, the anti-tumor effect of CAR-T cell therapy will be significantly enhanced. At present, there is no effective strategy to inhibit or delay T cell exhaustion at home and abroad. The development of small molecule inhibitors to delay T cell exhaustion will have great significance for tumor immunotherapy.

[0003] Blimp-1, also known as PRDM1 (PR domain containing 1, PR domain containing protein 1), is a transcription factor. Blimp-1 mediates the transcriptional program of various innate and adaptive immune tissue-resident T cell types, and regulates the expression of related genes. Studies have shown that Blimp-1 is a transcriptional regulator of T cell exhaustion and an inhibitor of memory T cell differentiation, and the increase of Blimp-1 expression is related to the inhibition of memory T cell differentiation and the increase of inhibitory receptor expression (characteristics of T cell exhaustion), and conditional knockout of PRDM1 reverses both characteristics of T cell exhaustion. Specific knockout of PRDM1 in regulatory T cells (Treg) can enhance anti-tumor immunity, delay tumor growth, and also enhance the effect of anti-PD-1 therapy. Knocking out the PRDM1 gene in CAR-T cells supports the maintenance of early memory phenotype and the secretion of multiple cytokines, and promotes the expansion of memory CAR-T cells with low differentiation degree in vivo, thereby enhancing the persistence of CAR-T cells and improving the therapeutic effect in various tumor models. In summary, Blimp-1 is an important target for delaying T cell exhaustion, and its small molecule inhibitors can enhance the anti-tumor effect of CAR-T therapy. However, so far, there is no small molecule inhibitor targeting Blimp-1 reported at home and abroad.

[0004] Therefore, it is feasible, necessary and urgent to develop a Blimp-1 targeted inhibitory drug to delay T cell exhaustion. SUMMARY

[0005] The object of the present application is to provide a small molecule compound as a Blimp1 inhibitor and uses thereof.

[0006] The present application provides a compound shown in formula I, a pharmaceutically acceptable salt, a solvate or a stereoisomer thereof:

[0007] wherein,

[0008] Ring A is selected from substituted or unsubstituted 5-6 membered cycloalkyl, substituted or unsubstituted 5-6 membered heterocycloalkyl, substituted or unsubstituted 5-6 membered aryl, substituted or unsubstituted 5-6 membered heteroaryl; the substituents of cycloalkyl, heterocycloalkyl, aryl, heteroaryl in ring A are each independently selected from halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy;

[0009] n is 0 or 1;

[0010] R1, R2 are each independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl; or R1 and R2 are connected to form substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-13 membered heteroaryl;

[0011] the substituents of said alkyl are each independently selected from halogen, hydroxyl, C1-C6 alkoxy, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl;

[0012] the substituents of said cycloalkyl, heterocycloalkyl, aryl, heteroaryl are each independently selected from halogen, cyano, hydroxyl, amino, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, -(CR3’R4’) m C(O)R3, -C(O)(CR3’R4’) m R3, -C(O)(CR3’R4’) m OR3, -C(O)C(O)R3, -NR3’C(O)R3, -C(O)NR3’(CR3’R4’) m R3, -S(O)(O)R3, substituted or unsubstituted 3-10 membered cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl, -NR4R5; or two substituents on the same carbon atom form =O;

[0013] m is 0 or 1;

[0014] R3is selected from the group consisting of substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, substituted or unsubstituted C1-C6alkylthio, substituted or unsubstituted 3-10 membered cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl, -NR4R5;

[0015] R3', R4' are each independently selected from the group consisting of hydrogen, C1-C6alkyl;

[0016] R4, R5 are each independently selected from the group consisting of hydrogen, C1-C6alkyl, haloC1-C6alkyl, -C(O)R6;

[0017] R6is selected from the group consisting of C1-C6alkyl, substituted or unsubstituted 5-10 membered heteroaryl;

[0018] each substituent of said alkoxy, alkylthio is independently selected from the group consisting of halogen, substituted or unsubstituted 5-10 membered aryl.

[0019] Further, said compound is represented by formula IIA or formula IIB:

[0020] wherein,

[0021] n is 0 or 1 ;

[0022] R1, R2 are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl; or R1and R2are linked to form a substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-13 membered heteroaryl;

[0023] each substituent of said alkyl is independently selected from the group consisting of halogen, hydroxy, C1-C6alkoxy, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl;

[0024] each substituent of said cycloalkyl, heterocycloalkyl, aryl, heteroaryl is independently selected from the group consisting of halogen, cyano, hydroxy, amino, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, substituted or unsubstituted C1-C6alkylthio, -(CR3'R4') m C(O)R3, -C(O)(CR3'R4') m R3, -C(O)(CR3'R4') m OR3, -C(O)C(O)R3, -NR3'C(O)R3, -C(O)NR3'(CR3'R4') mR3, -S(O)(O)R3, substituted or unsubstituted 3-10 membered cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl, -NR4R5; or two substituents on the same carbon atom form =O;

[0025] m is 0 or 1;

[0026] R3is selected from the group consisting of substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, substituted or unsubstituted C1-C6alkylthio, substituted or unsubstituted 3-10 membered cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl, -NR4R5;

[0027] R3', R4' are each independently selected from the group consisting of hydrogen, C1-C6alkyl;

[0028] R4, R5are each independently selected from the group consisting of hydrogen, C1-C6alkyl, haloC1-C6alkyl, -C(O)R6;

[0029] R6is selected from the group consisting of C1-C6alkyl, substituted or unsubstituted 5-10 membered heteroaryl;

[0030] each substituent of said alkoxy, alkylthio is independently selected from the group consisting of halogen, substituted or unsubstituted 5-10 membered aryl;

[0031] R5' is a substituent on the phenyl ring at any position and is selected from the group consisting of hydrogen, halogen, C1-C6alkyl, haloC1-C6alkyl, C1-C6alkoxy, haloC1-C6alkoxy.

[0032] Further, the compound is represented by formula IIB-1:

[0033] wherein,

[0034] R5' is a substituent on the phenyl ring at any position and is selected from the group consisting of hydrogen, halogen, C1-C6alkyl, haloC1-C6alkyl, C1-C6alkoxy, haloC1-C6alkoxy.

[0035] X is O or NR7';

[0036] R6' is a substituent on the heterocyclic ring at any position and is selected from the group consisting of hydrogen, C1-C6alkyl;

[0037] R7' is selected from the group consisting of -C(O)R8';

[0038] R8' is selected from the group consisting of furanyl.

[0039] Further, the compound is represented by Formula IIA-1 or Formula IIA-2:

[0040] wherein,

[0041] R7is selected from -C(O)(CR3'R4') m R3, -C(O)(CR3'R4') m OR3, -S(O)(O)R3, -C(O)NR3'(CR3'R4') m R3, -NR3'C(O)R3;

[0042] m is 0 or 1 ;

[0043] R3', R4' are each independently selected from hydrogen, C1-C6alkyl;

[0044] R3is selected from substituted or unsubstituted C1-C6alkyl, 3-6 membered cycloalkyl, each of said substituents is independently selected from C1-C6alkyl, haloC1-C6alkyl, hydroxy-substituted C1-C6alkyl, -C(O)C1-C6alkyl, -N(H)C(O)C1-C6alkyl, C1-C6alkoxy, C1-C6alkylthio, -NR4R5, halogen, hydroxy, -(CH2) m1 C1-C6alkoxy, 3-6 membered cycloalkyl,

[0045] m1is selected from 1, 2 or 3;

[0046] R4, R5are each independently selected from hydrogen, C1-C6alkyl, haloC1-C6alkyl;

[0047] In Formula IIA-2, Y1, Y2are each independently selected from N or CH, m2is selected from 1, 2 or 3, and R8is selected from hydrogen, C1-C6alkyl, haloC1-C6alkyl.

[0048] Further,

[0049] R7is selected from

[0050] Further, the compound is selected from one of the following structures:

[0051] The present application also provides use of the aforementioned compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof in the preparation of a Blimp1 inhibitor.

[0052] The present application also provides use of the aforementioned compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof in the preparation of a medicament for preventing and / or treating an immune-related disease.

[0053] Preferably, the immune-related disease is systemic lupus erythematosus, rheumatoid arthritis, cancer, hemophagocytic syndrome.

[0054] The present application also provides a pharmaceutical preparation comprising the aforementioned compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof as an active ingredient, together with a pharmaceutically acceptable adjuvant.

[0055] The present application also provides a pharmaceutical composition comprising the aforementioned compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof.

[0056] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. Also, the relevant terms and laboratory operation steps used herein are the terms and conventional steps widely used in the corresponding field. At the same time, in order to better understand the present application, the definitions and explanations of the relevant terms are provided as follows.

[0057] As used herein and unless otherwise indicated, the term "about" or "approximately" means plus or minus 10% of the given value or range. In the case of integers, the term means plus or minus 10% of the given value or range, rounded to the nearest integer.

[0058] In the description herein, reference to "some embodiments", "some implementations" or "some aspects" describes a subset of all possible embodiments, but it is understood that "some embodiments" can be the same subset of all possible embodiments or different subsets, and can be combined with each other without conflict.

[0059] As used herein and unless otherwise specified, the terms "comprise", "include", "have", "contain", including their grammatical equivalents, should generally be understood to be open-ended and non-limiting, for example, not excluding other unlisted elements or steps.

[0060] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present application which is pharmaceutically acceptable and which has the pharmacological activity of the parent compound. Such salts include acid addition salts with inorganic acids such as nitric acid, phosphoric acid, carbonic acid, and the like; or with organic acids such as propionic acid, hexanoic acid, cyclopentanepropionic acid, ethane-1,2-diol, acetic acid, glycolic acid, lactic acid, glyceric acid, pyruvic acid, malonic acid, succinic acid, heptanoic acid, heptanoic acid, embonic acid, and the like; or salts of metal ions such as alkali metal ions or alkaline earth metal ions which replace the acidic protons present on the parent compound; or coordination compounds with organic bases such as ethanolamine and the like. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound which contains an acidic or basic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two. Generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, and the like, are preferred. In addition to salt forms, the compounds provided by the present application can exist in a prodrug form. Prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to become converted into the compounds of the present application. Additionally, prodrugs can be converted to the compounds of the present application by chemical or biochemical methods in an ex vivo environment.

[0061] As used herein, the term "solvate" refers to a compound of the present application in combination with a pharmaceutically acceptable solvent. Pharmaceutically acceptable solvents include acetic acid and the like. Solvates include stoichiometric amounts of solvent and non-stoichiometric amounts of solvent. Certain compounds of the present application can exist in unsolvated as well as solvated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present application.

[0062] As used herein, the term "stereoisomers" includes conformational isomers and configurational isomers, wherein configurational isomers include primarily cis-trans isomers and optical isomers. The compounds of the present application can exist in the form of stereoisomers and therefore encompass all possible stereoisomeric forms, including but not limited to, cis-trans isomers, tautomers, enantiomers, diastereomers, atropisomers, and the like, and the compounds of the present application can also exist in any combination or mixture of the foregoing stereoisomers, such as mesomers, racemates, equal mixtures of atropisomers, and the like. For example, a single enantiomer, a single diastereomer or a mixture thereof, or a single atropisomer or a mixture thereof. When the compounds of the present application contain an olefinic double bond, unless otherwise specified, it includes the cis and trans isomers, as well as any combination thereof. Atropisomers of the present application are stereoisomers based on restricted rotation within the molecule resulting in axial or planar chirality. And as a drug, the stereoisomer with superior activity is preferred. The compounds of the present application have optical isomers derived from asymmetric carbon and the like, and a single isomer can be obtained by resolution, if necessary, by a method known in the art, such as crystallization or chiral chromatography, and the like.

[0063] As used herein, the term "cycloalkyl" refers to saturated monocyclic or polycyclic cyclic hydrocarbon groups, including monocyclic cycloalkyl, spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl groups. The ring carbon atoms of the cycloalkyl groups described in the present application can optionally be substituted with 1, 2, or 3 oxo groups to form cyclic ketone structures. The term "C 3-8 cycloalkyl" refers to cycloalkyl groups having 3 to 8 ring carbon atoms, including monocyclic cycloalkyl, spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl groups.

[0064] As used herein, the term "heterocycloalkyl" refers to saturated or partially unsaturated monocyclic or polycyclic fused cyclic hydrocarbon groups, and the term "3 to 8 membered heterocycloalkyl" refers to saturated cyclic hydrocarbon groups having 3 to 8 ring atoms, wherein one or more, preferably 1, 2, 3, or 4, of the ring atoms are heteroatoms selected from nitrogen, oxygen, sulfur, and the remaining ring atoms are carbon.

[0065] As used herein, the term "aryl" or "aromatic ring" refers to all-carbon monocyclic, all-carbon polycyclic (rings connected by covalent bonds, non-fused), or all-carbon fused polycyclic (that is, sharing pairs of adjacent carbon atoms) groups, at least one ring of which is aromatic, that is, having a conjugated pi-electron system. The term "C 6-14 aryl" refers to aryl groups having 6 to 14 ring atoms. Preferably, C 6-10 aryl. In the present application, C 6-14 aryl includes monocyclic aryl, non-fused polycyclic aryl, and aromatic fused polycyclic rings, wherein examples of monocyclic aryl include phenyl, and examples of non-fused polycyclic aryl include biphenyl, and the like. The term "6 to 10 membered aromatic ring" refers to aromatic rings having 6 to 10 ring atoms.

[0066] As used herein, the term "heteroaryl" refers to a monocyclic or fused polycyclic (i.e., sharing pairs of adjacent ring atoms, which can be C-C or N-C) group in which ring atoms are replaced by at least one heteroatom independently selected from nitrogen, oxygen, or sulfur (=0) m (wherein m' is an integer of 0 to 2). The 5- to 10-membered heteroaryl group in the present application can be a monocyclic heteroaryl group or a fused bicyclic heteroaryl group.

[0067] As used herein, the term "5- or 6-membered heteroaryl" refers to a group having 5 or 6 ring atoms, wherein 1, 2, or 3 ring atoms are heteroatoms independently selected from nitrogen, oxygen, or sulfur (=0) m (wherein m' is an integer of 0 to 2). Specific examples of the heteroaryl group include, but are not limited to, thienyl, furanyl, thiazolyl, isothiazolyl, imidazolyl, oxazolyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, tetrazolyl, isoxazolyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, and the like.

[0068] The two carbon atoms to which the representative is attached are a pair of adjacent carbon atoms that are shared when fused with other rings.

[0069] In the present application, each of the above-mentioned heteroaryl groups can be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the substituent groups described in the present application.

[0070] As used herein, the term "effective amount" or "therapeutically effective amount" means an amount of a drug or pharmaceutical agent that is sufficient to achieve an intended effect. In the context of the present application, the amount of a given drug will depend on factors such as the particular dosage form being administered, the type and severity of the disease or condition, the identity of the recipient or host (e.g., weight), but the amount administered can be routinely determined by methods known in the art according to the particular circumstances, including, for example, the specific drug employed, the route of administration, the condition being treated, and the subject or host being treated. In general, for dosage

[0071] The compounds of the present application can be prepared using synthetic methods known in the art or using methods known in the art in combination with the methods described in the present application. The solvents, temperatures, and other reaction conditions given in the present application are exemplary and can be varied according to methods well known in the art. The compounds of the examples described in the present application can be synthesized according to the methods described in the examples using appropriate starting materials according to the specific structure, or can be synthesized using methods analogous to those described in the examples. The starting materials used to synthesize the compounds of the examples of the present application can be prepared by known synthetic methods or by analogous methods described in the literature, or obtained from commercial sources. The compounds of the examples can be further resolved into their stereoisomers, if desired, by methods well known in the art, for example, crystallization, chromatography, and the like, the resolution conditions of which are readily obtained by those skilled in the art by routine means or limited experimentation.

[0072] The present application provides a small molecule compound as a Blimp1 inhibitor. The compound of the present application can effectively inhibit Blimp1 activity and has excellent effect of delaying CAR-T cell exhaustion. Therefore, the compound of the present application can enhance the anti-tumor effect of CAR-T therapy, and the compound of the present application can also inhibit the progression of hemophagocytic syndrome. The compound of the present application is a compound suitable for clinical application and drug development, and has good application prospect.

[0073] Obviously, according to the above description of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical ideas of the present application.

[0074] The above summary of the application will be further explained in the detailed description of specific embodiments below. This summary should not be construed to mean that the application is only limited to these embodiments described below. Any technology based on the above summary of the application falls within the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0075] Figure 1 is a graph of CAR-T cell proliferation results.

[0076] Figure 2 is a graph of CAR-T cell apoptosis results.

[0077] Figure 3 is a graph of CAR-T cell memory phenotype results.

[0078] Figure 4 is a graph of cytokine secretion detection results.

[0079] Figure 5 is a graph of animal model characterization experiment process and tumor cell inhibition results of each group.

[0080] Figure 6 is a graph of tumor cell inhibition results of each group.

[0081] Figure 7 is a graph of the effects of each group on liver and spleen weight. DETAILED DESCRIPTION

[0082] The raw materials and equipment used in the specific embodiments of the application are known products, which can be obtained by purchasing commercially available products.

[0083] Example 1, preparation of compound 8

[0084] Step a: preparation of intermediate 1

[0085] Take a 250 mL single-necked round-bottom flask, add the substrate 2,4-dichloropyrido[2,3-d]pyrimidine (5 g, 25.00 mmol), then add 100 mL THF, stir to mix evenly. Then add the substrate 2-(trifluoromethyl)pyridine-3-methylamine (3.75 mL, 27.50 mmol), triethylamine (6.95 mL, 49.99 mmol) in turn, continue to stir at room temperature for 6 h. After the reaction is completed by TLC monitoring, evaporate the THF, add water, filter and dry to obtain a light yellow solid, which is intermediate 1, 7.2 g, yield 85%, MS (ESI) m / z: 340.06 [M+H] + .

[0086] Step b: preparation of compound 8

[0087] Take 50 mL reaction tube, add intermediate 1 (200 mg, 0.5887 mmol), add 3 mL super dry dimethyl sulfoxide to dissolve, finally add substrate 2-(thiophen-2-yl) ethylamine (104 μL, 0.8831 mmol) and triethylamine (164 μL, 0.1180 mmol), heat at 90 °C for 4 h. After monitoring the reaction is complete by TLC, add the system solution to water, a large amount of solid is precipitated, filter, and the filter cake is purified by column chromatography to obtain a light yellow solid, compound 8, 148 mg, yield 58%, MS (ESI) m / z: 431.13 [M+H] + .

[0088] Example 2, preparation of compound 60

[0089] Step a: preparation of intermediate 2

[0090] Take 50 mL reaction tube, add intermediate 1 (200 mg, 0.5887 mmol), add 3 mL super dry dimethyl sulfoxide to dissolve, finally add substrate 2-(thiophen-2-yl) ethylamine (104 μL, 0.8831 mmol) and triethylamine (164 μL, 0.1180 mmol), heat at 90 °C for 4 h. After monitoring the reaction is complete by TLC, add the system solution to water, a large amount of solid is precipitated, filter, and the filter cake is purified by column chromatography to obtain a light yellow solid, compound 8, 148 mg, yield 58%, MS (ESI) m / z: 431.13 [M+H] + .

[0091] Step b: preparation of intermediate 3

[0092] Take 50 mL reaction tube, add intermediate 1 (200 mg, 0.5887 mmol), add 3 mL super dry dimethyl sulfoxide to dissolve, finally add substrate 2-(thiophen-2-yl) ethylamine (104 μL, 0.8831 mmol) and triethylamine (164 μL, 0.1180 mmol), heat at 90 °C for 4 h. After monitoring the reaction is complete by TLC, add the system solution to water, a large amount of solid is precipitated, filter, and the filter cake is purified by column chromatography to obtain a light yellow solid, compound 8, 148 mg, yield 58%, MS (ESI) m / z: 431.13 [M+H]

[0093] Step c: preparation of compound 60

[0094] Take 10 mL reaction bottle, add intermediate 3 (200 mg, 0.4167 mmol), 5-acetyl thiophene-2-carboxylic acid (142 mg, 0.8334 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (160 mg, 0.8334 mmol), 1-hydroxybenzotriazole (113 mg, 0.8334 mmol), add 3 mL N, N-dimethylformamide, stir, then add triethylamine (290 μL, 2.08 mmol), react at room temperature overnight. After the reaction is completed, the system solution is added to water, a large amount of solid is precipitated, and the filter cake is purified by column chromatography to obtain a light yellow solid, i.e. compound 60, 170 mg, yield 68%, MS (ESI) m / z: 596.21 [M+H] + .

[0095] Example 3, preparation of compound 78

[0096] Step a: preparation of intermediate 4

[0097] Take intermediate 1 (3 g, 8.83 mmol), substrate 4-methyl piperidine carboxylate (1.9 g, 13.25 mmol) into a 250 mL single-necked round-bottom flask, then add 15 mL of ultra-dry dimethyl sulfoxide and triethylamine (2.46 mL, 17.66 mmol), heat at 90°C for 8 h. After the reaction is completed, the system solution is added to water, a large amount of solid is precipitated, and the filter cake is purified by column chromatography to obtain a light yellow solid, i.e. intermediate 4, 2.9 g, yield 75%, MS (ESI) m / z: 447.18 [M+H] + .

[0098] Step b: preparation of intermediate 5

[0099] Dissolve intermediate 4 (2 g, 4.48 mmol) in 18 mL of methanol, add aqueous sodium hydroxide solution (18 mL, 17.92 mmol, 1M), react at room temperature overnight. After the reaction is completed, the reaction liquid is concentrated, diluted with 30 mL of water, and then adjusted to pH 2-3 with 1M aqueous hydrochloric acid solution, a large amount of white solid is precipitated, and the filter cake is filtered to obtain intermediate 5, a light yellow solid, 1.8 g, yield 93%, MS (ESI) m / z: 433.16 [M+H] + .

[0100] Step c: preparation of intermediate 6

[0101] Into a 250 mL round bottom flask, intermediate 5 (1 g, 2.31 mmol) was taken, 50 mL of dry dichloromethane was added and the system was kept in ice bath, stirred to mix well, then oxalyl chloride (391 μL, 4.62 mmol) and 2 drops of dry N,N-dimethylformamide was added drop wise. The reaction was monitored by TLC and on completion, it was directly taken to the next step.

[0102] Step d: Preparation of compound 78

[0103] Into a 100 mL single necked round bottom flask, 5-amino-2-methoxypyridine (66 mg, 0.5323 mmol) was taken and 10 mL of dry dichloromethane was added and stirred to dissolve, then triethylamine (185 μL, 1.33 mmol) was added drop wise. After 10 min, 10 mL of dry dichloromethane solution containing intermediate 6 (208 mg, 0.4436 mmol) was added drop wise under ice bath and after completion of addition, the reaction was allowed to take place at room temperature. The reaction was monitored by TLC and on completion, water (20 mL) was added to the reaction flask, dichloromethane was used to extract the reaction, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated and column chromatography gave compound 78 as a white solid, 202 mg, 85% yield, MS (ESI) m / z: 539.21 [M+H]+.

[0104] Other target compounds of the present application were prepared by following the procedure of example 1-3. The numbers, structures and characterization data of the target compounds obtained are shown in table 1.

[0105] Table 1. Numbers, structures and characterization data of the target compounds of the present application

[0106] The advantageous effects of the present application are demonstrated below through specific test examples.

[0107] Test example 1, study of the inhibitory activity of the compounds of the present application on Blimp1

[0108] 1. Experimental method

[0109] The present application uses differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC) to evaluate the inhibitory activity of the compounds of the present application on Blimp1.

[0110] The differential scanning fluorimetry experiment uses a Bio-Rad CFX96 RT-PCR instrument to determine the thermal shift ΔTm. When configuring the sample, 9.8 μL of a mixture of Blimp1 protein and SYPRO Orange is added to each well, and 0.2 μL of a compound solution is added. The final test system is a buffer system of 20 mM HEPES pH 7.5, 150 mM NaCl. The final concentration of Blimp1 protein is 2 μM, and the final concentration of the compound to be tested is 100 μM, and the total volume is 10 μL. When testing, first, the sample is equilibrated at 25°C for 3 minutes; then heated from 25°C to 95°C at a rate of 1°C / min.

[0111] The isothermal titration calorimetry experiment uses PEAQ-ITC to determine the affinity K D value. All tests are carried out at 25°C using a buffer system of 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.4% DMSO. Before the experiment, the compound is directly diluted in the same batch of buffer. Each experiment is a reverse titration experiment (Blimp1 protein is sucked into the syringe and the small molecule is injected into the sample cell). The initial titration is 0.2 μL of protein, and then 2 μL of protein is titrated every 100 s for 19 consecutive drops. The experimental data are analyzed using ΔG = ΔH - TΔS = RTlnK D The thermodynamic parameters ΔG, ΔH and ΔS are the changes in free energy, enthalpy and entropy, respectively.

[0112] 2. Experimental results

[0113] The experimental results are shown in Table 2.

[0114] K D is the strength of the mutual combination between a single biomolecule (protein) and its ligand (drug or inhibitor). ΔTm: The melting temperature Tm1 of the protein changes after the drug molecule or inhibitor binds to the protein pocket. The melting temperature of the protein without the small molecule is Tm0, and ΔTm = Tm1 - Tm0. When the small molecule binds to the protein pocket, it prevents Blimp1 from binding to the substrate, thereby inhibiting the activity of Blimp1.

[0115] Table 2. Experimental results

[0116] From the experimental results in Table 2, it can be seen that some of the compounds of the present application have good inhibitory activity on Blimp1. Among them, compound 60 has the best inhibitory activity on Blimp1, KD The value is 0.098 μΜ.

[0117] Test Example 2, Delaying the CAR-T cell exhaustion cell characterization by the compound of the application

[0118] 1. Experimental method

[0119] The best active compound 60 in Test Example 1 was selected for further detection of the effect of the compound on the proliferation, apoptosis, memory phenotype, and cytokine secretion of CAR-T cells.

[0120] (1) Preparation of CAR-T cells: human peripheral blood lymphocytes were taken, and T cells were separated. After Anti-CD3 / CD28 magnetic beads and IL-2 stimulation for 48 h, the prepared CAR virus was used to infect T cells. After 24 h, 1 μΜ of compound 60 was added to the culture system, and cultured for 7 days.

[0121] (2) CAR-T cell proliferation detection: During the culture of CAR-T cells, the cell density of CAR-T cells with compound 60 and DMSO was detected every two days.

[0122] (3) CAR-T cell apoptosis detection: CAR-T cells in (1) were taken, and apoptosis kit was used to detect the apoptosis of CAR-T cells. After co-culturing CAR-T and tumor cells at E:T = 1:1 for two days, the apoptosis of CAR-T cells was detected. The detailed operation steps are as follows:

[0123] ① Collect the CAR-T cells treated with compound 60 and DMSO in a 15 mL sterile centrifuge tube, add 2 mL PBS for centrifugal washing, 1200 rpm, 10 min;

[0124] ② Resuspend the cell pellet with 1 × Binding buffer, and adjust the cell density to 1 × 10 6 / mL according to the counting results;

[0125] ③ Flow cytometry antibody staining, the staining system is 100 μL, according to the reagent instruction, add 5 μL APC-AnnnexinV and 5 μL 7-AAD, mix well. At the same time, set up control tubes: blank tube (without any treatment), 7-AAD single staining tube, APC-AnnexinV single staining tube;

[0126] ④ Incubate at room temperature for 20-30 min, add 400 μL 1 × Binding buffer, and analyze the apoptosis within 1 h.

[0127] (4) CAR-T memory phenotype detection

[0128] Take the CAR-T cells in (1), detect the memory phenotype of CAR-T, CAR-T and tumor cells are co-cultured for 6 days, 9 days after E:T=1:1, respectively, detect the memory phenotype of CAR-T, collect the CAR-T treated with compound 60 and DMSO and the CAR-T cells after co-culture in 15 mL sterile centrifuge tube, add 2 mL PBS centrifugal washing, 1200 rpm, 10 min; Add 0.5 μL Percp-CD8, FITC-MYC, BV510-CD2L, PE-CD45RO mix well. At the same time, set up control tube, blank tube (without any treatment), single dye tube; Incubate at room temperature for 20-30 min, add 2 mL PBS centrifugal washing, 1 h within machine analysis.

[0129] (5) Cytokine secretion detection

[0130] Take the CAR-T cells in (1), detect the cytokine secretion of CAR-T and tumor cells after co-culture for two days, collect the CAR-T cells co-cultured with tumor cells in 15 mL sterile centrifuge tube, add 2 mL PBS centrifugal washing, 1200 rpm, 10 min; Add 0.5 μL APC-CD4, FITC-MYC mix well, incubate at room temperature for 20-30 min, fix the cells, add 0.5 μL PE-IFNγ flow cytometry antibody, incubate at room temperature for 20-30 min, add 2 mL PBS centrifugal washing, 1 h within machine analysis. At the same time, set up control tube, blank tube (without any treatment), single dye tube.

[0131] 2, Experimental results

[0132] (1) CAR-T cell proliferation results

[0133] CAR-T cell proliferation results are shown in Figure 1, as shown in Figure 1: the addition of 1 μM of compound 60 in CAR-T cell culture system can significantly increase the proliferation ability of CAR-T cells.

[0134] (2) CAR-T cell apoptosis results

[0135] CAR-T cell apoptosis results are shown in Figure 2, as shown in Figure 2: the addition of 1 μM of compound 60 in CAR-T cell culture system can significantly reduce the apoptosis of CAR-T cells before and after co-culture.

[0136] (3) CAR-T cell memory phenotype results

[0137] CAR-T cell memory phenotype results are shown in Figure 3, as shown in Figure 3: the addition of 1 μM of compound 60 in CAR-T cell culture system can significantly increase the central memory cells TCM The proportion.

[0138] (4) Cytokine secretion detection results

[0139] The results of the cytokine secretion assay are shown in Figure 4. As can be seen from Figure 4, the addition of 1 μM of compound 60 to the CAR-T cell culture system can significantly increase the ability of co-cultured CAR-T cells to secrete IFNγ.

[0140] The above experimental results demonstrate that the compound of this invention can effectively delay CAR-T cell depletion.

[0141] Experimental Example 3: Characterization of the compound of the present invention in an animal model that delays CAR-T cell exhaustion

[0142] 1. Experimental Methods

[0143] (1) Inoculation with tumor cells: NCG mice were subcutaneously inoculated with 3×10 6 The tumor cells were observed to monitor tumor growth.

[0144] (2) Preparation of CAR-T cells: Human lymphocytes were collected and T cells were isolated. After stimulation with Anti-CD3 / CD28 magnetic beads and IL-2 for 48 h, the prepared CAR virus was added to infect the T cells. After 24 h, 1 μM of PRDM1 / Blimp1 inhibitor compound 60 was added to the culture system and cultured for 7 days.

[0145] (3) CAR-T cell reinfusion: NSG mice inoculated with tumor cells were divided into three groups of 5-9 mice each according to the size of the tumor. The first, second and third groups were infused with CAR-T cells treated with compound 60, CAR-T cells treated with DMSO and NT cells, respectively. The CAR positivity rate of the first and second groups was the same.

[0146] 2. Experimental Results

[0147] The experimental results are shown in Figure 5: In the CAR-T cell group treated with compound 60, tumor growth was significantly inhibited.

[0148] Example 4: Characterization of the compound of the present invention in an animal model combined with PD-1

[0149] 1. Experimental Methods

[0150] C57BL mice were subcutaneously injected with 3 × 10⁻⁶ mice. 6 MC38 cells were used to observe tumor growth; the tumor volume was 100 mm. 3On the 10th day, the mice were divided into 7 groups, and 40 mg / kg of compound 60, 40 mg / kg of compound 60 + anti-PD-1 antibody, 20 mg / kg of compound 60, 20 mg / kg of 60 + anti-PD-1 antibody, anti-PD-1 antibody, solvent, PBS + solvent were administered, respectively. Compound 60 was administered twice a day, and PD-1 was administered once every 3 days, with a dose of 100 μg per mouse.

[0151] 2. Experimental results

[0152] The experimental results are shown in Figure 6: 40 mg / kg of compound 60 combined with PD-1 significantly inhibited tumor growth.

[0153] Test Example 5, characterization of the compound of the application in an animal model of hemophagocytic syndrome

[0154] 1. Experimental method

[0155] C57BL male mice were divided into 8 groups, and the modeling group was injected intraperitoneally with 50 μg of CpG-ODN1826 on days 0, 2, 4, 6, 8, and 10, and the normal group was injected with the same volume of PBS. The administration group was designed as a normal group, a blank control group, a dexamethasone group, an etoposide group, a ruxolitinib group, a dexamethasone + ruxolitinib group, a compound 60 (40 mg / kg) group, and a dexamethasone + compound 60 (40 mg / kg) group. The compound 60 group was administered twice a day, the dexamethasone was administered intraperitoneally once a day, and the dose was 1.5 mg / kg. The etoposide was administered intraperitoneally twice a week, and the dose was 50 mg / kg. The ruxolitinib was administered orally twice a day, and the dose was 30 mg / kg. The mice were weighed every two days. After modeling for 10 days, the mice were taken from the orbital blood, liver, spleen, and femur, and the liver and spleen weights were observed and measured.

[0156] 2. Experimental results

[0157] The experimental results are shown in Figure 7: the 40 mg / kg compound 60 administration group and the 40 mg / kg compound 60 + dexamethasone combination group can significantly inhibit the improvement of the liver and spleen weights of the mice and inhibit the progression of hemophagocytic syndrome.

[0158] In summary, the present application provides a small molecule compound as a Blimp1 inhibitor. The compound of the present application can effectively inhibit the activity of Blimp1 and has excellent effect of delaying CAR-T cell exhaustion. Therefore, the compound of the present application can enhance the anti-tumor effect of CAR-T therapy, and the compound of the present application can also inhibit the progression of hemophagocytic syndrome. The compound of the present application is a compound suitable for clinical application and has good application prospect.

Claims

1. A compound of Formula I: ###0001### I or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. wherein, ring A is selected from substituted or unsubstituted 5-6 membered cycloalkyl, substituted or unsubstituted 5-6 membered heterocycloalkyl, substituted or unsubstituted 5-6 membered aryl, substituted or unsubstituted 5-6 membered heteroaryl; the substituents of cycloalkyl, heterocycloalkyl, aryl, heteroaryl in ring A are each independently selected from halogen, C1-C6 alkyl, halo-C1-C6 alkyl, C1-C6 alkoxy, halo-C1-C6 alkoxy; n is 0 or 1; R1, R2are each independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl; or R1and R2are linked to form substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-13 membered heteroaryl; the substituents of said alkyl are each independently selected from halogen, hydroxy, C1-C6 alkoxy, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl; the substituents of said cycloalkyl, heterocycloalkyl, aryl, heteroaryl are each, independently, selected from the group consisting of halogen, cyano, hydroxy, amino, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, substituted or unsubstituted C1-C6alkylthio, -(CR3'R4')pR2, -OR3, -C(O)R3, -C(O)(CR3'R4') m C(O)R3, -C(O)(CR3'R4') m R3, -C(O)(CR3'R4') m OR3, -C(O)C(O)R3, -NR3'C(O)R3, -C(O)NR3'(CR3'R4') m R3, -S(O)(O)R3, substituted or unsubstituted 3-10 membered cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl, -NR4R5; or two substituents on the same carbon atom form =O; m is 0 or 1; R3is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted 3-10 membered cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl, -NR4R5; R3', R4' are each independently selected from hydrogen, C1-C6 alkyl; R4, R5are each independently selected from hydrogen, C1-C6 alkyl, halo-C1-C6 alkyl, -C(O)R6; R6is selected from C1-C6 alkyl, substituted or unsubstituted 5-10 membered heteroaryl; the substituents of said alkoxy, alkylthio are each independently selected from halogen, substituted or unsubstituted 5-10 membered aryl.

2. The compound, pharmaceutically acceptable salt, solvate, or stereoisomer thereof of claim 1, wherein: The compound is shown in formula IIA or formula IIB: wherein, n is 0 or 1; R1, R2are each independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl; or R1and R2are linked to form substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-13 membered heteroaryl; the substituents of said alkyl are each independently selected from halogen, hydroxy, C1-C6 alkoxy, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl; the substituents of said cycloalkyl, heterocycloalkyl, aryl, heteroaryl are each, independently, selected from the group consisting of halogen, cyano, hydroxy, amino, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, substituted or unsubstituted C1-C6alkylthio, -(CR3'R4')pR2, -OR3, -C(O)R3, -C(O)(CR3'R4') m C(O)R3, -C(O)(CR3'R4') m R3, -C(O)(CR3'R4') m OR3, -C(O)C(O)R3, -NR3'C(O)R3, -C(O)NR3'(CR3'R4') m R3, -S(O)(O)R3, substituted or unsubstituted 3- to 10-membered cycloalkyl, substituted or unsubstituted 3- to 10-membered heterocycloalkyl, substituted or unsubstituted 5- to 10-membered aryl, substituted or unsubstituted 5- to 10-membered heteroaryl, -NR4R5; or two substituents on the same carbon atom form =O; m is 0 or 1; R3is selected from substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted 3-10 membered cycloalkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl, -NR4R5; R3', R4' are each independently selected from hydrogen, C1-C6 alkyl; R4, R5are each independently selected from hydrogen, C1-C6 alkyl, halo-C1-C6 alkyl, -C(O)R6; R6is selected from C1-C6 alkyl, substituted or unsubstituted 5-10 membered heteroaryl; the substituents of said alkoxy, alkylthio are each independently selected from halogen, substituted or unsubstituted 5-10 membered aryl. R5' is a substituent on the phenyl ring at any position selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy.

3. The compound, pharmaceutically acceptable salt, solvate, or stereoisomer thereof of claim 2, wherein: The compound is shown in formula IIB-1: wherein, R5' is a substituent on the phenyl ring at any position selected from the group consisting of hydrogen, halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy; X is O or NR7'; R6' is a substituent on the heterocyclic ring at any position selected from the group consisting of hydrogen, C1-C6 alkyl; R7' is selected from -C(O)R8'; R8' is selected from furanyl.

4. The compound, pharmaceutically acceptable salt, solvate, or stereoisomer thereof of claim 2, wherein: The compound is shown in formula IIA-1 or formula IIA-2: wherein, R7is selected from -C(O)(CR3'R4') m R3, -C(O)(CR3'R4') m OR3, -S(O)(O)R3, -C(O)NR3'(CR3'R4') m R3, -NR3'C(O)R3; m is 0 or 1; R3', R4' are each independently selected from hydrogen, C1-C6 alkyl; R3is selected from the group consisting of substituted or unsubstituted: C1-C6alkyl, 3- to 6-membered cycloalkyl, each of said substituents is independently selected from the group consisting of C1-C6alkyl, haloC1-C6alkyl, hydroxy-substituted C1-C6alkyl, -C(O)C1-C6alkyl, -N(H)C(O)C1-C6alkyl, C1-C6alkoxy, C1-C6alkylthio, -NR4R5, halogen, hydroxy, -(CH2) m1 C1-C6alkoxy, 3- to 6-membered cycloalkyl, m1 is selected from 1, 2 or 3; R4, R5 are each independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl; In formula IIA-2, Y1, Y2 are each independently selected from N or CH, m2 is selected from 1, 2 or 3, and R8 is selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl.

5. The compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof according to claim 4, characterized in that: R7is selected from 6. The compound, pharmaceutically acceptable salt, solvate, or stereoisomer thereof of any one of claims 1-5, wherein: The compound is selected from one of the following structures:

7. Use of a compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof according to any one of claims 1 to 6 for the manufacture of a Blimp1 inhibitor.

8. Use of a compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof according to any one of claims 1 to 6 for the manufacture of a medicament for the prevention and / or treatment of an immune-related disease. Preferably, the immune-related disease is systemic lupus erythematosus, rheumatoid arthritis, cancer, hemophagocytic syndrome.

9. A pharmaceutical preparation, characterized by: It is prepared from a compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof according to any one of claims 1 to 6 as an active ingredient, together with a pharmaceutically acceptable adjuvant.

10. A pharmaceutical composition, characterized by: It comprises a compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof according to any one of claims 1 to 6.

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