A small molecule compound as a Blimp1 inhibitor and its use

Abstract

The present invention provides a small molecule compound that acts as a Blimp1 inhibitor and its use, belonging to the technical field of organic synthetic drugs. 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 and has excellent effects in delaying CAR-T cell exhaustion. Therefore, the compound of the present invention can enhance the anti-tumor effect of CAR-T therapy and can also inhibit the progression of hemophagocytic syndrome. The compound of the present invention is a druggable compound suitable for clinical application and has good application prospects. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthetic drugs, and particularly relates to a small molecule compound as a Blimp1 inhibitor and a use thereof. Background Art

[0002] Chimeric antigen receptor T-cell therapy (CAR-T therapy) is a new type of tumor immunotherapy developed in recent years. It involves genetically engineering a patient's own T cells to enable them to recognize and attack tumor cells. Currently, CAR-T cell therapy has achieved good therapeutic effects in hematologic malignancies but less so in solid tumors. Even in hematologic malignancies, the complete remission rate is only approximately 50%. There are various reasons for the poor efficacy of CAR-T cell therapy, with CAR-T cell exhaustion being one of the most prominent. Studies have shown that inhibiting or delaying T cell exhaustion significantly enhances the anti-tumor efficacy of CAR-T cell therapy. Currently, there are no effective strategies for inhibiting or delaying T cell exhaustion, either domestically or internationally. The development of small molecule inhibitors that delay T cell exhaustion will be of great significance to tumor immunotherapy.

[0003] Blimp-1, also known as PRDM1 (PR domain containing protein 1), is a transcription factor. Blimp-1 mediates the transcriptional programs of various innate and adaptive immune tissue-resident T cell types, regulating 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. Elevated Blimp-1 expression is associated with suppressed memory T cell differentiation and increased expression of inhibitory receptors (characteristics of T cell exhaustion). Conditional knockout of PRDM1 reverses both of these characteristics of T cell exhaustion. Specific knockout of PRDM1 in regulatory T cells (Tregs) can enhance anti-tumor immunity, delay tumor growth, and enhance the efficacy of anti-PD-1 therapy. Knocking out the PRDM1 gene in CAR-T cells supports the maintenance of the early memory phenotype and the secretion of multifunctional cytokines, promoting the expansion of less differentiated memory CAR-T cells in vivo, thereby enhancing the persistence of CAR-T cells and improving therapeutic efficacy 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, to date, no small molecule inhibitors targeting Blimp-1 have been reported in China or abroad.

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

[0005] The purpose of the present invention is to provide a small molecule compound as a Blimp1 inhibitor and its use.

[0006] The present invention provides a compound represented by Formula I, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof:

[0007]

[0008] in,

[0009] 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 the cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups in Ring A are each independently selected from the following groups: halogen, C1-C6 alkyl, halo-substituted C1-C6 alkyl, C1-C6 alkoxy, halo-substituted C1-C6 alkoxy;

[0010] n is 0 or 1;

[0011] R1 and R2 are independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl; or R1 and R2 are linked to form a substituted or unsubstituted 3-10 membered heterocycloalkyl, or a substituted or unsubstituted 5-13 membered heteroaryl;

[0012] The substituents of the alkyl group are each independently selected from the following groups: halogen, hydroxyl, C1-C6 alkoxy, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl;

[0013] The substituents of the cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups are independently selected from the following groups: 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;

[0014] m is 0 or 1;

[0015] R3 is 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;

[0016] R3' and R4' are independently selected from hydrogen and C1-C6 alkyl;

[0017] R4 and R5 are independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, -C(O)R6;

[0018] R6 is selected from C1-C6 alkyl, substituted or unsubstituted 5-10 membered heteroaryl;

[0019] The substituents of the alkoxy group and the alkylthio group are independently selected from the following groups: halogen, and substituted or unsubstituted 5-10 membered aryl groups.

[0020] Furthermore, the compound is represented by Formula IIA or Formula IIB:

[0021]

[0022] in,

[0023] n is 0 or 1;

[0024] R1 and R2 are independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl; or R1 and R2 are linked to form a substituted or unsubstituted 3-10 membered heterocycloalkyl, or a substituted or unsubstituted 5-13 membered heteroaryl;

[0025] The substituents of the alkyl group are each independently selected from the following groups: halogen, hydroxyl, C1-C6 alkoxy, substituted or unsubstituted 5-10 membered aryl, substituted or unsubstituted 5-10 membered heteroaryl;

[0026] The substituents of the cycloalkyl, heterocycloalkyl, aryl and heteroaryl groups are independently selected from the following groups: 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') 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;

[0027] m is 0 or 1;

[0028] R3 is 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;

[0029] R3' and R4' are independently selected from hydrogen and C1-C6 alkyl;

[0030] R4 and R5 are independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, -C(O)R6;

[0031] R6 is selected from C1-C6 alkyl, substituted or unsubstituted 5-10 membered heteroaryl;

[0032] The substituents of the alkoxy and alkylthio groups are independently selected from the following groups: halogen, substituted or unsubstituted 5-10 membered aryl groups;

[0033] R5' is a substituent at any position on the benzene ring, selected from the following groups: hydrogen, halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy.

[0034] Furthermore, the compound is represented by Formula IIB-1:

[0035]

[0036] in,

[0037] R5' is a substituent at any position on the benzene ring, selected from the following groups: hydrogen, halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy;

[0038] X is O or NR7';

[0039] R6' is a substituent at any position on the heterocycle, selected from the following groups: hydrogen, C1-C6 alkyl;

[0040] R7′ is selected from -C(O)R8′;

[0041] R8' is selected from furyl.

[0042] Furthermore, the compound is represented by Formula IIA-1 or Formula IIA-2:

[0043]

[0044] in,

[0045] R7 is 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;

[0046] m is 0 or 1;

[0047] R3' and R4' are independently selected from hydrogen and C1-C6 alkyl;

[0048] R3 is selected from the following substituted or unsubstituted groups: C1-C6 alkyl, 3-6 membered cycloalkyl, The substituents are each independently selected from the following groups: C1~C6 alkyl, halogenated C1~C6 alkyl, hydroxy-substituted C1~C6 alkyl, -C(O)C1~C6 alkyl, -N(H)C(O)C1~C6 alkyl, C1~C6 alkoxy, C1~C6 alkylthio, -NR4R5, halogen, hydroxy, -(CH2) m1 C1-C6 alkoxy, 3-6 membered cycloalkyl,

[0049] m1 is selected from 1, 2 or 3;

[0050] R4 and R5 are independently selected from hydrogen, C1-C6 alkyl, and halogenated C1-C6 alkyl;

[0051] In formula IIA-2, Y1 and Y2 are independently selected from N or CH, m2 is selected from 1, 2 or 3, and R8 is selected from hydrogen, C1~C6 alkyl, and halogenated C1~C6 alkyl.

[0052] Further,

[0053] R7 is selected from

[0054] Furthermore, the compound is selected from one of the following structures:

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] The present invention also provides use of the aforementioned compound, its pharmaceutically acceptable salt, solvate or stereoisomer in the preparation of a Blimp1 inhibitor.

[0064] The present invention also provides the use of the aforementioned compound, its pharmaceutically acceptable salt, solvate or stereoisomer in the preparation of a medicament for preventing and / or treating immune-related diseases;

[0065] Preferably, the immune-related disease is systemic lupus erythematosus, rheumatoid arthritis, cancer, or hemophagocytic syndrome.

[0066] The present invention also provides a pharmaceutical preparation, which is prepared by using the aforementioned compound, its pharmaceutically acceptable salt, solvate, or stereoisomer as an active ingredient and pharmaceutically acceptable excipients.

[0067] The present invention also provides a pharmaceutical composition comprising the aforementioned compound, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

[0068] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the relevant terms and laboratory procedures used herein are those widely used in the relevant fields and routine procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0069] As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0070] In the description herein, references to “some embodiments,” “some implementation schemes,” or “some implementation plans” describe a subset of all possible embodiments, but it will be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0071] As used herein and unless otherwise specified, the terms "comprises," "includes," "has," "contains," and their grammatical equivalents should generally be understood as open-ended and non-limiting, e.g., not excluding other unlisted elements or steps.

[0072] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is pharmaceutically acceptable and possesses the pharmacological activity of the parent compound. Such salts include acid addition salts formed with inorganic or organic acids, such as nitric, phosphoric, and carbonic acids; organic acids, such as propionic, hexanoic, cyclopentylpropionic, glycolic, pyruvic, gluconic, stearic, and muconic acids; salts formed when an acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal or alkaline earth metal ion; or coordination compounds formed with an organic base, such as ethanolamine. Pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid or base groups by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water, an organic solvent, or a mixture of both. Generally, non-aqueous media, such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, are preferred. In addition to salt forms, the compounds provided herein also exist in prodrug form. Prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to convert to the compounds of the invention. Additionally, prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in an in vivo environment.

[0073] As used herein, the term "solvate" refers to a compound of the present invention in combination with a pharmaceutically acceptable solvent. Pharmaceutically acceptable solvents include acetic acid, etc. Solvates include stoichiometric solvates and non-stoichiometric solvates. Certain compounds of the present invention may exist in unsolvated or solvated forms. Generally speaking, solvated forms are equivalent to unsolvated forms and are encompassed by the present invention.

[0074] As used herein, the term "stereoisomer" includes conformational isomers and configurational isomers, wherein configurational isomers mainly include cis-trans isomers and optical isomers. The compounds of the present invention may 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, etc. The compounds of the present invention may also exist in the form of any combination or any mixture of the aforementioned stereoisomers, such as mesomorphs, racemates, equal mixtures of atropisomers, etc. For example, a single enantiomer, a single diastereomer or a mixture thereof, or a single atropisomer or a mixture thereof. When the compound of the present invention contains an olefin double bond, unless otherwise specified, it includes cis-isomers and trans-isomers, and any combination thereof. The atropisomers of the present invention are stereoisomers with axial or planar chirality based on restricted intramolecular rotation. And as a drug, stereoisomers with excellent activity are preferred. The compounds of the present invention have optical isomers arising from asymmetric carbon atoms, etc., and individual isomers can be resolved and obtained, if necessary, by methods known in the art, such as crystallization or chiral chromatography.

[0075] As used herein, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic hydrocarbon group, including, for example, monocyclic cycloalkyl, spirocyclic alkyl, fused cycloalkyl and bridged cycloalkyl. The ring carbon atoms of the cycloalkyl group described in the present invention may be optionally substituted with 1, 2 or 3 oxo groups to form a cyclic ketone structure. The term "C 3-8 The term "cycloalkyl" refers to a cycloalkyl group having 3 to 8 ring carbon atoms, including monocyclic cycloalkyl, spirocyclic cycloalkyl, fused cycloalkyl and bridged cycloalkyl.

[0076] As used herein, the term "heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic fused cyclic hydrocarbon group, and the term "3- to 8-membered heterocycloalkyl" refers to a saturated cyclic hydrocarbon group having 3 to 8 ring atoms, wherein one or more (preferably 1, 2, 3 or 4) ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, and the remaining ring atoms are carbon.

[0077] As used herein, the term "aryl" or "aromatic ring" refers to an all-carbon monocyclic, all-carbon non-fused polycyclic (rings are connected by covalent bonds and are not fused) or all-carbon fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group, in which at least one ring is aromatic, i.e., has a conjugated π electron system. The term "C 6-14 "Aryl" refers to an aromatic group having 6 to 14 ring atoms. Preferably, C 6-10 Aryl. In the present invention, C 6-14 The aryl group includes a monocyclic aryl group, a non-fused polycyclic aryl group, and an aromatic fused polycyclic group, wherein examples of the monocyclic aryl group include phenyl, and examples of the non-fused polycyclic aryl group include biphenyl, etc. The term "6- to 10-membered aromatic ring" refers to an aromatic ring having 6 to 10 ring atoms.

[0078] As used herein, the term "heteroaryl" refers to a monocyclic or fused polycyclic (i.e., shared adjacent pairs of ring atoms, which may be CC or NC) group, wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen atom may be optionally quaternized. The term "5- to 10-membered heteroaryl" refers to a group having 5 to 10 ring atoms, wherein 1, 2, 3 or 4 ring atoms are selected from nitrogen, oxygen or S(=O). m ' (wherein m' is an integer from 0 to 2). The 5- to 10-membered heteroaryl group in the present invention may be a monocyclic heteroaryl group or a condensed bicyclic heteroaryl group.

[0079] 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 selected from nitrogen, oxygen or S(=O). m m' (wherein m' is an integer from 0 to 2). Specific examples of heteroaryl groups include, but are not limited to, thiophene, furan, thiazole, isothiazole, imidazole, oxazole, pyrrole, pyrazole, triazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,5-triazole, 1,3,4-triazole, tetrazole, isoxazole, oxadiazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, thiadiazole, pyridine, pyridazine, pyrimidine, pyrazine, and the like.

[0080] The two carbon atoms represented as being connected are adjacent pairs of carbon atoms that are shared when fused to other rings.

[0081] In the present invention, the various heteroaryl groups mentioned above may be substituted or unsubstituted. When substituted, the substituents are preferably one or more substituent groups described in this application.

[0082] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a non-toxic but sufficient amount of a drug or medicament that achieves the desired effect. In embodiments of the present invention, when a patient is treated according to the present invention, the amount of a given drug depends on many factors, such as a specific dosing regimen, the type of disease or condition and its severity, the uniqueness (e.g., body weight) of the subject or host to be treated, but, according to specific surrounding circumstances, including, for example, the specific drug, route of administration, the condition to be treated, and the subject or host to be treated, the dosage can be conventionally determined by methods known in the art. Typically, for adult treatment, the dosage is typically in the range of 0.02-5000 mg / day, such as about 1-1500 mg / day. The desired dosage can be conveniently expressed as one dose, or simultaneously administered (or in a short period of time) or in divided doses at appropriate intervals, such as two, three, four, or more divided doses per day. It will be appreciated by those skilled in the art that, although the above-mentioned dosage range has been given, the specific effective amount can be appropriately adjusted according to the patient's condition and in conjunction with the physician's diagnosis.

[0083] The compounds of the present invention 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 invention. The solvents, temperatures and other reaction conditions given in the present invention are exemplary and can be varied according to methods well known in the art. The embodiment compounds described in the present invention can be synthesized according to the methods described in the embodiments using appropriate starting materials according to their specific structures, or can be synthesized using methods similar to those described in the embodiments. The starting materials for synthesizing the embodiment compounds of the present invention can be prepared by known synthetic methods or similar methods described in the literature or obtained from commercial sources. The embodiment compounds can be further split by methods well known in the art, such as crystallization, chromatography, etc., as needed to obtain their stereoisomers, and the splitting conditions are easily obtained by those skilled in the art through conventional means or limited experiments.

[0084] The present invention provides a small molecule compound that acts as a Blimp1 inhibitor. The compound can effectively inhibit Blimp1 activity and effectively delay CAR-T cell exhaustion. Therefore, the compound can enhance the anti-tumor effect of CAR-T therapy and inhibit the progression of hemophagocytic syndrome. The compound is suitable for clinical application and is druggable, with promising application prospects.

[0085] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0086] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 Figure 2 is the result of CAR-T cell proliferation.

[0088] Figure 2 Figure 2 is the result of CAR-T cell apoptosis.

[0089] Figure 3 This is a diagram showing the memory phenotype results of CAR-T cells.

[0090] Figure 4 Figure 2 is the result of cytokine secretion detection.

[0091] Figure 5 This figure represents the experimental process of the animal model and the results of each group's inhibition on tumor cells.

[0092] Figure 6 The graph shows the inhibition results of each group on tumor cells.

[0093] Figure 7 The graph shows the effects of each group on the weight of liver and spleen. DETAILED DESCRIPTION

[0094] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercial products.

[0095] Example 1. Preparation of Compound 8

[0096]

[0097] Step a: Preparation of Intermediate 1

[0098]

[0099] To a 250 mL single-necked round-bottom flask, add the substrate 2,4-dichloropyrido[2,3-d]pyrimidine (5 g, 25.00 mmol), followed by 100 mL of THF, and stir to mix thoroughly. Then, add the substrate 2-(trifluoromethyl)pyridine-3-methylamine (3.75 mL, 27.50 mmol) and triethylamine (6.95 mL, 49.99 mmol) sequentially, and continue stirring at room temperature for 6 h. After completion of the reaction, monitor the reaction by TLC, evaporate the THF, add water, filter, and dry to obtain a pale yellow solid, Intermediate 1, 7.2 g, 85% yield, MS (ESI) m / z: 340.06 [M+H]. + .

[0100] Step b: Preparation of compound 8

[0101]

[0102] To a 50 mL reaction tube, intermediate 1 (200 mg, 0.5887 mmol) was added, followed by dissolution in 3 mL of ultra-dry dimethyl sulfoxide. Finally, the substrates 2-(thiophen-2-yl)ethylamine (104 μL, 0.8831 mmol) and triethylamine (164 μL, 0.1180 mmol) were added, and the mixture was heated at 90°C for 4 h. After completion of the reaction, as monitored by TLC, the system solution was added to water, resulting in the precipitation of a large amount of solid. The solid was filtered, and the filter cake was purified by column chromatography to afford a pale yellow solid, compound 8, 148 mg, in a 58% yield. MS (ESI) m / z: 431.13 [M+H]. + .

[0103] Example 2: Preparation of Compound 60

[0104]

[0105] Step a: Preparation of Intermediate 2

[0106]

[0107] Intermediate 1 (3 g, 8.83 mmol) and the substrate 2-amino-7-Boc-7-azaspiro[3.5]nonane (3.18 g, 13.25 mmol) were weighed into a 250 mL single-necked round-bottom flask. 15 mL of ultra-dry dimethyl sulfoxide and triethylamine (2.46 mL, 17.66 mmol) were then added and heated at 90°C for 8 h. After completion of the reaction, as monitored by TLC, the system solution was added to water, resulting in the precipitation of a large amount of solid. The solid was filtered and the filter cake was purified by column chromatography to afford a light yellow solid, Intermediate 2, 3.4 g, in a 71% yield. MS (ESI) m / z: 544.26 [M+H]. + .

[0108] Step b: Preparation of Intermediate 3

[0109]

[0110] To a 100 mL single-necked round-bottom flask, add Intermediate 2 (2 g, 3.68 mmol). A small amount of methanol was added to dissolve the substrate, followed by 40 mL of a 4 M hydrochloric acid solution in dioxane. The reaction was allowed to react overnight at room temperature. After completion of the reaction, as monitored by TLC, the system was concentrated and dried to afford Intermediate 3, which was used directly in the next step without purification.

[0111] Step c: Preparation of compound 60

[0112]

[0113] To a 10 mL reaction flask, intermediate 3 (200 mg, 0.4167 mmol), 5-acetylthiophene-2-carboxylic acid (142 mg, 0.8334 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (160 mg, 0.8334 mmol), and 1-hydroxybenzotriazole (113 mg, 0.8334 mmol) were added. 3 mL of N,N-dimethylformamide was added and stirred, followed by the addition of triethylamine (290 μL, 2.08 mmol). The mixture was allowed to react overnight at room temperature. After completion of the reaction, as monitored by TLC, the solution was added to water, resulting in the precipitation of a large amount of solid. The solid was filtered and the filter cake was purified by column chromatography to afford a light yellow solid, compound 60, 170 mg, in a 68% yield. MS (ESI) m / z: 596.21 [M+H]. + .

[0114] Example 3: Preparation of Compound 78

[0115]

[0116] Step a: Preparation of intermediate 4

[0117]

[0118] Intermediate 1 (3 g, 8.83 mmol) and the substrate methyl 4-piperidinate (1.9 g, 13.25 mmol) were weighed into a 250 mL single-necked round-bottom flask. 15 mL of ultra-dry dimethyl sulfoxide and triethylamine (2.46 mL, 17.66 mmol) were then added and heated at 90°C for 8 h. After completion of the reaction, as monitored by TLC, the solution was added to water, resulting in the precipitation of a large amount of solid. The solid was filtered and the filter cake was purified by column chromatography to afford a pale yellow solid, Intermediate 4, 2.9 g, in a 75% yield. MS (ESI) m / z: 447.18 [M+H]. + .

[0119] Step b: Preparation of Intermediate 5

[0120]

[0121] Intermediate 4 (2 g, 4.48 mmol) was dissolved in 18 mL of methanol and aqueous sodium hydroxide solution (18 mL, 17.92 mmol, 1 M) was added. The mixture was allowed to react overnight at room temperature. After completion of the reaction, monitored by TLC, the reaction solution was concentrated and diluted with 30 mL of water. The pH was then adjusted to 2-3 with 1 M aqueous hydrochloric acid. A large amount of white solid precipitated and was filtered to obtain Intermediate 5 as a light yellow solid (1.8 g, 93% yield). MS (ESI) m / z: 433.16 [M+H] + .

[0122] Step c: Preparation of intermediate 6

[0123]

[0124] Intermediate 5 (1 g, 2.31 mmol) was added to a 250 mL round-bottom flask. 50 mL of ultra-dry dichloromethane was added and the mixture was placed in an ice bath. Stirring was performed to mix thoroughly. Oxalyl chloride (391 μL, 4.62 mmol) and 2 drops of ultra-dry N,N-dimethylformyl were then added dropwise. After TLC monitoring, the reaction was carried out directly to the next step.

[0125] Step d: Preparation of compound 78

[0126]

[0127] To a 100 mL single-necked round-bottom flask, 5-amino-2-methoxypyridine (66 mg, 0.5323 mmol) and 10 mL of ultra-dry dichloromethane were added and stirred to dissolve. Triethylamine (185 μL, 1.33 mmol) was then added dropwise. After 10 min, 10 mL of ultra-dry dichloromethane solution containing intermediate 6 (208 mg, 0.4436 mmol) was added dropwise under an ice bath. After the addition was complete, the reaction was allowed to react at room temperature. After completion of the reaction, as monitored by TLC, water (20 mL) was added to the reaction flask and extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to afford compound 78 as a white solid (202 mg, 85% yield). MS (ESI) m / z: 539.21 [M+H]+.

[0128] Other target compounds of the present invention were prepared by referring to the methods of Examples 1-3. The numbers, structures and characterization data of the obtained target compounds are shown in Table 1.

[0129] Table 1. Number, structure and characterization data of the target compounds of the present invention

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0147] Test Example 1: Study on the inhibitory activity of the compounds of the present invention on Blimp1

[0148] 1. Experimental methods

[0149] The present invention uses differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC) to evaluate the inhibitory activity of the compounds of the present invention on Blimp1.

[0150] Thermal shift (ΔTm) measurements were performed using a Bio-Rad CFX96 RT-PCR instrument using differential scanning fluorimetry. Samples were prepared by adding 9.8 μL of a mixture of Blimp1 protein and SYPRO Orange to each well, followed by 0.2 μL of the compound solution. The final assay buffer was 20 mM HEPES pH 7.5, 150 mM NaCl. The final concentrations of Blimp1 protein and the test compound were 2 μM and 100 μM, respectively, in a total volume of 10 μL. The samples were first equilibrated at 25°C for 3 minutes; then, they were heated from 25°C to 95°C at a rate of 1°C / min.

[0151] Isothermal titration calorimetry experiments using PEAQ-ITC to determine affinity K DDetermination of the value. All tests were performed at 25°C using a buffer system of 20mM HEPES (pH 7.5), 150mM NaCl, and 0.4% DMSO. Prior to the experiment, the compounds were diluted directly in the same batch of buffer. Each experiment was a reverse titration experiment (Blimp1 protein was aspirated into the syringe and the small molecule was injected into the sample cell). Initially, 0.2μL of protein was titrated, and then 2μL of protein was titrated every 100s for 19 consecutive drops. Experimental data were calculated using ΔG=ΔH-TΔS=RTlnK D Computational thermodynamic parameter analysis. ΔG, ΔH, and ΔS are the changes in free energy, enthalpy, and entropy, respectively.

[0152] 2. Experimental results

[0153] The experimental results are shown in Table 2.

[0154] K D This refers to the strength of the binding between a single biomolecule (protein) and its ligand (drug or inhibitor). ΔTm: When a drug or inhibitor binds to a protein pocket, it shifts the protein's melting temperature (Tm1). The melting temperature of the protein unbound by the small molecule is Tm0, where ΔTm = Tm1 - Tm0. When small molecules bind to the protein pocket, they prevent Blimp1 from binding to its substrate, inhibiting its activity.

[0155] Table 2. Experimental results

[0156]

[0157]

[0158]

[0159] The experimental results in Table 2 show that some compounds of the present invention have good inhibitory activity against Blimp1. Among them, compound 60 has the best inhibitory activity against Blimp1, K D The value is 0.098 μM.

[0160] Experimental Example 2: Characterization of the compounds of the present invention in delaying CAR-T cell exhaustion

[0161] 1. Experimental methods

[0162] Compound 60 with the best activity in Experimental Example 1 was selected, and its effects on the proliferation, apoptosis, memory phenotype, and cytokine secretion of CAR-T cells were further tested.

[0163] (1) Preparation of CAR-T cells: Human peripheral blood lymphocytes were collected and T cells were isolated. After 48 h of stimulation with anti-CD3 / CD28 magnetic beads and IL-2, the prepared CAR virus was added to infect the T cells. 24 h later, 1 μM compound 60 was added to the culture system and cultured for 7 days.

[0164] (2) CAR-T cell proliferation detection: During the culture of CAR-T cells, the cell density of CAR-T cells added with compound 60 and DMSO was detected every two days.

[0165] (3) CAR-T cell apoptosis detection: Take the CAR-T cells in (1) and use an apoptosis kit to detect the apoptosis of CAR-T cells. After CAR-T and tumor cells are co-cultured at an E:T ratio of (effector cells: target cells) of 1:1 for two days, the apoptosis of CAR-T cells is detected. The detailed operation steps are as follows:

[0166] ① CAR-T cells treated with compound 60 and DMSO were collected in a 15 mL sterile centrifuge tube, and washed by centrifugation at 1200 rpm for 10 min after adding 2 mL PBS.

[0167] ② Resuspend the cell pellet in 1× Binding buffer and adjust the cell density to 1×10 6 / mL;

[0168] ③ For flow cytometry antibody staining, add 5 μL of APC-Annnexin V and 5 μL of 7-AAD to a 100 μL sample according to the reagent instructions and mix thoroughly. Also set up control tubes: a blank tube (untreated), a 7-AAD single-stained tube, and an APC-Annexin V single-stained tube.

[0169] ④ Incubate at room temperature in the dark for 20-30 min, add 400 μL 1× Binding buffer, and analyze cell apoptosis within 1 h.

[0170] (4) CAR-T memory phenotype detection

[0171] The CAR-T cells in (1) were taken and the memory phenotype of CAR-T was detected. After 6 days and 9 days of co-culture of CAR-T and tumor cells at E:T=1:1, the memory phenotype of CAR-T was detected respectively. The CAR-T treated with compound 60 and DMSO or the CAR-T cells after co-culture were collected in a 15 mL sterile centrifuge tube, and 2 mL of PBS was added for centrifugation and washing at 1200 rpm for 10 min; 0.5 μL of Percp-CD8, FITC-MYC, BV510-CD2L, and PE-CD45RO were added and mixed. At the same time, a control tube, a blank tube (no treatment), and a single-stained tube were set up; incubated at room temperature in the dark for 20-30 min, 2 mL of PBS was added for centrifugation and washing, and the tube was analyzed within 1 hour.

[0172] (5) Cytokine secretion detection

[0173] Take the CAR-T cells in (1) and detect the cytokine secretion after two days of co-culture of CAR-T and tumor cells at E:T=1:1. Collect the CAR-T cells after co-culture with tumor cells in a 15mL sterile centrifuge tube, add 2mL PBS and centrifuge and wash at 1200rpm for 10min; add 0.5μL APC-CD4 and FITC-MYC and mix well, incubate at room temperature in the dark for 20-30min, fix the cells, add 0.5μL PE-IFNγ flow cytometry antibody, incubate at room temperature in the dark for 20-30min, add 2mL PBS and centrifuge and wash, and analyze within 1h. At the same time, set up a control tube, a blank tube (no treatment), and a single staining tube.

[0174] 2. Experimental results

[0175] (1) CAR-T cell proliferation results

[0176] CAR-T cell proliferation results Figure 1 As shown by Figure 1 It can be seen that adding 1 μM compound 60 to the CAR-T cell culture system can significantly increase the proliferation ability of CAR-T cells.

[0177] (2) CAR-T cell apoptosis results

[0178] CAR-T cell apoptosis results Figure 2 As shown by Figure 2 It can be seen that adding 1 μM compound 60 to the CAR-T cell culture system can significantly reduce the apoptosis of CAR-T cells before and after co-culture.

[0179] (3) CAR-T cell memory phenotype results

[0180] CAR-T cell memory phenotype results Figure 3 As shown by Figure 3 It can be seen that the addition of 1 μM compound 60 to the CAR-T cell culture system can significantly increase the central memory cell T of CAR-T cells before and after co-culture. CM proportion.

[0181] (4) Cytokine secretion test results

[0182] Cytokine secretion test results Figure 4 As shown by Figure 4 It can be seen that adding 1 μM of compound 60 to the CAR-T cell culture system can significantly increase the ability of CAR-T cells to secrete IFNγ after co-culture.

[0183] The above experimental results show that the compounds of the present invention can effectively delay CAR-T cell exhaustion.

[0184] Experimental Example 3: Characterization of the Compounds of the Invention in Delaying CAR-T Cell Exhaustion in Animal Models

[0185] 1. Experimental methods

[0186] (1) Tumor cell inoculation: NCG mice were subcutaneously inoculated with 3×10 6 of tumor cells and observe tumor growth.

[0187] (2) Preparation of CAR-T cells: Human lymphocytes were harvested and T cells were isolated. After 48 hours of stimulation with anti-CD3 / CD28 magnetic beads and IL-2, the prepared CAR virus was added to infect the T cells. 24 hours later, 1 μM of the PRDM1 / Blimp1 inhibitor compound 60 was added to the culture system and cultured for 7 days.

[0188] (3) CAR-T cell transfusion: NSG mice inoculated with tumor cells were divided into three groups according to tumor size, with 5-9 mice in each group. Groups 1, 2, and 3 were transfused with compound 60-treated CAR-T cells, DMSO-treated CAR-T cells, and NT cells, respectively. The CAR positivity rates in groups 1 and 2 were the same.

[0189] 2. Experimental results

[0190] The experimental results are as follows Figure 5 As shown: After the CAR-T cell group treated with compound 60 was reinfused, tumor growth was significantly inhibited.

[0191] Experimental Example 4: Characterization of the Compound of the Invention in Combination with PD-1 in Animal Models

[0192] 1. Experimental methods

[0193] C57BL mice were subcutaneously inoculated with 3 × 10 6MC38 cells were used to observe tumor growth. The tumor volume was 100 mm 3 At the same time, mice were divided into seven groups and administered 40 mg / kg of compound 60, 40 mg / kg of compound 60 plus anti-PD-1 antibody, 20 mg / kg of compound 60, 20 mg / kg of compound 60 plus anti-PD-1 antibody, anti-PD-1 antibody, vehicle, or PBS plus vehicle. Compound 60 was administered twice daily, and PD-1 was administered every three days at a dose of 100 μg per mouse.

[0194] 2. Experimental results

[0195] The experimental results are as follows Figure 6 As shown: 40 mg / kg compound 60 combined with PD-1 significantly inhibited tumor growth.

[0196] Test Example 5: Characterization of the compounds of the present invention in an animal model of hemophagocytic syndrome

[0197] 1. Experimental methods

[0198] C57BL male mice were divided into eight groups. The modeling group received intraperitoneal injections of 50 μg of CpG-ODN1826 on days 0, 2, 4, 6, 8, and 10, respectively, while the normal control group received the same volume of PBS. The treatment groups were designated as normal, blank, dexamethasone, etoposide, ruxolitinib, dexamethasone + ruxolitinib, compound 60 (40 mg / kg), and dexamethasone + compound 60 (40 mg / kg). Compound 60 was administered twice daily, while dexamethasone was administered intraperitoneally once daily at a dose of 1.5 mg / kg. Etoposide was administered intraperitoneally twice weekly at a dose of 50 mg / kg. Ruxolitinib was administered orally twice daily at a dose of 30 mg / kg. Mice were weighed every two days. After 10 days of modeling, orbital blood, liver, spleen, and femur were collected, and liver and spleen weights were observed and weighed.

[0199] 2. Experimental results

[0200] The experimental results are as follows Figure 7 As shown: both the 40 mg / kg Compound 60 administration group and the 40 mg / kg Compound 60 + dexamethasone combination group could significantly inhibit and improve the weight of the liver and spleen of mice and inhibit the progression of hemophagocytic syndrome.

[0201] In summary, the present invention provides a small molecule compound that acts as a Blimp1 inhibitor. The compound of the present invention can effectively inhibit Blimp1 activity and has excellent effects in delaying CAR-T cell exhaustion. Therefore, the compound of the present invention can enhance the anti-tumor effect of CAR-T therapy and can also inhibit the progression of hemophagocytic syndrome. The compound of the present invention is a druggable compound suitable for clinical application and has good application prospects.

Claims

1. A compound, a pharmaceutically acceptable salt or stereoisomer thereof, characterized in that: The compound is represented by formula IIB-1: in, R5' is a substituent at any position on the benzene ring, selected from the following groups: hydrogen, halogen; X is NR7'; R6' is a substituent at any position on the heterocycle, selected from the following groups: hydrogen, C1-C6 alkyl; R7′ is selected from -C(O)R8′; R8' is selected from furyl.

2. A compound, a pharmaceutically acceptable salt or stereoisomer thereof, characterized in that: The compound is represented by formula IIA-1: in, R7 is selected from -C(O)(CR3'R4') m R3, -C(O)(CR3'R4') m OR3, -S(O)(O)R3; m is 0 or 1; R3' and R4' are each independently selected from hydrogen; R3 is selected from the following substituted or unsubstituted groups: C1 to C6 alkyl, The substituents are each independently selected from the following groups: C1-C6 alkyl, halogenated C1-C6 alkyl, -C(O)C1-C6 alkyl, -N(H)C(O)C1-C6 alkyl, C1-C6 alkoxy, -NR4R5, halogen, 3-6 membered cycloalkyl; R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, and halogenated C1-C6 alkyl.

3. The compound according to claim 2, its pharmaceutically acceptable salt or stereoisomer, characterized in that: R7 is selected from 4. A compound, a pharmaceutically acceptable salt or stereoisomer thereof, characterized in that: The compound is represented by formula IIA-2: in, Y1 is selected from N, Y2 is independently selected from N or CH, m2 is selected from 1 or 2, R8 is selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl; R7 is selected from -C(O)(CR3'R4') m R3, -C(O)NR3'(CR3'R4') m R3, -NR3'C(O)R3; m is 0 or 1; R3' and R4' are each independently selected from hydrogen; R3 is selected from the following substituted or unsubstituted groups: C1-C6 alkyl, 3-6 membered cycloalkyl, The substituents are each independently selected from the following groups: C1-C6 alkyl, halogenated C1-C6 alkyl, -C(O)C1-C6 alkyl, C1-C6 alkoxy, and halogen.

5. The compound according to claim 4, its pharmaceutically acceptable salt or stereoisomer, characterized in that:

6. A compound, a pharmaceutically acceptable salt or stereoisomer thereof, characterized in that: The compound is selected from one of the following structures:

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

8. Use of the compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt or stereoisomer thereof, in the preparation of a medicament for preventing and / or treating immune-related diseases; The immune-related disease is hemophagocytic syndrome.

9. Use of a compound, a pharmaceutically acceptable salt or stereoisomer thereof in the preparation of a medicament for delaying CAR-T cell exhaustion, characterized in that: The structure of the compound is 10. A pharmaceutical preparation, characterized in that: The invention is prepared by taking the compound according to any one of claims 1 to 6, its pharmaceutically acceptable salt or stereoisomer as active ingredients and adding pharmaceutically acceptable auxiliary materials.

11. A pharmaceutical composition, characterized in that: It includes the compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt or stereoisomer thereof.

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