Small molecule compound as Blimp1 inhibitor and application thereof

Abstract

The invention provides a small molecule compound as a Blimp1 inhibitor and application thereof, and belongs to the technical field of organic synthetic drugs. The small molecule compound is a compound as shown in a formula I, and pharmaceutically acceptable salt, solvate or stereoisomer thereof. The compound provided by the invention can effectively inhibit the activity of Blimp1 and has an excellent effect of delaying CAR-T cell depletion. Therefore, the compound provided by the invention can enhance the anti-tumor effect of CAR-T therapy, and meanwhile, the compound provided by the invention can also inhibit the development of the hemophagocytosis syndrome. The compound provided by the invention is suitable for clinical application, can be used as a medicine compound, and has a good application prospect. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of organic synthetic drugs, and in particular 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, that is, by genetically engineering the patient's own T cells to enable them to recognize and attack tumor cells. At present, CAR-T cell therapy has achieved good therapeutic effects in blood tumors and poor effects in solid tumors, but even in blood tumors, its 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 efficacy 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 that delay T cell exhaustion will be of great significance to tumor immunotherapy.

[0003] Blimp-1, also known as PRDM1 (PR domain containing 1), is a transcription factor. Blimp-1 mediates the transcriptional programs 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. Increased Blimp-1 expression is associated with inhibition of 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 (Treg) 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 early memory phenotypes and the secretion of multifunctional cytokines, promotes the expansion of less differentiated memory CAR-T cells in vivo, thereby enhancing the persistence of CAR-T cells and improving the therapeutic efficacy of various tumor models. In short, 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 have been no reports of small molecule inhibitors targeting Blimp-1 at home and 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 in Ring A are each independently selected from the following groups: halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated 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 substituted or unsubstituted 3-10 membered heterocycloalkyl, 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 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 substituted or unsubstituted 3-10 membered heterocycloalkyl, 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 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 furanyl.

[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,

[0049] 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,

[0050] m1 is selected from 1, 2 or 3;

[0051] R4 and R5 are independently selected from hydrogen, C1-C6 alkyl, and halogenated C1-C6 alkyl;

[0052] 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, halogenated C1~C6 alkyl.

[0053] Further,

[0054] R7 is selected from

[0055]

[0056] Furthermore, the compound is selected from one of the following structures:

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] The present invention also provides use of the aforementioned compound, its pharmaceutically acceptable salt, solvate or stereoisomer in the preparation of Blimp1 inhibitors.

[0066] The present invention also provides the use of the aforementioned compound, its pharmaceutically acceptable salt, solvate or stereoisomer in the preparation of a drug for preventing and / or treating immune-related diseases;

[0067] Preferably, the immune-related disease is systemic lupus erythematosus, rheumatoid arthritis, cancer, or hemophagocytic syndrome.

[0068] 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 adding pharmaceutically acceptable excipients.

[0069] The present invention also provides a pharmaceutical composition, which comprises the aforementioned compound, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

[0070] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the relevant terms and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0071] As used herein and unless otherwise specified, 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.

[0072] In the description of this document, reference is made to “some embodiments”, “some implementation schemes” or “some implementation plans”, which describe a subset of all possible embodiments, but it can 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.

[0073] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

[0074] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is pharmaceutically acceptable and has the pharmacological activity of the parent compound. Such salts include: acid addition salts formed with inorganic acids or organic acids, such as nitric acid, phosphoric acid, carbonic acid, etc.; organic acids such as propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, gluconic acid, stearic acid, muconic acid, etc.; or salts formed when the acidic protons present on the parent compound are replaced by metal ions, such as alkali metal ions or alkaline earth metal ions; or coordination compounds formed with organic bases, such as ethanolamine, etc. The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. In general, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. In addition to the salt form, the compounds provided by the present invention also exist in the form of prodrugs. Prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to be converted 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.

[0075] As used herein, the term "solvate" refers to a substance formed by the combination of a compound of the present invention and a pharmaceutically acceptable solvent. Pharmaceutically acceptable solvents include acetic acid and the like. Solvates include stoichiometric solvates and non-stoichiometric solvates. Certain compounds of the present invention may exist in a non-solvated form or a solvated form. Generally speaking, the solvated form is comparable to the non-solvated form and is included within the scope of the present invention.

[0076] As used in the text, 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 cover all possible stereoisomer 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 meso, racemate, 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 of axial or planar chirality generated based on restricted intramolecular rotation. And as a drug, stereoisomers with excellent activity are preferred. The compounds of the present invention have optical isomers derived from asymmetric carbon atoms, etc. If necessary, single isomers can be separated and obtained by methods known in the art, such as crystallization or chiral chromatography.

[0077] As used herein, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic cyclic 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 by 1, 2 or 3 oxo groups to form a cyclic ketone structure. 3-8 The term "cycloalkyl" refers to a cycloalkyl group having 3 to 8 ring carbon atoms, including monocyclic cycloalkyl groups, spirocyclic alkyl groups, fused cycloalkyl groups and bridged cycloalkyl groups.

[0078] As used herein, the term "heterocycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic condensed 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.

[0079] 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, at least one ring in the group 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 In the present invention, C 6-14 The aryl group includes a monocyclic aryl group, a non-condensed polycyclic aryl group and an aromatic condensed polycyclic group, wherein examples of the monocyclic aryl group include phenyl, and examples of the non-condensed 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.

[0080] As used herein, the term "heteroaryl" refers to a monocyclic or fused polycyclic (i.e., sharing adjacent pairs of ring atoms, which may be CC or NC) group whose ring atoms are substituted by at least one heteroatom independently selected from nitrogen, oxygen or sulfur, wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen atom may be optionally quaternized. The term "5- to 10-membered hetero ... independently selected from nitrogen, oxygen or S(=O) m The 5- to 10-membered heteroaryl group in the present invention may be a monocyclic heteroaryl group or a condensed bicyclic heteroaryl group.

[0081] 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 of the ring atoms are selected from nitrogen, oxygen or S(=O). m ' (wherein m' is an integer from 0 to 2). Specific examples of heteroaryl 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.

[0082] The two carbon atoms represented as being connected are adjacent pairs of carbon atoms that are shared when fused with other rings.

[0083] In the present invention, the above-mentioned various heteroaryl groups may be substituted or unsubstituted. When substituted, the substituent is preferably one or more substituent groups described in the present application.

[0084] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a non-toxic drug or medicament that can achieve the desired effect. In embodiments of the present invention, when treating a patient according to the present invention, the amount of a given drug depends on many factors, such as a specific dosing regimen, a disease or condition type and its severity, the uniqueness of the subject or host to be treated (e.g., body weight), but, according to specific surrounding conditions, including, for example, a specific drug, route of administration, condition to be treated, and the subject or host to be treated, the dosage can be routinely determined by methods known in the art. Usually, with respect to the dosage used 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 dose can be conveniently expressed as a single dose, or a dose administered simultaneously (or in a short period of time) or in a divided dose 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 is given, the specific effective amount can be appropriately adjusted according to the patient's condition and in conjunction with the physician's diagnosis.

[0085] 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 used to synthesize 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 separated by methods well known in the art, such as crystallization, chromatography, etc. to obtain their stereoisomers as needed, and the separation conditions are easily obtained by those skilled in the art through conventional means or limited experiments.

[0086] The present invention provides a small molecule compound as a Blimp1 inhibitor, which can effectively inhibit Blimp1 activity and has an excellent effect of delaying CAR-T cell exhaustion. Therefore, the compound of the present invention can enhance the anti-tumor effect of CAR-T therapy, and the compound of the present invention 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.

[0087] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0088] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 This is a graph showing the proliferation results of CAR-T cells.

[0090] Figure 2 This is the result of CAR-T cell apoptosis.

[0091] Figure 3 This is a diagram of the CAR-T cell memory phenotype results.

[0092] Figure 4 This is a graph showing the results of cytokine secretion detection.

[0093] Figure 5 The figure represents the experimental process of the animal model and the inhibition results of each group on tumor cells.

[0094] Figure 6 The graphs show the inhibition results of each group on tumor cells.

[0095] Figure 7 The graph shows the effects of each group on liver and spleen weights. DETAILED DESCRIPTION

[0096] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0097] Example 1. Preparation of Compound 8

[0098]

[0099] Step a: Preparation of Intermediate 1

[0100]

[0101] Take a 250mL single-mouth round-bottom flask, add the substrate 2,4-dichloropyrido[2,3-d]pyrimidine (5g, 25.00mmol), then add 100mL THF, and stir to mix evenly. Then add the substrate 2-(trifluoromethyl)pyridine-3-methylamine (3.75mL, 27.50mmol) and triethylamine (6.95mL, 49.99mmol) in sequence, and continue stirring at room temperature for 6h. After the reaction is completed by TLC monitoring, evaporate THF, add water, filter, and dry to obtain a light yellow solid, that is, intermediate 1, 7.2g, yield 85%, MS (ESI) m / z: 340.06 [M+H] + .

[0102] Step b: Preparation of compound 8

[0103]

[0104] Take a 50mL reaction tube, add intermediate 1 (200mg, 0.5887mmol), add 3mL ultra-dry dimethyl sulfoxide to dissolve, and finally add substrate 2-(thiophen-2-yl)ethylamine (104μL, 0.8831mmol) and triethylamine (164μL, 0.1180mmol), and heat at 90°C for 4h. After the reaction is completed by TLC monitoring, the system solution is added to water, a large amount of solid is precipitated, filtered, and the filter cake is purified by column chromatography to obtain a light yellow solid, i.e., compound 8, 148mg, yield 58%, MS (ESI) m / z: 431.13 [M+H] + .

[0105] Example 2: Preparation of Compound 60

[0106]

[0107] Step a: Preparation of intermediate 2

[0108]

[0109] Intermediate 1 (3 g, 8.83 mmol) and substrate 2-amino-7-Boc-7-azaspiro[3.5]nonane (3.18 g, 13.25 mmol) were weighed and added to a 250 mL single-mouth round-bottom flask, and then 15 mL of ultra-dry dimethyl sulfoxide and triethylamine (2.46 mL, 17.66 mmol) were added and heated at 90 ° C for 8 h. After the reaction was completed by TLC monitoring, the system solution was added to water, and a large amount of solid was precipitated. The solid was filtered and the filter cake was purified by column chromatography to obtain a light yellow solid, namely intermediate 2, 3.4 g, with a yield of 71%, MS (ESI) m / z: 544.26 [M+H] + .

[0110] Step b: Preparation of intermediate 3

[0111]

[0112] Take a 100mL single-mouth round-bottom flask, add intermediate 2 (2g, 3.68mmol), add a small amount of methanol to dissolve the substrate, then add 40mL hydrochloric acid dioxane solution (4M), and react at room temperature overnight. After the reaction is completed by TLC monitoring, the system is concentrated and dried to obtain intermediate 3, which is directly used in the next step without purification.

[0113] Step c: Preparation of compound 60

[0114]

[0115] Take a 10mL reaction bottle, add intermediate 3 (200mg, 0.4167mmol), 5-acetylthiophene-2-carboxylic acid (142mg, 0.8334mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (160mg, 0.8334mmol), 1-hydroxybenzotriazole (113mg, 0.8334mmol), add 3mL N,N-dimethylformamide, stir, then add triethylamine (290μL, 2.08mmol), react at room temperature overnight. After the reaction is completed by TLC monitoring, the system solution is added to water, a large amount of solid is precipitated, filtered, and the filter cake is purified by column chromatography to obtain a light yellow solid, i.e., compound 60, 170mg, yield 68%, MS (ESI) m / z: 596.21 [M+H] + .

[0116] Example 3. Preparation of Compound 78

[0117]

[0118] Step a: Preparation of intermediate 4

[0119]

[0120] Weigh the intermediate 1 (3 g, 8.83 mmol) and the substrate methyl 4-piperidinic acid ester (1.9 g, 13.25 mmol) into a 250 mL single-mouth round-bottom flask, then add 15 mL of ultra-dry dimethyl sulfoxide and triethylamine (2.46 mL, 17.66 mmol), and heat at 90 ° C for 8 h. After the reaction is completed by TLC monitoring, the system solution is added to water, a large amount of solid is precipitated, filtered, and the filter cake is purified by column chromatography to obtain a light yellow solid, namely intermediate 4, 2.9 g, yield 75%, MS (ESI) m / z: 447.18 [M+H] + .

[0121] Step b: Preparation of intermediate 5

[0122]

[0123] Intermediate 4 (2 g, 4.48 mmol) was dissolved in 18 mL of methanol, and sodium hydroxide aqueous solution (18 mL, 17.92 mmol, 1 M) was added, and the reaction was allowed to react overnight at room temperature. After the reaction was completed by TLC monitoring, the reaction solution was concentrated, diluted with 30 mL of water, and then adjusted to pH 2-3 with 1 M hydrochloric acid aqueous solution. A large amount of white solid precipitated, and intermediate 5 was obtained by filtration as a light yellow solid, 1.8 g, with a yield of 93%, MS (ESI) m / z: 433.16 [M+H] + .

[0124] Step c: Preparation of intermediate 6

[0125]

[0126] Add intermediate 5 (1 g, 2.31 mmol) to a 250 mL round-bottom flask, add 50 mL of ultra-dry dichloromethane and place the system in an ice bath, stir to mix evenly, then drop oxalyl chloride (391 μL, 4.62 mmol) and 2 drops of ultra-dry N,N-dimethylformyl. After the reaction is completed by TLC monitoring, directly proceed to the next step.

[0127] Step d: Preparation of compound 78

[0128]

[0129] Take a 100mL single-mouth round-bottom flask, add 5-amino-2-methoxypyridine (66mg, 0.5323mmol) and 10mL ultra-dry dichloromethane, stir to dissolve, and then add triethylamine (185μL, 1.33mmol) dropwise. After 10 minutes, add 10mL ultra-dry dichloromethane solution containing intermediate 6 (208mg, 0.4436mmol) dropwise under an ice bath, and after the addition is complete, react at room temperature. After the reaction is completed by TLC monitoring, water (20mL) is added to the reaction bottle, and dichloromethane is extracted. The organic phases are combined, dried over anhydrous sodium sulfate, concentrated and column chromatographed to obtain compound 78 as a white solid, 202mg, yield 85%, MS (ESI) m / z: 539.21 [M+H] +.

[0130] Referring to the methods of Examples 1-3, other target compounds of the present invention were prepared. The numbers, structures and characterization data of the obtained target compounds are shown in Table 1.

[0131] Table 1. Number, structure and characterization data of the target compounds of the present invention

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0149] Experimental Example 1. Study on the inhibitory activity of the compounds of the present invention on Blimp1 1. Experimental method

[0150] 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.

[0151] The differential scanning fluorescence experiment uses a Bio-Rad CFX96RT-PCR instrument to measure the thermal shift ΔTm. When preparing the sample, add 9.8μL of a mixture of Blimp1 protein and SYPRO Orange to each well, and then add 0.2μL of the compound solution. The final test system is a buffer system of 20mM HEPES pH 7.5 and 150mM NaCl. The final concentration of Blimp1 protein is 2μM, the final concentration of the test compound is 100μM, and the total volume is 10μL. During the test, the sample is first equilibrated at 25℃ for 3 minutes; then heated from 25℃ to 95℃ at a rate of 1℃ / min.

[0152] Isothermal titration calorimetry experiments using PEAQ-ITC for affinity K D Determination of the value. All tests were performed at 25°C using a buffer system of 20 mM HEPES (pH 7.5), 150 mM 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 sucked into the syringe and small molecules were injected into the sample cell). Initially 0.2 μL of protein was titrated, and then 2 μL of protein was titrated every 100 s for 19 consecutive drops. The experimental data were calculated using ΔG = ΔH - TΔS = RTlnKD Computational thermodynamic parameter analysis. ΔG, ΔH, and ΔS are the changes in free energy, enthalpy, and entropy, respectively.

[0153] 2. Experimental results

[0154] The experimental results are shown in Table 2.

[0155] K D Refers to the strength of the binding between a single biological molecule (protein) and its ligand (drug or inhibitor). ΔTm: The drug molecule or inhibitor binds to the protein pocket, causing the protein's melting temperature Tm1 to change. The protein melting temperature without the small molecule is Tm0, ΔTm = Tm1-Tm0. When the small molecule binds to the protein pocket, it prevents Blimp1 from binding to the substrate, inhibiting the activity of Blimp1.

[0156] Table 2. Experimental results

[0157]

[0158]

[0159]

[0160] From the experimental results in Table 2, it can be seen 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 0.098μM.

[0161] Experimental Example 2: Characterization of the compounds of the present invention in delaying CAR-T cell exhaustion

[0162] 1. Experimental methods

[0163] The compound 60 with the best activity in Experimental Example 1 was selected, and the effects of the compound on the proliferation, apoptosis, memory phenotype, and cytokine secretion of CAR-T cells were further tested.

[0164] (1) Preparation of CAR-T cells: Human peripheral blood lymphocytes were obtained 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 T cells. After 24 hours, 1 μM of compound 60 was added to the culture system and cultured for 7 days.

[0165] (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.

[0166] (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:

[0167] ① Collect the CAR-T cells treated with compound 60 and DMSO into a 15 mL sterile centrifuge tube, add 2 mL PBS and centrifuge for washing at 1200 rpm for 10 min;

[0168] ② Resuspend the cell pellet in 1×Binding buffer and adjust the cell density to 1×10 6 / mL;

[0169] ③ Flow antibody staining, the staining system is 100μL, according to the reagent instructions, add 5μL APC-AnnnexinV and 5μL 7-AAD, mix well. At the same time, set up control tubes: blank tube (no treatment), 7-AAD single staining tube, APC-AnnexinV single staining tube;

[0170] 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.

[0171] (4) CAR-T memory phenotype detection

[0172] Take the CAR-T cells in (1) and detect the memory phenotype of CAR-T. After CAR-T and tumor cells were co-cultured for 6 and 9 days at an E:T ratio of 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 PBS was added for centrifugation and washing at 1200 rpm for 10 min; 0.5 μL Percp-CD8, FITC-MYC, BV510-CD2L, PE-CD45RO were added and mixed. At the same time, a control tube, a blank tube (without any treatment), and a single-stained tube were set up; incubated at room temperature in the dark for 20-30 min, 2 mL PBS was added for centrifugation and washing, and the tube was analyzed within 1 hour.

[0173] (5) Cytokine secretion detection

[0174] 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 for washing at 1200rpm for 10min; add 0.5μL APC-CD4 and FITC-MYC to mix, 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 for washing, and analyze on the machine within 1h. At the same time, set up a control tube, a blank tube (no treatment), and a single staining tube.

[0175] 2. Experimental results

[0176] (1) CAR-T cell proliferation results

[0177] CAR-T cell proliferation results Figure 1 As shown by Figure 1 It can be seen that adding 1 μM of compound 60 to the CAR-T cell culture system can significantly increase the proliferation ability of CAR-T cells.

[0178] (2) CAR-T cell apoptosis results

[0179] CAR-T cell apoptosis results Figure 2 As shown by Figure 2 It can be seen that adding 1 μM of compound 60 to the CAR-T cell culture system can significantly reduce the apoptosis of CAR-T cells before and after co-culture.

[0180] (3) CAR-T cell memory phenotype results

[0181] 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.

[0182] (4) Cytokine secretion test results

[0183] 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.

[0184] The above experimental results indicate that the compounds of the present invention can effectively delay CAR-T cell exhaustion.

[0185] Experimental Example 3: Characterization of the compounds of the present invention in delaying CAR-T cell exhaustion in animal models

[0186] 1. Experimental methods

[0187] (1) Tumor cell inoculation: NCG mice were subcutaneously inoculated with 3×10 6 of tumor cells and observe the tumor growth.

[0188] (2) Preparation of CAR-T cells: Human lymphocytes were obtained 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 T cells. After 24 h, 1 μM PRDM1 / Blimp1 inhibitor compound 60 was added to the culture system and cultured for 7 days.

[0189] (3) Re-infusion of CAR-T cells: NSG mice inoculated with tumor cells were divided into three groups according to the size of the tumor, with 5-9 mice in each group. The first, second and third groups were re-infused with CAR-T cells treated with compound 60, CAR-T cells treated with DMSO and NT cells, respectively. The CAR positive rates of the first and second groups were the same.

[0190] 2. Experimental results

[0191] The experimental results are as follows Figure 5 As shown: In the CAR-T cell group treated with compound 60, tumor growth was significantly inhibited.

[0192] Experimental Example 4: Characterization of the compound of the present invention in combination with PD-1 in an animal model

[0193] 1. Experimental methods

[0194] C57BL mice were subcutaneously inoculated with 3 × 10 6 MC38 cells were used to observe tumor growth. The tumor volume was 100 mm 3 At the same time, the mice were divided into 7 groups and given 40 mg / kg compound 60, 40 mg / kg compound 60 + anti-PD-1 antibody, 20 mg / kg compound 60, 20 mg / kg 60 + anti-PD-1 antibody, anti-PD-1 antibody, vehicle, and PBS + vehicle. Compound 60 was administered twice a day, and PD-1 was administered once every 3 days, with a dose of 100 μg / mouse.

[0195] 2. Experimental results

[0196] The experimental results are as follows Figure 6 As shown: 40 mg / kg compound 60 combined with PD-1 significantly inhibited tumor growth.

[0197] Experimental Example 5: Characterization of the compounds of the present invention in an animal model of hemophagocytic syndrome

[0198] 1. Experimental methods

[0199] C57BL male mice were divided into 8 groups. The modeling group was injected with 50 μg CpG-ODN1826 on days 0, 2, 4, 6, 8, and 10, and the normal group was injected with the same volume of PBS. The drug-dosing groups were designed as normal group, blank control group, dexamethasone group, etoposide group, ruxolitinib group, dexamethasone + ruxolitinib group, compound 60 (40 mg / kg) group, and dexamethasone + compound 60 (40 mg / kg) group. The compound 60 group was administered twice a day, and dexamethasone was administered once a day by intraperitoneal injection at a dose of 1.5 mg / kg. Etoposide was injected intraperitoneally twice a week at a dose of 50 mg / kg. Ruxolitinib was administered by gavage twice a day at a dose of 30 mg / kg. The mice were weighed every two days. After 10 days of modeling, the orbital blood, liver, spleen, and femur of the mice were taken, and the weight of the liver and spleen was observed and weighed.

[0200] 2. Experimental results

[0201] 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.

[0202] In summary, the present invention provides a small molecule compound as a Blimp1 inhibitor, which can effectively inhibit Blimp1 activity and has an excellent effect in delaying CAR-T cell exhaustion. Therefore, the compound of the present invention can enhance the anti-tumor effect of CAR-T therapy, and the compound of the present invention 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 of formula I, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof: in, 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 in Ring A are each independently selected from the following groups: halogen, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy; n is 0 or 1; 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 substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-13 membered heteroaryl; 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; The substituents of the cycloalkyl, heterocycloalkyl, aryl and heteroaryl 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; m is 0 or 1; 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; R3' and R4' are independently selected from hydrogen and C1-C6 alkyl; R4 and R5 are independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, -C(O)R6; R6 is selected from C1-C6 alkyl, substituted or unsubstituted 5-10 membered heteroaryl; 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.

2. The compound according to claim 1, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, characterized in that: The compound is represented by Formula IIA or Formula IIB: in, n is 0 or 1; 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 substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-13 membered heteroaryl; 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; The substituents of the cycloalkyl, heterocycloalkyl, aryl and heteroaryl 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; m is 0 or 1; 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; R3' and R4' are independently selected from hydrogen and C1-C6 alkyl; R4 and R5 are independently selected from hydrogen, C1-C6 alkyl, halogenated C1-C6 alkyl, -C(O)R6; R6 is selected from C1-C6 alkyl, substituted or unsubstituted 5-10 membered heteroaryl; The substituents of the alkoxy and alkylthio groups are independently selected from the following groups: halogen, substituted or unsubstituted 5-10 membered aryl groups; 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.

3. The compound according to claim 2, or a pharmaceutically acceptable salt, solvate 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, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy; X is O or 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 furanyl.

4. The compound according to claim 2, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, characterized in that: The compound is represented by Formula IIA-1 or Formula IIA-2: in, 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; m is 0 or 1; R3' and R4' are independently selected from hydrogen and C1-C6 alkyl; 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, m1 is selected from 1, 2 or 3; R4 and R5 are independently selected from hydrogen, C1-C6 alkyl, and halogenated C1-C6 alkyl; 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, halogenated C1~C6 alkyl.

5. The compound according to claim 4, or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, characterized in that: R7 is selected from 6. The compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, solvate 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, solvate 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, solvate or stereoisomer thereof in the preparation of a medicament for preventing and / or treating immune-related diseases; Preferably, the immune-related disease is systemic lupus erythematosus, rheumatoid arthritis, cancer, or hemophagocytic syndrome.

9. A pharmaceutical preparation, characterized in that: The invention is prepared by using the compound described in any one of claims 1 to 6, its pharmaceutically acceptable salt, solvate, or stereoisomer as active ingredients and adding pharmaceutically acceptable auxiliary materials.

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

Images