Disclosure of Invention
The invention aims to provide a small molecule compound serving as a Blimp1 inhibitor and application thereof.
The present invention provides a compound of formula I, a pharmaceutically acceptable salt, solvate or stereoisomer thereof:
Wherein, the
The substituent groups of the cyclic alkyl, the heterocyclic alkyl, the aryl and the heteroaryl in the cyclic A are independently selected from halogen, C1～C6 alkyl, halogenated C1～C6 alkyl, C1～C6 alkoxy and halogenated C1～C6 alkoxy;
n is 0 or 1;
R1、R2 is independently selected from hydrogen, substituted or unsubstituted C1～C6 alkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, or R1 and R2 are connected to form substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-13 membered heteroaryl;
The substituent groups of the alkyl are independently selected from halogen, hydroxyl, C1～C6 alkoxy, substituted or unsubstituted 5-10 membered aryl and substituted or unsubstituted 5-10 membered heteroaryl;
The substituent groups of the cycloalkyl, heterocycloalkyl, aryl and heteroaryl are each independently selected from halogen, cyano, hydroxyl, amino, substituted or unsubstituted C1～C6 alkyl, substituted or unsubstituted C1～C6 alkoxy, substituted or unsubstituted C1～C6 alkylthio 、-(CR3'R4')mC(O)R3、-C(O)(CR3'R4')mR3、-C(O)(CR3'R4')mOR3、-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 substituent groups 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'、R4' is independently selected from hydrogen, C1～C6 alkyl;
R4、R5 is 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 substituent groups of the alkoxy and the alkylthio are respectively and independently selected from halogen and substituted or unsubstituted 5-10 membered aryl.
Further, the compound is represented by formula IIA or formula IIB:
Wherein, the
N is 0 or 1;
R1、R2 is independently selected from hydrogen, substituted or unsubstituted C1～C6 alkyl, substituted or unsubstituted 3-10 membered heterocycloalkyl, or R1 and R2 are connected to form substituted or unsubstituted 3-10 membered heterocycloalkyl, substituted or unsubstituted 5-13 membered heteroaryl;
The substituent groups of the alkyl are independently selected from halogen, hydroxyl, C1～C6 alkoxy, substituted or unsubstituted 5-10 membered aryl and substituted or unsubstituted 5-10 membered heteroaryl;
The substituent groups of the cycloalkyl, heterocycloalkyl, aryl and heteroaryl are each independently selected from halogen, cyano, hydroxyl, amino, substituted or unsubstituted C1～C6 alkyl, substituted or unsubstituted C1～C6 alkoxy, substituted or unsubstituted C1～C6 alkylthio 、-(CR3'R4')mC(O)R3、-C(O)(CR3'R4')mR3、-C(O)(CR3'R4')mOR3、-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 substituent groups 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'、R4' is independently selected from hydrogen, C1～C6 alkyl;
R4、R5 is 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 substituent groups of the alkoxy and the alkylthio are respectively and independently selected from halogen and substituted or unsubstituted 5-10 membered aryl;
R5' is a substituent at any position on the benzene ring selected from the group consisting of hydrogen, halogen, C1～C6 alkyl, halogenated C1～C6 alkyl, C1～C6 alkoxy, halogenated C1～C6 alkoxy.
Further, the compound is represented by a formula IIB-1:
Wherein, the
R5' is a substituent at any position on the benzene ring selected from the group consisting of hydrogen, halogen, C1～C6 alkyl, halogenated C1～C6 alkyl, C1～C6 alkoxy, halogenated C1～C6 alkoxy;
X is O or NR7';
R6' is substituent at any position on the heterocycle selected from the group consisting of hydrogen, C1～C6 alkyl;
R7 'is selected from-C (O) R8';
r8' is selected from furyl.
Further, the compound is represented by formula IIA-1 or formula IIA-2:
Wherein, the
R7 is selected from -C(O)(CR3'R4')mR3、-C(O)(CR3'R4')mOR3、-S(O)(O)R3、-C(O)NR3'(CR3'R4')mR3、-NR3'C(O)R3;
M is 0 or 1;
R3'、R4' is independently selected from hydrogen, C1～C6 alkyl;
r3 is selected from the group consisting of substituted or unsubstituted C1～C6 alkyl, 3-to 6-membered cycloalkyl,The substituents are each independently selected from the group consisting of C1～C6 alkyl, halo 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)m1C1～C6 alkoxy, 3-6 membered cycloalkyl,
M1 is selected from 1,2 or 3;
R4、R5 is independently selected from hydrogen, C1～C6 alkyl, halogenated C1～C6 alkyl;
In the formula IIA-2, Y1、Y2 is independently selected from N or CH, m2 is selected from 1, 2 or 3, R8 is selected from hydrogen, C1～C6 alkyl and halogenated C1～C6 alkyl.
Further, the method comprises the steps of,
R7 is selected from
Further, the compound is selected from one of the following structures:
The invention also provides application of the compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof in preparing Blimp1 inhibitor.
The invention also provides application of the compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof in preparing medicines for preventing and/or treating immune related diseases;
Preferably, the immune related disorder is systemic lupus erythematosus, rheumatoid arthritis, cancer, hemophagous syndrome.
The invention also provides a pharmaceutical preparation which is prepared by taking the compound, pharmaceutically acceptable salt, solvate or stereoisomer thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
The invention also provides a pharmaceutical composition comprising the aforementioned compound, a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, the relative terms and laboratory procedures used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
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 integers are required, the term refers to rounding up or down to the nearest integer within plus or minus 10% of a given value or range.
In the description herein, reference is made to "some embodiments," "some implementations," or "some implementations," which describe a subset of all possible embodiments, but it is to be understood that "some embodiments" may be the same subset or different subsets of all possible embodiments and may be combined with one another without conflict.
As used herein and unless otherwise indicated, the terms "comprising," "including," "having," "containing," and their grammatical equivalents are generally understood to be open-ended and not to be limiting, e.g., not to exclude other, unrecited elements or steps.
As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the invention that is pharmaceutically acceptable and has the pharmacological activity of the parent compound. Such salts include salts with inorganic acids such as nitric acid, phosphoric acid, carbonic acid, etc., or with organic acids such as propionic acid, caproic acid, cyclopentapropionic acid, glycolic acid, pyruvic acid, gluconic acid, stearic acid, muconic acid, etc., or salts formed when acidic protons present on the parent compound are replaced with metal ions, e.g., alkali metal ions or alkaline earth metal ions, or complex compounds formed with organic bases such as ethanolamine, etc. Pharmaceutically acceptable salts of the invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. Typically, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. In addition to salt forms, the compounds provided herein exist in prodrug forms. Prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to convert to the compounds of the invention. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an in vivo environment.
As used herein, the term "solvate" refers to a substance formed by the combination of a compound of the invention with a pharmaceutically acceptable solvent. Pharmaceutically acceptable solvents include acetic acid and the like. Solvates include stoichiometric amounts of solvates and non-stoichiometric amounts of solvates. Certain compounds of the invention may exist in unsolvated forms or solvated forms. In general, solvated forms, which are equivalent to unsolvated forms, are intended to be encompassed within the scope of the present invention.
As used herein, the term "stereoisomers" includes conformational isomers and configurational isomers, wherein configurational isomers include predominantly cis-trans isomers and optical isomers. The compounds of the present invention may exist as stereoisomers and thus encompass all possible stereoisomeric forms, including but not limited to cis, trans, tautomers, enantiomers, diastereomers, atropisomers and the like, as well as any combination or mixture of any of the aforementioned stereoisomers, for example, meso, racemates, equal amounts of the atropisomers and the like. For example, a single enantiomer, a single diastereomer or a mixture thereof, or a single atropisomer or a mixture thereof. When the compounds of the present invention contain olefinic double bonds, they include cis-isomers and trans-isomers, as well as any combination thereof, unless specified otherwise. The atropisomers of the present invention are stereoisomers of axial or planar chirality based on limited intramolecular rotation. And stereoisomers having excellent activity are preferable as the drugs. The compounds of the present invention have optical isomers derived from asymmetric carbons and the like, and if necessary, single isomers can be resolved by methods known in the art, such as crystallization or chiral chromatography.
As used herein, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic cyclic hydrocarbon group, including, for example, monocyclic cycloalkyl, spirocycloalkyl, fused ring alkyl, and bridged cycloalkyl. The ring carbon atoms of the cycloalkyl groups described in the present invention may be optionally substituted with 1,2 or 3 oxo groups to form a cyclic ketone structure. The term "C3-8 cycloalkyl" refers to cycloalkyl groups having 3 to 8 ring carbon atoms, including monocyclic cycloalkyl, spirocycloalkyl, fused ring alkyl, and bridged cycloalkyl groups.
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, sulfur, the remaining ring atoms being carbon.
As used herein, the term "aryl" or "aromatic ring" refers to an all-carbon monocyclic, all-carbon non-fused polycyclic (rings attached to the rings by covalent bonds, non-fused), or all-carbon fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group, at least one of the rings of which is aromatic, i.e., has a conjugated pi-electron system. The term "C6-14 aryl" refers to aryl groups having 6 to 14 ring atoms. Preferably a C6-10 aryl group. In the present invention, the C6-14 aryl group includes monocyclic aryl groups, non-condensed polycyclic aryl groups, and aromatic condensed polycyclic groups, wherein examples of monocyclic aryl groups include phenyl groups, examples of non-condensed polycyclic aryl groups include biphenyl groups, and the like. The term "6 to 10 membered aromatic ring" refers to an aromatic ring having 6 to 10 ring atoms.
As used herein, the term "heteroaryl", "refers to a monocyclic or fused polycyclic (i.e., sharing pairs of adjacent ring atoms, which may be C-C or N-C) group in which the ring atoms are substituted with at least one heteroatom independently selected from nitrogen, oxygen, or sulfur, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen atom may optionally be quaternized. The term "5-to 10-membered heteroaryl" refers to heteroaryl groups having 5 to 10 ring atoms, wherein 1,2,3 or 4 ring atoms are heteroatoms selected from nitrogen, oxygen or S (=o)m '(wherein m' is an integer from 0 to 2). The 5 to 10 membered heteroaryl groups in the present invention may be monocyclic heteroaryl groups or fused bicyclic heteroaryl groups.
As used herein, the term "5-or 6-membered heteroaryl" refers to heteroaryl groups having 5 or 6 ring atoms, wherein 1,2 or 3 ring atoms are heteroatoms selected from nitrogen, oxygen or S (=o)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.
Representative of two carbon atoms attached are pairs of adjacent carbon atoms that are shared when fused with other rings.
In the present application, each of the above heteroaryl groups may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the substituent groups described in the present application.
As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a drug or agent that is non-toxic but achieves the desired effect. In embodiments of the invention, the amount of a given drug in treating a patient according to the invention will depend on a number of factors, such as the particular dosing regimen, the type of disease or condition and its severity, the uniqueness of the subject or host in need of treatment (e.g., body weight), but depending on the particular circumstances, including, for example, the particular drug employed, the route of administration, the condition being treated, and the subject or host being treated, the dosage administered can be routinely determined by methods known in the art. Generally, for dosages used in adult treatment, the dosage administered is typically in the range of 0.02-5000 mg/day, for example about 1-1500 mg/day. The desired dosage may conveniently be presented as a single dose, or as divided doses administered simultaneously (or in short time periods) or at appropriate intervals, for example two, three, four or more divided doses per day. It will be appreciated by those skilled in the art that, although the above dosage ranges are given, the specific effective amount may be suitably adjusted depending on the patient's condition in combination with a physician's diagnosis.
The compounds of the present invention may be prepared using synthetic methods known in the art or using methods known in the art in combination with the methods described herein. The solvents, temperatures, and other reaction conditions set forth herein are exemplary and may vary according to methods well known in the art. The compounds of the examples described in the present invention can be synthesized by the methods described in the examples using appropriate starting materials according to their specific structures, or by the methods similar to those described in the examples. The starting materials for the synthesis of the compounds of the examples of the present invention may be prepared by known synthetic methods or similar methods described in the literature or obtained from commercial sources. The compounds of the examples may be further resolved, as desired, by methods well known in the art, such as crystallization, chromatography, etc., to give stereoisomers thereof, the resolution conditions of which are readily available to those skilled in the art by conventional means or limited experimentation.
The invention provides a small molecular compound serving as a Blimp1 inhibitor, which can effectively inhibit the activity of Blimp1 and has excellent effect of delaying the depletion of CAR-T cells. Thus, the compounds of the invention may enhance the anti-tumor effect of CAR-T therapies, while also inhibiting the progression of hemophagia syndrome. The compound of the invention is a patentable compound suitable for clinical application and has good application prospect.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Detailed Description
The materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
Example 1 preparation of Compound 8
Step a preparation of intermediate 1
A250 mL single neck round bottom flask was taken and the substrate 2, 4-dichloropyrido [2,3-d ] pyrimidine (5 g,25.00 mmol) was added followed by 100mL THF and stirred to mix well. The substrate 2- (trifluoromethyl) pyridine-3-methylamine (3.75 mL,27.50 mmol) and triethylamine (6.95 mL,49.99 mmol) were then added in this order and stirring was continued at room temperature for 6h. After completion of TLC monitoring, THF was evaporated, water was added, filtered and dried to give intermediate 1,7.2g as a pale yellow solid in 85% yield, MS (ESI) m/z:340.06[ M+H ]+.
Step b preparation of Compound 8
50ML of the reaction tube was taken, intermediate 1 (200 mg,0.5887 mmol) was added, 3mL of ultra-dry dimethyl sulfoxide was added to dissolve, and finally substrate 2- (thiophen-2-yl) ethylamine (104. Mu.L, 0.8831 mmol) and triethylamine (164. Mu.L, 0.1180 mmol) were added and heated at 90℃for 4h. After the TLC monitoring reaction is finished, the system solution is added into water, a large amount of solids are separated out, the solid is filtered, and the filter cake is purified by column chromatography to obtain pale yellow solid, namely compound 8,148mg, yield 58%, MS (ESI) m/z:431.13[ M+H ]+.
Example 2 preparation of Compound 60
Step a preparation of intermediate 2
Intermediate 1 (3 g,8.83 mmol), substrate 2-amino-7-Boc-7-azaspiro [3.5] nonane (3.18 g,13.25 mmol) was weighed into a 250mL single neck round bottom flask, followed by 15mL of ultra dry dimethyl sulfoxide and triethylamine (2.46 mL,17.66 mmol) and heated at 90 ℃ for 8h. After the TLC monitoring reaction is finished, the system solution is added into water, a large amount of solids are separated out, the solid is filtered, and the filter cake is purified by column chromatography to obtain pale yellow solid, namely intermediate 2,3.4g, the yield is 71%, MS (ESI) m/z is 544.26[ M+H ]+.
Step b preparation of intermediate 3
A100 mL single neck round bottom flask was taken, intermediate 2 (2 g,3.68 mmol) was added, a small amount of methanol was added to dissolve the substrate, and then 40mL dioxane solution (4M) of hydrochloric acid was added and reacted overnight at room temperature. After TLC monitoring the reaction was completed, the system was concentrated and dried to give intermediate 3, which was used directly in the next reaction without purification.
Step c preparation of Compound 60
A10 mL reaction flask was charged with 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), 1-hydroxybenzotriazole (113 mg,0.8334 mmol), 3mL of N, N-dimethylformamide was added thereto, stirred, and triethylamine (290. Mu.L, 2.08 mmol) was then added thereto, and the reaction was carried out overnight at room temperature. After the TLC monitoring reaction is finished, the system solution is added into water, a large amount of solids are separated out, the filtration is carried out, and the filter cake is purified by column chromatography to obtain a pale yellow solid, namely compound 60,170mg, yield 68%, MS (ESI) m/z:596.21[ M+H ]+.
Example 3 preparation of Compound 78
Step a preparation of intermediate 4
Intermediate 1 (3 g,8.83 mmol) and substrate methyl 4-piperidinecarboxylate (1.9 g,13.25 mmol) were weighed into a 250mL single neck round bottom flask, followed by 15mL of overdry dimethyl sulfoxide and triethylamine (2.46 mL,17.66 mmol) and heated at 90℃for 8h. After the TLC monitoring reaction is finished, the system solution is added into water, a large amount of solid is separated out, the solid is filtered, and the filter cake is purified by column chromatography to obtain pale yellow solid, namely intermediate 4,2.9g, yield 75%, MS (ESI) m/z 447.18[ M+H ]+.
Step b preparation of intermediate 5
Intermediate 4 (2 g,4.48 mmol) was dissolved in 18mL of methanol and aqueous sodium hydroxide (18 mL,17.92mmol, 1M) was added and reacted overnight at room temperature. After the TLC monitoring reaction was completed, the reaction solution was concentrated, diluted with 30mL of water, then pH was adjusted to 2-3 with 1M aqueous hydrochloric acid, a large amount of white solid was precipitated, and intermediate 5 was obtained by filtration as pale yellow solid, 1.8g, yield 93%, MS (ESI) M/z:433.16[ M+H ]+.
Step c preparation of intermediate 6
Intermediate 5 (1 g,2.31 mmol) was added to a 250mL round bottom flask, 50mL of overdry dichloromethane was added and the system was placed in an ice bath and stirred to mix well, followed by dropwise addition of oxalyl chloride (391. Mu.L, 4.62 mmol) and 2 drops of overdry N, N-dimethylformamide. After the completion of the TLC monitoring reaction, the reaction was directly taken into the next step.
Step d preparation of Compound 78
A100 mL single-necked round bottom flask was taken, 5-amino-2-methoxypyridine (66 mg,0.5323 mmol) and 10mL of ultra-dry dichloromethane were added and stirred to dissolve, followed by dropwise addition of triethylamine (185. Mu.L, 1.33 mmol). After 10min, 10mL of an ultra-dry dichloromethane solution containing intermediate 6 (208 mg,0.4436 mmol) was added dropwise under ice bath, and after the addition was completed, the reaction was carried out at room temperature. After completion of TLC monitoring the reaction, water (20 mL) was added to the reaction flask, dichloromethane extraction, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated and column chromatographed to give compound 78 as a white solid, 202mg, 85% yield, MS (ESI) m/z 539.21[ M+H ] +.
Other target compounds of the present invention were prepared by the method of examples 1-3. The numbers, structures and characterization data of the resulting target compounds are shown in table 1.
TABLE 1 numbering, structure and characterization data for the target Compounds of the invention
The beneficial effects of the present invention are demonstrated by specific test examples below.
Test example 1 investigation of the inhibitory Activity of the Compound of the present invention on Blimp1
1. Experimental method
The present invention uses Differential Scanning Fluorescence (DSF) and Isothermal Titration Calorimetry (ITC) to evaluate the inhibitory activity of the compounds of the invention on Blimp 1.
Differential scanning fluorescence experiments thermal shift ΔTm was determined using a Bio-Rad CFX96RT-PCR instrument. When the samples were prepared, 9.8. Mu.L of a mixture of Blimp1 protein and SYPRO Orange was added to each well, followed by 0.2. Mu.L of the compound solution. The final test system was 20mM HEPES pH 7.5,150mM NaCl buffer system. The final concentration of Blimp1 protein was 2. Mu.M, the final concentration of the compound to be tested was 100. Mu.M, and the total volume was 10. Mu.L. The test was performed by first equilibrating the sample at 25℃for 3 minutes and then heating from 25℃to 95℃at a rate of 1℃/min.
Isothermal titration quantitative thermal experiments the determination of affinity KD values was performed using PEAQ-ITC. All assays were performed at 25℃using a buffer system of 20mM HEPES (pH 7.5), 150mM NaCl,0.4% DMSO. The compounds were diluted directly in the same batch of buffer prior to the experiment. Each experiment was a back titration experiment (inhalation of Blimp1 protein in a syringe and injection of small molecules into a sample cell). Initial titration of 0.2. Mu.L protein followed by titration of 2. Mu.L protein every 100s, followed by 19 consecutive drops. Experimental data were analyzed using calculated thermodynamic parameters of Δg=Δh-tΔs= RTlnKD. Δg, Δh, and Δs are changes in free energy, enthalpy, and entropy, respectively.
2. Experimental results
The experimental results are shown in table 2.
KD refers to the strength of the interaction between a single biomolecule (protein) and its ligand (drug or inhibitor). Δtm: the drug molecule or inhibitor binds to the protein pocket, changing the melting temperature Tm1 of the protein, and the melting temperature of the unbound small molecule is Tm0, Δtm=tm1-Tm 0. When the small molecule binds to the protein pocket, the binding of Blimp1 to the substrate is prevented, inhibiting the activity of Blimp 1.
TABLE 2 experimental results
As shown in the experimental results of Table 2, some of the compounds of the present invention have good inhibitory activity against Blimp 1. Of these, compound 60 has the best inhibitory activity against Blimp1, and the KD value is 0.098. Mu.M.
Test example 2, characterization of the inventive Compounds against CAR-T cell depleted cells
1. Experimental method
The best active compound 60 of test example 1 was selected and the effect of the compound on CAR-T cell proliferation, apoptosis, memory phenotype, cytokine secretion was further examined.
(1) Preparation of CAR-T cells, namely taking human peripheral blood lymphocytes and separating the T cells. After 48h of stimulation with Anti-CD3/CD28 magnetic beads and IL-2, T cells are infected by adding the prepared CAR virus. After 24 hours, 1. Mu.M of compound 60 was added to the culture system and cultured for 7 days.
(2) CAR-T cell proliferation assay cell density of CAR-T cells added with compound 60 and DMSO was measured every two days during CAR-T cell culture.
(3) CAR-T cell apoptosis detection CAR-T cells in (1) were taken, apoptosis of CAR-T cells was detected using an apoptosis kit, and CAR-T and tumor cells were co-cultured for two days with (effector cells: target cells) E: t=1:1, followed by detection of apoptosis of CAR-T cells. The detailed operation steps are as follows:
① Collecting the CAR-T cells treated by the compound 60 and the DMSO in a 15mL sterile centrifuge tube, adding 2mL PBS, and centrifugally washing at 1200rpm for 10min;
② The cell pellet is resuspended by 1×binding buffer, and the cell density is adjusted to 1×106/mL according to the counting result;
③ Flow antibody staining, staining system 100. Mu.L, according to the reagent instructions, adding 5. Mu.L APC-AnnnexinV and 5. Mu.L 7-AAD, mixing. Meanwhile, a control tube, namely a blank tube (without any treatment), a 7-AAD single-dyeing tube and an APC-annexin V single-dyeing tube, is arranged;
④ Incubation for 20-30min at room temperature and light shielding, adding 400 mu L of 1 Xbinding buffer, and performing on-line analysis on apoptosis in 1 h.
(4) CAR-T memory phenotype detection
Taking the CAR-T cells in the step (1), detecting the memory phenotype of the CAR-T, respectively detecting the memory phenotype of the CAR-T after the CAR-T and tumor cells are co-cultured for 6 days and 9 days according to E:T=1:1, collecting the CAR-T treated by the compound 60 and the DMSO or the CAR-T cells after co-culture in a 15mL sterile centrifuge tube, adding 2mL PBS for centrifugal washing at 1200rpm for 10min, and adding 0.5 mu L of Percp-CD8, FITC-MYC and BV510-CD2L, PE-CD45RO for uniform mixing. Meanwhile, a control tube, a blank tube (without any treatment) and a single-dyeing tube are arranged, incubated at room temperature for 20-30min in a dark place, added with 2mL PBS for centrifugal washing, and analyzed in a 1-hour period.
(5) Cytokine secretion assay
Taking the CAR-T cells in the step (1), detecting cytokine secretion conditions of the CAR-T and tumor cells after co-culturing for two days with E:T=1:1, collecting the CAR-T cells after co-culturing with the tumor cells in a 15mL sterile centrifuge tube, adding 2mL PBS for centrifugal washing at 1200rpm for 10min, adding 0.5 mu L of APC-CD4 and FITC-MYC for uniformly mixing, incubating for 20-30min at room temperature in a dark place, fixing the cells, adding 0.5 mu L of PE-IFNgamma streaming antibody, incubating for 20-30min at room temperature in a dark place, adding 2mL PBS for centrifugal washing, and performing on-machine analysis in 1 h. At the same time, a control tube, a blank tube (without any treatment), a single-dyeing tube was set.
2. Experimental results
(1) CAR-T cell proliferation results
The proliferation results of the CAR-T cells are shown in FIG. 1, and it is clear from FIG. 1 that the proliferation capacity of the CAR-T cells can be significantly increased by adding 1. Mu.M of compound 60 to the CAR-T cell culture system.
(2) Results of CAR-T cell apoptosis
The apoptosis results of the CAR-T cells are shown in figure 2, and the figure 2 shows that the addition of 1 mu M of compound 60 into the CAR-T cell culture system can significantly reduce the apoptosis of the CAR-T cells before and after co-culture.
(3) CAR-T cell memory phenotype results
The results of the memory phenotype of the CAR-T cells are shown in FIG. 3, and it is clear from FIG. 3 that the addition of 1. Mu.M compound 60 to the CAR-T cell culture system significantly increases the ratio of the central memory cells TCM of the CAR-T cells before and after co-culture.
(4) Cytokine secretion assay results
The cytokine secretion test results are shown in FIG. 4, and it is clear from FIG. 4 that the addition of 1. Mu.M compound 60 to the CAR-T cell culture system significantly increases the IFNγ secretion ability of the CAR-T cells after co-culture.
The experimental results show that the compound provided by the invention can effectively delay the exhaustion of CAR-T cells.
Test example 3 characterization of the inventive Compounds on animal models for delayed CAR-T cell depletion
1. Experimental method
(1) Tumor cell inoculation NCG mice are inoculated with tumor cells of 3X 106 subcutaneously, and the tumor growth is observed.
(2) Preparation of CAR-T cells human lymphocytes are taken and T cells are isolated. After 48h of stimulation with Anti-CD3/CD28 magnetic beads and IL-2, T cells are infected by adding the prepared CAR virus. After 24h, 1. Mu.M of PRDM1/Blimp1 inhibitor compound 60 was added to the culture system and incubated for 7 days.
(3) And (3) re-infusing the CAR-T cells, namely, dividing NSG mice inoculated with tumor cells into three groups of 5-9 NSG mice according to the tumor size, and re-infusing the CAR-T cells treated by the compound 60 in the first group, the second group and the third group, wherein the CAR-T cells treated by the DMSO and the NT cells are respectively. The first and second groups had the same CAR positive rate.
2. Experimental results
The experimental results are shown in figure 5, in which the CAR-T cell group treated with the reinfusion compound 60 has significantly inhibited tumor growth.
Test example 4 characterization of the Compounds of the invention in animal models for use in combination with PD-1
1. Experimental method
C57BL mice are inoculated with MC38 cells of 3X 106 subcutaneously, tumor growth is observed, and when the tumor volume is 100mm3, the mice are divided into 7 groups, and 40mg/kg of compound 60,40mg/kg of compound 60+anti-PD-1 antibody, 20mg/kg of compound 60,20mg/kg of 60+anti-PD-1 antibody, anti-PD-1 antibody, vehicle, PBS+vehicle are respectively administered. Compound 60 was administered twice daily and PD-1 was administered 1 time every 3 days at a dose of 100 μg/dose.
2. Experimental results
The experimental results are shown in figure 6, 40mg/kg of compound 60 and PD-1 combined, and the tumor growth is obviously inhibited.
Test example 5 characterization of the Compounds of the invention on animal models of hemophagous syndrome
1. Experimental method
C57BL male mice were divided into 8 groups, and the groups were intraperitoneally injected with 50. Mu.g CpG-ODN1826 on days 0, 2, 4, 6, 8, 10, respectively, and the normal groups were injected with the same volume of PBS. The dosing groups were designed as normal, placebo, dexamethasone, etoposide, ponciritinib, dexamethasone + ponciritinib, compound 60 (40 mg/kg), dexamethasone + compound 60 (40 mg/kg). The compound 60 groups were administered twice daily, and dexamethasone was administered once daily by intraperitoneal injection at a dose of 1.5mg/kg. Etoposide is injected intraperitoneally, and is twice weekly, and the dosage of the injection is 50mg/kg. The poncirtinib is administrated by stomach irrigation twice a day, and the administration dosage is 30mg/kg. The mice were weighed every two days. After modeling for 10 days, the orbital blood, liver, spleen and femur of the mice were taken, and the weights of the liver and spleen were observed and weighed.
2. Experimental results
As shown in the experimental results in FIG. 7, the 40mg/kg compound 60 administration group and the 40mg/kg compound 60+dexamethasone combination group can obviously inhibit and improve the weight of the liver and spleen of the mice, and inhibit the progress of hemophagia syndrome.
In conclusion, the invention provides a small molecular compound serving as a Blimp1 inhibitor, which can effectively inhibit Blimp1 activity and has excellent effect of delaying CAR-T cell exhaustion. Thus, the compounds of the invention may enhance the anti-tumor effect of CAR-T therapies, while also inhibiting the progression of hemophagia syndrome. The compound of the invention is a patentable compound suitable for clinical application and has good application prospect.