Small molecule inhibitor targeting BOB1 / OCT1 interface

Abstract

The present invention relates to compounds of formula (I) or pharmaceutically acceptable salts and / or solvates thereof. The invention also relates to the use of the compounds of the invention as immunomodulators. The invention also relates to the use of the compounds of the invention for the treatment of autoimmune diseases, transplant organ rejection, graft versus host disease and BOB1-related diseases. # imgabs0 #

Description

Field of the Invention

[0001] The present invention relates to compounds of formula (I) targeting the BOB1 / OCT1 interface for use as immunomodulators. The compounds of the present invention are particularly suitable for treating autoimmune diseases, transplanted organ rejection, graft-versus-host-disease and BOB1-related diseases. Background of the Invention

[0003] The transcriptional coactivator BOB1 is expressed exclusively in lymphocytes and plays a crucial role in initiating T cell-dependent immune responses and establishing immune memory. BOB1 acts synergistically with the POU-domain proteins OCT1 and OCT2. The interaction of BOB1 with the OCT1 or OCT2 transcription factors leads to the formation of a ternary BOB1-OCT1-DNA complex and activates transcription of target genes. In addition, abnormal expression of BOB1 is associated with the development of autoimmune diseases and chronic lung transplant rejection. Conversely, the absence of functional BOB1 protein prevents mice from developing such conditions.

[0004] In the setting of autoimmunity or organ transplantation, when an immune response is mounted against self-antigens, the balance between effector and regulatory lymphocytes determines the outcome that favors the persistence of autoimmune disease / allogeneic transplant rejection or tolerance. Recent therapies for autoimmune diseases include targeting proinflammatory cytokines such as TNF and IL-1b, inhibiting signaling pathways, blocking co-stimulatory molecules, or therapeutic vaccination using regulatory T cells. For solid organ transplantation, there is no treatment other than retransplantation. Therefore, the ability to control pathogenic lymphocyte responses would provide new therapeutic strategies to combat autoimmune diseases and transplant organ rejection.

[0005] Based on the idea that abnormally high expression of BOB1 promotes the formation of an overactive DNA-OCT1-BOB1 ternary complex, thereby triggering pathogenic lymphocyte responses and autoimmune tissue inflammation, the inventors developed new compounds to inhibit the biological activity of the BOB1-OCT1 axis.

[0006] The inventors surprisingly determined that the compounds of the present invention can efficiently target the BOB1 interaction interface within the POU domain of OCT1, thereby inhibiting BOB1 / OCT1-dependent transcription. Advantageously, the inventors demonstrated that the compounds of the present invention interfere with BOB1 function and elicit selective biological responses by inhibiting effector B and T lymphocytes without affecting the regulatory capacity of B cells.

[0007] Therefore, the compounds of the present invention represent valuable potential therapeutic agents for the treatment of autoimmune diseases, solid transplant rejection, graft-versus-host disease, and BOB1-related diseases, as well as useful tools for further studying the function of BOB1 in human health and disease. Summary of the invention

[0008] The present invention relates to a compound of formula (I)

[0009]

[0010] or a pharmaceutically acceptable salt and / or solvate thereof;

[0011] Where X, Y and R 1 -R 8 As defined below.

[0012] The present invention also relates to a pharmaceutical composition comprising a compound according to the present invention and at least one pharmaceutically acceptable carrier.

[0013] The present invention also relates to a compound according to the present invention or a pharmaceutical composition according to the present invention for use as a medicament. The present invention also relates to a compound according to the present invention or a pharmaceutical composition according to the present invention for use in the treatment of a disease selected from autoimmune diseases, transplant organ rejection, graft-versus-host disease and BOB1-related diseases. In one embodiment, the disease to be treated is selected from rheumatoid arthritis, type 1 diabetes, multiple sclerosis, primary biliary cirrhosis, end-stage chronic respiratory diseases (e.g., chronic obstructive pulmonary disease, COPD, cystic fibrosis, CF and pulmonary hypertension, PAH), germinal center-derived lymphomas and Waldenstrom macroglobulinemia.

[0014] The present invention also relates to a process for preparing a compound according to the present invention, comprising:

[0015] (a) in the presence of a base, reacting cyanuric chloride with the NH secondary amine functional group in the ring of the compound of formula (A)

[0016]

[0017] Steps for obtaining a monosubstituted 1,3,5-triazine of formula (B)

[0018]

[0019] (b) in the presence of a base, reacting compound (B) with a compound of the formula NHR 5 R 6 (c) reacting the obtained disubstituted 1,3,5-triazine with an amine of formula NHR in the presence of a base 7 R 8 (b) reacting compound (B) with an amine of formula (NHR) in the presence of a base to obtain a trisubstituted 1,3,5-triazine; or 7 R 8 (c) reacting the obtained disubstituted 1,3,5-triazine with an amine of formula NHR in the presence of a base. 5 R 6 The step of reacting with an amine to obtain a trisubstituted 1,3,5-triazine; and

[0020] (d) optionally, at least one step of deprotecting at least one alcohol, carboxylic acid and / or amine group present in the resulting trisubstituted 1,3,5-triazine; thereby obtaining a compound of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof.

[0021] definition

[0022] In the present invention, the following terms have the following meanings.

[0023] In cases where a chemical substituent is a combination of chemical groups, the point of attachment of the substituent to the molecule is through the last chemical group to the right of the substituent name. For example, an arylalkyl substituent is attached to the rest of the molecule through the alkyl portion, and it can be represented as follows: "aryl-alkyl-".

[0024] Unless otherwise stated, these compounds were used Professional 15.0 (PerkinElmer) named.

[0025] "Alkoxy" refers to an alkyl-O- group.

[0026] "Alkyl" refers to a saturated straight or branched hydrocarbon chain, typically containing 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms, more preferably 1 to 4 carbon atoms. In the present invention, alkyl can be monovalent or polyvalent (i.e., "alkylene" as defined herein is included in the definition of "alkyl"), but alkyl is typically monovalent. Non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, pentyl and isomers thereof (e.g., n-pentyl, isopentyl) and hexyl and isomers thereof (e.g., n-hexyl, isohexyl). Preferred alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl and tert-butyl.

[0027] "Alkylene" refers to a divalent alkyl group. Non-limiting examples of alkylene include methylene, ethylene, n-propylene, isopropylene, divalent butyl, divalent pentyl, and divalent hexyl. Preferred alkylene groups include methylene, ethylene, n-propylene, n-butylene, and n-butylene.

[0028] "Amine" refers to a derivative of ammonia (NH3) in which one or more hydrogen atoms have been replaced by a substituent (eg, an alkyl or aryl group).

[0029] "Aryl" refers to a cyclic polyunsaturated aromatic hydrocarbon radical containing at least one aromatic ring and containing 5 to 12 carbon atoms, preferably 6 to 10 carbon atoms. Aryl can have a single ring (e.g., phenyl) or multiple aromatic rings fused together (e.g., naphthyl) or covalently linked. The aromatic ring can optionally include one to two additional rings (or cycloalkyl, heterocycloalkyl, or heteroaryl) fused thereto. This definition of "aryl" includes partially hydrogenated derivatives of the carbocyclic ring systems listed herein, as long as at least one ring is an aromatic ring. Non-limiting examples of aryl groups include phenyl, biphenyl, biphenylenyl, 5- or 6-tetralinyl, 1- or 2-naphthyl, 4-, 5-, 6- or 7-indenyl, 1-, 2-, 3-, 4- or 5-acenaphthylenyl, 3-, 4- or 5-acenaphthenyl, 1- or 2-pentalenyl, 4- or 5-indanyl, 5-, 6-, 7- or 8-tetrahydronaphthyl, 1, 2, 3, 4-tetrahydronaphthyl, 1, 4-dihydronaphthyl, 1-, 2-, 3-, 4- or 5-pyrenyl. A particularly preferred aryl group is phenyl.

[0030] "Cycloalkyl" refers to a cyclic monovalent alkyl group as defined herein containing 3 to 11 carbon atoms, preferably 4 to 9 carbon atoms, more preferably 5 to 7 carbon atoms. This definition of "cycloalkyl" includes multicyclic cycloalkyls (eg, bicyclic) and bridged cycloalkyl structures.

[0031] According to the common terminology in the chemical field, the “C x -C y " or "(C x -C y )" means that the group contains x to y carbon atoms.

[0032] "Haloalkyl" refers to an alkyl group as defined herein substituted with at least one halogen atom, ie, wherein at least one hydrogen atom is replaced with a halogen atom. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, and trifluoromethyl.

[0033] "Heteroalkyl" refers to an alkyl group as defined herein, wherein one or more carbon atoms are replaced by heteroatoms selected from oxygen, nitrogen and sulfur. In heteroalkyl, heteroatoms are only combined with carbon atoms along the alkyl chain, i.e., each heteroatom is separated from any other heteroatom by at least one carbon atom. Nitrogen and sulfur heteroatoms may be optionally oxidized, and nitrogen heteroatoms may be optionally quaternized. Heteroalkyl may also include one or more =O and / or =S groups. Heteroalkyl is only combined with another group or molecule through a carbon atom, i.e., the combined atom is not selected from the heteroatoms contained therein. Non-limiting examples of heteroalkyl include alkoxy, ethers and polyethers, secondary and tertiary amines, and polyamines, thioethers and polythioethers, and combinations thereof.

[0034] "Heterocycloalkyl" refers to a cyclic monovalent heteroalkyl group, typically containing 2 to 7 carbon atoms, preferably 3 to 6 carbon atoms, more preferably 4 to 5 carbon atoms. Heterocycloalkyl is typically 3- to 7-membered, preferably 5- or 6-membered. Heterocycloalkyl is typically monocyclic or bicyclic, preferably monocyclic. This definition includes polycyclic heterocycloalkyl (e.g., bicyclic) and bridged heterocycloalkyl structures. Non-limiting examples of heterocycloalkyl include monovalent or divalent aziridine, pyrrolidine, piperidine, piperazine, morpholine, thiomorpholine, tetrahydrofuran, and tetrahydropyran.

[0035] "Prodrug" refers to a pharmacologically acceptable derivative of a therapeutic agent (e.g., a compound of the invention), the biotransformation product of which is a therapeutic agent (active drug) in vivo. Typical characteristics of prodrugs are increased bioavailability and easy metabolism to active compounds in vivo. Non-limiting examples of prodrugs include amide prodrugs and carboxylate prodrugs, particularly alkyl esters, cycloalkyl esters, and aryl esters.

[0036] "Solvate" refers to a molecular complex comprising a compound and one or more molecules of one or more solvents, typically a pharmaceutically acceptable solvent such as ethanol, in stoichiometric or substoichiometric amounts. The term "hydrate" refers to a solvate when the solvent is water (H2O).

[0037] "About" in this article means approximately, roughly, about, or within a range thereof. The term "about" before a number means plus or minus 10% of the numerical value. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below 10% of the listed values.

[0038] "Administration" or variations thereof (e.g., "administering") means providing a therapeutic agent (e.g., a compound of the invention), either alone or as part of a pharmaceutically acceptable composition, to a subject, particularly to a patient for whom the condition, symptom or disease is to be treated.

[0039] "BOB1" generally refers to the human protein designated NP_006226.2 in the NCBI database as of December 12, 2021 (corresponding to the protein of sequence SEQ ID NO: 1), or any of the predicted isoforms XP_005271650.1, XP_005271651.1, XP_006718922.1, XP_006718923.1, XP_016873421.1. In the NCBI database (https: / / www.ncbi.nlm.nih.gov), the reference human POU class 2 homeobox-associated factor 1 (POU2AF1) gene sequence encoding the BOB1 protein corresponds to NCBI gene ID: 5450, updated on March 13, 2022. The human BOB1 gene consists of 8 exons on chromosome 11q23.1. The BOB1 transcript includes 2875 nucleotides and encodes a 256 amino acid protein. Alternative names for BOB1 include "POU domain class 2 associated factor 1", "B cell-specific coactivator OBF-1", "OCA-B", "OCT binding factor 1", "OBF1", "POU class 2 homeobox associated factor 1", "POU class 2 associated factor 1", "BOB-1", "OBF-1", and "OCAB". BOB1 is a transcriptional coactivator that specifically associates with POU2F1 / OCT1 or POU2F2 / OCT2. It enhances promoter activity mediated by POU2F1 / OCT1 and, to a lesser extent, by POU2F2 / OCT2. It has no intrinsic DNA binding activity. It recognizes the POU domains of POU2F1 / OCT1 and POU2F2 / OCT2.

[0040]

[0041] "BOB1 -associated disease" refers to any disease in which BOB1 is abnormally expressed.

[0042] According to common patent application writing terminology, "comprise" or its variants (e.g., "comprises", "comprising") are used herein. Thus, "comprise" is preceded by an object and followed by an ingredient, which means that the presence of one ingredient in the object (usually as a component of the composition) is required, but does not exclude the presence of any other ingredients in the object. In addition, unless otherwise specified, any "comprise" or its variants appearing herein also include the narrow expression "essentially consisting of...", the further narrow expression "consist of..." and any variants thereof (e.g., "consists of...", "consisting of..."), and can be replaced thereby.

[0043] "Human" refers to male or female human subjects at any stage of development, including neonates, infants, minors, adolescents, and adults.

[0044] "Kit" or "Kit of parts" are synonyms and refer to any article of manufacture (e.g., package or container) containing a pharmaceutical composition of a compound according to the invention. The kit can be promoted, distributed or sold as a unit for practicing the method of the invention.

[0045] "OCT1" generally refers to the human protein designated as NP_001185712.1 in the NCBI database on February 13, 2022 (corresponding to the protein of sequence SEQ ID NO: 2), or any of the isoforms NP_002688.3, ​​NP_001185715.1, NP_001352777.1, NP_001352778.1, or any of the predicted isoforms XP_011507955.1, XP_011507956.1, XP_011507957.1, XP_016856997.1, XP_024303495.1. In the NCBI database (https: / / www.ncbi.nlm.nih.gov), the reference human POU class 2 homeobox 1 (POU2F1) gene sequence encoding the OCT1 protein corresponds to NCBI gene ID: 5451, updated on March 13, 2022. The human OCT1 gene consists of 23 exons on chromosome 1q24.2. The OCT1 transcript includes 14007 nucleotides and encodes a 755 amino acid protein. Alternative names for OCT1 include "POU class 2 homeobox 1", "OTF1", "POU domain, class 2, transcription factor 1", "octamer-binding transcription factor 1", "octamer-binding protein 1", "NF-A1", "OTF-1", "Oct-1", "Oct-1B", and "Oct1Z". OCT1 is a transcription factor that binds to an octamer motif (5'-ATTTGCAT-3') and activates the promoters of some small nuclear RNA (snRNA) genes and genes such as histone H2B and immunoglobulins.

[0046]

[0047]

[0048] "OCT2" generally refers to the human protein designated as NP_001381305.1 in the NCBI database as of December 20, 2021 (the protein corresponding to the sequence SEQ ID NO: 3), or the isoforms NP_002689.1, NP_001193954.1, NP_001193955.1, NP_001234923.1, NP_001380863.1, NP_001380864.1, NP_001380865.1, NP_001381306.1, NP_001381307.1, or the predicted isoforms XP_011525343.1, XP_011525344.1, XP_011525345.2, XP_01688 Any one of XP_016882374.1, XP_016882373.1, XP_016882375.1, XP_016882376.1, XP_016882377.1, XP_016882378.1, XP_016882379.1, XP_016882380.1, XP_016882381.1, XP_016882383.1, XP_016882384.1, XP_016882385.1, XP_024307314.1, XP_024307315.1. In the NCBI database (https: / / www.ncbi.nlm.nih.gov), the reference human POU class 2 homeobox 2 (POU2F2) gene sequence encoding the OCT2 protein corresponds to NCBI gene ID: 5452, updated on January 25, 2022. The human OCT2 gene consists of 21 exons on chromosome 19q13.2. The OCT2 transcript includes 7057 nucleotides and encodes a 624 amino acid protein. Alternative names for OCT2 include "POU class 2 homeobox 2", "OTF2", "octamer-binding transcription factor 2", "lymphoid-restricted immunoglobulin octamer-binding protein NF-A2", "POU domain, class 2, transcription factor 2", "octamer-binding protein 2", "POU domain class 2, transcription factor 2", "homeobox protein", and "OTF-2". OCT2 is a transcription factor that specifically binds to the octamer motif (5'-ATTTGCAT-3'), a common transcription factor binding site in immunoglobulin gene promoters.

[0049]

[0050]

[0051] A "patient" is a subject who is awaiting medical care or is receiving medical care, or who has been / is / will be the subject of a medical procedure, or who is being monitored for the development of a targeted disease or condition, such as an autoimmune disease, transplant rejection, graft-versus-host disease, or BOB1-related disease.

[0052] "Pharmaceutically acceptable" means that the ingredients of the composition are compatible with each other and not deleterious to a patient to whom the composition is to be administered.

[0053] "Pharmaceutically acceptable carrier" refers to an excipient that does not produce adverse, allergic or other adverse reactions when administered to animals, preferably humans. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc. For human administration, the preparation should meet the sterility, pyrogenicity, general safety and purity standards required by regulatory agencies (such as the U.S. Food and Drug Administration or the European Medicines Agency). Examples of pharmaceutically acceptable carriers include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (such as sodium carboxymethyl cellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin.

[0054] According to common patent application writing terms, "selected from" is used herein to introduce a series of elements, in which one or more items are selected. Any "selected from" appearing in the specification can be replaced with "selected from the group containing ..." or "selected from the group consisting of ...", and they are replaced with each other without changing their meaning.

[0055] "Subject" refers to an animal, usually a warm-blooded animal, preferably a mammal. The term "mammal" refers here to any mammal, including humans, livestock and farm animals, as well as zoos, sports or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a primate, more preferably a human. In one embodiment, the subject is a "patient" as defined herein. In one embodiment, the subject is an adult (e.g., a subject over 18 years old). In one embodiment, the subject is a child (e.g., a subject under 18 years old). In one embodiment, the subject is a male. In one embodiment, the subject is a female. In one embodiment, the subject is affected by autoimmune diseases, transplant rejection, graft-versus-host disease, or BOB1-related diseases, and is preferably diagnosed with these diseases. In one embodiment, the subject has the risk of developing autoimmune diseases, transplant rejection, graft-versus-host disease, or BOB1-related diseases. Examples of risk factors include, but are not limited to, genetic susceptibility; family history of autoimmune diseases, transplant rejection, graft-versus-host disease, or BOB1-related diseases.

[0056] "Therapeutic agent," "active pharmaceutical ingredient," and "active ingredient" refer to compounds that are used for therapeutic purposes and are related to health. In particular, a therapeutic agent (e.g., a compound of the invention) may be designated for treating a disease (e.g., an autoimmune disease, transplant rejection, graft-versus-host disease, or BOB1-related disease). An active ingredient may also be designated to improve the therapeutic activity of another therapeutic agent.

[0057] "Therapeutically effective amount" ("effective amount" for short) refers to an amount of a therapeutic agent (e.g., a compound of the invention) sufficient to achieve the desired therapeutic, defensive or preventive effect in a patient to which it is administered without causing significant negative or adverse side effects to the patient. For defensive or preventive effects, a therapeutically effective amount may be administered prior to the onset of a disease, disorder or condition. Alternatively or additionally, for a therapeutic effect, a therapeutically effective amount may be administered after the onset of a disease, disorder or condition.

[0058] "Treating," "treatment," or "alleviation" refers to both therapeutic treatment and defensive or preventative measures; wherein the purpose is to prevent or slow down (mitigate) the targeted pathological condition or disorder (i.e., "disease"). Those in need of treatment include those already suffering from the disease, as well as those susceptible to the disease or those in need of prevention of the condition or disease. The patient's disease is successfully "treated" if the patient, after receiving a therapeutic amount of a therapeutic agent (e.g., a compound according to the invention), exhibits an observable and / or measurable reduction or disappearance of one or more of the following: a reduction in the percentage of the total number of pathogenic cells; and / or a reduction to some extent in one or more symptoms associated with a particular disease; reduced morbidity and mortality, and improved quality of life issues. The above parameters for assessing successful treatment and improvement of the disease can be readily measured by routine procedures familiar to physicians. DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention relates to a compound of formula (I)

[0061]

[0062] or a pharmaceutically acceptable salt and / or solvate thereof;

[0063] in

[0064] X represents -(CH2) n -, where n is 0 or 1;

[0065] Y represents -O- or -(CH2) m -, where m is 1 or 2;

[0066] R 1 and R 2 Each represents H; or R 1 and R 2 Together with the heterocyclic ring to which they are attached, they form a fused phenyl group;

[0067] R 3 and R 4 Each independently represents H, alkyl, -(CH2) p -NR 9 R 10 , aryl or arylalkyl,

[0068] Wherein p is an integer from 1 to 5,

[0069] Where R 9 and R 10 Each independently represents H or (C1-C4) alkyl; or R 9 and R 10Together with the nitrogen atom to which they are attached they form a heterocycloalkyl group;

[0070] wherein the aryl or arylalkyl is optionally substituted with at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy;

[0071] or R 3 and R 4 Together with the nitrogen atom to which they are attached they form a heterocycloalkyl group;

[0072] wherein the heterocycloalkyl is optionally substituted with at least one aryl or arylalkyl;

[0073] wherein the aryl or arylalkyl is optionally substituted with at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy;

[0074] R 5 represents an aryl group or an arylalkyl group;

[0075] wherein the aryl or arylalkyl is optionally substituted with at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy;

[0076] R 6 represents H or (C1-C4)alkyl; and

[0077] R 7 and R 8 Each independently represents H or an alkyl group substituted with at least one COOH or OH.

[0078] According to one embodiment, X represents a single bond, ie n is 0. According to one embodiment, X represents CH2, ie n is 1.

[0079] According to one embodiment, Y represents -O-. According to one embodiment, Y represents CH2, ie m is 1. According to one embodiment, Y represents CH2-CH2, ie m is 2.

[0080] In one embodiment, X represents a single bond and Y represents CH2. In one embodiment, X represents a single bond and Y represents CH2-CH2. In one embodiment, X represents CH2 and Y represents -O-. In one embodiment, X and Y each represent CH2.

[0081] According to one embodiment, R 1 and R 2 Each represents H. According to one embodiment, R 1 and R 2 Together with the heterocyclic ring to which they are attached they form a fused phenyl group.

[0082] According to a preferred embodiment, X represents CH2, Y represents -O-, and R 1 and R 2 Together with the heterocyclic ring to which they are attached they form a fused phenyl group.

[0083] According to one embodiment, R 3 and R 4 Each independently represents H, alkyl, -(CH2) p -NR 9 R 10 or aromatic group.

[0084] According to one embodiment, R 3 and R 4 Each independently represents H, alkyl or -(CH2) p -NR 9 R 10 ; wherein p is an integer in the range of 1 to 5. In one embodiment, R 3 Represents -(CH2) p -NR 9 R 10 , wherein p is an integer in the range of 1 to 5, and R 9 and R 10 Together with the nitrogen atom to which they are attached they form a heterocycloalkyl group, for example pyrrolidinyl (eg pyrrolidin-1-yl).

[0085] According to one embodiment, R 9 and R 10 Together with the nitrogen atom to which they are attached they form a heterocycloalkyl group, for example pyrrolidinyl (eg pyrrolidin-1-yl).

[0086] According to one embodiment, R 3 and R 4 Not all mean H.

[0087] According to one embodiment, R 3 and R 4 In one embodiment, R 3 and R 4 Not all represent alkyl groups.

[0088] According to one embodiment, R 3 represents an aryl group, wherein the aryl group is optionally substituted by at least one halogen, (C1-C4) alkyl, (C1-C4) haloalkyl, COOH, OH or (C1-C4) alkoxy. 3represents an aryl group, wherein the aryl group is optionally substituted by at least one halogen, (C1-C4) alkyl, (C1-C4) haloalkyl, COOH, OH or (C1-C4) alkoxy group, and R 4 represents H. In one embodiment, R 3 represents an aryl group, wherein the aryl group is optionally substituted by at least one halogen, (C1-C4) alkyl or (C1-C4) haloalkyl. In one embodiment, R 3 represents an aryl group, wherein the aryl group is optionally substituted by at least one Br, Cl, F, methyl or trifluoromethyl (CF3). In one embodiment, R 3 represents an aryl group, wherein the aryl group is optionally substituted by at least one Br, Cl, methyl or trifluoromethyl (CF3).

[0089] According to one embodiment, R 4 Indicates H.

[0090] According to a preferred embodiment, R 3 represents an aryl group, wherein the aryl group is optionally substituted by at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy group; and R 4 In a preferred embodiment, R 3 represents a phenyl group, wherein the phenyl group is optionally substituted by at least one Br, Cl, methyl or trifluoromethyl (CF3); and R 4 Indicates H.

[0091] According to one embodiment, R 3 and R 4 Together with the nitrogen atom to which they are attached, they form a heterocycloalkyl group, wherein the heterocycloalkyl group is optionally substituted with at least one aryl or arylalkyl group. 3 and R 4 Together with the nitrogen atom to which they are connected, a heterocycloalkyl is formed, wherein the heterocycloalkyl is optionally substituted by at least one arylalkyl. In one embodiment, the heterocycloalkyl is unsubstituted. In a preferred embodiment, the heterocycloalkyl is substituted by exactly one arylalkyl. In one embodiment, the heterocycloalkyl is morpholinyl or piperidinyl. In one embodiment, the heterocycloalkyl is morpholinyl (e.g., morpholine-4-yl). In a preferred embodiment, the heterocycloalkyl is piperidinyl (e.g., piperidin-1-yl). In one embodiment, aryl or arylalkyl is unsubstituted. In one embodiment, aryl or arylalkyl is phenyl or benzyl. In one embodiment, aryl is phenyl. In one embodiment, arylalkyl is benzyl.

[0092] According to a preferred embodiment, R 3 and R 4Together with the nitrogen atom to which they are attached, they form a heterocycloalkyl group, wherein the heterocycloalkyl group is substituted with at least one aryl group or arylalkyl group. 3 and R 4 Together with the nitrogen atom to which they are attached, they form a heterocycloalkyl group, wherein the heterocycloalkyl group is substituted by at least one arylalkyl group. In a preferred embodiment, the heterocycloalkyl group is a piperidinyl group (e.g., piperidin-1-yl). In a preferred embodiment, the arylalkyl group is a benzyl group.

[0093] According to a preferred embodiment, either: (i) R 3 represents an aryl group, wherein the aryl group is optionally substituted by at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy group; and R 4 represents H; or (ii) R 3 and R 4 Together with the nitrogen atom to which they are attached, they form a heterocycloalkyl group, wherein the heterocycloalkyl group is substituted with at least one aryl group or arylalkyl group. 3 The aryl group in the aryl group or arylalkyl group is phenyl.

[0094] According to one embodiment, R 5 represents an arylalkyl group, wherein the arylalkyl group is optionally substituted by at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy. In one embodiment, the arylalkyl group is optionally substituted by at least one halogen, (C1-C4)alkyl, or (C1-C4)haloalkyl. 1- In one embodiment, the arylalkyl is optionally substituted with at least one Br, Cl, F, methyl or trifluoromethyl (CF3). In one embodiment, the arylalkyl is optionally substituted with at least one F. In one embodiment, the arylalkyl is substituted with at least one F. In a specific embodiment, the arylalkyl is substituted with exactly one F. In one embodiment, the arylalkyl in R 5 The aryl portion of the alkyl group is substituted, i.e., the substituents are not on the alkyl portion thereof.

[0095] In one embodiment, R 5 represents phenyl-(CH2) q -, wherein q is an integer in the range of 1 to 5, and wherein the phenyl group is optionally substituted with at least one halogen, (C1-C4) alkyl, (C1-C4) haloalkyl, COOH, OH or (C1-C4) alkoxy. In one embodiment, the phenyl group is optionally substituted with at least one halogen, (C1-C4) alkyl or (C1-C4) alkoxy. 1-In one embodiment, phenyl is optionally substituted with at least one Br, Cl, F, methyl or trifluoromethyl (CF3). In one embodiment, phenyl is optionally substituted with at least one F. In one embodiment, phenyl is substituted with at least one F. In a specific embodiment, phenyl is substituted with exactly one F. In one embodiment, q is an integer in the range of 1 to 3. In a specific embodiment, q is 1. In a specific embodiment, q is 3.

[0096] According to one embodiment, R 6 Indicates H.

[0097] In one embodiment, R 5 represents an arylalkyl group, wherein the arylalkyl group is optionally substituted by at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy group; and R 6 Indicates H.

[0098] According to one embodiment, R 7 and R 8 Each independently represents an alkyl group substituted with at least one COOH or OH. 7 and R 8 Each independently represents an alkyl group substituted with at least one OH group. 7 and R 8 In one embodiment, R 7 and R 8 The alkyl group in is (C1-C4)alkyl. In a preferred embodiment, the alkyl group is ethyl.

[0099] According to one embodiment, R 7 represents an alkyl group substituted by at least one COOH or OH. According to one embodiment, R 7 represents an alkyl group substituted by at least one COOH or OH; and R 8 represents H. In one embodiment, R 7 represents an alkyl group substituted by at least one COOH group. 7 The alkyl group in is (C1-C4)alkyl. In a preferred embodiment, the alkyl group is ethyl.

[0100] According to one embodiment, R 8 Indicates H.

[0101] According to one embodiment, R 5represents phenyl-CH2-, wherein the phenyl group is optionally substituted by at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy; and R 7 and R 8 Each independently represents an alkyl group substituted with at least one OH group.

[0102] According to a preferred embodiment, R 5 represents phenyl-(CH2)3-, wherein the phenyl group is optionally substituted by at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy; R 7 represents an alkyl group substituted with at least one COOH group; and R 8 Indicates H.

[0103] According to one embodiment, the compound of formula (I) is a compound of formula (IA)

[0104]

[0105] or a pharmaceutically acceptable salt and / or solvate thereof;

[0106] Where R 1 , R 2 , R 3 , R 4 and R 5 are each independently as defined above under formula (I).

[0107] According to one embodiment, the compound of formula (I) is a compound of formula (IB)

[0108]

[0109] or a pharmaceutically acceptable salt and / or solvate thereof;

[0110] Where R 1 , R 2 , R 3 , R 4 and R 5 are each independently as defined above under formula (I).

[0111] According to a preferred embodiment, the compound of formula (I) is a compound of formula (IBa)

[0112]

[0113] or a pharmaceutically acceptable salt and / or solvate thereof;

[0114] Where R 3 , R4 and R 5 are each independently as defined above under formula (I).

[0115] According to one embodiment, the compound of formula (I) is selected from the compounds of Table 1 below and pharmaceutically acceptable salts and / or solvates thereof.

[0116]

[0117]

[0118]

[0119] Table 1

[0120] Compound 001 named above as “(R)-1-(4-(bis(2-hydroxyethyl)amino)-6-((3-fluorobenzyl)amino)-1,3,5-triazin-2-yl)-N,N-diethylpyrrolidine-2-carboxamide” can be alternatively named as “N-(4-(bis(2-hydroxyethyl)amino)-6-((3-fluorobenzyl)amino)-1,3,5-triazin-2-yl)-N′,N′-diethyl-L-prolinamide”.

[0121] In one embodiment, the compound of formula (I) is selected from the compounds of Table 2 below and pharmaceutically acceptable salts and / or solvates thereof.

[0122]

[0123]

[0124] Table 2: Bis(2-hydroxyethyl)amino / 3-fluorobenzylamino compounds

[0125] According to one embodiment, the compound of formula (I) is selected from the compounds of Table 3 below and pharmaceutically acceptable salts and / or solvates thereof.

[0126]

[0127] Table 3: 3-(4-Fluorophenyl)propylamino / γ-aminobutyric acid compounds

[0128] According to one embodiment, the compound of formula (I) is selected from compounds 004, 005 and 006 of Table 1 above, and pharmaceutically acceptable salts and / or solvates thereof.

[0129] All references to compounds of the invention herein (e.g., "compounds of Formula (I)") include references to their salts, solvates, multicomponent complexes, and liquid crystals. All references to compounds of the invention herein include references to their polymorphs and their crystal habits. All references to compounds of the invention herein include references to their isotopically labeled compounds, including deuterated compounds thereof. All references to compounds of the invention herein include references to their stereoisomers. All references to compounds of the invention herein include references to their pharmaceutically acceptable prodrugs.

[0130] In particular, the compounds of the present invention (eg, "compounds of formula (I)") may be in the form of pharmaceutically acceptable salts. According to one embodiment, the compounds of the present invention are pharmaceutically acceptable salts.

[0131] Pharmaceutically acceptable salts include acid addition salts and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, cyclamate, edisylate, ethanesulfonate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, toluenesulfonate, trifluoroacetate, and xinafoate. Suitable base salts are formed from bases which form non-toxic salts. Examples include aluminum, arginine, benzathine penicillin, calcium, choline, diethylamine, 2-(diethylamino)ethanol, diethanolamine, ethanolamine, glycine, 4-(2-hydroxyethyl)-morpholine, lysine, magnesium, meglumine, morpholine, ethanolamine, potassium, sodium, tromethamine and zinc salts. Hemi-salts of acids and bases can also be formed, such as hemi-sulfate and hemi-calcium salts. When the compound contains an acidic group as well as a basic group, the compound can also form an inner salt, and these compounds are within the scope of the present invention. When the compound contains a hydrogen-donating heteroatom (e.g., NH), the present invention also includes salts and / or isomers formed by transferring the hydrogen atom to a basic group or atom within the molecule. The pharmaceutically acceptable salts of the compounds of the present invention can be prepared by one or more of these methods: (i) by reacting the compound with a desired acid; (ii) by reacting the compound with a desired base; (iii) by removing an acid- or base-labile protecting group from a suitable precursor of the compound, or by opening a suitable cyclic precursor (e.g., a lactone or lactam) with a desired acid; and / or (iv) by converting one salt of the compound into another salt by reacting with a suitable acid or by means of a suitable ion exchange column. All of these reactions are typically carried out in solution. The salt can be precipitated from the solution and can be collected by filtration or can be recovered by evaporating the solvent. The degree of ionization in the salt can range from completely ionized to almost non-ionized.

[0132] In particular, the compounds of the present invention (e.g., "compounds of formula (I)") may be in the form of pharmaceutically acceptable solvates. According to one embodiment, the compounds of the present invention are pharmaceutically acceptable solvates. According to one embodiment, the compounds of the present invention are pharmaceutically acceptable salts and solvates.

[0133] In particular, the compounds of the invention (e.g., "compounds of formula (I)") may include at least one asymmetric center and may therefore exist in different stereoisomeric forms. Therefore, all references to compounds of the invention include all possible stereoisomers, including not only racemic compounds, but also individual enantiomers and non-racemic mixtures thereof. When a compound is required in single enantiomeric form, such single enantiomer may be obtained by stereospecific synthesis, by resolution of the final product or any convenient intermediate, or by chiral chromatographic methods (as each is known in the art). Resolution of the final product, intermediate, or starting material may be carried out by any suitable method known in the art.

[0134] The compounds of the present invention as described herein can be made by synthetic methods well known in the art.

[0135] The present invention also relates to processes for the manufacture of the compounds of the invention as described herein.

[0136] According to one embodiment, the method comprises:

[0137] (a) In the presence of a base, The cyanuric chloride reacts with the NH secondary amine functional group in the ring of the compound of formula (A)

[0138]

[0139] The steps of obtaining the monosubstituted 1,3,5-triazine of formula (B)

[0140]

[0141] (b) in the presence of a base, reacting compound (B) with a compound of the formula NHR 5 R 6 The step of reacting with an amine to obtain a disubstituted 1,3,5-triazine; and then

[0142] (c) in the presence of a base, reacting the obtained disubstituted 1,3,5-triazine with a compound of formula NHR 7 R 8 A step of reacting an amine with an amine to obtain a trisubstituted 1,3,5-triazine;

[0143] or

[0144] (b) in the presence of a base, reacting compound (B) with a compound of the formula NHR 7 R 8 The step of reacting with an amine to obtain a disubstituted 1,3,5-triazine; and then

[0145] (c) in the presence of a base, reacting the obtained disubstituted 1,3,5-triazine with a compound of formula NHR 5 R6 The step of reacting with an amine to obtain a trisubstituted 1,3,5-triazine; and

[0146] (d) optionally, at least one step of deprotecting at least one alcohol (OH), carboxylic acid (COOH) and / or amine (NH or NH2) group present in the resulting trisubstituted 1,3,5-triazine;

[0147] Thus, the compound of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof is obtained.

[0148] The method according to the invention may also comprise at least one purification and / or separation step known in the art.

[0149] In one embodiment, the method comprises (b) reacting compound (B) with a compound of formula NHR in the presence of a base 5 R 6 (c) reacting the obtained disubstituted 1,3,5-triazine with an amine of formula NHR in the presence of a base. 7 R 8 The invention relates to a step of reacting an amine with a substituted 1,3,5-triazine to obtain a trisubstituted 1,3,5-triazine.

[0150] In one embodiment, the base is a tertiary alkylamine, such as N,N-diisopropylethylamine (DIPEA).

[0151] In one embodiment, the formula NHR 6 R 7 The compounds include a carboxylic acid (COOH) group protected as an alkyl ester (COO-alkyl) group, such as a methyl ester.

[0152] Another object of the present invention is a composition comprising or essentially consisting of a compound according to the invention as described herein.

[0153] In one embodiment, the composition is a pharmaceutical composition, and further comprises at least one pharmaceutically acceptable carrier.

[0154] Therefore, another object of the present invention is a pharmaceutical composition comprising or essentially consisting of a compound according to the invention as described herein and at least one pharmaceutically acceptable carrier.

[0155] As used herein, the term "consisting essentially of" with respect to a composition, pharmaceutical composition or medicament means that at least one compound according to the invention is the only therapeutic or biologically active agent in the composition, pharmaceutical composition or medicament.

[0156] Another object of the present invention is a medicament comprising or essentially consisting of a compound according to the invention as described herein.

[0157] Another object of the invention is a kit comprising a compound according to the invention as described herein and instructions for use.

[0158] The present invention also relates to a compound, a composition or a pharmaceutical composition according to the invention as described herein for use as a medicament.

[0159] The present invention also relates to a compound, a composition or a pharmaceutical composition according to the invention as described herein for use in the treatment of an autoimmune disease, transplant organ rejection, graft-versus-host disease or a BOB1 related disease.

[0160] The present invention also relates to a method for treating an autoimmune disease, transplant organ rejection, graft-versus-host disease or BOB1-related disease in a subject in need thereof, comprising administering to the subject a compound, composition or pharmaceutical composition according to the present invention as described herein.

[0161] The present invention also relates to the use of a compound, composition or pharmaceutical composition according to the present invention as described herein for the preparation of a medicament for treating an autoimmune disease, transplant organ rejection, graft-versus-host disease or BOB1-related disease in a subject in need thereof.

[0162] The present invention also relates to the use of a compound, composition or pharmaceutical composition according to the present invention as described herein for treating an autoimmune disease, transplant organ rejection, graft-versus-host disease or a BOB1-related disease in a subject in need thereof.

[0163] According to one embodiment, the compound is selected from the compounds of Table 1 herein. In one embodiment, the compound is selected from the compounds of Table 2 herein. In one embodiment, the compound is selected from the compounds of Table 3 herein.

[0164] Examples of autoimmune diseases include, but are not limited to, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, primary biliary cirrhosis, Addison's disease, acquired immunodeficiency syndrome (AIDS), ankylosing spondylitis, anti-glomerular basement membrane disease, autoimmune hepatitis, dermatitis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura (HSP), juvenile arthritis, and leukemia. arthritis, juvenile myositis, Kawasaki disease, inflammatory bowel diseases (e.g., Crohn's disease and ulcerative colitis), polymyositis, pulmonary alveolar proteinosis, myasthenia gravis, neuromyelitis optica, Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS), psoriasis, psoriatic arthritis, Sjögren's syndrome syndrome, systemic sclerosis, systemic lupus erythematosus, thrombocytopenic purpura (TTP), type I diabetes mellitus, uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada Disease.

[0165] In one embodiment, the autoimmune disease is selected from the group comprising or consisting of rheumatoid arthritis, type 1 diabetes, multiple sclerosis and primary biliary cirrhosis.

[0166] Examples of BOB1-associated diseases include, but are not limited to, end-stage chronic respiratory diseases (e.g., chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), pulmonary arterial hypertension (PAH)), germinal center-derived lymphomas (Follicular lymphomas, Burkitt lymphomas, and Diffuse Large B-cell lymphomas), and Waldenstrom's macroglobulinemia.

[0167] Other examples of BOB1-associated diseases include, but are not limited to, solid cancers (eg, breast cancer, lung cancer, pancreatic cancer, colon cancer, kidney cancer, prostate cancer).

[0168] As used herein, the term "cancer" has its ordinary meaning in the art, and particularly refers to a disease caused by the uncontrolled division of abnormal cells.

[0169] Examples of solid cancers include, but are not limited to, adenoid cystic carcinoma, adrenocortical carcinoma, epithelial carcinoma, AIDS-related cancers, anal cancer, appendix cancer, astrocytoma, atypical teratoid tumor / rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumor, breast cancer, bronchial tumor, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, embryonal tumor, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, olfactory neuroblastoma, ovarian cancer ... Ewing sarcoma family of tumors, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, bone fibrous histiocytoma and osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), soft tissue sarcomas, germ cell tumors, gestational trophoblastic tumors, gliomas, hairy cell leukemia, heart cancer, hepatocellular (liver) cancer, histiocytosis, hypopharyngeal cancer, intraocular melanoma, islet cell tumors (endocrine pancreas), Kaposi sarcoma, kidney cancer, Langerhans cell Histiocytosis, Laryngeal, Lip, and Oral Cavity Cancer, Liver Cancer (Primary), Lobular Carcinoma in Situ (LCIS), Lung Cancer, Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone, Medulloblastoma, Medulloepithelioma, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary Midline Tract Cancer Involving the NUT Gene, Oral Cancer, Multiple Endocrine Neoplasm Syndrome, Multiple Myeloma / Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndrome, Myelodysplastic / Myeloproliferative Neoplasms, Multiple Myeloma, Myeloproliferative Disorders, Nasal and Paranasal Sinus Cancer , nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, pleuropulmonary blastoma and breast cancer, prostate cancer, rectal cancer, renal cell (kidney) cancer, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sezary syndrome (Sezary syndrome syndrome), small cell lung cancer, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, gastric cancer, supratentorial primitive neuroectodermal tumors, skin cancer, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, bile duct cancer, head and neck squamous cell carcinoma, glioblastoma, adrenocortical carcinoma, bladder cancer, kidney cancer, renal cancer, colorectal cancer, cervical cancer, gestational trophoblastic disease (GTD), primary peritoneal cancer.

[0170] In one embodiment, the compounds according to the present invention inhibit BOB1 / OCT1 driven transcription. In one embodiment, the compounds according to the present invention reduce plasmablast differentiation. In one embodiment, the compounds according to the present invention eliminate B cell activation. In one embodiment, the compounds according to the present invention inhibit B cell differentiation into plasma cells that secrete antibodies. In one embodiment, the compounds according to the present invention inhibit effector B cell proliferation. In one embodiment, the compounds according to the present invention inhibit effector T cell proliferation. In one embodiment, the compounds according to the present invention inhibit memory T cell response. In one embodiment, the compounds according to the present invention inhibit memory Th17 cell response. In one embodiment, the compounds according to the present invention do not change the regulatory function of B cells.

[0171] For administration to a subject, a medicament, composition or pharmaceutical composition according to the invention as described herein will be formulated.

[0172] In one embodiment, the medicament, composition or pharmaceutical composition is administered parenterally, orally, by inhalation, spray, rectally, nasally or via an implanted reservoir.

[0173] In one embodiment, the medicament, composition or pharmaceutical composition is administered by injection, including but not limited to subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0174] Examples of forms suitable for injection include, but are not limited to, solutions, such as sterile aqueous solutions; gels; dispersions; emulsions; suspensions; solid forms suitable for preparation of solutions or suspensions upon addition of liquid prior to use, such as powders; liposomes, etc.

[0175] In one embodiment, the medicament, composition or pharmaceutical composition will be administered to a subject in need thereof in a therapeutically effective amount.

[0176] However, it should be understood that the total daily dosage of the compounds, compositions, pharmaceutical compositions or drugs according to the present invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dosage level for any particular patient will depend on a variety of factors, including the disease being treated and the severity of the disease; the activity of the compound used; the age, weight, general health, sex and diet of the subject; the time of administration, route of administration and excretion rate of the specific therapeutic agent used; the duration of treatment; drugs used in combination or concurrently with the specific therapeutic agent used; and similar factors well known in the medical field. For example, those skilled in the art are fully capable of starting the dosage of the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increasing the dosage until the desired effect is achieved. The total dosage required for each treatment can be administered by multiple doses or a single dose.

[0177] In one embodiment, the dosage of the compound is generally about 0.01 to 500 mg / kg patient body weight / day, which can be administered in single or multiple doses. Preferably, the dosage level is about 0.1 to about 250 mg / kg / day; more preferably about 0.5 to about 100 mg / kg / day. Suitable dosage levels can be about 0.01 to 250 mg / kg / day, about 0.05 to 100 mg / kg / day, or about 0.1 to 50 mg / kg / day. Within this range, the dosage can be about 0.05 to 0.5 mg / kg / day, about 0.5 to 5 mg / kg / day, or about 5 to 50 mg / kg / day. For oral administration, the composition can be provided in the form of a tablet containing about 1.0 to 1000 mg of active ingredient, particularly about 1.0 mg, about 5.0 mg, about 10.0 mg, about 15.0 mg, about 20.0 mg, about 25.0 mg, about 50.0 mg, about 75.0 mg, about 100.0 mg, about 150.0 mg, about 200.0 mg, about 250.0 mg, about 300.0 mg, about 400.0 mg, about 500.0 mg, about 600.0 mg, about 750.0 mg, about 800.0 mg, about 900.0 mg and about 1000.0 mg of active ingredient to adjust the dosage of the patient to be treated according to the symptoms. The compound can be administered in a regimen of 1 to 4 times a day, preferably once or twice a day. However, it is understood that the specific dosage level and frequency of administration for any particular patient may vary and depend on a variety of factors, including the activity of the specific compound employed, the metabolic stability and duration of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host being treated.

[0178] BRIEF DESCRIPTION OF THE DRAWINGS

[0179] Figure 1A-C is a combination of graphs showing that compound 005 inhibits BOB1 / OCT1-mediated transcription in vitro. HEC293Ea1 cells known to express endogenous OCT1 were co-transfected with p6xW-tk-luc reporter gene, pEV-OBF1 (BOB1) effector, and pCMV-β-gal calibration plasmids. Luciferase and β-galactosidase activities were measured 48 hours after transfection. 100% luciferase activity was set as activity in the presence of vehicle (10 μM DMSO). Figure 1A Relative luciferase activity inhibition of compounds at 10 μM concentration is shown. Figure 1B Dose-dependent inhibition of BOB1 / OCT1-mediated transcription by Compound 005 over the indicated concentration range is shown (left panel). Figure 1C The corresponding "inhibitors vs. normalized responses with variable Hill slope coefficients" best fit curves are shown, with indicated IC50 values ​​(95% CI 4.261 to 9.422 μM). All experiments were performed at least in triplicate. Bar graphs represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0180] Figure 2A -D shows the characterization of compound 005-OCT1 interaction in vitro. Figure 2A Characterization of bacterially produced and purified His-tagged recombinant POU domain of OCT1 (POU1, 22 kDa) and BOB1 (33 kDa) on Coomassie R250-stained 12% SSDS-PAGE is shown; Figure 2B Electrophoretic mobility shift assays (EMSA) using biotinylated PORE DNA (bio-PORE) as probe, His-tagged BOB1 and POU1 recombinant proteins, and compound 005 (100 μM) or its vehicle (DMSO) are shown. Supershifts of monomeric POU1 with DNA, and complexes of BOB1 and bio-PORE are depicted; Figure 2C The values ​​of the binding constant (Kb), dissociation constant (Kd), Gibbs energy change (ΔG), and number of binding sites (n) of the complex of compound 005 with POU1 at 298.15 K are shown. Figure 2D Representative results of thermal shift assay (TSA) of the complex of compound 005 with POU1 and with BOB1 (as control) are shown; the results are expressed as mean±SD (n=3); statistical analysis was performed using t-test, n=3 (technical replicates), *P<0.05.

[0181] Figure 3A-Y is a combination of charts and FACS drawings, showing that compound 005 targets BOB1-OCT complexes in human B cell differentiation in vitro models and dose-dependently impairs B cell proliferation, activation and differentiation into plasmablasts. On the 0th day, the enriched memory B cells in healthy subjects (n=15) blood were stimulated with anti-F (ab') 2, CD40L, CpG and IL-2 for four days (activation stage). On the 4th day, cells were washed and stimulated with IL-2, IL-4 and IL-10 for another three days (differentiation stage). Compound 005 was added to the culture every day so that its final concentration was 0 (DMSO), 5, 10, 15, 20 μM as shown. Figure 3A Representative shifted histogram overlays showing median fluorescence intensity of gated live B cells stained with cell proliferation dye (CPD) and mAbs specific for CD27, CD38, and CD20 expression analyzed on day 4 are shown. Figure 3B CPD analysis on day 4 is shown 低 Corresponding summary data for the frequency of proliferating B cells. Figure 3C -H shows the CD27 analysis on day 4 + ( Figure 3C -D), CD38 + ( Figure 3E -F) and CD20 + ( Figure 3G -H)B cell frequency ( Figure 3C , E and G ) and relative mean fluorescence intensity (MFI; Figure 3D , F and H) of the corresponding summary data. Fig. 3I Representative offset histogram overlays showing median fluorescence intensity of gated live B cells stained with mAbs specific for CD27, CD38, and CD20 expression at day 7 are shown. Figure 3J CD20 at day 7 is shown 低 CD38 ++ Representative dot plots of plasmablasts. Figure 3K -P shows CD27 analyzed on day 7 + ( Figure 3K -L), CD38 + ( Figure 3M -N) and CD20 + ( Fig.3O -P) The corresponding summary data of the frequency (panels K, M and O) and relative mean fluorescence intensity (MFI; panels L, N and P) of B cells. Figure 3Q CD20 at day 7 is shown 低 CD38 ++ Corresponding summary data for plasmablast frequencies. Figure 3R -Y shows the effect of compound 005 on IgG1 ( Figure 3R )、IgG2( Figure 3S )、IgG3( Figure 3T )、IgG4( Figure 3U )、IgA( Figure 3V )、IgE( Figure 3W )、IgM( Figure 3X ) and IgD( Figure 3Y ) Effect of immunoglobulin subtype production levels. Error bars are mean ± SD. P values ​​were determined using one-way ANOVA with Geisser-Greenhouse multiple comparison test.

[0182] Figure 4A -AA is a combination of a heat map plot and a graph showing the expression of B cell related genes on days 4 and 7 of an in vitro model of human B cell differentiation in the presence of 0 μM and 10 μM of compound 005 (n=6). The experimental conditions are as described in FIG3 . Figure 4A Unsupervised hierarchical gene clustering of 46 B cell-related genes at days 4 and 7 is shown, with five gene categories highlighted on the heat map. Figure 4B -AA shows the proliferation rate on day 4 ( Figure 4B -D), plasma cell differentiation ( Figure 4E -I), immunoglobulin subtype switching ( Figure 4J -M), B cell identity ( Figure 4N -S) and B cell activation ( Figure 4T -AA)-related expression of selected genes.

[0183] Figure 5A -I is a combination of a graph and a FACS plot showing that compound 005 does not induce apoptosis in B cells. Figure 5A Representative FACS plots of caspase-3 expression in B cells cultured with different concentrations of Compound 005 on days 4 and 7 are shown. Figure 5B -C shows the 4th day ( Figure 5B ) and Day 7 ( Figure 5C ) Corresponding summary data for the frequency of apoptotic B cells assessed by active caspase-3 staining. Figure 5D Representative FACS plots showing the effect of compound 005 on apoptosis of differentiated CD20 cells via active caspase-3. 低 CD38 ++ Plasmablasts and undifferentiated CD38 + / - Non-plasmablasts were identified by staining. Figure 5E -F shows caspase-3 at day 7 + CD38 + / - Non-plasmablasts ( Figure 5E ) and caspase-3 + CD20 低CD38 ++ Plasmablasts ( Fig. 5F ) of the study population (n=8). Figure 5G The expression of IgD in cell cultures with the indicated concentrations of Compound 005 is shown. + CD27 - Representative FACS plots of naive B cell frequencies. Figure 5H The corresponding summary data are shown. Fig.5I The effect of compound 005 on IgD at day 7 is shown. + CD27 - Naive B cells and CD20 低 CD38 ++ Effect of the ratio of plasmablast frequency. Error bars are mean ± SD. P values ​​were determined by one-way ANOVA with Geisser-Greenhouse multiple comparison test.

[0184] Fig. 6A -K is a combination of a graph and a FACS plot showing that targeting the BOB1-OCT interaction by compound 005 does not affect the suppressive capacity of B cells. B cells were kept unstimulated (B resting) or stimulated (B expanded) for 24 hours with an expansion cocktail containing F(ab')2 anti-BCR, CpG ODN, CD40L, IL-2, and IL-21. Fig. 6A -B shows the frequency ( Fig. 6A ) and through MFI( Figure 6B ) The expression level of granzyme B measured. Figure 6C -G shows CD4 labeled with autologous CPD after washing with fresh medium and in the presence of anti-CD3 / CD28 Dynabeads. + CD25 - Effector T cells were co-cultured with resting and expanded B cells for 3 days. Compound 005 was added to B cells only during the 24-hour activation with the expansion mixture ( Figure 6C -D), and then supplemented daily during the three days of co-cultivation ( Fig. 6E -G). Representative shifted histogram overlays showing the median fluorescence intensity of gated live B cells stained with cell proliferation dye (CPD) ( Figure 6C , 6E and 6F). The percentage of proliferating cells is indicated for one representative donor. Fig.6D and 6GFigure 2 shows the expression of anti-CD3 / CD28 activated CD4 T cells co-cultured with resting (open circles) or expanded (closed circles) B cells in the absence of BOB1 inhibitor (DMSO treated control) or in the presence of 10 μM Compound 005. + CD25 - The corresponding summary data of the percentage of T cell proliferation inhibition. The bar graph represents the mean ± SD. **P < 0.01, ***P < 0.001. Figure 6H -J respectively encodes POU2AF1 for BOB1, OCT1 and OCT2 ( Figure 6H )、POU2F1( Fig.6I ) and POU2F2( Figure 6J ) gene expression values ​​extracted from scRNA-seq data of stimulated (expanded) or unstimulated (resting) B cells. Data are presented as mean + SD of non-zero values ​​in bar graphs or as percentage of positive cells in dot graphs ( Figure 6H -J) or the mean expression in each condition ( Figure 6K ). P values ​​were adjusted for the number of genes expressed in the dataset.

[0185] Fig. 7A -P is a combination of a graph and a FACS plot showing that compound 005 targets the BOB1-OCT interaction to inhibit dose-dependent T cell proliferation and selectively suppress memory Th17 responses. Fig. 7A ), representative shift histogram overlays showing anti-CD3 / CD28 activated CD4 cultured in the absence of BOB1 inhibitor (DMSO treated control) or in the presence of 5 μM, 10 μM or 15 μM Compound 005. + CD25 - Median fluorescence intensity of cell proliferation dye (CPD) in T cells. The percentage of proliferating cells is indicated for one representative donor. Figure 7B The corresponding summary data for inhibition of proliferation are shown. Figure 7C -O shows the in vitro recall (memory) T cell response during Th1-(IFNG( Figure 7C )、TNF( Fig.7D )、IL-2( Fig. 7E ) and TBX21( Figure 7F ))、Th2-(IL-4( Figure 7G ) and GATA3( Figure 7H ))、Th17-(IL-17A( Fig.7I )、IL-17F( Figure 7J )、IL-21( Figure 7K )、IL-22( Figure 7L ) and RORC( Figure 7M)), and Treg-(IL-10( Figure 7N ) and FOXP3( Fig.7O )) Expression of related cytokines and transcription factors. Figure 7P qPCR data confirmed at the protein level by intracellular staining of IL-17A are shown (n=8). Each point represents a biological replicate. The bar graph represents the mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001. Example

[0186] The present invention is further illustrated by the following examples.

[0187] Example 1: Synthesis of compounds

[0188] Materials and methods

[0189] All reagents were purchased from commercial sources and used as received. NMR spectral data in CDCl3 and DMSO-d6 were recorded using a 400 MHz spectrometer ( 1 H is 400.13MHz, 13 C is 100.61 MHz), and referenced to the residual solvent proton signal (δ H =7.26 and δ H =2.50) and solvent carbon signals (δ C =77.0 and δ C =39.5). All chemical shifts are reported in parts per million (ppm). The abbreviations used in the resonance descriptions are as follows: s (singlet), d (doublet), t (triplet), q (quartet), br (broad), m (multiplet). Coupling constants (J) are quoted to the nearest 0.1 Hz. Mass spectra were recorded using a HRMS-ESI-qTOF spectrometer (electrospray ionization mode). Melting points were determined using a melting point apparatus Stuart SMP 50 in an open capillary. Flash column chromatography was performed using Merk 60 grade silica gel (0.040-0.063 mm) 230-400 mesh (isocratic or gradient elution as indicated).

[0190] Synthesis method

[0191] Example compounds 001-006 were prepared starting from cyanuric chloride as shown in Scheme 1 below for the preparation of representative compound 005.

[0192]

[0193] Solution 1

[0194] The synthesis of 005 required the initial purification of benzoxazinamine 1 and the use of commercially available reagents 2 and 3. Sequential nucleophilic displacement of the three chlorine atoms in cyanuric chloride required increasingly strong conditions. Acidic hydrolysis of the ester group in the final step provided the target compound 005.

[0195] Example compounds 001-004 and 006 were prepared in a similar manner as described below.

[0196] result

[0197] Example Compound 005: 3-((4-(2-((3-chloro-4-methylphenyl)carbamoyl)-2,3-dihydro-4H-benzene 6-((3-(4-fluorophenyl)propyl)amino)-1,3,5-triazin-2-yl)amino 4-(2-amino)propionic acid (005)

[0198]

[0199] To a stirred mixture of 2-aminophenol (4.0 g, 37 mmol), NaOAc·3H2O (14.2 g, 105 mmol) and 40% (v / v) aqueous ethanol (100 mL) was added 2-nitrobenzenesulfonyl chloride (7.5 g, 35 mmol) in small portions over 20 minutes, then the mixture was refluxed, cooled, diluted with water (200 mL), and acidified with concentrated HCl. The dark precipitate was filtered off, washed with water, and treated with boiling ethanol (50 mL). The insoluble solid was discharged and the hot filtrate was diluted with water (50 mL). After cooling overnight, the precipitate was filtered off, washed with water and dried to give crude N-(2-hydroxyphenyl)-2-nitrobenzenesulfonamide (6.0 g, 57%) as a dark purple solid.

[0200] A mixture of crude N-(2-hydroxyphenyl)-2-nitrobenzenesulfonamide (5.9 g, 20 mmol), methyl 2,3-dibromopropionate (5.5 g, 2.2 mmol), K2CO3 (6.9 g, 50 mmol) and DMF (70 mL) was vigorously stirred at 50°C overnight, cooled, taken up in water (500 mL) and extracted with DCM (3×70 mL). The combined DCM phases were washed successively with water (3×70 mL), dried over K2CO3 and concentrated in vacuo to give methyl 4-((2-nitrophenyl)sulfonyl)-3,4-dihydro-2H-benzo[b][1,4]oxazine-2-carboxylate (4.91 g, 65%) as a red oil, which was used in the next step without any purification. 1H NMR (400MHz, CDCl3) δ7.94 (d, J=7.8Hz, 1H), 7.81-7.66 (m, 3H), 7.51 (dd, J=8.3, 1.0Hz, 1H), 7.21-7.12 (m, 1H), 7.05 (dd, J=8.2, 1.2Hz, 1H), 7. 01-6.92(m,1H),4.67(dd,J=8.4,3.0Hz,1H),4.52(dd,J=14.4,3.0Hz,1H),4.56-4.45(m,1H),3.86(s,3H),3.80-3.72(dd,J=8.4,3.0Hz,1H).

[0201] The ester thus obtained (4.8 g, 12.7 mmol) was dissolved in EtOH (70 mL) at 50°C and treated with 40 mL of aqueous KOH (1.9 g, 34 mmol) under stirring within 5 minutes. The resulting clear solution was diluted with water (100 mL) and acidified to pH <2 with concentrated HCl. The yellow precipitate was filtered off, washed with water and recrystallized from aqueous EtOH to give 4-((2-nitrophenyl)sulfonyl)-3,4-dihydro-2H-benzo[b][1,4]oxazine-2-carboxylic acid (3.93 g, 85%) in the form of a pale yellow powder. 1 HNMR (400 MHz, acetone-d6) δ11.7 (br.s., 1H), 8.07-7.96 (m, 3H), 7.94-7.85 (m, 1H), 7.44 (dd, J=8.3, 1.4 Hz, 1H), 7.20-7.11 (m, 1H), 7.02 (dd, J=8.2, 1.4 Hz, 1H), 6.95 (ddd, J=8.6, 7.4, 1.5 Hz, 1H), 4.80 (dd, J=7.5, 3.1 Hz, 1H), 4.45 (dd, J=14.2, 3.2 Hz, 1H), 4.02 (dd, J=14.2, 7.5 Hz, 1H).

[0202] To a suspension of the acid (3.8 g, 10.4 mmol) in DCM (10 mL) was added thionyl chloride (1 mL) and 1 drop of DMF and the resulting mixture was stirred at reflux until a clear solution and then stirred for an additional 20 min before being concentrated in vacuo to afford crude 4-((2-nitrophenyl)sulfonyl)-3,4-dihydro-2H-benzo[b][1,4]oxazine-2-carbonyl chloride as an orange oil which was used in the next step without any purification.

[0203] To a stirred solution of 3-chloro-4-methylaniline (780 mg, 5.5 mmol) and NMM (1 mL) in DCM (30 mL) was added a solution of the above acid chloride (1.9 g, 5.0 mmol) in DCM (15 mL) over 2 min. Stirring was continued for 30 min, then the resulting mixture was washed with 5% HCl (2 x 20 mL), water, 5% NaOH (10 mL), washed again with water, dried over K2CO3 and concentrated in vacuo. The residue was crystallized from Et2O-DCM to give N-(3-chloro-4-methylphenyl)-4-((2-nitrophenyl)sulfonyl)-3,4-dihydro-2H-benzo[b][1,4]oxazine-2-carboxamide (2.0 g, 75%) as a pale yellow solid. 1 H NMR (400MHz, CDCl3) δ8.28 (s, 1H), 7.70 (d, J = 2.0Hz, 1H), 7.36 (dd, J = 8.2, 2.1Hz, 1H),7.20(d,J=8.2Hz,1H),6.99(dd,J=8.0,1.1Hz,1H),6.88(td,J=7.6,1.2Hz,1 H),6.78(td,J=7.8,1.3Hz,1H),6.71(dd,J=7.8,1.3Hz,1H),4.78(dd,J=7.3,2.9 Hz, 1H), 3.80 (dd, J = 11.9, 2.9 Hz, 1H), 3.54 (dd, J = 11.9, 7.3 Hz, 1H), 2.36 (s, 3H).

[0204] A mixture of the previous amide (1.90 g, 3.9 mmol), thiophenol (1.29 g, 11.7 mmol), K2CO3 (2.29 g, 19.5 mmol) and DMF (20 mL) was stirred in a sealed tube at 40 ° C for 5 hours, then poured into 3% aqueous NaOH (60 mL) and extracted with DCM (3×20 mL). The combined organic phases were washed with 3% aqueous NaOH, water, and the product was extracted with 5% HCl (3×20 mL). The combined aqueous phases were washed with DCM and basified with 10% aqueous NaOH. The product was extracted with DCM (3×20 mL), washed with water, dried over MgSO 4 , and concentrated in vacuo to afford crude N-(3-chloro-4-methylphenyl)-3,4-dihydro-2H-benzo[b][1,4]oxazine-2-carboxamide 1, which was recrystallized from Et 2 O-DCM to afford the pure compound (907 mg, 77%) as a beige solid. 1H NMR (400MHz, CDCl3) δ8.28(br.s,1H),7.70(d,J=2.0Hz,1H),7.36(dd,J=8.2,2.1Hz,1 H),7.20(d,J=8.2Hz,1H),6.99(dd,J=8.0,1.1Hz,1H),6.88(td,J=7.6,1.2Hz,1H),6. 78(td,J=7.8,1.3Hz,1H),6.71(dd,J=7.8,1.3Hz,1H),4.78(dd,J=7.3,2.9Hz,1H),3. 80(dd,J=11.9,2.9Hz,1H), 3.54(dd,J=11.9,7.3Hz,1H), 3.3(br.s,1H), 2.36(s,3H).

[0205]

[0206] A mixture of cyanuric chloride (213 mg, 1.15 mmol), previous amine 1 (350 mg, 1.15 mmol), DIPEA (164 mg, 127 mmol) and acetonitrile (20 mL) was stirred at room temperature overnight. The solvent was evaporated in vacuo, the residue was dissolved in DCM (50 mL), the resulting solution was washed with saturated NaHCO3, dried over MgSO4, and concentrated in vacuo. The product was isolated by silica gel column chromatography eluting with EtOAc-DCM (1:1) to give N-(3-chloro-4-methylphenyl)-4-(4,6-dichloro-1,3,5-triazin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazine-2-carboxamide (301 mg, 58%) as a beige solid. 1 H NMR (400MHz, CDCl3) δ8.21(br.s,1H),8.03(dd,J=8.3,1.1Hz,1H),7.63(d,J=2.0Hz,1H),7.34(dd,J=8.2,2.0Hz,1H),7 .27-7.08(m,4H),5.00(dd,J=13.6,3.2Hz,1H),4.92(dd,J=7.7,3.2Hz,1H),4.18(dd,J=13.7,7.7Hz,1H),2.36(s,3H).

[0207]

[0208] A solution of the previous compound N-(3-chloro-4-methylphenyl)-4-(4,6-dichloro-1,3,5-triazin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazine-2-carboxamide (285 mg, 0.633 mmol), 3-(4-fluorophenyl)propan-1-amine 2 (107 mg, 699 mmol) and DIPEA (91 mg, 71 mmol) in acetonitrile (25 mL) was stirred at 50 °C for 2 h. The solvent was evaporated in vacuo and the residue was triturated with saturated aqueous NaHCO3 solution. The white solid product was filtered off, washed with water and a small amount of Et2O, and dried in air to give 320 mg (89%) of N-(3-chloro-4-methylphenyl)-4-(4-chloro-6-((3-(4-fluorophenyl)propyl)amino)-1,3,5-triazin-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazine-2-carboxamide. 1 H NMR (400 MHz, DMSO-d6) δ 10.23 (br.s, 1H), 8.25-8.05 (m, 1H), 8.17 (d, J = 45.8 Hz, 1H), 7.88 (br.t, 1H), 7.68-7.63 (m, 1H), 7.49-6.80 (m, 5H), 5.10-4.91 (m, 1H), 4.75-4.15 (br.m, 2H), 3.29-3.06 (m, 2H), 2.66-2.51 (m, 3H), 2.25 and 2.27 (s, 3H), 1.77 (m 2H). A mixture of two rotamers in a ratio of about 3:1.

[0209]

[0210] Methyl 3-((4-(2-((3-chloro-4-methylphenyl)carbamoyl)-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-6-((3-(4-fluorophenyl)propyl)amino)-1,3,5-triazin-2-yl)amino)propanoate. A mixture of (290 mg, 0.511 mmol), β-alanine methyl ester hydrochloride (143 mg, 1.02 mmol), DIPEA (199 mg, 1.54 mmol) in 1,4-dioxane (10 mL) was heated at 90 °C for 24 hours. Saturated NaHCO3 solution (30 mL) was added and the resulting suspension was extracted with DCM (3×30 mL). The combined organic extracts were washed with water (75 mL) and dried over solid anhydrous MgSO4. After filtration, the solvent was removed under reduced pressure and the residue was purified by flash column chromatography eluting with DCM-EtOAc (1:1) to give the title compound (210 mg, 65%) as a white amorphous solid. 1H NMR(400MHz,DMSO-d6)δ10.16(br.s,1H),8.07(br.s,1H),7.74(br.s,1H),7.43(br.s,1H),7.33-7.15(m,3H),7.15-6.69( m,7H),5.04-3.74(br.m,3H),3.59(s,3H),3.52-3.37(m,2H),3.25-3.10(m,2H),2.56(m,4H),2.27(s,3H),1.76(br.s,2H).

[0211] 3-((4-(2-((3-chloro-4-methylphenyl)carbamoyl)-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-6-((3-(4-fluorophenyl)propyl)amino)-1,3,5-triazin-2-yl)amino)propanoic acid (005). A mixture of the previous methyl ester (200 mg, 0.315 mmol), 6N aqueous HCl solution (3 mL) and THF (5 mL) was stirred at room temperature for 24 hours. After this time, the pH of the clear solution was adjusted to about 5 by adding 25% aqueous NH3 solution. The mixture was vigorously stirred for 1 hour, the precipitate formed was filtered off, washed with water and dried to give 110 mg (56%) of the title compound (005) as a white amorphous solid. 1 H NMR (400MHz, DMSO-d6) δ12.15 (br.s, 1H), 10.16 (br.s, 1H), 8.2-6.5 (br.m, 15H), 5.21-3.55 (m, 6H), 2.27 (s, 3H), 1.77 (br.s, 2H). Some signals overlap with water. HRMS (ESI-TOF): [C 31 H 31 ClFN7O4+H] + , measured value 620.2191; calculated value 620.2183.

[0212] Example Compound 002: 1-(4-(bis(2-hydroxyethyl)amino)-6-((3-fluorobenzyl)amino 1,3,5-triazine-2-yl)-N-(3-(pyrrolidin-1-yl)propyl)piperidine-3-carboxamide (002)

[0213]

[0214] N-(3-(Pyrrolidin-1-yl)propyl)piperidine-3-carboxamide was prepared from (±)-N-Boc-3-piperidinic acid and 1-(3-aminopropyl)pyrrolidine by a standard mixed anhydride procedure (IBCF, NMM, DCM; column chromatography) followed by Boc-cleavage (TFA, DCM; then aqueous NaHCO3) and used in the next step without any purification.

[0215] N-(4-chloro-6-(3-fluorobenzylamino)-1,3,5-triazine-2-yl)diethanolamine. To an ice-cooled suspension of cyanuric chloride (1.85 g, 10.0 mmol) in DCM (50 ml) was added a mixture of 3-fluorobenzylamine (1.25 g, 10.0 mmol) and DIPEA (1.42 g, 11.0 mmol) dissolved in DCM (25 ml) over 15 minutes. The resulting mixture was stirred for 2 hours under ice cooling and then allowed to warm to room temperature. An emulsion of diethanolamine (1.16 g, 11.0 mmol) in DCM (25 mL) was then added and the mixture was stirred at room temperature for 20 hours, washed quickly with saturated NaHCO3 solution (30 mL), then quickly washed with water (partial crystallization of the product), and concentrated under reduced pressure. The white product was crystallized using a DCM-Et2O mixture, filtered off and dried in air to give 2.20 g (65%) of N-(4-chloro-6-(3-fluorobenzylamino)-1,3,5-triazin-2-yl)diethanolamine. 1 H NMR (400MHz, DMSO-d6, 100℃) δ7.95(br.s,1H),7.35(td,J=7.9,6.2Hz,1H),7.16(d,J=7.7Hz,1H),7.10(br .d,J=10.2Hz,1H),7.02(td,J=8.5,2.3Hz,1H),4.48(d,J=6.3Hz,2H),4.38(br.s,2H),3.70-3.52(m,8H).

[0216] 1-(4-(Bis(2-hydroxyethyl)amino)-6-((3-fluorobenzyl)amino)-1,3,5-triazin-2-yl)-N-(3-(pyrrolidin-1-yl)propyl)piperidine-3-carboxamide (002). A mixture of the previous compound (250 mg, 0.731 mmol), crude N-(3-(pyrrolidin-1-yl)propyl)piperidine-3-carboxamide (296 mg, 1.24 mmol), DIPEA (482 mg, 3.74 mmol) and 1,4-dioxane (10 mL) was heated at 90 °C for 24 hours. Subsequent workup and isolation of the title product was carried out as described above for (005) as a white amorphous solid in a yield of 120 mg (30%). 1H NMR(400MHz,DMSO-d6)δ7.79(br.t,J=5.2Hz,1H),7.35-7.25(m,2H),7.13(d,J=7.7Hz ,1H),7.09(d,J=10.0Hz,1H),7.01(t,J=8.5Hz,1H),4.75-4.52(m,4H),4.39(d,J=6.1H z,2H),3.60-3.40(m,8H),3.06(dd,J=12.5,6.6Hz,2H),2.74(t,J=12.0Hz,2H),2.44- 2.25(m,7H),1.70-1.60(m,6H),1.58-1.50(m,2H),1.39(br.s,2H).HRMS(ESI-TOF):[C 27 H 41 FN8O3+H] + , measured value 545.3378; calculated value 545.3358.

[0217] Example Compound 006: 3-((4-(3-(4-benzylpiperidin-1-carbonyl)piperidin-1-yl)-6-((3-(4-fluoro Phenyl)propyl)amino)-1,3,5-triazin-2-yl)amino)propionic acid (006)

[0218]

[0219] The requisite amine (4-benzylpiperidin-1-yl)(piperidin-3-yl)methanone was prepared using the 2-nitrobenzenesulfonyl (2-Ns) strategy starting from ethyl (±)-3-piperidincarboxylate and 4-benzylpiperidine via 2-Ns-3-piperidincarboxylic acid and its acyl chloride, followed by removal of 2-Ns as described for compound (005) and used without chromatographic purification. 1 H NMR (400 MHz, CDCl3) δ 7.37-7.06 (m, 5H), 4.59 (d, J = 13.0 Hz, 1H), 3.91 (d, J = 13.1 Hz, 1H), 3.73 (br.s, 2H), 3.18-2.40 (m, 8H), 1.93-1.47 (m, 7H), 1.21-1.05 (m, 2H), about 90% purity.

[0220] 4-Benzylpiperidin-1-yl)(1-(4-chloro-6-((3-(4-fluorophenyl)propyl)amino)-1,3,5-triazin-2-yl)piperidin-3-yl)methanone was prepared using the same one-pot procedure from cyanuric chloride (231 mg, 1.25 mmol), the previous crude amine (400 mg) and finally 3-(4-fluorophenyl)propan-1-amine (191 mg, 1.25 mmol) in the presence of DIPEA (348 mg, 2.70 mmol, total). Yield 250 mg (32%).1 H NMR (400 MHz, CDCl3) δ 7.34-7.08 (m, 7H), 6.98 (t, J = 7.9 Hz, 2H), 5.67-4.93 (br. m, 1H), 4.88-4.53 (m, 3H), 4.11-3.84 (m, 1H), 3.50-3.25 (m, 2H), 3.10-2.80 (m, 3H), 2.70-2.45 (m, 6H), 1.99-1.70 (m, 9H), 1.20-1.12 (br. m., 2H). Mixture of rotamers.

[0221] 3-((4-(3-(4-benzylpiperidine-1-carbonyl)piperidin-1-yl)-6-((3-(4-fluorophenyl)-propyl)amino)-1,3,5-triazin-2-yl)amino)propanoic acid methyl ester was prepared as described for compound 002 from the previous triazinyl chloride (240 mg, 0.436 mmol), β-alanine methyl ester hydrochloride (160 mg, 1.14 mmol), DIPEA (207 mg, 1.60 mmol). Yield 155 mg (58%), white amorphous solid. 1 H NMR (400 MHz, CDCl3) δ 7.34-7.10 (m, 7H), 7.01-6.94 (m, 2H), 5.14 (br. s, 1H), 4.92-4.58 (m, 4H), 4.08-3.96 (m, 1H), 3.71 and 3.69 (s, total 3H), 3.69-3.61 (m, 2H), 3.45-3.31 (m, 2H), 3.03-2.82 (m, 2H), 2.80-2.43 (m, 9H), 1.94-1.68 (m, 9H), 1.24-1.12 (m, 2H). Mixture of rotamers.

[0222] The title compound (006) was prepared by acidic hydrolysis of the previous methyl ester (140 mg, 0.226 mmol) as described above. Yield 90 mg (67%), white amorphous solid. 1H NMR (400MHz, DMSO-d6, 100℃) δ8.0-7.5(br,1H),7.32-7.21(m,7H),7.06(t,J=8.7Hz,2H),4.51(t,J=11.2Hz,2H),4.13(s,2H),3.54(t,J=6.4Hz,2H ),3.35(br.t,J=5.6Hz,2H),3.11-2.91(m,2H),2.83-2.73(br,2H),2.66( t,J=7.5Hz,2H),2.58-2.45(m,7H),1.94-1.44(m,9H),1.18-1.12(m,2H). HRMS(ESI-TOF):[C 33 H 42 FN7O3+H] + , measured value 604.3435; calculated value 604.3406.

[0223] Example Compound 004: 1-(4-(bis(2-hydroxyethyl)amino)-6-((3-fluorobenzyl)amino 4-(4-bromo-3-(trifluoromethyl)phenyl)piperidine-2-carboxamide (004)

[0224]

[0225] The requisite amine N-(4-bromo-3-(trifluoromethyl)phenyl)piperidine-2-carboxamide was prepared using the 2-nitrobenzenesulfonyl strategy starting from (±)-piperidine-2-carboxylic acid ethyl ester and N-(4-bromo-3-(trifluoromethyl)aniline, which is closely related to compound 005. Cream-colored amorphous solid. 1 H NMR (400MHz, CDCl3) δ9.13(br.s,1H),7.91(d,J=2.5Hz,1H),7.75(dd,J=8.7,2.4Hz,1H),7.64(d,J=8.7Hz,1H),3.39(dd,J=9.3,3. 6Hz,1H),3.06(dt,J=11.5,3.6Hz,1H),2.85-2.75(m,1H),2.09-1.98(m,1H),1.93-1.80(m,1H),1.72(br.s,1H),1.67-1.42(m,5H).

[0226] N-(4-bromo-3-(trifluoromethyl)phenyl)-1-(4-chloro-6-((3-fluorobenzyl)amino)-1,3,5-triazin-2-yl)piperidine-2-carboxamide was prepared according to the one-pot procedure (002) mentioned above from cyanuric chloride (185 mg, 1.00 mmol), N-(4-bromo-3-(trifluoromethyl)phenyl)piperidine-2-carboxamide (420 mg, 1.20 mmol), then 3-fluorobenzylamine (139 mg, 1.10 mmol) in the presence of DIEPA (322 mg, 2.5 mmol total). Yield 320 mg (54%), beige foam. 1 H NMR (400 MHz, CDCl3) δ 8.7 and 8.1 (br.s, 1H), 7.95-7.70 (br.m, 1H), 7.66-7.58 (m, 2H), 7.48-7.29 (m, 1H), 7.25-6.24 (br.m, 4H), 5.62-5.32 (br.m, 1H), 4.82-4.49 (m, 3H), 3.02-2.87 (m, 1H), 2.45-2.30 (m, 1H), 2.00-1.43 (m, 5H). Mixture of rotamers.

[0227] The title compound (004) was prepared from the previous compound (280 mg, 0.478 mmol), diethanolamine (106 mg, 1.00 mmol) and DIEPA (140 mg, 1.09 mmol) in 1,4-dioxane (10 mL) as described for 005. Yield 102 mg (33%), white amorphous solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.3-10.2 (br.m, 1H), 8.20 and 8.05 (br.s, 1H), 7.91-7.56 (m, 2H), 7.48-6.65 (m, 5H), 5.32 (d, J = 61.8 Hz, 1H), 4.75-4.38 (br.m, 4H), 3.72-3.32 (m, 10H), 3.30-3.02 (br.m, 1H), 1.80-1.55 (m, 3H), 1.45-1.29 (m, 2H). Mixture of rotamers. HRMS (ESI-TOF): [C 27 H 30 F3N7O3+H] + , measured value 658.1625; calculated value 658.1582.

[0228] Example Compound 003: (1-(4-(bis(2-hydroxyethyl)amino)-6-((3-fluorobenzyl)amino)-1,3,5- Triazine-2-yl)piperidin-2-yl)(morpholino)methanone (003)

[0229]

[0230] The requisite amine morpholino(piperidin-2-yl)methanones were prepared from (±)-N-Boc-piperidine-2-carboxylic acid and morpholine using a standard mixed anhydride protocol (IBCF, NMM, DCM; column chromatography).

[0231] (1-(4-Chloro-6-((3-fluorobenzyl)amino)-1,3,5-triazin-2-yl)piperidin-2-yl)(morpholino)methanone was prepared according to the one-pot procedure (002) mentioned above from cyanuric chloride (185 mg, 1.00 mmol), the previous amine (220 mg, 1.11 mmol), then 3-fluorobenzylamine (139 mg, 1.10 mmol) in the presence of DIPEA (322 mg, 2.5 mmol total). Yield 165 mg (38%), white foam. 1 H NMR (400 MHz, DMSO-d6) δ 8.5-8.1 (four t, 1H), 7.43-7.32 (m, 1H), 7.20-6.99 (m, 3H), 5.45-5.35 (m, 1H), 4.77-4.22 (m, 3H), 3.74-3.07 (m, 14H), 1.96-1.28 (m, 6H). A mixture of four rotamers.

[0232] The title compound (003) was prepared from the previous compound (155 mg, 0.357 mmol), diethanolamine (106 mg, 1.00 mmol) and DIEPA (140 mg, 1.09 mmol) in 1,4-dioxane (10 mL) as described for 005. Yield 108 mg (60%), white amorphous solid. 1 H NMR (400MHz, DMSO-d6) δ7.37-7.28 (m, 1H), 7.19-6.97 (m, 4H), 5.43 (br.s, 1H), 4.78-4.20 (m, 5H), 3.74-3.06 (m, 17H), 1.94-1.21 (m, 6H). Mixture of rotamers. HRMS (ESI-TOF): [C 24 H 34 FN7O4+H] + , measured value 504.2747; calculated value 504.2729.

[0233] Example Compound 001: (R)-1-(4-(bis(2-hydroxyethyl)amino)-6-((3-fluorobenzyl)amino)-1,3, 5-Triazine-2-yl)-N,N-diethylpyrrolidine-2-carboxamide (001)

[0234]

[0235] The requisite amine L-Pro-NEt2 was prepared from Boc-L-Pro-OH and diethylamine using a standard mixed anhydride protocol (IBCF, NMM, DCM; crystallization from Et2O / hexanes).

[0236] The title compound (001) was prepared from N-(4-chloro-6-(3-fluorobenzylamino)-1,3,5-triazin-2-yl)diethanolamine (as described above for compound 002, 171 mg, 0.50 mmol) and the previous amine as described in this case (170 mg, 1.00 mmol). Yield 115 mg (48%), white amorphous solid. 1 H NMR (400MHz, DMSO-d6, 100℃) δ7.32(td,J=7.9,6.1Hz,1H),7.18-7.02(br.m,2H),6.98(td,J=8.4,2.2Hz,1H),7.0-6.4(br.,1H),4.80(dd,J=8.6 ,3.4Hz,1H),4.46(d,J=6.4Hz,2H),4.33(br.s,2H),3.72-3.14(m,14H) ,2.24(dq,J=12.1,8.2Hz,1H),2.03-1.71(m,3H),1.29-0.96(br.s,6H). HRMS(ESI-TOF):[C 23 H 34 FN7O3+H] + , measured value 476.2796; calculated value 476.2780.

[0237] Example 2: Bioassay

[0238] Materials and methods

[0239] Luciferase reporter gene assay

[0240] HEK293E1a cells were cultured at 1.2 x 10 cells per well in DMEM medium containing 10% fetal bovine serum. 4The density of 100 μg / mL was seeded on a 48-well plate. After 24 hours, cells were transfected with a mixture of plasmids (300 ng / well) containing a specified amount of p6xPORE-luc reporter gene, pEV-OBF1 and pCG-Oct1 effectors, pBluescript II KS+ vectors and pCMV-βgal (for standardization) as described above. Transfection was performed in three biological replicates using a 2:1 ratio of polyethyleneimine (PEI 25K, Polysciences Inc.) to DNA. After 48 hours, cells were washed with PBS, lysed, and luciferase activity was measured using the Bright-Glo luciferase assay system (Promega), and β-galactosidase activity was measured in a standard assay.

[0241] Protein expression and purification

[0242] Using human cDNA as template, PCR amplified DNA sequences encoding the POU domains of OCT1 (POU1) and BOB1. The following primers were used for amplification:

[0243] -5'-TTAGGATCCGAGGAGCCCAGTGACCTTGA-3' (SEQ ID NO: 4) -POU1 forward,

[0244] -5'-GCAAAGCTTAGTTGATTCTTTTTTCTTTCTGGCGG-3' (SEQ ID NO: 5)-POU1 reverse;

[0245] -5'-TTAGGATCCATGCTCTGGCAAAAACCCAC-3' (SEQ ID NO: 6) -BOB1 forward,

[0246] -5'-GCAAAGCTTCTAAAAGCCTTCCACAGAGAGAGT-3' (SEQ ID NO: 7)-BOB1 reverse.

[0247] The amplified fragment was cut with BamHI and HindIII and connected to the pET28 vector. The resulting construct contains an N-terminal histidine tag in the frame. Protein expression was induced with 1mM IPTG in E. coli BL21 cells for 4 hours. Cells were harvested by centrifugation and cracked in buffer A1 (8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl pH8.0, 5mM β-mercaptoethanol). Cell lysate was applied to Ni-NTA His bound resin (Novagen), washed with buffer A2 (8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl pH 6.3, 5mM β-mercaptoethanol), and eluted with buffer A3 (8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl pH 4.5, 5mM β-mercaptoethanol). The protein sample was dialyzed in four steps with buffer, with each step decreasing the urea concentration by two-fold and the pH increasing gradually. The final dialysis step was performed for 12 h in PBS containing 5 mM β-mercaptoethanol. The POU1 domain was additionally purified using a Hi-Trap heparin agarose column. The column was pre-equilibrated with buffer B (20 mM HEPES pH 7.6, 100 mM NaCl, 1 mM EDTA), the sample was loaded onto the column, and then eluted with a linear gradient of NaCl from 100 mM to 500 mM in the same buffer. BOB1 was further purified on a Superdex 75 column pre-equilibrated in PBS containing 5 mM β-mercaptoethanol.

[0248] Electrophoretic mobility shift assay (EMSA)

[0249] By annealing the oligonucleotide in 10 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM MgCl2 buffer: 5′-biotin-AAGTTAAAATCAC ATTTGAAATGCAAAT GGAAAAGCAAGGCCCG-3′ (SEQ ID NO:8), and 5′-CGGGCCTTGCTTTTCC ATTTGCATTTCAAATGTGATTTTAACTT-3′ (SEQ ID NO: 9) (Evrogen, Moscow), and then the solution was heated to 95°C for 5 minutes and then slowly cooled to room temperature in a water bath to prepare a dsDNA probe containing biotinylated PORE (underline). The biotinylated PORED dsDNA probe (20 ng) was mixed with purified protein samples (500 ng of BOB1, 400 ng of OCT1) in 10 μL of binding buffer (the binding buffer contained Tris-HCl pH 7.6, 10% glycerol, 0.14 M NaCl, 0.5 mM EDTA, 0.1 mg / mL bovine serum albumin (BSA), 0.1 mg / mL poly (dI-dC) (Amersham), 5 mM DDT and 1× protease inhibitor cocktail (Complete, Roche)), and then incubated at room temperature for 20 minutes. Samples were separated by electrophoresis in 6% polyacrylamide gels in 0.5× TBE buffer and transferred to nitrocellulose membranes. The membranes were blocked with 2.5% BSA in PBS for 30 min, incubated with 1:10,000 dilution of streptavidin-HRP (Sigma) in blocking buffer for 1 h at room temperature, washed with blocking buffer for 30 min, and washed twice with PBS-0.1% Tween-20 for 30 min each. Chemiluminescence was visualized using SuperSignal West Pico Chemiluminescent Substrate (ThermoScientific).

[0250] Thermal Shift Assay (TSA)

[0251] POU1 and BOB1 proteins (each at a final concentration of 5 μM), compound 005 (final concentration of 10 μM), and 1xProteOrange fluorescent dye (Lumiprobe, Russia) were mixed in triplicate in 100 μL of 0.1 M phosphate buffered saline (pH 7.4) and pipetted into a 96-well 0.2 mL thin-walled PCR plate (Roche LifeScience, Switzerland) sealed with an optical grade sealing film. Fluorescence data are presented in The fluorescence emission signals were collected on a 96 real-time PCR instrument (Roche Life Science, Switzerland) with an excitation range of 470-533 nm. The fluorescence emission signals at 533-5572 nm were measured for data analysis. The temperature was maintained at 30 seconds / degree, from 37°C to 98°C (heating rate was about 0.5°C / min). The derivative of the fluorescence signal with temperature change was used for further analysis.

[0252] Kd measurement

[0253] Using Varioskan TMThe binding of compound 005 to POU1 protein was studied using a LUX multimode microplate reader (Thermo Fisher Scientific, USA). The emission spectra were recorded at a temperature of 298.15 K and an excitation wavelength of 280 nm in the wavelength range λ = 310-450 nm with an accuracy of ±0.3 K. The POU1 concentration was 3 μM, while the compound 005 concentration was in the range of 3-39 μM with a step size of 6 μM. The calculations were based on three parallel measurements.

[0254] Primary B cell purification and enrichment

[0255] After obtaining written consent, blood samples were collected from the Sanquin Blood Supply Foundation (Amsterdam, The Netherlands) or the Etablissement Blood samples were obtained from healthy volunteers provided by Dr. du Sang (Nantes, France). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque gradient centrifugation on Lymphoprep (Nycomed). Total B cells were isolated from freshly purified PBMCs by negative selection on magnetic columns (B Cell Isolation Kit II; Miltenyi Biotech, 130-091-151) using an autoMACS Pro separator according to the manufacturer's instructions. Subsequently, CD19 B cells were isolated from negatively selected CD27 B cells by positive and negative selection using CD27 microbeads (Miltenyi Biotech, 130-051-601) using an autoMACS Pro separator according to the manufacturer's instructions. + Finally, MACS-isolated CD19 + B cells, CD19 + CD27 + IgD - Memory B cells and CD19 + CD27 - IgD + Purity of memory B cells: B cell purity>90%, memory B cell purity>65%, and naive B cell purity>85%. All cell cultures were performed in RPMI1640 medium (Thermo Fisher Scientific) supplemented with 10% FCS, L-glutamine, and penicillin / streptomycin. Cells were incubated at 37°C and 5% CO2 atmosphere.

[0256] Cell proliferation dye (CPD) labeling

[0257] The cells were resuspended in PBS, labeled with the cell proliferation dye eFluor450 (CPD) (eBioscience) at a final concentration of 10 μM for 10 minutes at 37°C, kept on ice for 5 minutes, and washed twice with 10 mL of ice-cold RPMI 1640 10% FCS medium.

[0258] In vitro plasmablast differentiation and Ig production

[0259] B cells were stimulated to differentiate into plasmablasts using a two-phase cell culture protocol as described previously. Briefly, B cells were cultured at 4 × 10 5 Cells / mL were seeded in 24-well plates and activated with 50 ng / mL of recombinant human soluble CD40L (R&D Systems), 2.5 μg / mL of CpG oligodeoxynucleotide (ODN) 2006 (Amidogen or Cayla Invivogen), 2 μg / mL of F(ab′)2 fragment goat anti-human IgA+IgG+IgM (H+L) polyclonal antibody (anti-BCR) (Jackson ImmunoResearch Laboratories) and 50 U / mL of human IL-2 (SARL Pharmaxie or Novartis) within 4 days. The activated B cells on day 4 were washed and cultured for another three days in fresh medium containing 50 U / mL of human IL-2, 10 ng / mL of human IL-4 (R&D Systems) and 10 ng / mL of human IL-10 (R&D System) at the same concentration to induce B cell differentiation into plasmablasts. On days 0, 4, and 7, B cells were stained with the viability marker LIVE / DEAD Fixable Aqua Dead Cell Stain (Thermofisher) to identify dead cells, and then extracellularly stained with anti-human mAbs: anti-CD20 FITC, anti-IgD BUV395, anti-CD27 BUV737, anti-CD38 BV605 (all from BD Biosciences), and analyzed by flow cytometry (FACS Celesta). TM In some experiments, B cell apoptosis was assessed by intracellular active caspase-3 staining (caspase-3-PE, BD Biosciences).

[0260] Immunoglobulin subtypes were quantified using the Human Immunoglobulin Typing LEGENDplex 8-plex Kit (BioLegend) according to the manufacturer's instructions. TMData were collected on an ELISA II flow cytometer and analyzed using LEGENDplex data analysis software.

[0261] High-throughput multiplex qPCR

[0262] mRNA was extracted from freshly thawed cell lysates using the RNEasy micro kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using oligo-dT and Omniscript RT (Qiagen) according to the manufacturer's instructions. TMThe gene expression levels of 46 B cell-related genes were evaluated by qRT-PCR using HD system and 46.48GE Dynamic Array (Fluidigm) according to the manufacturer's protocol and the following gene expression assays (all from ThermoFisher): AICDA (Hs00221068_m1), B2M (Hs99999907_m1), BACH2 (Hs00222364_m1), BCL2L1 (Hs00236329_m1), BCL6 (Hs00153368_m1), CCL3 (Hs00234142_m1), CCND1 (Hs00765553_m1), and CCND2 (Hs00153369_m1). 3380_m1), CD27(Hs00386811_m1), CD38(Hs01120071_m1), CD40(Hs003741 76_m1), CD79a (Hs00233566_m1), CD80 (Hs01045163_m1), CD86 (Hs9999910 4_m1), Ciita(Hs00172106_m1), CXCR4(Hs00607978_s1), ERN1(Hs0017638 5_m1), ETS1(Hs00428293_m1), EZH2(Hs00172783_m1), HPRT1(Hs02800695 _m1), ID2(Hs04187239_m1), ID3(Hs00171409_m1), IgHG1(Hs00378340_m1 ), IgHG3(Hs00941519_g1), IGHM(Hs00378435_m1), IL2RA(Hs00907779_m1 ), IL6(Hs00174131_m1), IRF4(Hs00180031_m1), IRF8(Hs01128710_m1), M EF2C(Hs00231149_m1), MKI67(Hs01032443_m1), MS4A1(Hs00544819_m1), MYC(Hs00153408_m1), PAX5(Hs00277134_m1), POU2AF1(Hs01573371_m1), POU2F2(Hs00922172_m1), PRDM1(Hs00153357_m1), RELA(Hs00153294_m1) , RELB(Hs00232399_m1), SDC1(Hs00896423_m1), SLAMF1(Hs00234149_m1), SPI1(Hs02786711_m1), SPIB(Hs00162150_m1), STAT3(Hs01047580_m1),SYK (Hs00895377_m1), TLR4 (Hs00370853_m1), TLR9 (Hs00152973_m1), XBP1s (Hs03929085_g1). The data were normalized using the geometric mean and δ / δCt methods of two housekeeping genes (HPRT1, B2M). Unsupervised hierarchical gene clustering based on Euclidean distance and full linkage was generated using the pheatmap (v1.0.12) package in R (v4.2.2).

[0263] GZM + B cell expansion and T cell suppression assays

[0264] Primary total human B cells (1×10 ) were cultured in the presence of vehicle (DMSO) or 10 μM of compound 005 using a previously described mixture containing 10 ng / mL IL-21 (Miltenyi Biotec), 5 μg / mL F(ab′)2 anti-BCRAbs (Jackson ImmunoResearchLaboratories Inc.), 50 ng / mL CD40L (R&D systems), 1 μg / mL CpG oligonucleotide (ODN 2006, Invivogen), and 50 U / mL IL-2 (Novartis). 6 cells / mL) for about 24 hours. On day 3, cells were stained with fixable viability dye (FVD) eFluor 450 to identify dead cells, and then stained for CD19 and intracellular granzyme B (GZMB) to evaluate B cell GZMB expression. The ability of B cells to inhibit the proliferation of effector T cells was assessed as previously described. For autologous T cell isolation, PBMCs from the same donor were maintained in culture medium overnight at 4°C. The next day, CD4+ T cells were isolated by negative selection (Human CD4+ T Cell Isolation Kit, Miltenyi Biotech, 130-096-533) on a magnetic separation column using an autoMACS Pro separator according to the manufacturer's instructions. Subsequently, CD25 microbeads (Miltenyi Biotech, 130-092-983) were used to separate CD4+ T cells from CD4+ T cells using an autoMACS Pro separator. + CD25 depletion in T cells + T cells to obtain CD4 + CD25 - The B cells were then washed, resuspended in fresh medium, and incubated with autologous CPD-labeled CD4 + CD25- Effector T cells were co-cultured for three days. A total of 1 × 10 T cells were co-cultured in a 96-well U-bottom plate in a total volume of 150 μL / well. 5 B cells and 0.5×10 5 T cell proliferation was evaluated as the percentage of low CPDT cells on day 3.

[0265] ScRNA sequencing

[0266] B cells from 2 healthy volunteers were isolated using the Human B Cell Isolation Kit II. Isolation was performed on an AutoMACS Pro separator according to the supplier's instructions. + Bregs were incubated with 50 U / mL rhIL-2 (Proleukine-Novartis); 10 ng / mL rhIL-21, R&D systems; 5 μg / mL goat anti-human IgG+A+M(H+L)F(ab)'2 (Jackson ImmunoResearch); 1 μg / mL CpG oligodeoxynucleotide ODN 2006-Invivogen); and 50 ng / mL recombinant human soluble CD40L (R&D systems) in complete medium at 37°C in a 5% CO2 incubator in 6-well plates at 5.10 °C. 6 Cells / 5mL / well were induced. At the end of the culture, cells were labeled with a viability dye (fixable viability dye eFluor450, 1 / 1000 in PBS without azide or protein, Invitrogen) for 25 minutes. Live cells were sorted on an ARIA III (BD Biosciences). According to the CITE-seq protocol (https: / / www.biorxiv.org / content / 10.1101 / 237693v1), live cells were labeled with anti-β2-microglobulin or anti-CD-293 coupled DNA sequences (HashTag oligonucleotides, HTO). Cells were then pooled at equal concentrations and a total of 20,000 cells were sequenced on the GenoBird platform (IRS-UN, CHUNantes) on the Nova-Seq 6000 (Illumina). Data were analyzed using R, Rstudio, Seurat v 4.0.2, and Graphpad Prism.

[0267] T cell recall assay

[0268] Total PBMCs were collected at 0.5 × 10 6Cells / well were plated into 96-well round-bottom plates and stimulated for 6 hours with anti-CD3 / CD28 Dynabeads (Gibco Life Technologies) at a bead / cell ratio of 0.2:1 in the presence of compound 005 (10 μM) or vehicle (DMSO) to induce polyclonal primary responses. To induce recall responses, cells were stimulated with beads for two days, allowed to rest for eight days in medium supplemented with IL-2 (10 U / mL), and restimulated for a final 6 hours with anti-CD3 / CD28 beads in the presence of compound 005 (10 mM) or vehicle. The expression of Th1-, Th2-, Th17-, and Treg-related cytokines and transcription factors was assessed by qPCR. For intracellular staining, cells were restimulated for 10 hours with anti-CD3 / CD28 beads, and Brefeldin A (10 μg / mL, Sigma) was added to the culture medium 4 hours after the start of restimulation. Using LIVE / Dead cells were excluded using a fixable aqueous dead cell stain kit (Life Technologies). Cells were then surface stained with CD3-BUV395, CD4-BUV737, CD8-Violet650 (all three from BD Horizon), and CD161-PE-Cy7 (from Biolegend) for 30 minutes at 4°C, then incubated with fixation / permeabilization working solution (eBioscience), washed with permeabilization buffer, and stained with IL-17A-PE and IFNγ-BV711 (both from BD Biosciences). Cells were resuspended in ice-cold FACS buffer (0.5% BSA, 2 mM EDTA, and 50 U / mL of penicillin / streptomycin in PBS). TM Flow cytometry data were collected and analyzed using FlowJo TM The cells were analyzed using FlowJo software (FlowJo, LLC).

[0269] qRT-PCR

[0270] Total RNA was extracted from PBMCs using the RNeasy micro kit (Qiagen) according to the manufacturer's protocol. RNA was processed for cDNA synthesis (Fermentas) and analyzed by qPCR and TaqMan Gene Expression Assays using the Real-Time PCR QuantStudio3 PCR System (Applied Biosystems) for IFNG (Hs00989291_m1), IL-2 (Hs00174114_m1), TNF (Hs00174128_m1), TBX21 (Hs00203436_m1), IL-4 (Hs00174122_m1), GATA3 (Hs00231122_m1), IL-17A ( Hs00174383_m1), IL-17F (Hs00369400_m1), IL-21 (Hs00222327_m1), IL-22 (Hs01574154_m1), RORC (Hs01076122_m1), IL-10 (Hs00961622_m1), FOXP3 (Hs01085834_m1), POU2AF1 (Hs01573371_m1) (Thermofisher). The expression of all genes was normalized to the expression of GAPDH (4310884E), a housekeeping gene, and expressed as fold change relative to the reference sample.

[0271] Statistical analysis

[0272] One-way ANOVA with Greenhouse-Geisser correction was used to analyze differences between groups, followed by Bonferroni correction for multiple comparisons (p<0.05). Paired ratio tests were performed for in vitro PBMC stimulation assays (p<0.05). Values ​​are expressed as mean and standard deviation (SD) or median and IQR range according to the criteria for (non)parametric analysis. Statistical tests were performed using Prism version 7 software (GraphPad Software).

[0273] result

[0274] Compound 005 antagonizes BOB1 / OCT1-dependent transcriptional activation

[0275] BOB1 is described as a coactivator that works in concert with the POU domain proteins OCT1 and OCT2, which bind to different DNA motifs in monomeric or dimeric configurations. A class of motifs in these motifs (called palindromic Oct recognition elements, or POREs) has been shown to act as a platform for OCT1 dimer assembly in a specific dimer configuration that allows BOB1 recruitment, thereby providing transcriptional activation. As an initial functional assay, we examined the ability of compounds according to the present invention to affect BOB1-OCT1-PORE-mediated transcriptional activation in transient transfection luciferase assays. To this end, we utilized previously generated 6xPORE-luc and reporter plasmids, in which the hexamer of PORE was cloned upstream of the thymidine kinase (tk) minimal promoter and luciferase gene. In preliminary tests, we found that transfection of HEK293 cells with BOB1 expression plasmids alone can drive luc reporter gene transcription in a dose-dependent manner (data not shown). Furthermore, addition of a plasmid expressing exogenous OCT1 resulted in an inhibitory effect on both basal and BOB1-mediated transcription (data not shown). This observation is likely due to the fact that HEK293 cells, like all other mammalian cell types, express OCT1, and further addition of exogenous OCT1 to these cells shifts the BOB1 / OT1 balance away from the optimal range, resulting in a competitive inhibition (squelching effect) and, in our experimental setting, a stimulatory effect of approximately 3-fold compared to endogenous OCT1 alone ( Figure 1A Among the six synthesized compounds, Compound 005, Compound 006, and Compound 004 at a concentration of 10 μM in the culture medium significantly attenuated BOB1 / OCT1-driven transcription to the level mediated by endogenous OCT1, while the other compounds showed no inhibitory activity at this concentration ( Figure 1A Compound 005 was selected for further detailed study. We have demonstrated that compound 005 inhibits transcriptional activation in a dose-dependent manner over a concentration range of 0.5 μM to 40 μM ( Figure 1B The inhibitory effect of Compound 005 was readily observed in the MTT assay starting at a concentration of 10 μM and at higher concentrations, while it remained nontoxic or mildly toxic to HEK293 cells up to 40 μM, whereas Compound 005 showed toxicity only at 80 μM and above for NIH3T3 cells and mouse embryonic fibroblasts (MEFs) (data not shown).

[0276] Compound 005 binds to OCT1 in vitro

[0277] To assess whether compound 005 binds to OCT1 in vitro, we expressed and purified 6xHis-tagged full-length BOB1 protein and the POU domain of OCT1 (POU1), which has been previously shown to mediate this interaction, in bacteria. Two tagged proteins with predicted molecular weights of 31 kDa and 22 kDa for BOB1 and POU1, respectively, were expressed, purified to near homogeneity using Ni-NTA resin, and refolded ( Figure 2A Subsequently, their ability to form ternary complexes was verified by electrophoretic mobility shift assay (EMSA) using dsDNA containing biotin-labeled PORE as a probe. POU1 forms both monomeric and dimeric complexes on this sequence and migrates significantly toward the latter ( Figure 2B , Lane 2). In the presence of BOB1, a ternary complex is formed ( Figure 2B , lane 3). Preliminary tests indicated that bacterially produced POU1 and BOB1 were properly refolded and acquired at least some of their biochemical activities. However, unexpectedly, the presence of compound 005 in the binding reaction, even at a low concentration (100 μM), did not affect the ternary BOB1 / POU1 / PORE complex ( Figure 2B , lane 5), suggesting that the EMSA conditions do not allow compound 005 to bind or that the mode of action of compound 005 does not involve disruption of the ternary complex, possibilities that are discussed below.

[0278] To determine the binding constant (Kb) and number of binding sites (n) of compound 005 in complex with POU1, the dependencies were plotted in the Hill coordinate system:

[0279]

[0280] The Kd value obtained is 10 -9 , which is a typical value for drug-target interactions, confirming that compound 005 is able to form a complex with POU1 in solution. The Gibbs energy change was calculated based on the Kb value. The negative value of ΔG obtained indicates that the binding is thermodynamically favorable ( Figure 2C Next, thermal shift assay (TSA) showed that the addition of compound 005 increased the melting temperature of POU1 by approximately 4°C ( Figure 2D ).

[0281] In summary, the results obtained provide a convincing argument that compound 005 binds OCT1 in vitro with an affinity typical of drug-target interactions. This set of data also provides a solid biochemical rationale for further investigation of the biological effects of compound 005 in vivo.

[0282] Compound 005 inhibits T-dependent B cell terminal differentiation in a dose-dependent manner

[0283] Previous studies have shown that the lack of functional BOB1 in T cell-dependent responses leads to profound functional consequences, such as deficient germinal center formation, blocked plasma cell differentiation, and severely reduced production of T cell-dependent antigen subtype-switched antibodies in vitro and in vivo. Therefore, we set out to investigate whether and how interference with BOB1 function by Compound 005 affects T cell-dependent B cell differentiation using a previously established in vitro model. This two-step human B cell differentiation model combines BCR signaling, TLR activation, T cell help, and cytokines to mimic the molecular dynamics of germinal center B cell maturation in vivo. During the activation phase (days 0-4), B cells were first cultured with F(ab′)2 anti-Ig (A+G+M), CD40L, CpG, and IL-2. On day 4, activated B cells were washed and then replated with IL-2, IL-4, and IL-10 in the absence of CD40L. Due to the association with CD27 - Compared with naive B cells, CD27 + Memory B cells have an intrinsic capacity to rapidly terminally differentiate into plasma cells, and we focused on this cell population. We analyzed the effects of compound 005 inhibition of BOB1 on B proliferation capacity at day 4 and differentiation into CD20 at day 7. lo CD38 hi Analysis of flow cytometry data showed that during the activation phase, the proliferation burst was distinct from that of most cells cultured under control conditions (DMSO) (mean ± SD 80.9 ± 9.7%) as assessed by dilution of cell proliferation dye on day 4 ( Figure 3A ). Control B cells acquired expression of the activation and maturation marker CD38 (data not shown), resulting in CD38 hi The precursor appears and in the second stage of the model, it is directed to CD20 lo CD38 hi Plasmablast differentiation (mean ± SD 74.8 ± 14.9%) ( Fig. 3I ). Compared with control B cells, the addition of compound 005 to the cell culture significantly inhibited the proliferation ability of B cells in a dose-dependent manner (5μM, 10μM, 15μM and 20μM of compound 005 were 66.3±4.8% (P=0.0063), 46.7±18.3% (P=0.0003), 34.9±19.2% (P=0.002), 20.4±19.0% (P=0.0002) respectively) ( Figure 3B We also observed a similar dose-dependent inhibitory effect of compound 005 on CD38 marker expression, resulting in CD38 +The mean expression and frequency of B cells decreased during the activation and differentiation stages (on day 4, the mean ± SD of the control group was 58.4 ± 18.4%, while the mean ± SD of 5 μM, 10 μM, 15 μM and 20 μM of compound 005 were 30.5 ± 25.6% (P = 0.069), 23.9 ± 18.3 (P = 0.0001), 4.3 ± 3.7% (P = 0.0019) and 2.5 ± 2.2 (P = 0.0003), respectively) ( Figure 3E -F) and (On day 7, the mean ± SD of the control group was 73.6 ± 13.9%, while the mean ± SD of 5 μM, 10 μM, 15 μM and 20 μM of compound 005 were 68.9 ± 10.2% (P = 0.7224), 55.5 ± 16.1 (P < 0.0001), 26.3 ± 13.9% (P < 0.0001) and 13.0 ± 12.9 (P < 0.0001), respectively) ( Figure 3M -N). In contrast, cells cultured in the presence of 10 μM (81.7 ± 12.4% (P = 0.001)) and 15 μM (85.4 ± 11.7% (P = 0.02)) of Compound 005 showed increased expression of the CD20 marker compared to control cells (69.8 ± 16.8%). Fig.3O -P). Therefore, on day 7, inhibition of BOB1 induced CD20 lo CD38 hi Significant dose-dependent reduction in plasmablastogenesis (median percentage reductions of 30.3% (P<0.0001), 70.5% (P<0.0001), and 85.2% (P<0.0001) for 10 μM, 15 μM, and 20 μM of Compound 005, respectively) ( Figure 3Q ).

[0284] The effect of Compound 005 on CD38 was rather specific, as we did not observe a similar reduction in CD27-positive cells in culture. In particular, only high concentrations of Compound 005 significantly reduced CD27 on day 4. + The frequency of cells (the control group was 29.9±22.5%, while the 15μM and 20μM compound 005 were 9.5±11.7% (P=0.015) and 6.4±6.4 (P=0.0009) respectively) ( Figure 3C -D). However, on day 7, in the presence of 5 μM and 10 μM of compound 005, CD27 +The relative proportion of cells increased, but decreased in cultures containing 20 μM of the inhibitor (36.9 ± 16.5% in the control group, 54.3 ± 15.9% in 5 μM of compound 0.005 (P = 0.037), 53.6 ± 14.2% in 10 μM of compound 005 (P < 0.0001), and 9.4 ± 8.1% in 20 μM of compound 0.005 (P = 0.01) ( Figure 3K -L).

[0285] To further validate the suppression of plasma cell differentiation upon BOB1 inhibition, we next evaluated CD20 lo CD38 hi The dose-dependent reduction in plasmablast frequency in response to Compound 005 inhibition of BOB1-OCT interaction was also associated with a reduction in antibody secretion. Multiplex immunoassays showed that in the presence of 15 μM and 20 μM Compound 005, titers of all immunoglobulin subclasses were strongly inhibited ( Figure 3R -Y). However, compound 005 at 10 μM had a different effect on the immunoglobulin class, as IgG1 ( Figure 3R )、IgG2( Figure 3S )、IgG3( Figure 3T ) and IgG4( Figure 3U ) was significantly reduced, but IgA ( Figure 3V )、IgE( Figure 3W )、IgM( Figure 3X ) and IgD( Figure 3Y ) has not decreased.

[0286] We used CD27 - Similar experiments were repeated with B cells. In this case, CD20 lo CD38 hi The percentage of cells was significantly lower (mean ± SD of 21.5 ± 15.6% for naive B cells and 72.2 ± 15.7% for memory B cells, P < 0.0001), which is consistent with the concept that naive B cells are not easy to differentiate into plasma cells. However, the results obtained showed that compound 005 had a considerable dose-dependent effect on proliferation, CD38 and CD20 expression, and plasmablast differentiation (data not shown).

[0287] To evaluate the transcriptional programs underlying the phenotypic and functional changes during B cell differentiation mediated by Compound 005, we evaluated the expression of B cell-related gene sets by high-throughput multiplex qPCR analysis. The separate clustering of Compound 005-treated and control-treated cells on days 4 and 7 indicated the presence of specific effects and the effects of the compound during the two stages of the plasma cell differentiation model. On day 4, Compound 005-treated B cells were the farthest from the other clusters, indicating that Compound 005 had the most significant effect in targeting the BOB1-OCT1 complex in the activation phase plasma cell differentiation model ( Figure 4A ). Clustering allowed the identification of gene modules associated with B cell identity, plasma cell identity, and B cell activation, and revealed that plasma cell-related genes were suppressed in B cells treated with compound 005. In contrast, genes associated with B cell identity and activation were upregulated. Significantly downregulated were MKI67, MEF2C, and STAT3, which encode proteins essential for B cell proliferation ( Figure 4B -D), genes related to plasma cell differentiation (PRDM1, IRF4, POU2AF1, CD38) ( Figure 4E -I) and isoform conversion (AICDA, IgHG1) ( Figure 4J -M). In contrast, genes associated with B cell identity, including IRF8 and BACH2, were upregulated ( Figure 4N -S).

[0288] To evaluate the effect of BOB1 inhibition on CD20 lo CD38 hi To determine whether the significant effects on cell proliferation and development were due to the induction of caspase-dependent apoptosis, we analyzed the percentage of caspase-3-positive B cells in culture. Consistent with previous reports, only a small fraction of control B cells (approximately 7%) showed caspase-3-positive staining during the activation phase (days 0-4). The presence of compound 005 (even at high concentrations) did not affect the frequency of caspase-3-positive cells ( Figure 5A During the second phase, B cells ceased proliferation and entered apoptosis, resulting in a significant increase in the percentage of the caspase-3-positive population in control conditions (mean ± SD 7.8 ± 2.7% on day 4 ( Figure 5B ), while on the 7th day it was 46.1±14.7%, P<0.0001( Figure 5C)). Surprisingly, the addition of compound 005 did not increase the frequency of caspase-3-positive B cells. In contrast, cells cultured with 5 μM, 10 μM, and 15 μM of compound 005 showed lower levels of cell death apoptosis on day 7, as evidenced by a significant decrease in the percentage of caspase-3-positive B cells (mean ± SD of 5 μM, 10 μM, and 15 μM compound 005 were 33.9 ± 15.5% (P = 0.027), 25.6 ± 7.5% (P = 0.0002), and 30.8 ± 7.9% (P = 0.0005), respectively) ( Figure 5C ). Detailed analysis showed that inhibition of BOB1 reduced CD20 lo CD38 hi Plasmablasts and CD20 + CD38 + / - The sensitivity of both non-plasmablast populations to apoptosis ( Figure 5D -F). Therefore, with increasing concentrations of Compound 005, the frequency of naive B cells increased significantly, resulting in a significantly higher naive B cell / plasmablast ratio ( Figure 5G -I).

[0289] Collectively, these data suggest that compound 005 attenuates the biological activity of the BOB1-OCT1-DNA ternary complex, abrogating B cell activation and differentiation into antibody-secreting plasma cells, the final effectors of the humoral immune response, in a dose-dependent manner.

[0290] Compound 005 does not affect the regulatory function of B cells in vitro

[0291] Different signals during T cell-dependent activation determine whether B cells differentiate into effector cells of the immune response or regulatory B cells with immunosuppressive properties. Therefore, we will next use a previously established in vitro model of human B cell expansion expressing granzyme B (GZMB) with regulatory properties to investigate whether compound 005 targeting the BOB1-OCT interaction can affect the regulatory function of B cells. First, we evaluated whether compound 005 would affect the ability of B cells to express GZMB. To this end, we cultured purified human B cells in an expansion mixture containing F(ab')2 anti-BCR, CpG ODN, CD40L, IL-2, and IL-21 (amplified B) for three days, including 24 hours of pre-amplification or leaving them unstimulated (resting B). 10 μM of compound 005 (or 0.1% v / v DMSO for vehicle control) was added to the B cell cultures every day. Flow cytometric analysis on day 3 showed that amplified B expressed significantly more GZMB protein than resting B ( Fig. 6A -B). The presence of compound 005 did not affect the expression of GZMB ( Fig. 6ASecond, we demonstrated that expanded B cells that had been pre-stimulated with the expansion mixture for 24 h were able to suppress anti-CD3 / CD28-activated autologous CD4 + CD25 - Proliferation of effector T cells (48.8±9.8% vs. 5.4±1.9%, P=0.002) ( Figure 6C -D), which is consistent with previous reports. Figure 6C and Fig.6D As shown, the addition of 10 μM compound 005 during the 24-hour pre-stimulation phase did not affect the inhibitory capacity of expanded B compared to vehicle control. Third, we examined whether long-term inhibition of the BOB1-OCT interaction would affect the inhibitory capacity of expanded B by adding compound 005 (or 0.1% DMSO) to the cultures daily during and after the 24-hour pre-stimulation phase. T cell proliferation analysis showed that expanded B but not resting B effectively inhibited T cell proliferation (48.8±9.8% vs. 5.9±3.7%, P=0.002) ( Fig. 6E -G), similar to previous experiments. Addition of 10 μM of compound 005 inhibitor to T cells alone or to co-cultures of T cells and resting B cells reduced T cell proliferation to approximately 50% (53.7 ± 5.8% and 52.1 ± 7.1%, respectively), indicating that inhibition of BOB1-OCT interaction directly limits T cell proliferation ( Fig. 6E -G). Consistent with these data, addition of 10 μM of compound 005 to co-cultures of T cells and expanded B cells resulted in greater inhibition of effector T cell proliferation than co-cultures with expanded B cells alone (79.7 ± 4.9 vs. 40.2 ± 15.5%, P = 0.01) ( Fig. 6E -G).

[0292] To address the question of why targeting the BOB1-OCT interaction with Compound 005 inhibits B cell differentiation into antibody-secreting plasmablasts but does not affect their ability to become GZMB-secreting cells with regulatory properties, we investigated the expression of BOB1 and its OCT partner in two in vitro models. Consistent with the in vitro inhibitory data demonstrating that BOB1 is required for plasma cell production (Figure 3), expression of the POU2AF1 gene encoding BOB1 increases during plasma cell differentiation (Figure 3). Figure 4A In stark contrast, single-cell RNA sequencing analysis revealed that GZMB, a regulator of + In B cells, POU2AF1 expression is downregulated ( Figure 6H These data further support the idea that BOB1 does not function in Bregs and therefore, targeting the BOB1-OCT interaction does not alter the suppressive potential of B cells.

[0293] Compound 005 inhibits dose-dependent T cell proliferation and selectively suppresses memory Th17 responses

[0294] To confirm and extend the observation that targeting BOB1-OCT interaction effectively inhibits effector T cell proliferation, we cultured CD4 + CD25 - We found that compound 005 inhibited anti-CD3 / CD28-induced T cell proliferation in a dose-dependent manner, with 5 μM, 10 μM, and 15 μM compound 005 producing 18.8±12.5% ​​(P=0.18), 50.5±10.2% (P=0.002), and 77.5±9.2% (P<0.0001) inhibition, respectively. Fig. 7A -B). OCT1 is constitutively expressed in T cells, whereas BOB1 is induced only upon activation. It has been shown that both proteins are selectively required for the generation of CD4 memory T cells and the expression of multiple genes, including Th1-, Th2-, and Th17-type cytokines, upon antigen re-encounter in mice. Therefore, we next determined the consequences of inhibiting the OCT1-BOB1 complex on the expression of Th1-, Th2-, and Th17-related cytokines during primary and secondary (memory) T cell responses in vitro ( Figure 7C -O). Data analysis showed that restimulated T cells showed IFNG ( Figure 7C )、IL-17A( Fig.7I )、IL-17F( Figure 7J )、IL-22( Figure 7L ) and IL-10( Figure 7N ) expression of cytokine encoding genes was significantly increased. After initial activation (6-hour stimulated cells), only the effect of compound 005 on TNF expression was observed, and in the presence of small molecule inhibitors, TNF expression was significantly reduced (data not shown). For restimulated cells, treatment with compound 005 induced a significant decrease in IL-17A (median percentage decreased by 43%, P = 0.002) and IL-17F (median percentage decreased by 27%, P = 0.004), but no decrease in mRNA levels of other cytokines and transcription factors, including the Th-17 lineage-specific transcription factor RORC ( Figure 7M Next, we investigated whether BOB1-OCT1 inhibition would also reduce IL-17A production at the protein level. Based on qPCR data, compound 005 inhibited CD4 + T cells express IL-17A ( Figure 7P ).

[0295] These data support the notion that compound 005 targets the BOB1-OCT1 complex to elicit selective biological responses by inhibiting memory Th17 responses in vitro.

Claims

1. Compounds of formula (I) or a pharmaceutically acceptable salt and / or solvate thereof, in X represents -(CH2) n -, where n is 0 or 1; Y represents -O- or -(CH2) m -, where m is 1 or 2; R 1 and R 2 Each represents H; or R 1 and R 2 Together with the heterocyclic ring to which they are attached, they form a fused phenyl group; R 3 and R 4 Each independently represents H, alkyl, -(CH2) p -NR 9 R 10 , aryl or arylalkyl, Wherein p is an integer from 1 to 5, Where R 9 and R 10 Each independently represents H or (C1-C4) alkyl; or R 9 and R 10 Together with the nitrogen atom to which they are attached they form a heterocycloalkyl group; wherein the aryl or arylalkyl is optionally substituted with at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy; or R 3 and R 4 Together with the nitrogen atom to which they are attached they form a heterocycloalkyl group; wherein the heterocycloalkyl is optionally substituted with at least one aryl or arylalkyl; wherein the aryl or arylalkyl is optionally substituted with at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy; R 5 represents an aryl group or an arylalkyl group; wherein the aryl or arylalkyl is optionally substituted with at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy; R 6 represents H or (C1-C4)alkyl; and R 7 and R 8 Each independently represents H or an alkyl group substituted with at least one COOH or OH.

2. The compound according to claim 1, wherein R 3 and R 4 Each independently represents H, alkyl or -(CH2) p -NR 9 R 10 ; wherein p is an integer in the range of 1 to 5, and wherein R 9 and R 10 Together with the nitrogen atom to which they are attached they form a heterocycloalkyl group, preferably a pyrrolidinyl group.

3. The compound according to claim 1, wherein R 3 represents an aryl group, wherein the aryl group is optionally substituted by at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy group; and R 4 Indicates H.

4. The compound according to claim 1, wherein R 3 and R 4 Together with the nitrogen atom to which they are attached they form a heterocycloalkyl group, wherein the heterocycloalkyl group is optionally substituted by at least one aryl or arylalkyl group, preferably phenyl or benzyl, more preferably benzyl.

5. A compound according to any one of claims 1 to 4, wherein R 5 represents an arylalkyl group, wherein the arylalkyl group is optionally substituted by at least one halogen, (C1-C4)alkyl, (C1-C4)haloalkyl, COOH, OH or (C1-C4)alkoxy, preferably at least one F.

6. A compound according to any one of claims 1 to 5, wherein R 6 Indicates H.

7. A compound according to any one of claims 1 to 6, wherein R 7 and R 8 Each independently represents an alkyl group substituted with at least one OH group.

8. A compound according to any one of claims 1 to 7, wherein R 7 represents an alkyl group substituted with at least one COOH group, and R 8 Indicates H.

9. A compound according to any one of claims 1 to 8, wherein the formula (I) is selected from the group consisting of formula (IA) and formula (IB) in R 1 -R 4 As defined in claim 1; and R 5 Represents phenyl-(CH2) q -, wherein q is an integer in the range of 1 to 5, and wherein the phenyl group is optionally substituted by at least one Br, Cl, F, methyl or CF3, preferably by at least one F.

10. The compound according to any one of claims 1 to 9, wherein the compound is selected from: and pharmaceutically acceptable salts and / or solvates thereof.

11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 10 and at least one pharmaceutically acceptable carrier.

12. A compound according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 11 for use as a medicament.

13. The compound according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11, for use in treating a disease selected from the group consisting of autoimmune diseases, transplant organ rejection, graft-versus-host disease and BOB1-related diseases.

14. A compound for use according to claim 13, wherein the disease is selected from rheumatoid arthritis, type 1 diabetes, multiple sclerosis, primary biliary cirrhosis, end-stage chronic respiratory diseases (such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and pulmonary arterial hypertension (PAH)), germinal center lymphoma and Waldenstrom's macroglobulinemia.

15. A method for producing a compound according to any one of claims 1 to 10, wherein the method comprises: (a) In the presence of a base, The cyanuric chloride reacts with the NH secondary amine functional group in the ring of the compound of formula (A) Steps for obtaining a monosubstituted 1,3,5-triazine of formula (B) (b) in the presence of a base, reacting the compound (B) with a 5 R 6 The step of reacting with an amine to obtain a disubstituted 1,3,5-triazine; and then (c) in the presence of a base, reacting the obtained disubstituted 1,3,5-triazine with a compound of formula NHR 7 R 8 A step of reacting an amine with an amine to obtain a trisubstituted 1,3,5-triazine; or (b) in the presence of a base, reacting the compound (B) with a 7 R 8 The step of reacting with an amine to obtain a disubstituted 1,3,5-triazine; and then (c) in the presence of a base, reacting the obtained disubstituted 1,3,5-triazine with a compound of formula NHR 5 R 6 The step of reacting with an amine to obtain a trisubstituted 1,3,5-triazine; and (d) optionally, at least one step of deprotecting at least one alcohol, carboxylic acid and / or amine group present in the resulting trisubstituted 1,3,5-triazine; Thus, the compound of formula (I) or its pharmaceutically acceptable salt and / or solvate is obtained.

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