Use of a class of cerebrosides as immunosuppressive agents

Abstract

The present application belongs to the field of medicine, and particularly relates to a class of cerebrosides and the use of the pharmaceutical composition thereof as an immunosuppressive agent in the preparation of a medicine for preventing or treating immune-related diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more specifically to the use of a class of cerebroside compounds and their pharmaceutical compositions as immunosuppressants in the prevention or treatment of immune-related diseases. Background Technology

[0002] Cerebrosides (or glycoceramides) belong to the glycosphingolipid class and are mainly found in the animal kingdom, but also in plants and microorganisms. Cerebrosides consist of three parts: a glycosyl group, a fatty acid chain, and a sphingosine chain. Although all compounds in this class are composed of these three parts, they exhibit structural diversity due to variations in the type of sugar, glycosidic bonds, the length and degree of branching of the fatty acid and sphingosine chains, and the number and position of hydroxyl groups and double bonds (J.Nat.Prod.2017,80,6,1734–1741). Different compound structures often result in different types or degrees of pharmacological activity; therefore, cerebroside compounds have been reported to possess varying degrees of antitumor, antiviral, antimicrobial, and neuroprotective activities (Xu Jie, Isolation, Purification, Structural Analysis, and Bioactivity Study of Sea Cucumber Cerebrosides, Doctoral Dissertation, Ocean University of China, 2011).

[0003] Cordyceps sinensis is a dried complex of the stroma of the fungus Cordyceps sinensis (Ber K.) Sacc. (family Clavicipitaceae) parasitizing the larvae of insects in the family Hepialidae (family Hepialidae) and the larval corpse. It possesses various pharmacological effects, including immune regulation, anti-tumor activity, antioxidant activity, and anti-aging. Literature reports that the main chemical components of Cordyceps sinensis include polysaccharides, proteins, sugar alcohols, nucleosides, amino acids, sterols, and organic acids. Extracting new active ingredients from Cordyceps sinensis can provide a scientific basis for the comprehensive quality evaluation of Cordyceps sinensis and the development and application of related products. Summary of the Invention

[0004] The inventors of this invention conducted an in-depth analysis of the ethyl acetate fraction of Cordyceps sinensis and discovered that it contains cerebroside components. They then separated and purified these components to obtain seven cerebroside monomers. Furthermore, they conducted immunomodulatory activity studies on these cerebroside monomers and found that they possess excellent immunosuppressive activity.

[0005] The purpose of this invention is to provide a class of cerebroside compounds and their pharmaceutical compositions as immunosuppressants.

[0006] On one hand, the present invention provides the use of a compound as an immunosuppressant, wherein the compound is a compound represented by formula (IA) or (IB), or a stereoisomer, tautomer, solvate, metabolite, pharmaceutically acceptable salt, or prodrug thereof.

[0007]

[0008] in,

[0009] Each m1 and m2 is independently 3, 4, 5, or 6;

[0010] Each n1 and n2 is independently 5, 6, 7, 8, 9, or 10;

[0011] R 1a and R 1b Each independently represents H and C. 1-6 Alkyl or C 2-6 alkenyl; wherein the C 1-6 Alkyl and C 2-6 The alkenyl group can be independently and optionally selected from F, Cl, Br, I, CF3, CN, or C. 3-6 The cycloalkyl group is substituted by one, two, or three substituents;

[0012] Each R 2 Independently, it is H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl;

[0013] Each R 3 Independently for H and C 1-6 Alkyl or C 2-6 alkenyl; wherein the C 1-6 Alkyl and C 2-6 The alkenyl group can be independently and optionally selected from F, Cl, Br, I, CF3, CN, or C. 3-6 The cycloalkyl group is substituted by one, two, or three substituents;

[0014] R 4 For H, OH, C 1-6 Alkyl or C 2-6 alkenyl; wherein the C 1-6 Alkyl and C 2-6 The alkenyl group can be independently and optionally selected from F, Cl, Br, I, CF3, CN, or C. 3-6 It is substituted by one, two or three substituents in the cycloalkyl group.

[0015] In some implementation schemes, R 1a and R 1bEach of the following is independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, -CH=CH2, -CH2CH=CH2, -CH2C(CH3)=CH2, -CH2CH=CH(CH3), -CH2CH=C(CH3)2, -CHCH3CH=C(CH3)2 or -CH2C(CH3)=C(CH3)2; wherein the methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl Butyl, tert-butyl, -CH=CH2, -CH2CH=CH2, -CH2C(CH3)=CH2, -CH2CH=CH(CH3), -CH2CH=C(CH3)2, -CHCH3CH=C(CH3)2 and -CH2C(CH3)=C(CH3)2 can be independently and optionally replaced by 1, 2 or 3 substituents selected from F, Cl, Br, I, CF3, CN, cyclopropyl, cyclopentyl or cyclohexyl.

[0016] In some implementation schemes, each R 3 Independently, it can be H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, -CH=CH2, -CH2CH=CH2, -CH2C(CH3)=CH2, -CH2CH=CH(CH3), -CH2CH=C(CH3)2, -CHCH3CH=C(CH3)2 or -CH2C(CH3)=C(CH3)2; wherein the methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl The methyl, tert-butyl, -CH=CH2, -CH2CH=CH2, -CH2C(CH3)=CH2, -CH2CH=CH(CH3), -CH2CH=C(CH3)2, -CHCH3CH=C(CH3)2 and -CH2C(CH3)=C(CH3)2 can be independently and optionally replaced by 1, 2 or 3 substituents selected from F, Cl, Br, I, CF3, CN, cyclopropyl, cyclopentyl or cyclohexyl.

[0017] In some implementation schemes, R 4The substituted group is H, OH, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, -CH=CH2, -CH2CH=CH2, -CH2C(CH3)=CH2, -CH2CH=CH(CH3), -CH2CH=C(CH3)2, -CHCH3CH=C(CH3)2 or -CH2C(CH3)=C(CH3)2; wherein the methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, -CH=CH2, -CH2CH=CH2, -CH2C(CH3)=CH2, -CH2CH=CH(CH3), -CH2CH=C(CH3)2, -CHCH3CH=C(CH3)2 and -CH2C(CH3)=C(CH3)2 may be independently and optionally substituted by substituents selected from F, Cl, Br, I, CF3, CN, cyclopropyl, cyclopentyl or cyclohexyl.

[0018] In some embodiments, the compound is a compound of formula (IIA) or (IIB), or a solvate, metabolite, pharmaceutically acceptable salt thereof, or a prodrug thereof.

[0019]

[0020] Among them, each m1, n1, m2, n2, R 1a R 1b R 2 R 3 R 4 It has the definition as described in this invention.

[0021] In some embodiments, the compound has any one of the following structures, or a solvate, metabolite, pharmaceutically acceptable salt thereof, or a prodrug thereof.

[0022]

[0023]

[0024]

[0025] In some embodiments, the compounds of the present invention and pharmaceutically acceptable excipients are prepared into a pharmaceutical composition; the pharmaceutical composition is used as an immunosuppressant for the prevention or treatment of immune-related diseases.

[0026] In some embodiments, the pharmaceutical composition further comprises other adjunctive therapeutic agents selected from chemotherapeutic agents or antiproliferative agents, anti-inflammatory drugs, immunomodulators or immunosuppressants, neurotrophic factors, active agents for the treatment of cardiovascular diseases, active agents for the treatment of diabetes, and active agents for the treatment of autoimmune diseases.

[0027] In some implementations, the immune-related disease is organ transplant rejection or an autoimmune disease, wherein the autoimmune disease is lupus, multiple sclerosis, amyotrophic lateral sclerosis, rheumatoid arthritis, psoriasis, complications from organ transplantation, foreign body transplantation, diabetes, asthma, atopic dermatitis, autoimmune thyroid disease, ulcerative colitis, Crohn's disease, Alzheimer's disease, leukemia, or lymphoma.

[0028] In some embodiments, the compounds described in this invention can be used as immunosuppressants in the preparation of drugs for the prevention or treatment of immune-related diseases.

[0029] In some implementations, the immune-related disease is organ transplant rejection or an autoimmune disease, wherein the autoimmune disease is lupus, multiple sclerosis, amyotrophic lateral sclerosis, rheumatoid arthritis, psoriasis, type I diabetes, complications from organ transplantation, foreign body transplantation, diabetes, asthma, atopic dermatitis, autoimmune thyroid disease, ulcerative colitis, Crohn's disease, Alzheimer's disease, leukemia, or lymphoma.

[0030] Specifically, the salt is a pharmaceutically acceptable salt. The term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with other components of the preparation and / or the mammals to which it is treated.

[0031] The compounds of the present invention, including their salts, can also be obtained in their hydrated form, or include other solvents used for their crystallization. The compounds of the present invention can be inherently or by design formed into solvates having pharmaceutically acceptable solvents (including water); therefore, the present invention also includes both solvated and unsolvated forms.

[0032] The foregoing description only outlines certain aspects of the invention, but is not limited to these aspects. These and other aspects will be described in more detail below.

[0033] Detailed Description of the Invention

[0034] Certain embodiments of the invention will now be described in detail, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to cover all alternatives, modifications, and equivalents, all of which are included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials can be used to practice the invention. The invention is by no means limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0035] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention, for brevity, have been described in a single embodiment, but may also be provided individually or in any suitable sub-combination.

[0036] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0037] As described in this invention, the compounds of this invention can be independently and optionally substituted by one or more substituents, such as the general formula compounds above, or as the specific examples, subclasses, and classes of compounds included in this invention, as described in the embodiments. It should be understood that the term "independently and optionally substituted" is used interchangeably with the term "substituted or unsubstituted." Generally, the term "substituted" means that one or more hydrogen atoms in the given structure are substituted by a specific substituent. Unless otherwise indicated, an optional substituent group may be substituted at each substituted position of the group. When more than one position in the given structural formula can be substituted by one or more substituents selected from a specific group, the substituents may be substituted at the same or different positions.

[0038] Additionally, it should be noted that, unless otherwise explicitly stated, the descriptive terms “each…independently is”, “…each independently is”, and “…independently is” used in this invention are interchangeable and should be interpreted broadly. They can mean that the specific options expressed by the same symbols in different groups do not affect each other, or that the specific options expressed by the same symbols in the same group do not affect each other.

[0039] In various parts of this specification, the substituents of the compounds disclosed herein are disclosed according to the type or scope of the groups. In particular, the invention includes every independent secondary combination of the various members of these group types and scopes. For example, the term "C..." 1-6 "Alkyl" specifically refers to independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0040] The term "alkyl" refers to a saturated straight-chain or branched monovalent hydrocarbon group, wherein the alkyl group may optionally be substituted by one or more substituents described in this invention. In one embodiment of the invention, the alkyl group contains 1-6 carbon atoms; in another embodiment, the alkyl group contains 1-4 carbon atoms; and in yet another embodiment, the alkyl group contains 1-3 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), isobutyl (i-Bu, -CH2CH(CH3)2), sec-butyl (s-Bu, -CH(CH3)CH2CH3), tert-butyl (t... -Bu、-C(CH3)3), n-pentyl(-CH2CH2CH2CH2CH3), 2-pentyl(-CH(CH3)CH2CH2CH3), 3-pentyl(-CH(CH2CH3)2), 2-methyl-2-butyl(-C(CH3)2CH2CH3), 3-methyl-2-butyl(-CH(CH3)CH(CH3)2), 3-methyl-1-butyl(-CH2CH2CH(CH3)2), 2- Methyl-1-butyl (-CH2CH(CH3)CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)) CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), etc.

[0041] The term "alkenyl" refers to a straight-chain or branched monovalent hydrocarbon group with 2-12 carbon atoms, or 2-8 carbon atoms, or 2-6 carbon atoms, or 2-4 carbon atoms, wherein at least one C or C atom is sp. 2The double bond, wherein the alkenyl group may be independently unsubstituted or substituted by one or more substituents described in this invention, including groups with "cis" "trans" or "Z" "E" orientation. In embodiments of this invention, the alkenyl group contains 2-6 carbon atoms. Specific examples include, but are not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2), CH2C(CH3)=CH2, -CH2CH=CH(CH3), -CH2CH=C(CH3)2, -CHCH3CH=C(CH3)2 or -CH2C(CH3)=C(CH3)2, etc.

[0042] The term "cycloalkyl" refers to a saturated monovalent carbocyclic system. In embodiments of the invention, the cycloalkyl group contains 3-6 carbocyclic atoms, with specific examples including cyclopropyl, cyclopentyl, and cyclohexyl.

[0043] In this invention, "solvent" refers to an association formed by one or more solvent molecules and the compound of this invention. Solvents forming solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term "hydrate" refers to an association formed when the solvent molecules are water.

[0044] "Metabolic products" refer to the products obtained from the metabolism of a specific compound or its salt in vivo. The metabolites of a compound can be identified using techniques known in the art, and their activity can be characterized by experimental methods as described in this invention. Such products can be obtained by administering the compound through oxidation, reduction, hydrolysis, acylation, deacylation, esterification, defatting, enzymatic cleavage, etc. Accordingly, this invention includes the metabolites of compounds, including metabolites produced by sufficiently exposing the compounds of this invention to mammals for a period of time.

[0045] As used in this invention, "pharmaceutically acceptable salts" refers to the organic and inorganic salts of the compounds of this invention. Pharmaceutically acceptable salts are well-known in the field, as described in the literature: SMBerge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. Salts formed from pharmaceutically acceptable non-toxic acids include, but are not limited to, inorganic acid salts formed by reactions with amino groups, such as hydrochlorides, hydrobromic acids, phosphates, sulfates, and perchlorates, and organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, or these salts obtained by other methods described in the literature, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, transbutenedioic acid, glucono-p-gluconate, glyceryl phosphate, gluconate, hemisulfate, heptanate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pyruvate, pectinate, persulfate, 3-phenylpropionate, picrate, pentanoate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts obtained by means of appropriate bases include alkali metals, alkaline earth metals, ammonium, and nitrogen. + (C 1-4 Salts of alkyl groups (4). This invention also contemplates quaternary ammonium salts formed from any compound containing an N group. Water-soluble or oil-soluble or dispersed products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts, and amine cations resistant to the formation of equilibrium ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C 1-8 Sulfonates and aromatic sulfonates.

[0046] The term "prodrug" as used in this invention refers to the conversion of a compound into the compound represented by formula (I) in vivo. Such conversion is influenced by the hydrolysis of the prodrug in the blood or its enzymatic conversion into the parent structure in the blood or tissues. The prodrug compounds of this invention can be esters; among existing inventions, esters that can serve as prodrugs include phenyl esters and aliphatic (C) esters. 1-24Esters, acyloxymethyl esters, carbonates, carbamates, and amino acid esters. For example, one compound in this invention contains a hydroxyl group, meaning it can be acylated to yield a prodrug form. Other prodrug forms include phosphate esters, such as those obtained by phosphorylation of a parent compound with a hydroxyl group. For a complete discussion of prodrugs, please refer to the following literature: T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the ACSSymposium Series; Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987; J. Rautio et al., Prodrugs: Design and Clinical Applications, Nature Review Drug Discovery, 2008, 7, 255-270; and SJ Hecker et al., Prodrugs of Phosphates and Phosphonates, Journal of Medicinal Chemistry, 2008, 51, 2328-2345.

[0047] The term "treatment" as used in this invention refers to both therapeutic and preventative treatment. For example, therapeutic treatment includes reducing or improving the progression, severity, and / or duration of immune-mediated symptoms, or improving one or more symptoms (specifically, one or more identifiable symptoms) of immune-mediated symptoms by administering one or more therapies (e.g., one or more therapeutic agents, such as the compounds and compositions of this invention). In certain embodiments, therapeutic treatment includes improving at least one measurable physical parameter of immune-mediated symptoms. In other embodiments, therapeutic treatment includes suppressing the progression of immune-mediated symptoms physically by stabilizing identifiable symptoms or physiologically by stabilizing physical parameters, or both. In other embodiments, therapeutic treatment includes reducing or stabilizing the severity of immune-mediated disease. The medicaments of this invention can be used in the community to treat individuals already suffering from immune diseases to reduce the severity of symptoms and the number of days they are ill.

[0048] Description of the compounds of the present invention

[0049] This invention provides the use of a class of cerebroside compounds and their pharmaceutical compositions as immunosuppressants.

[0050] On the one hand, this invention provides the use of a class of compounds as immunosuppressants, wherein the compounds are those represented by general formula (IA) or (IB), or their stereoisomers, tautomers, solvates, metabolites, pharmaceutically acceptable salts, or prodrugs.

[0051]

[0052] Among them, each m1, n1, m2, n2, R 1a R b R 2 R 3 R 4 It has the definition as described in this invention.

[0053] In some implementations, m1 is 3, 4, 5, or 6.

[0054] In some implementations, m2 is 3, 4, 5, or 6.

[0055] In some implementations, n1 is 5, 6, 7, 8, 9, or 10.

[0056] In some implementations, n2 is 5, 6, 7, 8, 9, or 10.

[0057] In some implementation schemes, R 1a and R 1b Each independently represents H and C. 1-6 Alkyl or C 2-6 alkenyl; wherein the C 1-6 Alkyl and C 2-6 The alkenyl group can be independently and optionally selected from F, Cl, Br, I, CF3, CN, or C. 3-6 It is substituted by one, two or three substituents in the cycloalkyl group.

[0058] In some implementation schemes, each R 2 It can be H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl independently.

[0059] In some implementation schemes, each R 3 Independently for H and C 1-6 Alkyl or C 2-6 alkenyl; wherein the C 1-6 Alkyl and C 2-6 The alkenyl group can be independently and optionally selected from F, Cl, Br, I, CF3, CN, or C. 3-6 It is substituted by one, two or three substituents in the cycloalkyl group.

[0060] In some implementation schemes, R 4 For H, OH, C 1-6Alkyl or C 2-6 alkenyl; wherein the C 1-6 Alkyl and C 2-6 The alkenyl group can be independently and optionally selected from F, Cl, Br, I, CF3, CN, or C. 3-6 It is substituted by one, two or three substituents in the cycloalkyl group.

[0061] In some implementation schemes, R 1a and R 1b Each of the following can be independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, -CH=CH2, -CH2CH=CH2, -CH2C(CH3)=CH2, -CH2CH=CH(CH3), -CH2CH=C(CH3)2, -CHCH3CH=C(CH3)2 or -CH2C(CH3)=C(CH3)2; among which methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl The methyl, tert-butyl, -CH=CH2, -CH2CH=CH2, -CH2C(CH3)=CH2, -CH2CH=CH(CH3), -CH2CH=C(CH3)2, -CHCH3CH=C(CH3)2 and -CH2C(CH3)=C(CH3)2 are independently and optionally replaced by 1, 2 or 3 substituents selected from F, Cl, Br, I, CF3, CN, cyclopropyl, cyclopentyl or cyclohexyl.

[0062] In some implementation schemes, each R 3 Independently, it can be H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, -CH=CH2, -CH2CH=CH2, -CH2C(CH3)=CH2, -CH2CH=CH(CH3), -CH2CH=C(CH3)2, -CHCH3CH=C(CH3)2 or -CH2C(CH3)=C(CH3)2; wherein methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl , tert-butyl, -CH=CH2, -CH2CH=CH2, -CH2C(CH3)=CH2, -CH2CH=CH(CH3), -CH2CH=C(CH3)2, -CHCH3CH=C(CH3)2 and -CH2C(CH3)=C(CH3)2 are independently and optionally replaced by 1, 2 or 3 substituents selected from F, Cl, Br, I, CF3, CN, cyclopropyl, cyclopentyl or cyclohexyl.

[0063] In some implementation schemes, R 4The substituents are H, OH, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, -CH=CH2, -CH2CH=CH2, -CH2C(CH3)=CH2, -CH2CH=CH(CH3), -CH2CH=C(CH3)2, -CHCH3CH=C(CH3)2, or -CH2C(CH3)=C(CH3)2; wherein methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, -CH=CH2, -CH2CH=CH2, -CH2C(CH3)=CH2, -CH2CH=CH(CH3), -CH2CH=C(CH3)2, -CHCH3CH=C(CH3)2, and -CH2C(CH3)=C(CH3)2 may be independently and optionally substituted by substituents from F, Cl, Br, I, CF3, CN, cyclopropyl, cyclopentyl, or cyclohexyl.

[0064] In some embodiments, the compound is a compound of formula (IIA) or (IIB), or a solvate, metabolite, pharmaceutically acceptable salt thereof, or a prodrug thereof.

[0065]

[0066] Among them, each m1, n1, m2, n2, R 1a R 1b R 2 R 3 R 4 It has the definition as described in this invention.

[0067] In some embodiments, the compound has any one of the following structures, or a solvate, metabolite, pharmaceutically acceptable salt thereof, or a prodrug thereof.

[0068]

[0069]

[0070] In some embodiments, compounds 1, 2, 3, 4, 5, 6, and 7 of the present invention are extracted from Cordyceps sinensis.

[0071] In some embodiments, pharmaceutical compositions prepared from the compounds of the present invention and pharmaceutically acceptable excipients are used as immunosuppressants in the preparation of medicaments for the prevention or treatment of immune-related diseases.

[0072] In some embodiments, the pharmaceutical composition further comprises other adjunctive therapeutic agents selected from chemotherapeutic agents or antiproliferative agents, anti-inflammatory drugs, immunomodulators or immunosuppressants, neurotrophic factors, active agents for the treatment of cardiovascular diseases, active agents for the treatment of diabetes, and active agents for the treatment of autoimmune diseases.

[0073] In some implementations, the immune-related disease is organ transplant rejection or an autoimmune disease, wherein the autoimmune disease is lupus, multiple sclerosis, amyotrophic lateral sclerosis, rheumatoid arthritis, psoriasis, type I diabetes, complications from organ transplantation, foreign body transplantation, diabetes, asthma, atopic dermatitis, autoimmune thyroid disease, ulcerative colitis, Crohn's disease, Alzheimer's disease, leukemia, or lymphoma.

[0074] In some embodiments, the compounds described in this invention can be used as immunosuppressants in the preparation of drugs for the prevention or treatment of immune-related diseases.

[0075] In some implementations, the immune-related disease is organ transplant rejection or an autoimmune disease, wherein the autoimmune disease is lupus, multiple sclerosis, amyotrophic lateral sclerosis, rheumatoid arthritis, psoriasis, type I diabetes, complications from organ transplantation, foreign body transplantation, diabetes, asthma, atopic dermatitis, autoimmune thyroid disease, ulcerative colitis, Crohn's disease, Alzheimer's disease, leukemia, or lymphoma.

[0076] Specifically, the salt is a pharmaceutically acceptable salt. The term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with other components of the preparation and / or the mammals to which it is treated.

[0077] The compounds of the present invention also include other salts of such compounds, which are not necessarily pharmaceutically acceptable salts and can be used as intermediates for the preparation and / or purification of the compounds of the present invention and / or for the isolation of enantiomers of the compounds of the present invention.

[0078] Pharmaceutically usable acid addition salts can form with inorganic and organic acids, such as acetates, aspartates, benzoates, benzenesulfonates, bromides / hydrobromoates, bicarbonates / carbonates, hydrogen sulfates / sulfates, camphor sulfonates, chlorides / hydrochlorides, theophylline salts, citrates, ethanedisulfonates, fumarates, gluconate, gluconate, glucuronide, hippurate, hydroiodide / iodide, hydroxyethyl sulfonate, lactates, lacturonide, lauryl sulfate, malates, maleates, malonates, mandelates, methanesulfonates, methyl sulfates, naphthates, naphthalenesulfonates, nicotinates, nitrates, stearates, oleates, oxalates, palmitates, pyrates, phosphates / hydrogen phosphates / dihydrogen phosphates, polygalactosates, propions, stearates, succinates, sulfosalicylates, tartrates, toluenesulfonates, and trifluoroacetates.

[0079] Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid.

[0080] Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, sulfosalicylic acid, etc.

[0081] The compounds of the present invention, including their salts, can also be obtained in their hydrated form, or include other solvents used for their crystallization. The compounds of the present invention can be inherently or by design formed into solvates having pharmaceutically acceptable solvents (including water); therefore, the present invention also includes both solvated and unsolvated forms.

[0082] Any structural formulas given in this invention are also intended to represent these compounds in their unlabeled and isotopically labeled forms. Isotopically labeled compounds have the structures described by the general formulas given in this invention, except that one or more atoms are replaced by atoms having a chosen atomic weight or mass number. Exemplary isotopes that may be introduced into the compounds of this invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as... 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 36 S, 37 Cl or 125 I.

[0083] Compositions, formulations, and administration of the compounds of the present invention

[0084] This invention provides a pharmaceutical composition comprising a compound of formula (I) or (II) or a solvate thereof, a metabolite, a pharmaceutically acceptable salt thereof, or a prodrug thereof. The pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient, and optionally, other therapeutic and / or preventative ingredients. In some embodiments, the pharmaceutical composition comprises an effective amount of at least one pharmaceutically acceptable excipient.

[0085] As described in this invention, the pharmaceutical compositions or pharmaceutically acceptable compositions of this invention further comprise pharmaceutically acceptable excipients, including any solvent, diluent, liquid excipient, dispersant, suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid binder, or lubricant, etc., suitable for the specific target dosage form, as applied in this invention. Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. D.B. Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York, discloses various carriers used in formulating pharmaceutically acceptable compositions and their known preparation methods. Any other conventional carrier media and their uses are also within the scope of this invention, except for conventional carrier media incompatible with the compounds of this invention, such as those that produce adverse biological effects or interact harmfully with any other component in the pharmaceutically acceptable composition.

[0086] The compounds or compositions of the present invention can be administered by any suitable manner, and can be given to humans or other animals orally, rectally, parenterally, intracerebrospinal, vaginally, intraperitoneally, topically (as in powder, ointment or drops), or orally as a mouth or nasal spray, depending on the severity of the infection being treated.

[0087] The compounds used in the methods of the present invention can be formulated into unit dosage forms. The term "unit dosage form" refers to a physical discrete unit suitable as a unit dose for a treated patient, each unit containing a predetermined amount of active substance calculated to produce the intended therapeutic effect, optionally combined with a suitable pharmaceutical adjuvant. Unit dosage forms can be used as a single daily dose or as a multiple daily dose (e.g., about 1-4 times or more daily). When using multiple daily doses, the unit dosage form for each dose can be the same or different.

[0088] Uses of the compounds and compositions of the present invention

[0089] The compounds and pharmaceutical compositions provided by this invention can be used as immunosuppressants to prepare drugs for the prevention, treatment or alleviation of immune-related diseases in patients. Preferably, the immune-related diseases are organ transplant rejection reactions or autoimmune diseases. Preferably, the autoimmune diseases are lupus, multiple sclerosis, amyotrophic lateral sclerosis, rheumatoid arthritis, psoriasis, type I diabetes, complications caused by organ transplantation, foreign body transplantation, diabetes, asthma, atopic dermatitis, autoimmune thyroid disease, ulcerative colitis, Crohn's disease, Alzheimer's disease, leukemia or lymphoma.

[0090] This invention provides a method for treating, preventing, or alleviating immune-related diseases in patients, the method comprising administering to the patient a therapeutically effective amount of the compound or pharmaceutical composition thereof described in this invention. Furthermore, the compound or pharmaceutical composition thereof provided in this invention can be administered in combination with other therapies or therapeutic agents. Administration can be simultaneous, sequential, or at time intervals. The other therapies or therapeutic agents are selected from chemotherapeutic agents or antiproliferative agents, anti-inflammatory drugs, immunomodulators or immunosuppressants, neurotrophic factors, active agents for treating cardiovascular diseases, active agents for treating diabetes, and active agents for treating autoimmune diseases.

[0091] The dosage of the compound or pharmaceutical composition required to achieve therapeutic, preventive, or delaying effects typically depends on the specific compound being administered, the patient, the specific disease or condition and its severity, the route of administration, and the frequency, and needs to be determined by the attending physician based on the specific circumstances. For example, when administering the compound or pharmaceutical composition provided by this invention via intravenous route, it can be administered once a week or even at longer intervals.

[0092] The compounds and pharmaceutical compositions of the present invention, in addition to their therapeutic benefits for humans, can also be used in veterinary treatment of mammals, including pets, introduced breeds of animals, and farm animals. Other examples of animals include horses, dogs, and cats. Herein, the compounds of the present invention include their pharmaceutically acceptable derivatives. Detailed Implementation

[0093] The embodiments of the present invention are described in detail below. It should be noted that the described embodiments are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0094] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention, and other methods for preparing the compounds of the present invention are considered to be within the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0095] In some specific embodiments of the present invention, compounds 1, 2, 3, 4, 5, 6, and 7 provided by the present invention are prepared by extraction from Cordyceps sinensis.

[0096] 1. Source of medicinal materials

[0097] Cordyceps sinensis originates from Yichang Shancheng Shuidu Cordyceps sinensis Co., Ltd., Hubei Province.

[0098] 2. Experimental apparatus

[0099] EYELA rotary evaporator N-1001 (Rikakikai Corporation, Tokyo); Lisui low-pressure liquid chromatography system (Shanghai Lisui Chemical Technology Co., Ltd.); Dionex analytical and preparative high-performance liquid chromatographs (Thermo Fisher Scientific, USA); Phenomenex Gemini C 18Analytical (4.6 × 250 mm, 5 μm), semi-preparative (10.0 × 250 mm, 5 μm), and preparative (20.0 × 250 mm, 5 μm) chromatographic columns (Phenomenex, USA); Phenomenex Biphenyl analytical (4.6 × 250 mm, 5 μm) and semi-preparative (10.0 × 250 mm, 5 μm) chromatographic columns (Phenomenex, USA); JASCO V-550 UV spectrometer (Jasco International, Japan); JASCO FT / IR-480plus IR spectrometer (Jasco International, Japan); JASCO P-1020 optical rotation spectrometer (Jasco International, Japan); Bruker AV-400 / 600MHz NMR spectrometer (Bruker BioSpin, Switzerland); APCI ion source low-resolution mass spectrometer (Bruker Daltonics, USA); Waters Synapt G2 TOF high-resolution mass spectrometer (Waters, USA); HERA cell The 150i cell culture incubator was purchased from Thermo Fisher Scientific, USA; the Model 680 multi-functional microplate reader was purchased from Bio-Rad Laboratories, USA.

[0100] 3. Experimental Materials

[0101] Analytical grade petroleum ether, cyclohexane, ethyl acetate, and ethanol were purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; deuterated pyridine was purchased from Sigma-Aldrich, Germany; and chromatographic grade methanol was purchased from Shandong Yuwang Industrial Co., Ltd.

[0102] Silica gel was purchased from Qingdao Ocean Chemical Co., Ltd.; ODS (50μm) was purchased from YMC Corporation of Japan; HP-20 macroporous resin was purchased from Mitsubishi Chemical Co., Ltd. of Japan.

[0103] Male BABL / C mice were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.; 96-well plates were purchased from Corning (Shanghai) Management Co., Ltd.; Con A, Hank's solution, and erythrocyte lysis buffer were purchased from Sigma-Aldrich Chemical Company, USA; and Cell Counting Kit-8 was purchased from Dojindo Molecular Technologies, Japan.

[0104] The following abbreviations are used throughout the instruction manual:

[0105] t R Retention time

[0106] Pyr-d5: Deuterated pyridine

[0107] δ H Chemical shift of hydrogen

[0108] δ C : Carbon compound displacement

[0109] Unless otherwise specified, the detection wavelength for chromatographic analysis in this invention is 205 nm, and the column temperature is 30 °C.

[0110] Example 1: Isolation and preparation of cerebroside fraction (CSE-E7) from Cordyceps sinensis

[0111] 1.1 Preparation method of CSE-E from the ethyl acetate fraction of Cordyceps sinensis

[0112] 15 kg of Cordyceps sinensis was pulverized and extracted three times by reflux with 120 L of water for 2 hours each time. The extracts were filtered and combined to obtain the aqueous extract. The residue was then extracted three times by reflux with 120 L of 95% ethanol for 2 hours each time. The extracts were filtered and combined to obtain the 95% ethanol extract. The aqueous extract was adsorbed onto 20 kg of HP-20 macroporous resin and eluted with 250 L of 95% ethanol. The fraction not adsorbed by the HP-20 macroporous resin was concentrated under reduced pressure to obtain the extract labeled CSW (yield 27.6%). The fraction eluted with 95% ethanol and the 95% ethanol extract were combined and concentrated under reduced pressure to obtain the extract labeled CSE (yield 20.9%). The CSE fraction was added to water and stirred, then extracted sequentially with cyclohexane and ethyl acetate to obtain the cyclohexane fraction CSE-C (extraction yield 55.0%) and the ethyl acetate fraction CSE-E (extraction yield 1.2%).

[0113] 1.2 Preparation method of CSE-E7 fraction of cerebrosides from Cordyceps sinensis

[0114] CSE-E (38.10 g) was adsorbed and mixed with 50 g silica gel (100-200 mesh, Qingdao Ocean Chemical Co., Ltd., Qingdao, China), eluted with 280 mL petroleum ether, and the remaining sample still adsorbed by silica gel was evaporated and connected to an ODS column (500 g) (10.0 × 250 mm) (50 μm, YMC Co. Ltd, Tokyo, Japan) for medium-low pressure separation. Elution was performed sequentially with 5%, 10%, 30%, 50%, 70%, 90%, and 100% methanol-water, 5 L for each gradient. The 100% methanol eluent was combined and concentrated under reduced pressure to obtain the extract CSE-E7 (1.4 g).

[0115] Example 2: Isolation and identification of monomeric compounds 1-7 from the CSE-E7 fraction of Cordyceps sinensis cerebrosides

[0116] CSE-E7 (1.4 g) was dissolved in methanol and analyzed using a preparative high-performance liquid chromatography (HPLC) system. Separation was performed using a medium-low pressure ODS column (3.1 × 20.0 cm, YMC Corporation, Japan). The mobile phase was eluted with a gradient of 85%–100% (v / v) methanol-water for 3 hours. The eluent was collected in graduated centrifuge tubes, approximately 30 mL per tube, and every 15 tubes were combined into one fraction, yielding seven sub-fractions: F7.1, F7.2, F7.3, F7.4, F7.5, F7.6, and F7.7. Fraction F7.4 (10.8 mg) was separated using a semi-preparative column, Phenomenex Biphenyl (10.0 × 250 mm, 5 μm), eluted isocratically with a mobile phase of 85% methanol-water (v / v) at a flow rate of 3 mL / min to obtain compound 2 (t). R :43.8 min, 3.5 mg). Fraction F7.5 (93.8 mg) was separated using a semi-preparative chromatographic column Phenomenex Gemini C18 (10.0 × 250 mm, 5 μm) with isocratic elution in a mobile phase of 92% methanol-water (v / v) at a flow rate of 3 mL / min to obtain compound 3 (t R :51.6 min, 4.1 mg). Fraction F7.6 (523.4 mg) was separated using a preparative chromatographic column Phenomenex Gemini C18 (20.0 × 250 mm, 5 μm) with isocratic elution of 93% methanol-water (v / v) at a flow rate of 8 mL / min to obtain fraction F7.6.1 (t R :50.0~55.0min), F7.6.2(t R :60.0~65.0min), F7.6.3(t R :66.0~70.0min) and F7.6.4(t R (74.0~78.0 min). Fractions F7.6.1, F7.6.2, F7.6.3, and F7.6.4 were obtained sequentially. Fraction F7.6.1 (356.5 mg) was separated using a semi-preparative chromatographic column Phenomenex Gemini C18 (10.0 × 250 mm, 5 μm) with a mobile phase of 93% methanol-water (v / v) at a flow rate of 3 mL / min, yielding compound 5 (t). R :50.9 min, 319.9 mg). Fraction F7.6.2 (20.7 mg) was separated using a semi-preparative chromatographic column Phenomenex Biphenyl (10.0 × 250 mm, 5 μm) with isocratic elution in a mobile phase of 87% methanol-water (v / v) at a flow rate of 3 mL / min to obtain compound 1 (t R:31.6min, 9.3mg) and compound 7 (t R :29.3 min, 8.7 mg). Fraction F7.6.3 (8.8 mg) was separated using a semi-preparative chromatographic column Phenomenex Biphenyl (10.0 × 250 mm, 5 μm) with isocratic elution in a mobile phase of 87% methanol-water (v / v) at a flow rate of 3 mL / min to obtain compound 4 (t R :29.2 min, 2.5 mg). Fraction F7.6.4 (15.6 mg) was separated using a semi-preparative chromatographic column Phenomenex Gemini C18 (10.0 × 250 mm, 5 μm) with isocratic elution in a mobile phase of 93% methanol-water (v / v) at a flow rate of 3 mL / min to obtain compound 6 (t R (71.1 min, 12.9 mg). The structural and identification data of compounds 1-7 are shown below:

[0117] Compound 1:

[0118]

[0119] Compound 1: White powder; +50.5(c 0.40,MeOH); UV(MeOH)λ max (logε)202(4.22)nm;IR(KBr)v max 3283,2921,1639,1531,1466,1081,1029,968cm -1 ; APCI-MS / MS(positive)m / z740,722,560,294,276; HRESIMS(positive)m / z 740.5668[M+H] + (calcd.for C 42 H 78 NO9,740.5671); 1 H and 13 The C NMR values ​​are shown in Table 1.

[0120] Compound 2:

[0121]

[0122] Compound 2: White powder; +43.4(c 0.60,MeOH); UV(MeOH)λ max (logε)203(4.09)nm;IR(KBr)v max 3332,2915,1659,1539,1466,1073cm-1 ;APCI-MS / MS(positive)m / z 712,694,532,294,276; HRESIMS(positive)m / z 712.5357[M+H] + (calcd.for C 40 H 74 NO9,712.5358); 1 H and 13 The C NMR values ​​are shown in Table 1.

[0123] Compound 3:

[0124]

[0125] Compound 3: White powder; +33.1(c 0.60,MeOH); UV(MeOH)λ max (logε)202(4.02)nm;IR(KBr)v max 3218,2921,1639,1539,1466,1078cm -1 ;APCI-MS / MS(positive)m / z 712,694,532,280,262; HRESIMS(positive)m / z 712.5354[M+H] + (calcd.for C 40 H 74 NO9,712.5358); 1 H and 13 The C NMR values ​​are shown in Table 2.

[0126] Compound 4:

[0127]

[0128] Compound 4: White powder; +80.3(c 0.30,MeOH); UV(MeOH)λ max (logε)202(3.36)nm;IR(KBr)v max 3172,2918,1720,1542,1466,1079cm -1 ;APCI-MS / MS(positive)m / z 712,694,532,294,276; HRESIMS(positive)m / z 712.5721[M+H] + (calcd.for C 41 H 78NO8,712.5722); 1 H and 13 The C NMR values ​​are shown in Table 2.

[0129] Compound 5:

[0130]

[0131] Compound 5: White powder; APCI-MS / MS (positive) m / z 726,708,546,294,276; HRESIMS (positive) m / z 726,5505 [M+H] + (calcd.for C 41 H 76 NO9,726.5515); 1 H and 13 The C NMR values ​​are shown in Table 3.

[0132] Compound 6:

[0133]

[0134] Compound 6: White powder; APCI-MS / MS (positive) m / z 754,736,574,294,276; HRESIMS (positive) m / z 754,5830 [M+H] + (calcd.for C 43 H 80 NO9,754.5828); 1 H and 13 The C NMR values ​​are shown in Table 3.

[0135] Compound 7:

[0136]

[0137] Compound 7: White powder; APCI-MS / MS (positive) m / z 728, 710, 548, 294, 276; HRESIMS (positive) m / z 728, 5668 [M+H] + (calcd.For C 41 H 78 NO9,728.5671); 1 H and 13 The C NMR values ​​are shown in Table 3.

[0138] Table 1. NMR data assignments for compounds 1 and 2 (600 MHz, in Pyr-d5).

[0139]

[0140]

[0141] * This indicates that signals belonging to the same column may be interchangeable.

[0142] Table 2. NMR data assignments for compounds 3 and 4 (600 MHz, in Pyr-d5).

[0143]

[0144]

[0145] * This indicates that signals belonging to the same column may be interchangeable.

[0146] Table 3. NMR data assignments for compounds 5, 6, and 7 (400 MHz, in Pyr-d5).

[0147]

[0148]

[0149] * This indicates that signals belonging to the same column may be interchangeable.

[0150] Example 3: Immunosuppressive activity test of compounds 1-7

[0151] 3.1 Solution Preparation

[0152] Culture medium: 89% culture medium + 10% fetal bovine serum + 1% antibiotics, store at 2-8℃.

[0153] PBS: Dissolve one packet of PBS completely in 1L of pure water, dispense into smaller containers, and sterilize at high temperature.

[0154] 10% CCK-8: Add 1 mL of CCK8 to 9 mL of culture medium to prepare a 10% CCK-8 detection solution. Prepare and use immediately.

[0155] Concanavalin A (ConA): Accurately weigh 5 mg of concanavalin A, add 1 mL of sterile PBS and dissolve thoroughly to prepare a 5 mg / mL stock solution. Aliquot into 50 μL tubes and store at -20°C. When using, thaw and prepare a working solution with complete culture medium to a concentration of 150 μg / mL.

[0156] 3.2 Experimental Methods

[0157] 3.2.1 Cell Acquisition: After exsanguination and bloodletting of BABL / C male mice, the mice were euthanized and soaked in 75% alcohol for 10 min. The spleen was aseptically harvested in a laminar flow hood and placed in a culture dish containing PBS. The spleen surface and interior were rinsed with PBS using a syringe until the spleen turned white. The spleen was gently torn apart with forceps and then gently crushed on a steel mesh using a 5 mL syringe needle to prepare a single-cell suspension. The suspension was filtered through a 200-mesh sieve and centrifuged for 10 min (1000 rpm / min). After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in PBS and centrifuged again for 10 min (1000 rpm / min). This process was repeated once. 2 mL of erythrocyte lysis buffer was added to the cell pellet, mixed, and allowed to stand for 5 min. Then, 8 mL of PBS was added, mixed, and centrifuged at 1000 rpm / min for 5 min. After centrifugation, discard the supernatant, resuspend the cell pellet in PBS, and centrifuge again for 10 min (1000 rpm / min). Repeat this process once more. After centrifugation, discard the supernatant, resuspend the cell pellet in 1 mL of RPMI-1640 complete medium (containing 10% fetal bovine serum and 1% penicillin-dextrose antibody), add 2-3 mL of medium to dilute, and count the new cell type using a cell counter. Adjust the cell concentration to 2*10⁻⁶ cells / mL with complete medium. 6 per mL.

[0158] 3.2.2 Sample preparation: Dissolve appropriate amounts of compounds 1, 2, 3, 4, 5, 6, and 7 in DMSO to prepare 64 mM stock solutions. Then dilute with culture medium to obtain solutions with a concentration of 128 μM. Then, use culture medium for serial dilution to obtain sample diluents with concentrations of 64 μM, 32 μM, 16 μM, 8 μM, and 4 μM, respectively.

[0159] 3.2.3 Cell viability assay: The prepared cell suspension was added to 96-well plates, and the cell density was 2*102. 5 Cells were added to each well at a volume of 100 μL. Compounds 1, 2, 3, 4, 5, 6, and 7 were added to each well at 100 μL / well, with five replicates for each concentration. The wells were then incubated at 37°C in a 5% CO2 incubator for 60 h. Afterward, the cells were incubated with serum-free medium containing 10% CCK-8 for 4–5 h at 37°C. The absorbance (A) of each well was measured at 450 nm using a microplate reader. A control group was included: cells + 1640 medium. Results showed that, compared to the control group, compounds 1–7 at all concentrations (64, 32, 16, 8, 4, and 2 μM) showed no cytotoxic effect on splenic lymphocytes.

[0160] 3.2.4 Cell proliferation inhibition assay: The prepared cell suspension was added to a 96-well plate, and the cell density was 2*102. 5Cells were added to each well at a volume of 100 μL. Samples were added at 100 μL per well, with five replicates per concentration. The wells were then incubated at 37°C with 5% CO2 for 12 h. After carefully aspirating 10 μL of supernatant, 10 μL of ConA at a concentration of 150 μg / mL (final concentration 7.5 μg / mL) was added to the corresponding group. The wells were then incubated at 37°C with 5% CO2 for 48 h. Finally, serum-free medium with 10% CCK-8 was added, and the wells were incubated at 37°C for 4–5 h. The absorbance (A) of each well was measured at 450 nm using a microplate reader. One negative control group and one model control group were set up: cells + 1640 medium and cells + ConA + 1640 medium, respectively.

[0161] 3.2.5 Data Processing:

[0162] The proliferation rate (viability) of spleen lymphocytes in the sample was calculated using the following formula:

[0163] Proliferation rate % = [As / Ac] × 100%

[0164] As: Absorbance of sample group (spleen lymphocytes + test sample + CCK-8)

[0165] Ac: Absorbance of control group (splenic lymphocytes + CCK-8)

[0166] The inhibition rate of spleen lymphocyte proliferation by the sample was calculated using the following formula:

[0167] Proliferation inhibition rate % = [(Am-As) / (Am-Ac)] × 100%

[0168] Am: Absorbance of the model group control (splenic lymphocytes + ConA + CCK-8)

[0169] As: Absorbance of sample group (spleen lymphocytes + test sample + ConA + CCK-8)

[0170] Ac: Absorbance of negative control group (splenic lymphocytes + CCK-8)

[0171] 3.3 Experimental Results

[0172] The cell proliferation inhibition rates of compounds 1-7 on Con A-induced splenic lymphocyte proliferation are shown in Table 4 below. The IC50 was calculated by taking the logarithmic value of the concentration and then performing a linear fit between the logarithmic value (x) and the inhibition rate (y). 50 IC50 corresponding to compounds 1, 2, 3, 4, 5, 6, and 7 50 The values ​​were 4.0, 10.4, 19.5, 1.5, 15.7, 28.6, and 24.4 μM, respectively.

[0173] Table 4. Inhibition rate (%) of compounds 1-7 on Con A-induced splenic lymphocyte proliferation.

[0174]

[0175] ***: p < 0.001 compared to the model group; **: p < 0.01 compared to the model group; *: p < 0.05 compared to the model group.

[0176] As shown in Table 4, all compounds of the present invention exhibit good immunosuppressive activity in inhibiting Con A-induced proliferation of mouse spleen lymphocytes.

[0177] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "some implementations," "other implementations," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0178] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the concept of the present invention, and these improvements and modifications should also be considered within the protection scope of the present invention.

Claims

1. Use of a compound in the preparation of an immunosuppressant, wherein said compound has any one of the following structures, or a pharmaceutically acceptable salt thereof, 、 、 、 、 、 ,or 。 2. The use according to claim 1, wherein, The compound and pharmaceutically acceptable excipients are prepared into a pharmaceutical composition; the pharmaceutical composition is used as an immunosuppressant for the prevention or treatment of immune-related diseases.

3. The use according to claim 2, wherein, The pharmaceutical composition further comprises other adjunctive therapeutic agents selected from chemotherapeutic agents or antiproliferative agents, anti-inflammatory drugs, immunosuppressants, neurotrophic factors, active agents for the treatment of cardiovascular diseases, and active agents for the treatment of diabetes.

4. The use according to claim 2, wherein, The immune-related diseases mentioned are organ transplant rejection or autoimmune diseases, including lupus, multiple sclerosis, amyotrophic lateral sclerosis, rheumatoid arthritis, psoriasis, type I diabetes, atopic dermatitis, or autoimmune thyroid disease.

5. The use according to claim 2, wherein, The immune-related diseases include complications resulting from organ transplantation.

Images