Preparation and use of fluorescent probe targeting GPR84 receptor

Abstract

Disclosed in the present invention are preparation and a use of a fluorescent probe targeting a GPR84 receptor. Specifically, the structure of the fluorescent probe is as shown in formula (I), wherein in the formula, the definitions of substituents are as stated in the description. The compound of formula (I) of the present invention can be used as a fluorescent probe for a GPR84 receptor on a cell membrane surface. The probe can specifically target a GPR84 receptor on a cell membrane surface, and emit specific fluorescence in a fat-soluble environment by means of a Nile red fluorescent group. The probe compound is simple to synthesize and has high yield, can quickly target a corresponding membrane receptor and generate signals, and achieves good specificity and a high signal-to-noise ratio.

Description

Preparation and application of a fluorescent probe targeting GPR84 receptor Technical Field

[0001] The present invention relates to a class of fluorescent probes for G protein-coupled receptor 84 (GPR84), specifically the use of binaphthol phosphate derivatives as probes for cell membrane surface receptors. Specifically, it relates to the use of a binaphthol phosphate-Nile Red compound as a fluorescent probe for the GPR84 receptor on the cell membrane surface. The probes described herein can specifically target the GPR84 receptor on the cell membrane surface, emitting specific fluorescence using the Nile Red fluorescent group in combination with a lipid-soluble environment. These probe compounds are simple to synthesize, have high yields, can rapidly target the corresponding membrane receptors, and generate signals with good specificity and a high signal-to-noise ratio. Background Art

[0002] GPR84 is a member of the rhodopsin-like receptor family of G protein-coupled receptors (GPCRs) and can be activated by medium-chain fatty acids (MCFAs) with carbon chain lengths of C9-C14. The coding sequence for GPR84 was first discovered in human peripheral blood neutrophils in 2001 (Journal of Molecular Biology, 2001, 307(3):799-813.). The human GPR84 sequence is located at position 13 of chromosome 12, consists of 396 amino acids, and does not contain introns. GPR84 is expressed in various tissues and organs in the human body, such as the heart, lungs, kidneys, liver, bone marrow, and fat. It is particularly highly expressed in myeloid immune cells, such as peripheral blood monocytes, macrophages, neutrophils, and microglia in the central nervous system. In the resting state, GPR84 in tissues is at a low level, but stimulation by inflammatory factors (such as lipopolysaccharide LPS, tumor necrosis factor TNFα, etc.) will lead to an increase in the expression level of GPR84. A significant increase in the expression level of GPR84 has been observed in many inflammatory diseases, such as inflammatory bowel disease (IBD. Acta Pharmacologica Sinica, 2022, 43(8): 2042-2054.), multiple sclerosis (MS. Glia, 2007, 55(8): 790-800.), fibrosis (fibrosis. JCI Insight, 2023, 8(2). Journal of Clinical Medicine, 2020, 9(4): 1140.), and pulmonary inflammation (pulmonary inflammation. Acta Pharmacologica Sinica, 2023: 1-11.). GPR84 regulates the chemotaxis and infiltration of immune cells and promotes the secretion of pro-inflammatory cytokines and chemokines by immune cells in a Gi protein-dependent manner. These studies confirm that GPR84 plays a crucial role in the human inflammatory response. Its expression level is closely correlated with the severity of inflammation and serves as a key indicator of inflammatory diseases. Therefore, the development of fluorescent probes targeting GPR84 for quantitative tracking is of great significance. However, to date, there are no literature or patent reports on small molecule fluorescent probes targeting GPR84.

[0003] Nan Fajun et al. reported a class of highly active GPR84 antagonists (CN110680823B) with a biphenyl phosphate structure. These compounds effectively inhibit GPR84 receptor activation and are potentially useful in treating diseases caused by GPR84 overexpression or hyperexcitability, such as inflammatory bowel disease, liver fibrosis, pulmonary fibrosis, and arthritis. These compounds also possess strong molecular rigidity and low cell membrane permeability, which reduces their nonspecific effects and makes them ideal small molecule fluorescent probes.

[0004] The Nile Red fluorescent group was discovered by Greenspan et al. in 1985 (The Journal of Cell Biology, 1985, 100(3): 965-973.) and was initially used to detect intracellular lipid droplets in fluorescence microscopy or flow cytometry. The group has high brightness and a fluorescence range suitable for monitoring (yellow-red light), and also has the property of solvatochromism. Solvatochromism means that the group basically does not emit any fluorescence in a highly polar environment. When the polarity of the solvent gradually decreases, the fluorescence intensity of Nile Red increases significantly, and the maximum emission wavelength shifts to the blue. The difference in the maximum emission wavelength between polar and non-polar solvents can reach more than 100nm, and the difference in the maximum fluorescence intensity can reach 10 4 This polarity-dominated "turn-on" mechanism can reduce its nonspecific luminescence, and rational design can obtain highly specific fluorescent probe compounds.

[0005] Currently, people can only locate GPR84 at the cellular and tissue levels through antibody means, and lack direct and effective small molecule tool probes for positioning. The expression level of GPR84 receptor is closely related to the occurrence and development of inflammatory diseases. Therefore, the development of small molecule fluorescent probes targeting GPR84 has important physiological and pathological significance.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a novel GPR84 small molecule fluorescent probe and its preparation method and application.

[0008] In a first aspect of the present invention, there is provided a compound represented by formula (I), or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof,

[0009] Where, M is H;

[0010] L1 is none, substituted or unsubstituted C1~C 10 Alkylene, -O (substituted or unsubstituted C1~C 10Alkylene)-, -OCO (substituted or unsubstituted C1~C 10 alkylene)-, substituted or unsubstituted-(OCH2CH2) r -, substituted or unsubstituted C2~C 10 Alkenylene, -O-substituted or unsubstituted C2~C 10 Alkenylene, substituted or unsubstituted C2~C 10 Alkynylidene, -O-substituted or unsubstituted C2~C 10 Alkynylidene, -CO-, -NHCO-, -C(=CH2)-, -N(substituted or unsubstituted C1~C4 alkyl)-, substituted or unsubstituted C3~C6 cycloalkyl- or substituted or unsubstituted C3~C6 heterocyclyl-; wherein the substitution means that one or more hydrogen atoms (e.g., 2, 3, 4, 5 or 6 atoms) on the group are independently replaced by substituents selected from the group consisting of amino, hydroxyl, halogen, cyano, nitro, carboxyl, C1~C6 alkyl, C1~C6 haloalkyl, C1~C6 alkoxy, C1~C6 haloalkoxy; wherein r is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0011] R1 is one or more substituents on the connected benzene ring, wherein each R1 is independently selected from the following group: H, halogen, OH, NH2, SH, CN, COOH, substituted or unsubstituted C1~C 10 Alkoxy, substituted or unsubstituted C1~C 10 Alkyl, substituted or unsubstituted C1~C 10 Halogenated alkyl, substituted or unsubstituted C1~C 10 Halogenated alkoxy, substituted or unsubstituted C2~C 10 Alkenyl, -O-substituted or unsubstituted C2~C 10 Alkenyl, substituted or unsubstituted C2~C 10 Alkynyl, -O-substituted or unsubstituted C2~C 10 Alkynyl, substituted or unsubstituted C3-12 cycloalkyl, substituted or unsubstituted C6-C 14 aryl, monosubstituted or disubstituted amino, substituted or unsubstituted 3-10 membered heterocyclic group, substituted or unsubstituted 5-10 membered heteroaryl; wherein the substitution means that one or more hydrogen atoms (such as 2, 3, 4, 5 or 6) on the group are independently replaced by substituents selected from the group: amino, hydroxyl, halogen, cyano, nitro, carboxyl, C1-C6 alkyl, C1-C6 haloalkyl, C6-C 10 Aromatic ring, C1~C6 alkoxy, C1~C6 haloalkoxy, 3-6 membered cycloalkyl, -OC p H 2p -OC q H 2q+1, oxo (=O), -C(O)OC1~C6 alkyl, -O-C6-C 10 aryl;

[0012] wherein p is 1, 2, 3, 4, 5 or 6, and q is 1, 2, 3, 4, 5 or 6.

[0013] In another preferred embodiment, R1 is independently H or -OC1-C3 alkyl, preferably -OCH3.

[0014] In another preferred embodiment, L1 is independently -O(C1~C 10 Alkylene)-、-(OC2H4) r -; wherein each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0015] In another preferred embodiment, the compound is the axial chiral enantiomer of the compound of formula I.

[0016] In another preferred embodiment, the compound is selected from the following group:

[0017] In another preferred embodiment, the pharmaceutically acceptable salt is a metal ion salt of the compound or a salt formed with an organic base. Preferably, the metal is selected from the group consisting of Li, Na, K, Ca, Mg, Cu, Fe, Zn, Al and Mn; and the organic base is selected from the group consisting of NH3, arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and imidazole.

[0018] A second aspect of the present invention provides a method for fluorescence detection of GPR84 receptors, comprising the steps of:

[0019] The compound according to claim 1, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt is contacted with the GPR84 receptor in an isolated cell or tissue sample, and fluorescence detection is performed.

[0020] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0021] In another preferred embodiment, the method is a quantitative analysis method or a qualitative analysis method.

[0022] In another preferred embodiment, the method is used to detect the expression level of GPR84 receptor.

[0023] The third aspect of the present invention provides a method for preparing the compound described in the first aspect, comprising the following steps:

[0024] (a) Compound S1 undergoes a coupling reaction with H-L1-NH-X3 to generate compound S2;

[0025] (b) Compound S2 removes HO-X2 through hydrolysis and generates compound S3 through phosphorylation;

[0026] (c) Compound S3 removes HO-X3 through hydrolysis reaction and undergoes condensation reaction with Nile red group to form compound P1;

[0027] In each formula, R1, L1, and M are defined as above;

[0028] X1 is Cl, Br or I;

[0029] Each X2 is independently methyl, methoxymethyl, or benzyl;

[0030] X3 is benzyloxycarbonyl or tert-butyloxycarbonyl.

[0031] The fourth aspect of the present invention provides a GPR84 receptor fluorescent probe reagent, which comprises the compound described in the first aspect of the present invention, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt.

[0032] A fifth aspect of the present invention provides a pharmaceutical composition comprising:

[0033] The compound described in the first aspect, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.

[0034] The sixth aspect of the present invention provides a use of the compound described in the first aspect or its enantiomer, diastereomer, racemate or pharmaceutically acceptable salt, or the composition described in the fifth aspect, wherein the use is selected from the group consisting of:

[0035] (i) Used to prepare GPR84 receptor (fluorescent) probe;

[0036] (ii) used as a GPR84 receptor fluorescent probe;

[0037] (iii) for preparing a GPR84 receptor antagonist;

[0038] (iv) For the preparation of drugs for preventing or treating diseases caused by overexpression or hyperexcitability of GPR84 receptors, such as inflammatory bowel disease, liver fibrosis, pulmonary fibrosis, and arthritis.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0040] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 shows the fluorescence emission spectra of probe compounds F1-F11 in solvents of different polarities. As the polarity of the solvent decreases, the maximum emission wavelength decreases significantly and the fluorescence intensity increases significantly.

[0042] Figure 2 shows the difference in fluorescence imaging of probe compounds F6, F7, F8, and F9 in HEK293 / Gα16 cells that highly express the humanized GPR84 receptor and in HEK293 / Gα16 cells that do not express human GPR84. Probe compound F8 can selectively label the cell membrane of HEK293 / Gα16 cells that highly express hGPR84, but does not produce a fluorescent signal in HEK293 / Gα16 cells that do not express human GPR84.

[0043] Figure 3 shows the competitive imaging results of probe compounds F6, F7, F8, and F9 on HEK293 / Gα16 cells that highly express humanized GPR84 receptors. Adding an excess of the GPR84 antagonist 17S can completely inhibit the generation of specific fluorescence signals. DETAILED DESCRIPTION

[0044] After extensive and in-depth research, the inventors of this application have developed a novel small-molecule, three-fragment fluorescent probe targeting the GPR84 receptor. This probe is composed of a GPR84 antagonist fragment with a binaphthol phosphate structure, linker fragments of varying types and lengths, and a fluorescent fragment derived from a Nile Red group. This probe compound specifically binds to the GPR84 receptor on the cell membrane and further recognizes the lipid-soluble environment near the receptor, thereby emitting a specific fluorescent signal. This is the basis for the present invention.

[0045] the term

[0046] In the present invention, unless otherwise specified, the terms used have the general meanings commonly known to those skilled in the art.

[0047] When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes chemically equivalent substituents that would result if the formula were written from right to left. For example, -CH2O- is equivalent to -OCH2-.

[0048] Throughout the specification, the terms "optionally substituted" or "may be substituted" and the like are used to indicate that the group may or may not be further substituted or fused (to form a polycyclic system) with one or more non-hydrogen substituents. Suitable chemically suitable substituents for a particular functional group will be readily apparent to those skilled in the art.

[0049] The prefix "Cu-v" indicates that the following group has from u to v carbon atoms. For example, "C 1-10 "Alkyl" means an alkyl group having 1 to 10 carbon atoms. "Cu-v" includes all positive integers of C atoms between u and v, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0050] As used herein, the term "alkyl" by itself or as part of a group refers to a straight or branched chain alkyl group containing several carbon atoms, wherein "C1-C 10 "Alkyl" refers to a straight or branched chain alkyl group having 1 to 10 carbon atoms, including alkyl groups of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Typical "alkyl" groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, isobutyl, Examples of the alkyl group include pentyl, isopentyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, and decyl. In the present invention, the term "alkyl" also includes substituted alkyl groups. "Substituted alkyl" refers to an alkyl group with one or more substituents, particularly 1 to 4 substituents, which may be substituted at any position.

[0051] As used herein, the term "C1-C 10 "Alkoxy" refers to a straight or branched chain alkoxy group having 1 to 10 carbon atoms, with a C1-C 10 Alkyl-O-structure, C1-C 10 Alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0052] As used herein, the term "alkenyl" refers to a straight or branched chain hydrocarbon having at least 2 carbon atoms and at least one double bond. An alkenyl group may include any number of carbon atoms, wherein "C2-C10 alkenyl" refers to a straight or branched chain hydrocarbon having 2-10 carbon atoms and at least one double bond, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. An alkenyl group may have any suitable number of double bonds, including but not limited to 1, 2, 3, or more. Examples of alkenyl groups include, but are not limited to, ethenyl (vinyl group), propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl.

[0053] As used herein, the term "alkynyl" refers to a straight or branched chain hydrocarbon having at least 2 carbon atoms and at least one triple bond. An alkynyl group can include any number of carbon atoms. "C2-C10 alkynyl" refers to a straight or branched chain hydrocarbon having 2-10 carbon atoms and at least one triple bond, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. An alkynyl group can have any suitable number of double bonds, including but not limited to 1, 2, 3, or more. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentenyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl.

[0054] The term "aryl" refers to an aromatic cyclic hydrocarbon compound radical. "C6-C14 aryl" refers to an aromatic cyclic hydrocarbon compound radical containing 6 to 14 ring carbon atoms, having 1-3 rings, and particularly refers to monocyclic and bicyclic groups, such as phenyl or naphthyl. Where there are two or more aromatic rings (e.g., bicyclic), the aromatic rings of the aryl group may be connected by a single bond (e.g., biphenyl) or fused (e.g., naphthalene, anthracene, etc.). "Substituted aryl" refers to an aryl group that is substituted at one or more positions, particularly 1-3 substituents, and substitutions may occur at any position.

[0055] As used herein, the term "heteroaryl" refers to a heteroaromatic system containing 1-3 atoms selected from N, O, and S. "5-10 membered heteroaryl" refers to a 5-12 membered heteroaromatic system containing 1-3 atoms selected from N, O, and S. Heteroaryl is preferably a 5- to 10-membered ring, more preferably a 5- or 6-membered ring. Heteroaryl includes, but is not limited to, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazolyl, and tetrazolyl.

[0056] As used herein, the term "cycloalkyl" refers to a fully saturated cyclic hydrocarbon group having a number of carbon atoms, wherein "C3-C 12 "Cycloalkyl" refers to a fully saturated cyclic hydrocarbon group having 3-12 carbon atoms, preferably a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.

[0057] As used herein, the term "heterocyclyl" refers to a fully saturated or partially unsaturated cyclic group having a number (greater than or equal to 3) of ring atoms and 1-3 heteroatoms, wherein "3-12 membered heterocyclyl" refers to a fully saturated or partially unsaturated cyclic group having 3-12 ring atoms and 1-3 heteroatoms (including but not limited to 3-7 membered monocyclic, 6-10 membered bicyclic, or 8-12 membered tricyclic ring systems). The nitrogen or sulfur atom may be oxidized, and the nitrogen atom may be quaternized. The heterocyclic group may be attached to the residue of any heteroatom or carbon atom of the ring or ring system molecule. Typical monocyclic heterocycles include, but are not limited to, azetidinyl, pyrrolidinyl, oxetanyl, pyrazolinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, hexahydroazepinyl, 4-piperidonyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxanyl, and tetrahydro-1,1-dioxythiophene, and the like.

[0058] When the substituent is a non-terminal substituent or a related group removes a H atom, it is a subunit of the corresponding group, usually a divalent group, for example, an alkyl group removes a H atom to form an alkylene group (for example: methylene, ethylene, propylene, isopropylene (such as ), butylene (such as ), pentylene (such as ), hexamethylene (such as ), heptylene (such as ) etc.), cycloalkyl corresponds to cycloalkylene (such as: etc.), heterocyclic groups corresponding to heterocyclic groups (such as: ), cycloalkyl corresponding heterocyclyl (such as: etc.), alkoxy corresponds to alkyleneoxy (-CH2O-, -CH2CH2-O-CH2-, -CH2OCH2CH2CH2-), etc.

[0059] As used herein, the term "plurality" refers to two or more, such as 2, 3, 4, 5 or 6.

[0060] As described herein, the compounds of the present invention may be substituted with any number of substituents or functional groups to expand their scope. Generally, the term "substituted", whether it appears before or after the term "optional", includes the general formula of substituents in the present invention, and refers to the use of a specified structural substituent to replace the hydrogen radical. When multiple positions in a specific structure are substituted by multiple specific substituents, the substituents can be the same or different at each position. The term "substituted" as used herein includes all allowed substitutions of organic compounds. In a broad sense, allowed substituents include non-cyclic, cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic organic compounds. In the present invention, heteroatom nitrogen can have a hydrogen substituent or any allowed organic compound described above to supplement its valence state. In addition, the present invention is not intended to limit the allowed substitution of organic compounds in any way.

[0061] As used herein, the term "substituted" means that any one or more hydrogens on the designated atom are replaced with a substituent selected from the designated substituent, provided that the normal valence of the designated atom is not exceeded and that the resulting compound is stable, i.e., a compound that can be isolated, characterized, and tested for biological activity.

[0062] Unless otherwise specified, the substitution refers to that one or more H groups on each group are independently replaced by a substituted or unsubstituted group selected from the following groups: amino, hydroxyl, halogen, nitro, C1~C6 alkyl, C1~C6 haloalkyl, C1~C6 alkoxy, C1~C6 haloalkoxy.

[0063] Unless otherwise indicated, the term "halo" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom. In addition, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "C 1-4 "Haloalkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0064] For the compounds provided herein, a bond from a substituent (typically an R group) to the center of an aromatic ring (e.g., benzene, pyridine, etc.) will be understood to mean a bond providing attachment at any available vertex of the aromatic ring. In some embodiments, this description also includes attachment to a ring fused to an aromatic ring. For example, a bond drawn to the center of an indole benzene moiety will represent a bond attached to any available vertex of a six-membered or five-membered ring moiety of indole.

[0065] Unless otherwise specified, all compounds mentioned in the present invention are intended to include all possible optical isomers, such as single chiral compounds or mixtures of various chiral compounds (i.e., racemates). In all compounds of the present invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.

[0066] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers, and individual isomers (e.g., isolated enantiomers) are intended to be encompassed within the scope of the present invention. When compounds provided herein have defined stereochemistry (denoted as R or S, or indicated by dashed or wedge-shaped bonds), those skilled in the art will understand that those compounds are substantially free of other isomers (e.g., at least 80%, 90%, 95%, 98%, 99%, and up to 100% free of other isomers).

[0067] As used herein, the terms "comprising", "including" or "comprising" indicate that various components can be used together in the mixture or composition of the present invention. Therefore, the terms "consisting essentially of" and "consisting of" are included in the term "comprising".

[0068] Compounds of formula I

[0069] The GPR84 probe provided by the present invention is a compound having the structure of Formula I shown above.

[0070] Wherein, the definitions of M, L1 and R1 are as described in the present invention.

[0071] In the present invention, such as -O(C1~C 10 There is no special restriction on the connection mode of groups such as -alkylene)-, -N(C1~C4 alkyl)-, -C(=CH2)-, etc., and the atoms or groups connected thereto are connected from left to right or from right to left. For example, for -L1-NH-, when L1 is -O(C1~C 10 Alkylene)-, represents -O-(C1~C 10 Alkylene)-NH- or -NH-O-(C1~C 10In another preferred embodiment, when L1 is defined as the above group, the left end of the above group is connected to the phenyl group of the compound, and the right end is connected to NH.

[0072] The present invention also includes the enantiomers, diastereomers, racemates, or pharmaceutically acceptable salts thereof of the compound.

[0073] The present invention also provides pharmaceutically acceptable salts thereof, including salts obtained by reacting a compound of Formula I with an inorganic base or an organic base compound, particularly salts formed by the removal of the H at the M position as H+. Salts derived from inorganic bases include, but are not limited to, aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganic salts, manganous salts, potassium salts, sodium salts, zinc salts, and the like. Lithium salts, ammonium salts, calcium salts, magnesium salts, potassium salts, and sodium salts are particularly preferred. Salts derived from pharmaceutically acceptable organic non-toxic bases, including but not limited to salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, imidazole and the like.

[0074] Composition and use

[0075] The compound of the present invention has excellent GPR84 antagonistic activity and can selectively label the GPR84 receptor and emit fluorescence.

[0076] Therefore, the present invention provides a pharmaceutical composition comprising: the compound described in the first aspect of the present invention, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be used to prevent or treat diseases related to GPR84 receptor overexpression or hyperexcitability, such as inflammatory bowel disease, liver fibrosis, pulmonary fibrosis, and arthritis.

[0077] "Pharmaceutically acceptable carrier", "pharmaceutically acceptable carrier" or "pharmaceutically usable carrier" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredient of the present invention (the compound of formula I or its pharmaceutically acceptable salt) and with each other without significantly reducing the efficacy of the active ingredient. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0078] The compounds and compound compositions of the present invention may be in various forms, such as capsules, tablets, granules, solutions, powders, granules, or syrups, for oral administration or parenteral administration in the form of injections. The compounds and compound compositions of the present invention may be present in suitable solid or liquid carriers and suitable sterile apparatus for injection or instillation. Such preparations may be prepared by conventional synthetic methods.

[0079] Furthermore, the present invention also provides a GPR84 receptor fluorescent probe reagent, comprising the compound described in the first aspect of the present invention, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof. The compound of Formula I, as a small molecule fluorescent probe targeting the GPR84 receptor, can specifically bind to the GPR84 receptor on the cell membrane and further recognize the lipid-soluble environment near the receptor, thereby emitting a specific fluorescent signal, and can be used to characterize the expression level of the GPR84 receptor at the cellular and tissue levels.

[0080] The present invention also provides uses of the compound or its enantiomer, diastereomer, racemate or pharmaceutically acceptable salt, or the composition of the fifth aspect, wherein the uses are selected from the group consisting of:

[0081] (i) Used to prepare GPR84 receptor (fluorescent) probe;

[0082] (ii) used as a GPR84 receptor fluorescent probe;

[0083] (iii) for preparing a GPR84 receptor antagonist;

[0084] (iv) For the preparation of drugs for preventing or treating diseases caused by overexpression or hyperexcitability of GPR84 receptors, such as inflammatory bowel disease, liver fibrosis, pulmonary fibrosis, and arthritis.

[0085] Fluorescence detection method for GPR84 receptor

[0086] The present invention also provides a method for fluorescence detection of GPR84 receptors, comprising the steps of:

[0087] The compound according to claim 1, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt is contacted with the GPR84 receptor in an isolated cell or tissue sample, and fluorescence detection is performed.

[0088] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0089] In another preferred embodiment, the method is a quantitative analysis method or a qualitative analysis method.

[0090] In another preferred embodiment, the method is used to detect the expression level of GPR84 receptor.

[0091] This method uses a fluorescent probe, the compound described in the first aspect. Due to the lysochromic nature of the fluorescent group, this compound emits little fluorescence in aqueous solution. However, the GPR84 ligand fragment in the probe binds to the GPR84 receptor expressed on the cell surface, anchoring the probe. The Nile Red fluorescent group at the other end of the probe then inserts into the cell membrane near the receptor, creating a lipid-soluble environment and emitting a specific fluorescent signal. This method can specifically reflect the expression level of GPR84 in cells and tissues by measuring the intensity of the fluorescent signal, allowing for preliminary characterization of GPR84-related inflammation.

[0092] Preparation method

[0093] The compound of formula I can be obtained by the following route:

[0094] (a) Compound S1 undergoes a coupling reaction with H-L1-NH-X3 to generate compound S2;

[0095] (b) Compound S2 removes HO-X2 through hydrolysis and generates compound S3 through phosphorylation;

[0096] (c) Compound S3 removes HO-X3 through hydrolysis reaction and undergoes condensation reaction with Nile red group to form compound P1;

[0097] In each formula, R1, L1, and M are defined as above;

[0098] X1 is Cl, Br or I;

[0099] Each X2 is independently methyl, methoxymethyl, or benzyl;

[0100] X3 is benzyloxycarbonyl or tert-butyloxycarbonyl.

[0101] More specifically, the first step reaction is carried out in a solvent such as ethylene glycol dimethyl ether (DME), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc) or toluene; the catalyst used is tetrakistriphenylphosphine palladium (Pd(PPh3)4), bis(triphenylphosphine)palladium dichloride (Pd(PPh3)2Cl2) or other palladium catalytic reagents, and the reaction temperature is 60°C to 110°C; the reaction time is about 6 to 24 hours; after the reaction is completed, the system is diluted with distilled water and extracted with solvents such as ethyl acetate (EtOAc), diethyl ether (Et2O), dichloromethane (CH2Cl2), chloroform (CHCl3), and the organic phase concentrate is purified by column chromatography to obtain intermediate S2. The second step is first carried out in a solvent such as ethyl acetate, dichloromethane, or 1,4-dioxane. The acidic reagent used is trifluoroacetic acid or hydrogen chloride. The reaction time is approximately 6 to 48 hours. The product is then concentrated under reduced pressure and redissolved in a solvent such as dichloromethane or N,N-dimethylformamide. Phosphorus oxychloride is used as a reaction reagent, and an organic base such as triethylamine (Et3N) or diisopropylethylamine (DIPEA) is used as an acid-binding agent. The reaction temperature is 0°C to 30°C. The reaction time is approximately 6 to 24 hours. After completion of the reaction, the product is diluted with distilled water and extracted with a solvent such as ethyl acetate (EtOAc), diethyl ether (Et2O), dichloromethane (CH2Cl2), or chloroform (CHCl3). The organic phase concentrate is subjected to column chromatography to obtain the target intermediate S3. The third step is first carried out in a solvent such as acetic acid. The acidic reagent used is hydrogen bromide, hydrogen chloride, etc. The reaction time is about 2 to 8 hours. After the reaction is completed, the system is diluted with distilled water and extracted with solvents such as ethyl acetate (EtOAc), diethyl ether (Et2O), dichloromethane (CH2Cl2), and chloroform (CHCl3). The organic phase concentrate is washed with diethyl ether (Et2O) and filtered to obtain a solid. The solid is redissolved in N,N-dimethylformamide (DMF) and then eluted with benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP), (3 The activating agent is tri-1-pyrrolidinyl hexafluorophosphate (PyAOP), and reagents such as triethylamine (Et3N) and diisopropylethylamine (DIPEA) provide an alkaline environment. The reaction temperature ranges from 0°C to 30°C, and the reaction time is approximately 2 to 24 hours. After completion, the reaction is diluted with distilled water and extracted with solvents such as ethyl acetate (EtOAc), diethyl ether (Et2O), dichloromethane (CH2Cl2), and chloroform (CHCl3). The concentrated organic phase is purified by column chromatography to yield product P1. The product's structure was identified and characterized by proton nuclear magnetic resonance (PMR), carbon nuclear magnetic resonance (CMR), and mass spectrometry (MS).

[0102] Wherein, R1, L1, and M are as defined above; X1 is Cl, Br, or I; each X2 is independently methyl, methoxymethyl, or benzyl; and X3 is benzyloxycarbonyl or tert-butyloxycarbonyl.

[0103] The features described above, or in the embodiments, may be combined in any combination. All features disclosed in this specification may be used in any combination, and any feature disclosed in this specification may be replaced by any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are merely general examples of equivalent or similar features.

[0104] The main advantages of the present invention include:

[0105] The inventors have developed a new type of small molecule three-fragment fluorescent probe targeting the GPR84 receptor, the structure of which is composed of a GPR84 antagonist fragment with a binaphthol phosphate structure, a connecting chain fragment, and a fluorescent fragment of the Nile Red group. The probe compound of the present application can specifically bind to the GPR84 receptor on the cell membrane with its GPR84 antagonist fragment, and then the Nile Red group at the other end of its connecting chain is further inserted into the cell membrane structure near the receptor and obtains a fat-soluble environment. The solvatochromic property of the Nile Red group causes its fluorescence intensity to increase significantly, causing the probe compound to emit a specific fluorescent signal. This dual-functional recognition feature enables this type of probe compound to have better fluorescence specificity than conventional membrane receptor fluorescent probes. It will only emit a fluorescent signal after recognizing the GPR84 receptor, and can recognize and respond to GPR84 receptors at different expression levels in cells and tissues.

[0106] In summary, the compounds provided by the present invention can well recognize the membrane receptor GPR84 and emit specific fluorescent signals, and the recognition mode of its bifunctional fragment can be simply and effectively applied to the field of other membrane receptor probes.

[0107] In addition, the compound of the present invention is simple to synthesize and has a high yield, can quickly target the corresponding membrane receptors and generate signals, has good specificity and a high signal-to-noise ratio.

[0108] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0109] In the following examples, NMR measurements were performed using Bruker AVANCE III 400M and AVANCE III 600M instruments, with NMR calibrations of δH 7.26 ppm (CDCl3), 2.50 ppm (DMSO-d6), and 3.15 ppm (CD3OD). Reagents were primarily provided by Shanghai Chemical Reagent Company. TLC thin-layer chromatography silica gel plates were produced by Shandong Yantai Huiyou Silica Gel Development Co., Ltd., model HSGF 254. The normal-phase column chromatography silica gel used for compound purification was produced by Shandong Qingdao Ocean Chemical Plant Branch, model zcx-11, 200-300 mesh.

[0110] Example 1

[0111] Preparation of compound F1

[0112] Synthesis of intermediate G3-1. Raw material G1-1 (384 mg, 0.63 mmol) was placed in a flask and dissolved in ethylene glycol dimethyl ether (DME, 4 mL). The atmosphere was then replaced with argon. Pd(PPh3)4 (73 mg, 0.06 mmol) was quickly added to the system with stirring. After the addition was complete, G2-1 (369 mg, 0.70 mmol) and Na2CO3 solution (2 M in H2O, 1 mL) were added all at once, stirring at room temperature for about 10 min. The atmosphere was replaced with argon again, and the system was heated under reflux at 100°C for about 16 h before TLC monitoring. After the reaction was complete, distilled water (30 mL) was added to the system for dilution. The mixture was extracted with ethyl acetate (3×30 mL) and the organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was separated by Flash column chromatography (PE / EtOAc = 20 / 1 to 10 / 1) to give G3-1 (485 mg, yield 64%) as a yellow oily liquid.

[0113] Synthesis of Intermediate G4-1: G3-1 (184 mg, 0.22 mmol) was dissolved in 2M HCl in ethyl acetate (20 mL) and stirred at room temperature for approximately 4 h before TLC monitoring. After complete conversion of the starting material, the solvent was removed by concentration under reduced pressure. The remaining product was redissolved in dry dichloromethane (5 mL). Phosphorus oxychloride (0.022 mL, 0.24 mmol) and triethylamine (0.073 mL, 0.53 mmol) were slowly added dropwise. The reaction was stirred at room temperature for approximately 16 h. After the reaction, the solvent was removed under reduced pressure, and a mixture of THF:H2O:Et3N = 1:1:0.1 (5 mL) was added to redissolve the product. The reaction was stirred at room temperature for 20 min. After 20 minutes, ethyl acetate (30 mL) and distilled water (30 mL) were added for extraction. The mixture was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The mixture was separated by Flash column chromatography (DCM / MeOH = 25 / 1 to 10 / 1) to obtain a white solid powder G4-1 (74 mg, yield 39%).

[0114] Synthesis of Product F1. Intermediate G4-1 (96 mg, 0.12 mmol) was dissolved in 33% HBr in acetic acid (15 mL). The reaction was stirred at room temperature for approximately 2 hours and monitored by TLC. After the complete disappearance of the starting material, ethyl acetate (30 mL) and distilled water (30 mL) were added for extraction. The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The remaining solid was redissolved in a minimal amount of dichloromethane and recrystallized from glacial ether to yield an off-white solid. This product, a Nile Red fragment, and benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP) were placed in a flask protected from light. N,N-dimethylformamide (DMF) was added to dissolve the product. DIPEA was then slowly added dropwise to the system with stirring. The reaction was allowed to proceed at room temperature in the dark. TLC monitoring was initiated after approximately 1.5 hours. After completion of the reaction, ethyl acetate (30 mL) and distilled water (30 mL) were added for extraction. The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was separated using a Flash column chromatography system (DCM / MeOH / TFA = 25 / 1 / 0.01 to 10 / 1 / 0.01) to obtain a purple-black powder product F1 (62 mg, yield 58%).

[0115] The following compounds were synthesized using the same method

[0116] Example 2

[0117] Preparation of compound F6

[0118] Synthesis of intermediate G3-6. Raw material G1-6 (197 mg, 0.31 mmol) was placed in a flask and dissolved in ethylene glycol dimethyl ether (DME, 2 mL). The atmosphere was then replaced with argon. Pd(PPh3)4 (32 mg, 0.03 mmol) was quickly added to the system with stirring. After the addition was complete, G2-1 (182 mg, 0.35 mmol) and Na2CO3 solution (2M in H2O, 0.5 mL) were added all at once, stirred at room temperature for about 10 min, and the atmosphere was replaced with argon again. The system was heated under reflux at 100°C for about 16 h before TLC monitoring. After the reaction was complete, distilled water (20 mL) was added to the system for dilution. The mixture was extracted with ethyl acetate (3×20 mL) and the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was separated by Flash column chromatography (PE / EtOAc = 20 / 1 to 10 / 1) to give G3-6 (192 mg, yield 55%) as a colorless oily liquid.

[0119] Synthesis of Intermediate G4-6: G3-6 (192 mg, 0.23 mmol) was dissolved in 2M HCl in ethyl acetate (15 mL) and stirred at room temperature for approximately 4 h before TLC monitoring. After complete conversion of the starting material, the solvent was removed by concentration under reduced pressure. The remaining product was redissolved in dry dichloromethane (4 mL). Phosphorus oxychloride (0.023 mL, 0.25 mmol) and triethylamine (0.075 mL, 0.54 mmol) were slowly added dropwise. The reaction was stirred at room temperature for approximately 16 h. After the reaction, the solvent was removed under reduced pressure and redissolved in a mixture of THF:H2O:Et3N = 1:1:0.1 (5 mL). The reaction was stirred at room temperature for 20 min. After 20 minutes, ethyl acetate (30 mL) and distilled water (30 mL) were added for extraction. The mixture was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The mixture was separated by Flash column chromatography (DCM / MeOH = 25 / 1 to 10 / 1) to obtain a white solid powder G4-1 (72 mg, yield 36%).

[0120] Synthesis of Product F6. Intermediate G4-6 (72 mg, 0.10 mmol) was dissolved in 33% HBr in acetic acid (15 mL). The reaction was stirred at room temperature for approximately 2 hours and monitored by TLC. After the complete disappearance of the starting material, ethyl acetate (30 mL) and distilled water (30 mL) were added for extraction. The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The remaining solid was redissolved in a minimal amount of dichloromethane and recrystallized from glacial ether to yield an off-white solid. This product, Nile Red fragment, and benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP) were placed in a flask protected from light. N,N-dimethylformamide (DMF) was added to dissolve the product. DIPEA was then slowly added dropwise to the system with stirring. The system was allowed to react at room temperature in the dark. TLC monitoring was initiated after approximately 1.5 hours. After completion of the reaction, ethyl acetate (30 mL) and distilled water (30 mL) were added for extraction. The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was separated by Flash column chromatography (DCM / MeOH / TFA = 25 / 1 / 0.01 to 10 / 1 / 0.01) to obtain a purple-black powder product F6 (22 mg, yield 32%).

[0121] The following compounds were synthesized using the same method

[0122] Example 3

[0123] (1) Experimental purpose

[0124] The compounds of the present invention were tested for their GPR84 antagonistic activity.

[0125] (2) Source of materials

[0126] The human GPR84 cell line was generated by transfecting HEK293 cells with plasmids encoding GPR84 and Gα16 proteins. The fluorescent dye Fluo-4 AM was purchased from Invitrogen.

[0127] (3) Test principle

[0128] Intracellular Ca 2+ Ion is a very important second messenger in G protein coupled receptor signaling pathway. 16 After the protein-coupled GPR84 binds to the agonist, intracellular Ca 2+ Ion concentration can be significantly increased. Fluo-4 is a Ca 2+ Ion-specific fluorescent probe that can react with Ca 2+The ions bind quantitatively and emit fluorescence. Therefore, the agonist or antagonist activity of the compound is measured using fluorescence detection in 96-well or 384-well flat-bottom microplates.

[0129] Detection of the inhibitory effect of GPR84 antagonists on receptors: After incubating GPR84 cells with the fluorescent dye Fluo-4, different concentrations of antagonist compounds were added and incubated for a period of time to allow them to occupy the binding site between the agonist and GPR84 (antagonist binding site). Then, a certain concentration of agonist (6-n-octylaminouracil, 6-OAU) was added to compete with the antagonist for the binding site. At the same time, the light source with a wavelength of 485nm was used for excitation and the change in dye fluorescence intensity caused by the change in intracellular calcium ion concentration was detected at 525nm. The half-maximal inhibitory concentration (IC50) of the compound was calculated using GraphPad PRISM software. 50 ).

[0130] (4) Experimental process

[0131] Prepare HBSS: 0.4 g / L KCl (5.4 mM), 0.12 g / L Na2HPO4·12H2O (0.3 mM), 0.06 g / L KH2PO4 (0.4 mM), 0.35 g / L NaHCO3 (4.2 mM), 0.14 g / L CaCl2 (1.3 mM), 0.10 g / L MgCl2·6H2O (0.5 mM), 0.05 g / L MgSO4 (0.6 mM), 8.0 g / L NaCl (137 mM). Weigh each of the above ingredients and dissolve them in ultrapure water. Adjust the pH to 7.4 with hydrochloric acid or NaOH solution. Filter and store at 4°C for one month.

[0132] Prepare calcium flux buffer: First, prepare a 560 mM D-glucose (100X) stock solution and a 250 mM 1,2-diphenyl-4-(2-phenylsulfinyl)ethyl-3,5-pyrazolidinedione (1000X) stock solution. Then, add BSA (0.5 g), 1 mL of the 560 mM D-glucose stock solution, and 100 μL of the 250 mM 1,2-diphenyl-4-(2-phenylsulfinyl)ethyl-3,5-pyrazolidinedione stock solution to 100 mL of HBSS to a final concentration of 0.5% BSA, 5.6 mM D-glucose, and 250 μM 1,2-diphenyl-4-(2-phenylsulfinyl)ethyl-3,5-pyrazolidinedione, respectively. Mix thoroughly and use immediately.

[0133] Preparation of dye: 2 mM Fluo-4 AM (1000X, purchased from Invitrogen) in DMSO (1 μL) was mixed with 3% Cremophor EL (100X, purchased from Sigma-Aldrich) in PBS (10 μL), and then 1 mL of calcium flux buffer was added and mixed.

[0134] The cells were plated at 4 × 10 4 The cells were seeded at a density of 100 μg / well in a 96-well plate. After culturing for 24 hours, the culture medium was removed and 40 μL of Fluo-4 AM fluorescent dye was added to each well and incubated in a 37°C incubator for 40 minutes. After aspirating the dye, 50 μL of the test compound was added and incubated at room temperature for 10 minutes. Then, 25 μL of 6-OAU (the effective concentration after dilution was within the EC value of the agonist) was added using a Flex Station III microplate reader. 80 The fluorescence intensity of Fluo-4AM dye caused by the change of intracellular calcium ion flow was read in real time (excitation wavelength 485 nm, detection wavelength 525 nm).

[0135] (5) Experimental results

[0136] Table 1. IC values ​​of probe compounds F1-F11 50

[0137] (6) Results analysis

[0138] The results in Table 1 show that the compound IC 50 The values ​​were all less than 2 μM, and the IC 50 The values ​​are between 0.1–0.3 μM, indicating that the compounds of the present invention have good antagonistic activity and can effectively bind to and localize with the GPR84 receptor at the cellular or tissue level.

[0139] Example 4

[0140] (1) Experimental purpose

[0141] The fluorescence emission spectra of compounds F1, F2, F3, F4, F5, F6, F7, F8, F9, F10 and F11 of the present invention in solvents of different polarity were measured to investigate their solvatochromic characteristics under different polarity environments.

[0142] (2) Test principle

[0143] The solvatochromic property of this type of probe compound is an important property to ensure its fluorescence specificity. The difference in fluorescence intensity in solvents of different polarity determines the fluorescence specificity of the probe compound.

[0144] (3) Experimental process

[0145] The test compound was dissolved in 1,4-dioxane, methanol (MeOH), ethanol (EtOH), acetonitrile (Acetonitrile), acetone (Acetone) and water (H2O) at a concentration of 0.1 μM. Each solvent was dissolved in triplicate. The excitation wavelength was 520 nm, and the fluorescence emission spectrum of each compound was recorded in the range of 550 nm to 750 nm.

[0146] (4) Experimental results

[0147] All probe compounds exhibit significant solvatochromic properties. As shown in Figure 1, compound F1 emits virtually no fluorescence in the highly polar solvent water (H2O). However, its fluorescence intensity increases significantly as the solvent polarity decreases, reaching its peak in the least polar solvent, 1,4-dioxane. Furthermore, its emission wavelength undergoes a significant blue shift with decreasing solvent polarity, with the maximum emission wavelength varying by up to 50 nm. Figure 1 shows the fluorescence emission spectra of probe compounds F1-F11, demonstrating that the probe compounds described in this invention all exhibit excellent fluorescence specificity.

[0148] Example 5

[0149] (1) Experimental purpose

[0150] The imaging results of the compounds F6, F7, F8 and F9 of the present invention at the cellular level were measured to investigate the specificity of the probe compounds to the fluorescence signal of the GPR84 receptor.

[0151] (2) Test principle

[0152] 1) Compare the fluorescence imaging results of the probe compounds in HEK293 / Gα16 cells that overexpress hGPR84 and control HEK293 / Gα16 cells that are empty transfected;

[0153] 2) Competitive antagonists of the probe compounds were added to examine changes in the fluorescence signals of the probe compounds in hGPR84-HEK293 / Gα16 cells.

[0154] (3) Experimental process

[0155] 1) The probe compounds were incubated at a concentration of 1 μM with HEK293 / Gα16 cells overexpressing hGPR84 and control group empty-transfected HEK293 / Gα16 cells for 15 min, and the differences were observed under a fluorescence microscope.

[0156] 2) The probe compound was divided into two groups and incubated with HEK293 / Gα16 cells that highly express hGPR84 at a concentration of 1 μM for 15 min. One group was also incubated with 10 μM of the competitive inhibitor 17S, and the differences were observed under a fluorescence microscope.

[0157] (4) Experimental results

[0158] 1) The probe compounds exhibited differential fluorescence signals in both HEK293 / Gα16 cells overexpressing hGPR84 and in a control, empty-transfected HEK293 / Gα16 cell system. As shown in Figure 2, probe compound F8 produced a specific fluorescence signal on the membrane of HEK293 / Gα16 cells overexpressing hGPR84, whereas no specific fluorescence was observed in the empty-transfected control cells. Figure 2 shows the comparative experimental results of probe compounds F6-F9 in a GPR84 overexpressing cell system and an empty-transfected cell system, demonstrating that these probe compounds can selectively label the GPR84 receptor and emit fluorescence.

[0159] 2) The specific signal of the probe compound on the HEK293 / Gα16 cell membrane that highly expresses hGPR84 can be completely competed

[0160] As shown in Figure 3, probe compound F8 can specifically label the membrane of HEK293 / Gα16 cells that highly express hGPR84, but the fluorescence signal completely disappears under competition with 17S, indicating that probe compound F8 specifically labels the GPR84 receptor on the cell membrane surface, causing it to emit fluorescence. Figure 3 shows the results of a competitive imaging experiment with probe compounds F6-F9, demonstrating that the fluorescence signals of these probe compounds are highly GPR84-selective.

[0161] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A compound represented by formula (I), or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof, Wherein, M is H; L1 is zero, substituted or unsubstituted C1~C 10 Alkylene, -O (substituted or unsubstituted C1~C 10 Alkylene)-, -OCO (substituted or unsubstituted C1~C 10 Alkylene)-, substituted or unsubstituted-(OCH2CH2) r -, substituted or unsubstituted C2~C 10 Alkenylene, -O-substituted or unsubstituted C2~C 10 Alkenylene, substituted or unsubstituted C2~C 10 Alkyne, -O-substituted or unsubstituted C2~C 10 Alkyne, -CO-, -NHCO-, -C(=CH2)-, -N(substituted or unsubstituted C1-C4 alkyl)-, substituted or unsubstituted C3-C6 cycloalkyl- or substituted or unsubstituted C3-C6 heterocyclyl-; wherein, The substitution means that one or more hydrogen atoms (such as 2, 3, 4, 5 or 6 atoms) on the group are independently replaced by a substituent selected from the group consisting of amino, hydroxy, halogen, cyano, nitro, carboxyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy; wherein r is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; R1 is one or more substituents on the connected benzene ring, wherein each R1 is independently selected from the following group: H, halogen, OH, NH2, SH, CN, COOH, substituted or unsubstituted C1~C 10 Alkoxy, substituted or unsubstituted C1~C 10 Alkyl, substituted or unsubstituted C1~C 10 Halogenated alkyl, substituted or unsubstituted C1~C 10 Halogenated alkoxy, substituted or unsubstituted C2~C 10 Alkenyl, -O-substituted or unsubstituted C2~C 10 Alkenyl, substituted or unsubstituted C2~C 10 Alkynyl, -O-substituted or unsubstituted C2~C 10 Alkynyl, substituted or unsubstituted C3-12 cycloalkyl, substituted or unsubstituted C6-C 14 aryl, monosubstituted or disubstituted amino, substituted or unsubstituted 3-10 membered heterocyclic group, substituted or unsubstituted 5-10 membered heteroaryl; wherein the substitution means that one or more hydrogen atoms (such as 2, 3, 4, 5 or 6 atoms) on the group are independently replaced by substituents selected from the group consisting of amino, hydroxyl, halogen, cyano, nitro, carboxyl, C1-C6 alkyl, C1-C6 haloalkyl, C6-C 10 Aromatic ring, C1-C6 alkoxy, C1-C6 haloalkoxy, 3-6 membered cycloalkyl, -OC p H 2p -OC q H 2q+1 , oxo (=O), -C(O)OC1~C6 alkyl, -O-C6-C 10 Aryl; wherein p is 1, 2, 3, 4, 5 or 6, and q is 1, 2, 3, 4, 5 or 6.

2. The compound according to claim 1, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that: R1 is independently H or -OC1-C3 alkyl, preferably -OCH3.

3. The compound according to claim 1, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that: L1 is independently -O(C1~C 10 Alkylene)-, -(OC2H4) r -; wherein each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

4. The compound according to claim 1, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from the group consisting of:

5. The compound according to claim 1, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof, characterized in that: The pharmaceutically acceptable salt is a metal ion salt of the compound or a salt formed with an organic base. Preferably, the metal is selected from the group consisting of Li, Na, K, Ca, Mg, Cu, Fe, Zn, Al and Mn; the organic base is selected from the group consisting of NH3, arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and imidazole.

6. A method for fluorescence detection of GPR84 receptor, comprising the steps of: The compound according to claim 1, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt is contacted with the GPR84 receptor in an isolated cell or tissue sample, and fluorescence detection is performed.

7. The method for preparing the compound according to claim 1, comprising the following steps: (a) Compound S1 undergoes a coupling reaction with H-L1-NH-X3 to generate compound S2; (b) Compound S2 removes HO-X2 through hydrolysis and generates compound S3 through phosphorylation; (c) Compound S3 removes HO-X3 through hydrolysis reaction and undergoes condensation reaction with the Nile red group to generate compound P1; In each formula, R1, L1, and M are defined as above; X1 is Cl, Br or I; Each X2 is independently methyl, methoxymethyl, or benzyl; X3 is benzyloxycarbonyl or tert-butyloxycarbonyl.

8. A GPR84 receptor fluorescent probe reagent, characterized in that: The reagent includes the compound described in the first aspect of the present invention, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt.

9. A pharmaceutical composition comprising: the compound according to claim 1, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

10. Use of the compound according to claim 1 or its enantiomer, diastereomer, racemate or pharmaceutically acceptable salt thereof or the composition according to the fifth aspect, wherein the use is selected from the group consisting of: (i) Used to prepare GPR84 receptor (fluorescent) probe; (ii) used as a GPR84 receptor fluorescent probe; (iii) for preparing a GPR84 receptor antagonist; (iv) Used for preparing drugs for preventing or treating diseases caused by high expression or excessive excitability of GPR84 receptors, such as inflammatory bowel disease, liver fibrosis, pulmonary fibrosis, and arthritis.

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