Preparation and application of targeting GPR84 receptor fluorescent probe

Abstract

The invention discloses preparation and application of a targeting GPR84 receptor fluorescent probe. Specifically, the structure of the fluorescent probe is as shown in formula (I), and in the formula, the definition of each substituent group is as shown in the specification. The compound shown in the formula I can be used as a fluorescent probe of a GPR84 receptor on the surface of a cell membrane, and the probe can specifically target the GPR84 receptor on the surface of the cell membrane and emit specific fluorescence by combining a nile red fluorophore with a fat-soluble environment. The probe compound is simple to synthesize, high in yield, capable of quickly targeting a corresponding membrane receptor and generating a signal, good in specificity and high in signal-to-noise ratio. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to a class of fluorescent probes for G protein-coupled receptor 84 (GPR84), and relates to the application of binaphthol phosphate derivatives as cell membrane surface receptor probes. In particular, it refers to the application of a binaphthol phosphate-Nile red compound as a cell membrane surface GPR84 receptor fluorescent probe. The probe involved in the present invention can specifically target the GPR84 receptor on the cell membrane surface, and emit specific fluorescence by combining the Nile red fluorescent group with the fat-soluble environment. This type of probe compound is simple to synthesize and has a high yield, can quickly target the corresponding membrane receptor and generate signals, and has 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 a carbon chain length of C9-C14. The coding sequence of 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 intron sequences. GPR84 is expressed in a variety of tissues or organs in the human body, such as the heart, lungs, kidneys, liver, bone marrow, fat, etc., especially 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 can participate in regulating the chemotaxis and infiltration of immune cells, and can promote the secretion of pro-inflammatory cytokines and chemokines by immune cells in a Gi protein-dependent manner. The above studies have confirmed that GPR84 plays an important role in the human inflammatory response. Its expression level is closely related to the severity of inflammation and is an important indicator of inflammatory diseases. Therefore, it is of great significance to develop fluorescent probes targeting GPR84 for quantitative tracing. However, there are still 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 with biphenyl phosphate structures (CN110680823B), which can effectively inhibit the activation of GPR84 receptors and can be used to treat related diseases caused by high expression or excessive excitability of GPR84, such as inflammatory bowel disease, liver fibrosis, pulmonary fibrosis, arthritis, etc. At the same time, the structure of this type of compound has strong molecular rigidity and low cell membrane permeability, which is conducive to reducing its nonspecific effects and is a good property as a small molecule fluorescent probe.

[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 originally 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 significantly increases, and the maximum emission wavelength blue-shifts. The maximum emission wavelength difference between polar and non-polar solvents can reach more than 100nm, and the maximum fluorescence intensity difference can reach 10 4 This polarity-dominated "turn-on" mechanism can reduce its nonspecific luminescence, and rational design can produce highly specific fluorescent probe compounds.

[0005] At present, people can only locate GPR84 at the cellular and tissue levels through antibodies, and lack direct and effective small molecule tool probes for localization. The expression level of GPR84 receptor is closely related to the occurrence and development of inflammatory diseases, so the development of small molecule fluorescent probes targeting GPR84 has important physiological and pathological significance. Summary of the invention

[0006] The purpose of the present invention is to provide a novel GPR84 small molecule fluorescent probe and a preparation method and application thereof.

[0007] In a first aspect of the present invention, there is provided a compound represented by formula (I), or an enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof,

[0008]

[0009] Wherein, M is H;

[0010] L1 is zero, 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 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 hydrogens (such as 2, 3, 4, 5 or 6) on the group are independently replaced by substituents selected from the following group: 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 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;

[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]

[0018]

[0019]

[0020] 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 following group: Li, Na, K, Ca, Mg, Cu, Fe, Zn, Al and Mn; the organic base is selected from the following group: 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.

[0021] The second aspect of the present invention provides a method for fluorescence detection of GPR84 receptor, comprising the steps of:

[0022] 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.

[0023] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0024] In another preferred embodiment, the method is a quantitative analysis method or a qualitative analysis method.

[0025] In another preferred embodiment, the method is used to detect the expression level of GPR84 receptor.

[0026] The third aspect of the present invention provides a method for preparing the compound described in the first aspect, comprising the following steps:

[0027]

[0028] (a) Compound S1 undergoes a coupling reaction with H-L1-NH-X3 to generate compound S2;

[0029] (b) Compound S2 removes HO-X2 through hydrolysis and generates compound S3 through phosphorylation;

[0030] (c) Compound S3 removes HO-X3 through hydrolysis reaction and undergoes condensation reaction with the Nile red group to generate compound P1;

[0031] In each formula, R1, L1, and M are defined as above;

[0032] X1 is Cl, Br or I;

[0033] Each X2 is independently methyl, methoxymethyl, or benzyl;

[0034] X3 is benzyloxycarbonyl or tert-butyloxycarbonyl.

[0035] 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.

[0036] A fifth aspect of the present invention provides a pharmaceutical composition comprising:

[0037] The compound described in the first aspect, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0038] 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 following group:

[0039] (i) Used to prepare GPR84 receptor (fluorescent) probe;

[0040] (ii) used as a GPR84 receptor fluorescent probe;

[0041] (iii) for preparing a GPR84 receptor antagonist;

[0042] (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.

[0043] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0044] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The fluorescence emission spectra of probe compounds F1-F11 in solvents of different polarities are shown. As the polarity of the solvent decreases, the maximum emission wavelength decreases significantly and the fluorescence intensity increases significantly.

[0046] Figure 2 The fluorescence imaging differences of probe compounds F6, F7, F8 and F9 on HEK293 / Gα16 cells that highly express humanized GPR84 receptor and empty HEK293 / Gα16 cells are shown. Probe compound F8 can selectively label the cell membrane of highly expressed hGPR84-HEK293 / Gα16 cells but does not produce fluorescence signals on empty HEK293 / Gα16 cells.

[0047] Figure 3 The competitive imaging results of probe compounds F6, F7, F8 and F9 on HEK293 / Gα16 cells that highly express humanized GPR84 receptors are shown. Adding an excess of GPR84 antagonist 17S can completely inhibit the generation of specific fluorescence signals. DETAILED DESCRIPTION

[0048] The inventors of the present application have developed a novel small molecule three-fragment fluorescent probe targeting the GPR84 receptor through extensive and in-depth research, the structure of which is composed of a GPR84 antagonist fragment with a binaphthol phosphate structure, a connecting chain fragment of different types and lengths, and a fluorescent fragment of a Nile red group. The probe compound of the present application can specifically bind to the GPR84 receptor on the cell membrane and further recognize the fat-soluble environment near the receptor to emit a specific fluorescent signal. On this basis, the present invention was completed.

[0049] the term

[0050] In the present invention, unless otherwise specified, the terms used have the general meanings well known to those skilled in the art.

[0051] When substituents are described by conventional chemical formulas written from left to right, the substituents also include chemically equivalent substituents that would result if the formula were written from right to left. For example, -CH2O- is equivalent to -OCH2-.

[0052] Throughout the specification, the term "optionally substituted" or "may be substituted" etc. is 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 apparent to those skilled in the art.

[0053] 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 having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0054] As used herein, the term "alkyl" by itself or as part of a group refers to a straight or branched chain alkyl group containing a number of carbon atoms, wherein "C1-C 10 "Alkyl" refers to a straight or branched chain alkyl group having 1-10 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, The term "alkyl" refers to alkyl radicals such as pentyl, isopentyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, etc. In the present invention, alkyl radicals also include substituted alkyl radicals. "Substituted alkyl radicals" refer to alkyl radicals that are substituted at one or more positions, especially 1 to 4 substituents, which can be substituted at any position.

[0055] 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.

[0056] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon having at least 2 carbon atoms and at least one double bond. Alkenyl may include any number of carbon atoms, wherein "C2-C10 alkenyl" refers to a straight or branched hydrocarbon having 2-10 carbon atoms and at least one double bond, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Alkenyl 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.

[0057] As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon having at least 2 carbon atoms and at least one triple bond. Alkynyl groups may include any number of carbon atoms, and "C2-C10 alkynyl" refers to a straight or branched hydrocarbon having 2-10 carbon atoms and at least one triple bond, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Alkynyl groups may 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.

[0058] The term "aryl" refers to an aromatic cyclic hydrocarbon compound group, wherein "C6-C14 aryl" refers to an aromatic cyclic hydrocarbon compound group containing 6 to 14 ring carbon atoms, having 1-3 rings, especially monocyclic and bicyclic groups, such as phenyl or naphthyl. Where there are two or more aromatic rings (bicyclic, etc.), the aromatic rings of the aryl group can be connected by a single bond (such as biphenyl) or fused (such as naphthalene, anthracene, etc.). "Substituted aryl" means that one or more positions in the aryl group are substituted, especially 1-3 substituents, which can be substituted at any position.

[0059] As used herein, the term "heteroaryl" refers to a heteroaromatic system containing 1-3 atoms selected from N, O, and S, wherein "5-10 membered heteroaryl" refers to a 5-12 membered heteroaromatic system containing 1-3 atoms selected from N, O, and S. The heteroaryl is preferably a 5- to 10-membered ring, more preferably a 5- or 6-membered ring, and includes, but is not limited to, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazolyl, tetrazolyl, and the like.

[0060] 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 of 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Cycloalkyl includes but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.

[0061] 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 system). Among them, the nitrogen atom or sulfur atom can be oxidized, and the nitrogen atom can also be quaternized. The heterocyclic group can 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.

[0062] 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 become 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 correspond to heterocyclic groups (such as: ), cycloalkyl corresponding to heterocyclyl (such as: etc.), alkoxy corresponds to alkyleneoxy (-CH2O-, -CH2CH2-O-CH2-, -CH2OCH2CH2CH2-), etc.

[0063] As used herein, the term "plurality" refers to two or more, such as 2, 3, 4, 5 or 6.

[0064] As described herein, the compounds of the present invention can be replaced with any number of substituents or functional groups to expand their scope of inclusion. Generally, the term "substituted" appears before or after the term "optional", and the general formula of the substituent included in the present invention refers to the use of a specified structural substituent to replace the hydrogen free radical. When multiple positions in a specific structure are replaced by multiple specific substituents, each position of the substituent can be the same or different. The term "substituted" used herein includes all allowed organic compound substitutions. In a broad sense, allowed substituents include non-cyclic, cyclic, branched non-branched, 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.

[0065] 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 valency of the designated atom is not exceeded and that the compound produced by the substitution is stable, i.e., a compound that can be isolated, characterized, and tested for biological activity.

[0066] Unless otherwise specified, the substitution refers to that one or more H on each group is 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.

[0067] 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 or 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.

[0068] For 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 a connection at any available vertex of the aromatic ring. In some embodiments, this description also includes connections on a ring fused to an aromatic ring. For example, a bond drawn to the center of an indole benzene moiety will represent a bond connected to any available vertex of a six-membered or five-membered ring moiety of indole.

[0069] Unless otherwise specified, in the present invention, all compounds appearing are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). Among all compounds of the present invention, each chiral carbon atom can be optionally in R configuration or S configuration, or a mixture of R configuration and S configuration.

[0070] 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 all intended to be included within the scope of the present invention. When compounds provided herein have defined stereochemistry (expressed as R or S, or indicated by dashed or wedge-shaped bonds), those compounds will be understood by those skilled in the art to be substantially free of other isomers (e.g., at least 80%, 90%, 95%, 98%, 99% and up to 100% free of other isomers).

[0071] 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".

[0072] Compounds of formula I

[0073] The GPR84 probe provided by the present invention is a compound having the structure of Formula I shown above.

[0074]

[0075] Wherein, the definitions of M, L1 and R1 are as described in the present invention.

[0076] In the present invention, such as -O(C1~C 10 There is no particular restriction on the connection mode of groups such as -alkylene)-, -N(C1-C4 alkyl)-, and -C(=CH2)-. The atoms or groups connected are connected from left to right or from right to left. For example, for -L1-NH-, when L1 is -O(C1-C4 alkyl)-, 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.

[0077] The present invention also includes enantiomers, diastereomers, racemates, or pharmaceutically acceptable salts thereof of the compound.

[0078] The present invention also provides pharmaceutically acceptable salts thereof, including salts obtained by reacting the compound of formula I with an inorganic base or an organic base compound, especially salts formed after H at the M position leaves in the form of H+. Salts obtained 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, etc. 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.

[0079] Composition and use

[0080] The compound of the present invention has excellent GPR84 antagonistic activity and can selectively label the GPR84 receptor and emit fluorescence.

[0081] Therefore, the present invention provides a pharmaceutical composition comprising: the compound described in the first aspect of the present invention, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be used to prevent or treat inflammatory bowel disease, liver fibrosis, pulmonary fibrosis, arthritis and other related diseases caused by high expression or excessive excitability of GPR84 receptors.

[0082] "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 (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 ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0083] The compounds and compound compositions of the present invention may be in various forms, such as capsules, tablets, granules, solutions, powders, granules or syrups, or may be administered orally or non-orally in the form of injections, and 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. The above-mentioned preparations may be prepared by conventional synthetic methods.

[0084] Furthermore, the present invention also provides a GPR84 receptor fluorescent probe reagent, the reagent comprising the compound described in the first aspect of the present invention, or its enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt. The compound of formula I is a small molecule fluorescent probe targeting the GPR84 receptor, which can specifically bind to the GPR84 receptor on the cell membrane and further recognize the fat-soluble environment near the receptor to emit a specific fluorescent signal, and can be used to characterize the expression level of the GPR84 receptor at the cell and tissue levels.

[0085] The present invention also provides the use of the compound or its enantiomer, diastereomer, racemate or pharmaceutically acceptable salt or the composition of the fifth aspect, wherein the use is selected from the following group:

[0086] (i) for preparing GPR84 receptor (fluorescence) probe;

[0087] (ii) used as a fluorescent probe for the GPR84 receptor;

[0088] (iii) for preparing a GPR84 receptor antagonist;

[0089] (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.

[0090] Fluorescence detection method of GPR84 receptor

[0091] The present invention also provides a method for fluorescence detection of GPR84 receptors, comprising the steps of:

[0092] 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.

[0093] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0094] In another preferred embodiment, the method is a quantitative analysis method or a qualitative analysis method.

[0095] In another preferred embodiment, the method is used to detect the expression level of GPR84 receptor.

[0096] The fluorescent probe used in this method is the compound described in the first aspect. This type of compound hardly emits fluorescence in aqueous solution due to the solvochromic properties of its fluorescent group, but the GPR84 ligand fragment in the probe will bind to the GPR84 receptor expressed on the cell surface and anchor the probe, and then the Nile red fluorescent group at the other end of the probe will insert into the cell membrane near the receptor, obtain a fat-soluble environment and emit a specific fluorescent signal. This method can specifically reflect the expression level of GPR84 in cells and tissues by the intensity difference of the fluorescent signal, and the method can be used to preliminarily characterize the level of inflammation related to GPR84.

[0097] Preparation method

[0098] The compound of formula I can be prepared by the following route:

[0099]

[0100] (a) Compound S1 undergoes a coupling reaction with H-L1-NH-X3 to generate compound S2;

[0101] (b) Compound S2 removes HO-X2 through hydrolysis and generates compound S3 through phosphorylation;

[0102] (c) Compound S3 removes HO-X3 through hydrolysis reaction and undergoes condensation reaction with the Nile red group to generate compound P1;

[0103] In each formula, R1, L1, and M are defined as above;

[0104] X1 is Cl, Br or I;

[0105] Each X2 is independently methyl, methoxymethyl, or benzyl;

[0106] X3 is benzyloxycarbonyl or tert-butyloxycarbonyl.

[0107] 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, 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 reaction 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 about 6 to 48 hours. It is 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) and diisopropylethylamine (DIPEA) is used as an acid-binding agent. The reaction temperature is 0°C to 30°C; the reaction time is about 6 to 24 hours. After the reaction is completed, it is diluted with distilled water and extracted with a solvent such as ethyl acetate (EtOAc), diethyl ether (Et2O), dichloromethane (CH2Cl2), chloroform (CHCl3), etc. The organic phase concentrate is subjected to column chromatography to obtain the target intermediate S3. The third step is firstly carried out in a solvent such as acetic acid, and 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), ether (Et2O), dichloromethane (CH2Cl2), chloroform (CHCl3), etc. The organic phase concentrate is washed with ether (Et2O) and filtered to obtain a solid, which is redissolved in N,N-dimethylformamide (DMF) and then mixed with benzotriazole-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate (PyBOP), (3 H-1,2,3-triazolo[4,5-b]pyridine-3-oxyl)tri-1-pyrrolidinyl hexafluorophosphate (PyAOP) and the like are used as activators, triethylamine (Et3N), diisopropylethylamine (DIPEA) and other reagents provide an alkaline environment, the reaction temperature is 0℃~30℃; the reaction time is about 2~24h, after the reaction is completed, it is diluted with distilled water, extracted with solvents such as ethyl acetate (EtOAc), ether (Et2O), dichloromethane (CH2Cl2), chloroform (CHCl3), etc., and the organic phase concentrate is subjected to column chromatography to obtain the product P1. The product structure is identified and characterized by instruments such as nuclear magnetic resonance hydrogen spectrum (PMR), nuclear magnetic resonance carbon spectrum (CMR) and mass spectrometry (MS).

[0108] 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.

[0109] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All features disclosed in the specification of this case can be used in any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0110] The main advantages of the present invention include:

[0111] The inventors have developed a new type of small molecule three-fragment fluorescent probe targeting the GPR84 receptor, whose structure is composed of a GPR84 antagonist fragment with a binaphthol phosphate structure, a connecting chain fragment, and a fluorescent fragment of a 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 solvatochromicity of the Nile red group causes its fluorescence intensity to increase significantly, so that the probe compound emits 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, and only emits a fluorescent signal after recognizing the GPR84 receptor, and can recognize and respond to GPR84 receptors at different expression levels in cells and tissues.

[0112] 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 the bifunctional fragment can be simply and effectively applied to the field of other membrane receptor probes.

[0113] In addition, the compounds of the present invention are simple to synthesize with high yield, can quickly target corresponding membrane receptors and generate signals, have good specificity and high signal-to-noise ratio.

[0114] 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 present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0115] In the following examples, NMR was measured using AVANCE III 400M and AVANCE III 600M instruments produced by Bruker, and NMR calibration was: δH 7.26ppm (CDCl3), 2.50ppm (DMSO-d6), 3.15ppm (CD3OD); reagents were mainly 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; 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.

[0116] Example 1

[0117] Preparation of compound F1

[0118]

[0119] Synthesis of intermediate G3-1. The raw material G1-1 (384 mg, 0.63 mmol) was placed in a flask, dissolved with ethylene glycol dimethyl ether (DME, 4 mL) and replaced with argon protection. Pd(PPh3)4 (73 mg, 0.06 mmol) was quickly added to the system under stirring. After the addition was completed, G2-1 (369 mg, 0.70 mmol) and Na2CO3 solution (2M inH2O, 1 mL) were added at once, stirred at room temperature for about 10 min, and argon protection was replaced again after the addition was completed. The system was heated at 100°C for reflux reaction for about 16 h and then TLC monitoring was started. After the reaction is complete, distilled water (30 mL) is added to the system for dilution, and the organic phases are combined after extraction with ethyl acetate (3×30 mL), washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product is separated by Flash column chromatography purification system (PE / EtOAc=20 / 1~10 / 1) to obtain a yellow oily liquid G3-1 (485 mg, yield 64%).

[0120]

[0121] Synthesis of intermediate G4-1. G3-1 (184 mg, 0.22 mmol) was dissolved in 2M HCl ethyl acetate solution (20 mL), stirred at room temperature for about 4 h, and then TLC monitoring was started. After the raw material was completely converted, the solvent was concentrated under reduced pressure to remove the solvent, and 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 thereto. After the addition, the system was stirred at room temperature for about 16 h. After the reaction was completed, the solvent was removed under reduced pressure, and a system of THF:H2O:Et3N=1:1:0.1 (5 mL) was added to redissolve the product, and 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, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was separated by Flash column chromatography purification system (DCM / MeOH=25 / 1-10 / 1) to obtain a white solid powder G4-1 (74 mg, yield 39%).

[0122]

[0123] Synthesis of product F1. The intermediate G4-1 (96 mg, 0.12 mmol) was dissolved in 33% HBr acetic acid solution (15 mL), and the system was stirred at room temperature for about 2 hours and TLC monitoring was started. After the raw material disappeared completely, 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 with a minimum amount of dichloromethane and recrystallized with ice ether to obtain an off-white solid product. The product, Nile red fragment, and benzotriazole-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate (PyBOP) were placed in a flask and protected from light, and N,N-dimethylformamide (DMF) was added to dissolve it. Then, DIPEA was slowly added dropwise to the system under stirring. After the addition, the system was reacted at room temperature and protected from light. TLC monitoring of the reaction was started after about 1.5 hours. After the reaction was completed, 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 purification system (DCM / MeOH / TFA=25 / 1 / 0.01-10 / 1 / 0.01) to obtain a purple-black powder product F1 (62 mg, yield 58%).

[0124] The following compounds were synthesized in the same way

[0125]

[0126]

[0127] Example 2 Preparation of Compound F6

[0128]

[0129] Synthesis of intermediate G3-6. The raw material G1-6 (197 mg, 0.31 mmol) was placed in a flask, dissolved with ethylene glycol dimethyl ether (DME, 2 mL) and replaced with argon protection. Pd(PPh3)4 (32 mg, 0.03 mmol) was quickly added to the system under stirring. After the addition was completed, G2-1 (182 mg, 0.35 mmol) and Na2CO3 solution (2M inH2O, 0.5 mL) were added at once, stirred at room temperature for about 10 min, and argon protection was replaced again after the addition was completed. The system was heated at 100°C for reflux reaction for about 16 h and then TLC monitoring was started. After the reaction is complete, distilled water (20 mL) is added to the system for dilution, and the organic phases are combined after extraction with ethyl acetate (3×20 mL), washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product is separated by Flash column chromatography purification system (PE / EtOAc=20 / 1~10 / 1) to obtain colorless oily liquid G3-6 (192 mg, yield 55%).

[0130]

[0131] Synthesis of intermediate G4-6. G3-6 (192 mg, 0.23 mmol) was dissolved in 2M HCl ethyl acetate solution (15 mL), stirred at room temperature for about 4 h, and then TLC monitoring was started. After the raw material was completely converted, the solvent was concentrated under reduced pressure to remove the solvent, and 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 thereto. After the addition, the system was stirred at room temperature for about 16 h. After the reaction was completed, the solvent was removed under reduced pressure, and a system of THF:H2O:Et3N=1:1:0.1 (5 mL) was added to redissolve the product, and 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, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was separated by Flash column chromatography purification system (DCM / MeOH=25 / 1-10 / 1) to obtain a white solid powder G4-1 (72 mg, yield 36%).

[0132]

[0133] Synthesis of product F6. The intermediate G4-6 (72 mg, 0.10 mmol) was dissolved in 33% HBr acetic acid solution (15 mL), and the system was stirred at room temperature for about 2 hours and TLC monitoring was started. After the raw material disappeared completely, 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 with a minimum amount of dichloromethane and recrystallized with ice ether to obtain an off-white solid product. The product, Nile red fragment, and benzotriazole-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate (PyBOP) were placed in a flask and protected from light, and N,N-dimethylformamide (DMF) was added to dissolve it, and then DIPEA was slowly added dropwise to the system under stirring. After the dropwise addition, the system was reacted at room temperature and protected from light, and TLC monitoring of the reaction was started after about 1.5 hours. After the reaction was completed, 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 F6 (22 mg, yield 32%) was separated by Flash column chromatography purification system (DCM / MeOH / TFA = 25 / 1 / 0.01 to 10 / 1 / 0.01) to obtain a purple-black powder product F6 (22 mg, yield 32%).

[0134] The following compounds were synthesized in the same way

[0135]

[0136]

[0137] Example 3

[0138] (1) Experimental purpose

[0139] The compounds of the present invention were tested for their GPR84 antagonistic activity.

[0140] (2) Sources

[0141] The human GPR84 cell line was obtained by transfecting the HEK293 cell line with plasmids encoding GPR84 and Gα16 proteins. Fluorescent dye Fluo-4 AM was purchased from Invitrogen.

[0142] (3) Test principle

[0143] Intracellular Ca 2+ Ion is a very important second messenger in the G protein-coupled receptor signaling pathway. 16 After the protein-coupled GPR84 binds to the agonist, intracellular Ca 2+ The ion concentration can be significantly increased. Fluo-4 is a Ca 2+ Ion-specific fluorescent probe that can react with Ca2+ The ions bind quantitatively and emit fluorescence. Therefore, the agonist or antagonist activity of the compound is tested in a 96-well or 384-well flat-bottom microplate using fluorescence detection.

[0144] 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 the fluorescence intensity of the dye caused by the change in the intracellular calcium ion concentration was detected at 525nm. The half inhibitory concentration (IC50) of the compound was calculated using GraphPad PRISM software. 50 ).

[0145] (4) Experimental process

[0146] Prepare HBSS: 0.4g / L KCl (5.4mM), 0.12g / L Na2HPO4·12H2O (0.3mM), 0.06g / L KH2PO4 (0.4mM), 0.35g / L NaHCO3 (4.2mM), 0.14g / L CaCl2 (1.3mM), 0.10g / L MgCl2·6H2O (0.5mM), 0.05g / L MgSO4 (0.6mM), 8.0g / L NaCl (137mM). 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.

[0147] Preparation of calcium flux buffer: First, prepare 560mM D-glucose (100X) aqueous stock solution and 250mM 1,2-diphenyl-4-(2-phenylsulfinyl)ethyl-3,5-pyrazolidinedione (1000X) stock solution. Then add BSA (0.5g), 560mM D-glucose stock solution (1mL) and 250mM 1,2-diphenyl-4-(2-phenylsulfinyl)ethyl-3,5-pyrazolidinedione stock solution (100μL) to 100mL HBSS, so that the final concentrations are 0.5% BSA, 5.6mM D-glucose, and 250μM 1,2-diphenyl-4-(2-phenylsulfinyl)ethyl-3,5-pyrazolidinedione, respectively. Mix well and use immediately.

[0148] 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.

[0149] The cells were plated at 4 × 10 4 The cells were inoculated 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 Fluo-4 AM fluorescent dye was added to each well and incubated in a 37°C incubator for 40 minutes. After the dye was discarded, 50 μL of the test compound was added and incubated at room temperature for 10 minutes. Then, 25 μL 6-OAU (the effective concentration after dilution was within the EC value of the agonist) was added using the 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 485nm, detection wavelength 525nm).

[0150] (5) Experimental results

[0151] Table 1. IC of probe compounds F1-F11 50

[0152] Compound <![CDATA[IC 50 (μM)]]> Compound <![CDATA[IC 50 (μM)]]> F1 0.396 F6 0.168 F2 0.458 F7 0.334 F3 1.89 F8 0.206 F4 0.293 F9 0.129 F5 1.95 F10 1.38 F11 1.78

[0153] (6) Results Analysis

[0154] The results in Table 1 show that the compounds 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.

[0155] Example 4

[0156] (1) Experimental purpose

[0157] 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 polar environments.

[0158] (2) Test principle

[0159] The solvatochromicity 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.

[0160] (3) Experimental process

[0161] 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, and each solvent was dissolved in triplicate. The excitation wavelength was 520 nm, and the fluorescence emission spectrum of each compound in the range of 550 nm to 750 nm was recorded.

[0162] (4) Experimental results

[0163] All probe compounds have significant solutochromic properties, such as Figure 1 As shown in the figure, compound F1 basically does not emit fluorescence in the highly polar solvent water (H2O), but as the polarity of the solvent decreases, its fluorescence intensity increases significantly, reaching the strongest in the least polar 1,4-dioxane solvent. Its emission wavelength also undergoes a significant blue shift as the polarity of the solvent decreases, and the maximum emission wavelength difference can reach 50nm. Figure 1 The fluorescence emission spectra of probe compounds F1-F11 indicate that the probe compounds described in this invention have good fluorescence specificity.

[0164] Example 5

[0165] (1) Experimental purpose

[0166] 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.

[0167] (2) Test principle

[0168] 1) Compare the fluorescence imaging results of the probe compounds on HEK293 / Gα16 cells that overexpress hGPR84 and control group empty HEK293 / Gα16 cells;

[0169] 2) Competitive antagonists of the probe compounds were added to examine the changes in the fluorescence signals of the probe compounds in hGPR84-HEK293 / Gα16 cells.

[0170] (3) Experimental process

[0171] 1) The probe compounds were incubated at a concentration of 1 μM with HEK293 / Gα16 cells overexpressing hGPR84 and control group empty HEK293 / Gα16 cells for 15 min, and the differences were observed under a fluorescence microscope.

[0172] 2) The probe compounds were divided into two groups and incubated with HEK293 / Gα16 cells that highly expressed 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.

[0173]

[0174] (4) Experimental results

[0175] 1) The probe compound showed fluorescence signal differences in both the HEK293 / Gα16 cells with high expression of hGPR84 and the control group of empty HEK293 / Gα16 cells. Figure 2 As shown, the probe compound F8 can generate specific fluorescence signals on the membrane of HEK293 / Gα16 cells that highly express hGPR84, but there is no specific fluorescence in the empty cells of the control group. Figure 2 The comparative experimental results of the high-expression GPR84 cell system and the empty-transfection cell system of the probe compounds F6-F9 indicate that this type of probe compound can selectively label the GPR84 receptor and emit fluorescence.

[0176] 2) The specific signal of the probe compound on the membrane of HEK293 / Gα16 cells that highly express hGPR84 can be completely competed

[0177] The inhibitor 17S inhibits. Figure 3 As shown, the probe compound F8 can specifically label the HEK293 / Gα16 cell membrane that highly expresses hGPR84, but the fluorescence signal completely disappears under the competition of 17S, indicating that the probe compound F8 specifically labels the GPR84 receptor on the cell membrane surface to emit fluorescence. Figure 3 The competitive imaging experiment results of probe compounds F6-F9 indicate that the fluorescence signals of this type of probe compounds are highly GPR84 selective.

[0178] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

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 fluorescent probe for the GPR84 receptor; (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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