GPR84 RECEPTOR ANTAGONIST AND USE THEREOF

Abstract

The present invention relates to a GPR84 receptor antagonist and use thereof. The GPR84 receptor antagonist of the present invention has a structure as represented by formula (I), the definitions of R1, R2, R3, R4, L.sub.1, L.sub.2, L.sub.3, L.sub.4, L.sub.5, L.sub.6, Y, Z, and rings A, B, C, and D are as described in the description and claims. The GPR84 receptor antagonist of the present invention can competitively inhibit the activation of the receptor caused by an agonist of GPR84, and can be used in the preparation of a medicament for treating related diseases caused by high expression or high excitability of GPR84 receptor, the diseases including multiple sclerosis, inflammatory bowel disease, arthritis and the like.

##STR00001##

Claims

1. A compound or a pharmaceutically acceptable salt having the structure of formula I, ##STR00086## wherein, Y is O or S; Z is H, or an ion of the following metal: Li, Na, K, Ca, Mg, Cu, Fe, Zn, Al, Mn, or a conjugated acid of the following base: NH.sub.3, arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, aminoglucose, histidine, hydroxycobalamin, isopropylamine, lysine, methyl glucosamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, trometamol; L.sub.3 and L.sub.6 are each independently O, S, NH or CH.sub.2; each of rings A, B, C, and D is independently a C.sub.6-C.sub.10 aromatic ring, a C.sub.3-C.sub.10 cycloalkane ring, a C.sub.3-C.sub.10 heterocycloalkane ring, or a C.sub.3-C.sub.10 heteroaryl ring; R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are each independently 1 to 4 substituents on rings A, B, C, and D, and each substituent is independently absent, hydroxyl, mercapto, amino, F, Cl, Br, I, C.sub.rH.sub.2r-L.sub.7-C.sub.sH.sub.2s+1, C.sub.rH.sub.2rN(C.sub.tH.sub.2t+1)C.sub.sH.sub.2s+1, substituted or unsubstituted C.sub.1-C.sub.6 alkyl, substituted or unsubstituted C.sub.3-C.sub.6 cycloalkyl, substituted or unsubstituted C.sub.6-C.sub.10 aryl, substituted or unsubstituted C.sub.3-C.sub.10 heteroaryl, the above substitution means there is one or more substituents selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, halogen, hydroxyl, amino, COOC.sub.1-C.sub.6 alkyl, COOH; L.sub.7 is each independently 0, S, NH, each r is independently an integer of 0-6, each s is independently an integer of 0-6, and each t is independently an integer of 1-6; L.sub.2 and L.sub.5 are each independently absent, CH, N; L.sub.1 and L.sub.4 are each independently absent, CH, O, S, SO, SO.sub.2, CHCH, CO, C(CH.sub.2), substituted or unsubstituted C.sub.1-C.sub.6 alkylidene, NH, N(C.sub.1-C.sub.4 alkyl)-, C.sub.3-C.sub.6 cycloalkyl or C.sub.3-C.sub.6 heterocycloalkyl, said substituted means that there is one or more substituents selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, halogen, hydroxy; represents a single bond or a double bond.

2. The compound according to claim 1, wherein each of rings A, B, C and D is independently a benzene ring, a C.sub.3-C.sub.6 cycloalkane ring, a C.sub.3-C.sub.6 heterocycloalkane ring or a C.sub.3-C.sub.6 heteroaryl ring.

3. The compound according to claim 1, wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are each independently one, two or three substituents on rings A, B, C and D, and each substituent is independently absent, substituted or unsubstituted C.sub.1-C.sub.4 alkyl, C.sub.rH.sub.2r-L.sub.7-C.sub.sH.sub.2s+1, C.sub.rH.sub.2rN(C.sub.tH.sub.2t+1)C.sub.sH.sub.2s+1, hydroxyl, mercapto, amino, F, Cl, Br, I; the above substitution means there is one or more substituents selected from the group consisting of halogen, hydroxyl, amino, COOC.sub.1-C.sub.6 alkyl, COOH; L.sub.7 is each independently O, S, NH, each r is independently an integer of 0-4, each s is independently an integer of 0-4, and each t is independently an integer of 1-4.

4. The compound according to claim 1, wherein L.sub.1 and L.sub.4 are each independently absent, CH, O, S, SO, SO.sub.2, CHCH, CO, C(CH.sub.2), substituted or unsubstituted C.sub.1-C.sub.4 alkylidene, NH, N(C.sub.1-C.sub.3 alkyl)-, C.sub.3-C.sub.6 cycloalkyl or C.sub.3-C.sub.6 oxa-cycloalkyl, said substitution means there is one or more substituents selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, halogen, hydroxyl.

5. The compound according to claim 1, wherein, a carbon attached to L.sub.3 and/or L.sub.6 and a carbon of ring B and/or ring D form CHCH when L.sub.2 and/or L.sub.5 are absent; a carbon attached to L.sub.3 and/or L.sub.6 and a carbon attached to L.sub.2 and/or L.sub.5 form CHCH or CHN when L.sub.2 and/or L.sub.5 are CH or N.

6. The compound according to claim 1, wherein said compound is: ##STR00087## ##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095## ##STR00096## ##STR00097##

7. A method for preparing the compound according to claim 1, wherein the method comprises the following step: ##STR00098## reacting a compound of formula S1, a compound of formula S2 and a compound of formula S3 as starting materials to obtain the compound of formula I, wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, L.sub.1, L.sub.2, L.sub.3, L.sub.4, L.sub.5, L.sub.6, Y, Z, ring A, B, C, and D are as defined in claim 1, and represents a single bond or a double bond; X is F, Cl, Br or I.

8. A method for preparing the compound according to claim 1, wherein the method comprises the following steps: ##STR00099## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, L.sub.1, L.sub.2, L.sub.3, L.sub.4, L.sub.5, L.sub.6, Y, Z, ring A, B, C, and D are as defined in claim 1, and represents a single bond or a double bond; X is F, Cl, Br or I.

9. A pharmaceutical composition, comprising the compound or the pharmaceutically acceptable salt according to claim 1; and a pharmaceutically acceptable carrier.

10. Use of the compound or the pharmaceutically acceptable salt according to claim 1, (i) for the preparation of a GPR84 antagonist; (ii) as a GPR84 antagonist; (iii) for the preparation of a medicament for the treatment of a related disease caused by hyper-excitability or high expression of GPR84 receptor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0049] The inventors of the present application have extensively and intensively studied to develop a novel GPR84 antagonist, which can competitively inhibit the activation of the receptor by GPR84 agonist, and can be used for the preparation of a medicament for treating a related disease caused by hyper-excitability or high expression of GPR84 receptor including multiple sclerosis, inflammatory bowel disease, arthritis, and the like. On the basis of this, the present invention has been completed.

Definition

[0050] In the present invention, C.sub.6-C.sub.10 means there are 6 to 10 carbon atoms, and C.sub.3-C.sub.6 means there are 3 to 6 carbon atoms, and so on.

[0051] An integer of 0-4 means 0, 1, 2, 3, 4; and 6-10 carbon atoms means 6, 7, 8, 9, 10 carbon atoms, and so on.

[0052] In the present invention, terms such as an aromatic ring, a cycloalkane ring, an alkyl and the like have the same meanings as those familiar to those skilled in the art unless otherwise specified. For example, alkyl refers to a saturated linear or branched hydrocarbyl; for example, CH.sub.3 or CH(CH.sub.3).sub.2; alkylidene refers to a remaining moiety formed by formally removing two one-valence hydrogens from a saturated hydrocarbyl, including but not limited to methene (CH.sub.2), ethylene (CH.sub.2CH.sub.2), and the like. Alkoxy means O-(alkyl), including but not limited to OCH.sub.3, OCH.sub.2CH.sub.3 and the like. Cycloalkyl refers to a saturated cyclic hydrocarbyl such as a cyclohexyl. Heterocycloalkyl refers to a saturated cyclic hydrocarbyl containing at least one hetero atom (e.g., N, O or S). Heteroaryl refers to an aromatic ring containing at least one hetero atom.

[0053] Unless otherwise stated, the aromatic ring, heteroaryl ring, cycloalkane ring, alkyl, alkylidene, alkoxy, cycloalkyl, heterocycloalkyl, and the like described herein include both substituted and unsubstituted moiety, and possible substituent includes, but is not limited to, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.3-C.sub.20 cycloalkyl, C.sub.3-C.sub.20 cycloalkenyl, C.sub.1-Cao heterocycloalkyl, C.sub.1-C.sub.20 heterocycloalkenyl, alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, hydroxy, halogen, mercapto, cyano, nitro, carboxyl and carboxylate group.

GPR84 Antagonist

[0054] The GPR84 antagonist provided by the present invention is a compound having the structure of formula I:

##STR00016##

[0055] Each substituent is as defined above.

[0056] Most preferably, the compounds of formula I according to the present invention are compounds prepared in the examples.

[0057] The invention also provides a pharmaceutically acceptable salt thereof, comprising a salt formed from the reaction of the compound of formula I and an inorganic or organic base compound.

[0058] 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, manganese salts, manganite salts, potassium salts, sodium salts, zinc salts, etc. Ammonium salts, calcium salts, magnesium salts, potassium salts and sodium salts are particularly preferred.

[0059] Salts are derived from pharmaceutically acceptable organic non-toxic bases including, but not limited to, salts of primary, secondary and tertiary amines, substituted amines include naturally occurring substituted amines, cyclic amines and basic ions exchange resin such as arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, aminoglucose, histidine, hydroxocobalamin, isopropylamine, lysine, methyl glucosamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, trometamol, and the like.

Preparation Method

[0060] The compound of formula I can be achieved by any of the following routes 1 to 6:

Route 1

[0061] The reaction is carried out in pyridine; the reaction temperature is from 60 C. to 100 C.; the reaction time is about 1 to 24 hours; after the reaction is completed, the reaction mixture is extracted with a solvent such as AcOEt, Et.sub.2O, CH.sub.2C.sub.12, CHCl.sub.3 and the like, washed with saturated brine, dried, and subjected to low temperature and reduced pressure to remove the solvent, and the concentrate is subjected to column chromatography to give the desired product confirmed by NMR or the like.

[0062] When the starting material S1=S3, two equivalents of the starting material S1 are required for the reaction, and the obtained product P1 has a symmetrical structure. When the starting material S1S3, the reaction produces a mixture of three products (P1 to P3), and each product is separated by column chromatography.

##STR00017##

[0063] Each substituent is as defined above.

Route 2

[0064] The reaction is carried out in toluene; the base used is hexamethyldisilazane, the reaction temperature is 80 C. to 120 C.; the reaction time is about 1 to 24 hours; after the reaction is completed, the reaction mixture is extracted with a solvent such as AcOEt, Et.sub.2O, CH.sub.2Cl.sub.2, CHC.sub.13 and the like, washed with a saturated brine, dried, and subjected to low temperature and reduced pressure to remove the solvent, and the concentrate is subjected to column chromatography to give the desired product confirmed by NMR or the like.

##STR00018##

[0065] When the starting material S4=S6, two equivalents of the starting material Si are required for the reaction, and the obtained product P1 has a symmetrical structure. When the starting material S4S6, the reaction produces a mixture of three products (P1 to P3), and each product is separated by column chromatography.

[0066] Each substituent is as defined above.

Route 3: Salt Formation

[0067] ##STR00019##

[0068] The starting material P2 is dissolved in EA, and washed twice with an aqueous solution, into which a base (the conjugate base of M, the hydroxide of M or the carbonic acid compound of M) is added. The aqueous layer is back-extracted with EA, the EA layer is concentrated, and the crude product is subjected to silica gel column chromatography to obtain the product P4.

[0069] M is a cation of the following metals: Li, Na, K, Ca, Mg, Cu, Fe, Zn, Al, Mn;

[0070] or a conjugated acid of the following base: NH3, arginine, betaine, caffeine, choline, N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, aminoglucose, histidine, hydroxocobalamin, isopropylamine, lysine, methyl glucosamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, trometamol, and the like; and the other substituents are as defined above.

Route 4: Dealkylation

[0071] ##STR00020##

[0072] The starting material P5 is dissolved in anhydrous DCM under N.sub.2, and a solution of BBr.sub.3 in DCM is added dropwise under dry ice-acetone cooling, then gradually warmed to room temperature and stirred overnight. The reaction solution is diluted with water and extracted with EA, EA layer is washed with brine, and the organic phase is concentrated and subjected to silica gel column chromatography to obtain P6.

[0073] R1 is a C.sub.1-C.sub.4 alkyl, and the other substituents are as defined above.

Route 5: Hydrolysis

[0074] ##STR00021##

[0075] The starting material P7 is dissolved in a mixed solution of DCM/MeOH, and an aqueous NaOH solution is added thereto under stirring at room temperature, and reacted at room temperature. After the reaction is completed, the reaction solution is concentrated under reduced pressure to remove MeOH, and then diluted with water. The pH was adjusted to pH 2 with 1N HCl, then the mixture is extracted with EA, washed with brine, and the organic phase is dried over anhydrous Na.sub.2SO.sub.4, filtrated, and concentrated to obtain a crude product which is recrystallized from PE/DCM to give the hydrolysate P8.

[0076] R.sub.5 is a C.sub.1-C.sub.4 alkyl, and L.sub.7 is a C.sub.1-C.sub.4 alkylidene, and the other substituents are as defined above.

Route 6: Oxidation

[0077] ##STR00022##

[0078] The starting material P9 is dissolved in MeOH, and a solution of Oxone in H.sub.2O is added dropwise with stirring at room temperature overnight. The reaction solution is diluted with water, extracted with EA and washed with brine. The organic phase is concentrated and subjected to silica gel column chromatography to obtain P10 and P11.

Use

[0079] The compound of the formula I, as an antagonist of GPR84, is capable of competitively inhibiting the activation of the receptor by an agonist of GPR84, and can be used for the preparation of a medicament for treating a disease caused by hyper-excitability or high expression of GPR84 receptor. The diseases include multiple sclerosis, inflammatory bowel disease, arthritis and the like.

Pharmaceutical Composition

[0080] The pharmaceutical composition of the present invention comprises a therapeutically effective amount of the compound of formula I or the pharmaceutically acceptable salt thereof, and one or more medicinal carriers.

[0081] Medicinal carrier, pharmaceutically acceptable carrier or pharmacologically available carrier means one or more compatible solid or liquid filler or gel material which is suitable for human use and which must have sufficient purity and low toxicity. By compatibility, it is meant herein that each component in the composition is capable of intermixing with the active ingredient of the present invention (the compound of formula I or a pharmaceutically acceptable salt thereof) without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricant (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (Tween), wetting agent (such as sodium dodecyl sulfate), colorant, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water and the like.

[0082] The compounds and pharmaceutical compositions of the present invention may be in various forms, and may be administered orally in the form of, such as capsule, tablet, granule, solution, powder, pulvis or syrup, or administered in the non-oral form, such as an injection. The compounds and pharmaceutical compositions may be presented in a suitable solid or liquid vehicle and in a suitable sterilizing device for injection or drip. The above formulations can be prepared by conventional pharmaceutical methods.

[0083] The compounds and pharmaceutical compositions of the present invention are useful for clinical use in mammals, including humans and animals, and can be administered by the oral, nasal or gastrointestinal routes. The most preferred route of administration is oral.

[0084] The above features mentioned in the present invention, or the features mentioned in the embodiments, may be arbitrarily combined. All of the features disclosed in the present specification can be used in combination with any of the compositions, and each of the various features disclosed in the specification can be replaced by any alternative feature that provides the same, equal or similar purpose. Therefore, unless otherwise stated, the disclosed features are only general examples of equal or similar features.

[0085] The invention is further illustrated below in conjunction with specific examples. It is to be understood that the examples are for illustrative purposes only and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions suggested by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0086] In the following examples, NMR was measured using a Mercury-VX 300M or Mercury-VX400M instrument manufactured by Varian (NMR calibration: H 7.26 ppm (CDCl.sub.3), 2.50 ppm (DMSO-d.sub.6), 3.15 ppm (CD.sub.3OD)). The reagents were mainly provided by Shanghai Chemical Reagent Co., Ltd. TLC thin layer chromatography silica gel plate (model, HSGF 254) is produced by Shandong Yantai Huiyou Silicone Development Co., Ltd. The normal phase column chromatography silica gel used for compound purification (model zcx-11, 200-300 mesh) is produced by Branch Factory of Shandong Qingdao Marine Chemical Plant.

EXAMPLE 1

Preparation of compound CLH514

[0087] ##STR00023##

[0088] G1 was synthesized by referring to J. Med. Chem. 2010, 53, 7021-7034; and PCT Int. Appl., 2009016085, 05 Feb. 2009. Some analogs of G1 can be purchased from reagent companies, and the others were prepared using the same or similar routes as for the synthesis of G1.

[0089] The starting material G1 (510 mg, 2.25 mmol) was dissolved in dry pyridine (2.5 mL), and then double-distilled POCl.sub.3 (207 L, 2.25 mmol) was added dropwise under N.sub.2 and reacted at 130 C. for 3 h. The reaction solution was cooled to room temperature, diluted with water, adjusted to pH2 with 6 N HCl and then extracted with EA. The aqueous layer was back-extracted three times, dried over anhydrous Na.sub.2SO.sub.4, filtrated, concentrated, and then subjected to silica gel column chromatography (DCM/MeOH=20/110/1) to obtain the desired compound CLH514 (230 mg, 40%, white solid). .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 7.63 (d, J=7.2 Hz, 2H), 7.41-7.50 (m, 4H), 7.20-7.40 (m, 12H), 3.56 (br, 1H).

[0090] The following compounds were synthesized in the same manner:

TABLE-US-00001 No. Structure formula .sup.1H NMR data CLH478 [00024] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.64 (d, J = 7.5 Hz, 2H), 7.25 (s, 2H), 6.78-7.10 (m, 14H), 3.36 (s, 4H), 3.00 (br, 1H). CLH482 [00025] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.43 (d, J = 7.5 Hz, 2H), 6.90-7.10 (m, 12H), 6.69 (t, J = 6.9 Hz, 4H), 3.59 (br, 1H). CLH508 [00026] .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 7.35-7.55 (m, 2H), 7.10-7.35 (m, 4H), 6.80-7.10 (m, 12H), 3.34 (s, 1H), 3.17 (s, 3H), 3.12 (s, 3H).

EXAMPLE 2

Preparation of compound CLH536

[0091] ##STR00027##

[0092] The starting material G1 (2.635 g, 11.64 mmol) was dissolved in dry pyridine (10 mL), and then double-distilled POCl.sub.3 (0.610 mL, 6.64 mmol) was added dropwise under N.sub.2 and reacted at 130 C. for 3 h. The reaction solution was cooled to room temperature, diluted with water, adjusted to pH2 with 6 N HCl and then extracted with EA. The EA layer was washed with saturated Na.sub.2CO.sub.3 solution. The aqueous layer was back-extracted three times, dried over anhydrous Na.sub.2SO.sub.4, filtrated, concentrated, and then subjected to silica gel column chromatography (DCM/MeOH=10/1) to obtain the desired compound CLH536 (2.247 g, 72%, white solid). .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 7.72 (dd, J=5.4 Hz, 1.8 Hz, 2H), 7.40-7.44 (m, 4H), 7.15-7.35 (m, 12H).

[0093] The following compounds were synthesized in the same manner.

TABLE-US-00002 No. Structure formula .sup.1H NMR data CLH472 [00028] .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 8.76 (d, J = 8.4 Hz, 2H), 8.65-8.72 (m, 2H), 8.27 (d, J = 8.1 Hz, 2H), 7.85 (s, 2H), 7.70-7.75 (m, 2H), 7.65 (t, J = 6.9 Hz, 2H), 7.45-7.60 (m, 6H). CLH472a [00029] .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 8.73 (d, J = 8.4 Hz, 4H), 8.33 (s, 2H), 8.06 (d, J = 8.4 Hz, 4H), 7.48 (t, J = 7.2 Hz, 4H), 7.39 (t, J = 7.2 Hz, 4H) CLH524 [00030] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.35 (d, J = 8.1 Hz, 1H), 7.20-7.28 (m, 1H), 6.72-7.10 (m, 16H), 6.50-6.70 (m, 4H). CLH524a [00031] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.60-7.70 (m, 2H), 7.00-7.20 (m, 10H), 6.80-7.00 (m, 6H), 4.80-5.00 (m 4H). CLH528 [00032] .sup.1H NMR (CDCl.sub.3, 300 MHz): 8.13-8.16 (m, 2H), 8.01-8.04 (m, 2H), 7.60-7.75 (m, 6H), 7.40-7.50 (m, 2H), 7.28-7.40 (m, 4H), 6.80 s, 2H). CLH528a [00033] .sup.1H NMR (CDCl.sub.3, 400 MHz): 7.35-7.40 (m, 4H), 7.15-7.25 (m, 8H), 7.10-7.16 (m, 4H), 6.40-6.60 (br, 2H), 4.20 (q, J = 6.6 Hz, 2H), 1.62 (s, 6H). XYF528 [00034] .sup.1H NMR (CDCl.sub.3, 400 MHz): 7.27 (s, 2H), 6.86-6.97 (m, 14H), 6.75 (s, 2H), 3.04-3.14 (m, 8H). CLH544 [00035] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.47-7.72 (br, 2H), 7.01-7.25 (br, 2H), 6.71-7.69 (br, 2H), 6.36-6.62 (br, 2H), 3.70 (s, 6H), 3.00-3.20 (br, 4H), 2.26-2.56 (br, 4H), 1.20-1.60 (m, 8H). CLH544a [00036] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.55-7.47 (m, 4H), 7.30-7.15 (m, 4H), 6.72 (s, 2H), 2.41 (t, J = 12.5 Hz, 4H), 2.32 (t, J = 12.0 Hz, 4H), 1.80-1.67 (m, 4H), 1.65-1.52 (m, 4H). CLH548 [00037] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.28-7.35 (m, 2H), 7.08-7.18 (m, 8H), 7.02-7.08 (m, 2H), 6.77 (s, 2H). CLH552 [00038] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.72 (d, J = 6.6 Hz, 1H), 7.60 (d, J = 6.6 Hz, 1H), 7.34 (s, 2H), 7.16 (s, 4H), 6.88-7.10 (m, 6H), 6.60-6.88 (m, 4H), 1.16-1.36 (br, 3H), 1.11 (t, J = 6.3 Hz, 1H), 0.35-0.60 (m, 2H), 0.15-0.35 (br, 2H). CLH560 [00039] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.20-7.30 (m, 4H), 7.13 (d, J = 7.8 Hz, 2H), 6.90-7.06 (m, 4H), 6.66-6.86 (m, 4H), 6.57 (d, J = 8.1 Hz, 2H), 3.66 (s, 6H), 3.40-3.58 (br, 4H). XYF560 [00040] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.85-8.1 (m, 4H), 6.50-7.10 (m, 6H). CLH572a [00041] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.30-8.00 (br, 5H), 6.10-7.20 (m, 11H). CLH572b [00042] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.15-8.00 (br, 6H), 6.60-7.13 (m, 7H), 6.00-6.60 (br, 3H). CLH572c [00043] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.30-8.50 (m, 6H), 6.10-7.20 (m, 10H). XYF573 [00044] .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 7.71 (d, J = 7.2 Hz, 2H), 7.15-7.42 (m, 14H). XYF573a [00045] .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 7.71 (d, J = 9.0 Hz, 2H), 7.17-7.48 (m, 12H), 7.02 (d, J = 7.8 Hz, 2H). XYF573b [00046] .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 7.71 (d, J = 6.9 Hz, 2H), 7.32-7.48 (m, 10H), 7.06-7.10 (m, 2H), 6.97 (d, J = 10.2 Hz, 2H). XYF573c [00047] .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 7.73 (d, J = 7.8 Hz, 2H), 7.15-7.46 (m, 14H). CLH596a [00048] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.40 (d, J = 8.4 Hz, 2H), 7.30 (s, 2H), 7.22 (d, J = 7.8 Hz, 2H), 7.03 (t, J = 7.2 Hz, 4H), 6.88-7.00 (m, 4H), 6.59 (d, J = 7.2 Hz, 2H), 3.67 (s, 6H). CLH596b [00049] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.30-7.70 (br, 4H), 7.10-7.30 (br, 2H), 6.50-7.10 (m, 8H), 6.00-6.50 (br, 2H), 3.49 (s, 6H). CLH596c [00050] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.32-7.60 (br, 2H), 7.17-7.30 (br, 2H), 6.85-7.17 (m, 8H), 6.70-6.85 (br, 2H), 6.45-6.65 (br, 2H), 3.34 (s, 6H). XYF608 [00051] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.15-7.23 (m, 4H), 6.86-6.96 (m, 4H), 6.58-6.63 (m, 4H), 3.67 (s, 6H) CLH616a [00052] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.46-7.52 (m, 4H), 7.28-7.44 (m, 8H), 7.12-7.22 (m, 4H), 6.75 (s, 2H), 4.15-4.35 (m, 8H). CLH624 [00053] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.30-7.52 (m, 4H), 7.12-7.23 (m, 2H), 6.94-7.12 (m, 4H), 6.73-6.93 (m, 4H), 6.50-6.68 (m, 2H), 3.90 (q, J = 6.6 Hz, 4H), 1.37 (t, J = 6.6 Hz, 6H). XYF628 [00054] .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 7.55 (d, J = 7.5 Hz, 2H), 7.31-7.36 (m, 6H), 7.16-7.23 (m, 6H), 2.94 (q, J = 7.5 Hz, 4H), 1.17 (t, J = 7.5 Hz, 3H). XYF628a [00055] .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 7.55 (d, J = 7.5 Hz, 2H), 7.16-7.40 (m, 10H), 7.03 (d, J = 9.6 Hz, 2H), 2.97 (q, J = 7.5 Hz, 4H), 1.18 (t, J =7.5 Hz, 3H). XYF628b [00056] .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 7.54-7.56 (m, 4H), 7.31-7.34 (m, 4H), 7.20 (t, J = 7.5 Hz, 2H), 7.09 (t, J = 8.4 Hz, 2H), 6.95 (d, J = 9.6 Hz, 2H), 2.93 (q, J = 7.5 Hz, 4H), 1.16 (t, J = 7.5 Hz, 3H). XYF628c [00057] .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 7.56 (d, J = 7.2 Hz, 2H), 7.15-7.43 (m, 12H), 2.93 (q, J = 7.5 Hz, 4H), 1.17 (t, J = 7.5 Hz, 3H). XYF632 [00058] .sup.1H NMR (CDCl.sub.3, 400 MHz): 7.21 (s, 2H), 7.08-7.13 (m, 4H), 6.84-6.91 (m, 4H), 6.61 (d, J = 8.0 Hz, 2H), 6.50-6.58 (m, 2H), 3.69 (s, 6H). XYF632a [00059] .sup.1H NMR (d.sub.6-DMSO, 400 MHz): 7.35 (s, 2H), 7.27-7.30 (m, 6H), 7.14 (t, J = 8.8 Hz, 2H), 7.07 (dd, J = 2.0 Hz, 7.6 Hz, 2H), 6.98 (d, J = 8.0 Hz, 2H), 3.82 (s, 6H). XYF632b [00060] .sup.1H NMR (d.sub.6-DMSO, 400 MHz): 7.14-7.29 (m, 12H), 7.05 (d, J = 8.0 Hz, 2H), 3.81 (s, 6H). XYF632c [00061] .sup.1H NMR (d.sub.6-DMSO, 400 MHz): 7.39 (s, 2H), 7.17-7.32 (m, 10H), 7.06 (d, J = 8.0 Hz, 2H), 3.81 (s, 6H). CLH652 [00062] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.29-7.46 (d, J = 6.9 Hz, 4H), 7.15-7.25 (m, 2H), 6.96-7.10 (m, 4H), 6.75-6.96 (m, 4H), 6.58-6.73 (d, J = 6.6 Hz, 2H), 4.15-4.39 (m, 2H), 1.26 (d, J = 6.0 Hz, 12H). CLH656 [00063] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.40 (s, 2H), 7.20-7.34 (m 2H), 6.80-7.10 (m, 8H), 6.72 (s, 2H), 3.75 (s, 6H), 3.28 (s, 6H). CLH656a [00064] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.43-7.46 (m, 2H), 7.06-7.23 (m, 8H), 6.59-6.74 (m, 4H), 3.74 (s, 3H), 3.73 (s, 3H), 3.60 (s, 6H). XYF656 [00065] .sup.1H NMR (CDCl.sub.3, 400 MHz): 7.25-7.28 (m, 4H), 6.92-6.95 (m, 6H), 6.59 (d, J = 4.0 Hz, 2H), 6.45 (d, J = 4.0 Hz, 2H), 3.70 (s, 6H), 3.63 (s, 6H). XYF656a [00066] .sup.1H NMR (d.sub.6-DMSO, 400 MHz): 7.30 (s, 2H), 7.28 (d, J = 1.6 Hz, 2H), 7.17-7.22 (m, 4H), 7.02 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 8.0 Hz, 2H), 6.73 (d, J = 8.0 Hz, 2H), 3.80 (s, 12H). XYF656b [00067] .sup.1H NMR (d.sub.6-DMSO, 400 MHz): 7.30-7.34 (m, 6H), 7.21 (t, J = 8.0 Hz, 2H), 7.03 (d, J = 8.0 Hz, 2H), 6.77 (dd, J = 4.0 Hz, 12.0 Hz, 2H), 6.66 (d, J = 4.0 Hz, 2H), 3.81 (s, 12H), (3.68, 2H). CLH736 [00068] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.57 (d, J = 7.5 Hz, 2H), 7.20-7.40 (m, 6H), 6.90-7.15 m, 8H), 3.93 (q, J = 7.2 Hz, 4H), 3.44 (q, J = 7.2 Hz, 2H), 1.24 (d, J = 7.2 Hz, 6H), 1.04 (t, J = 7.2 Hz, 6H). XYF536c [00069] 1H NMR (d6-DMSO, 300 MHz): 7.65 (d, J = 7.5 Hz, 2H), 7.14-7.34 (m, 12H) 6.98-7.04 (m, 2H), 3.59 (s, 4H) XYF604 [00070] 1H NMR (d6-DMSO, 500 MHz): 7.71 (d, J = 7.5 Hz, 2H), 7.45-7.49 (m, 4H), 7.34-7.41 (m, 6H), 7.26 (t, J = 7.5, 2H), 7.15 (d, J = 8.0 Hz, 2H) XYF604a [00071] 1H NMR (d6-DMSO, 500 MHz): 7.70 (d, J = 8.0 Hz, 2H), 7.51 (dd, J = 7.5 Hz, 1.0 Hz, 2H), 7.30-7.39 (m, 8H), 7.20 (d, J = 8.5 Hz, 2H) XYF604b [00072] 1H NMR (d6-DMSO, 300 MHz): 7.71 (d, J = 7.8 Hz, 2H), 7.28-7.45 (m, 12H), 7.17 (d, J = 2.1, 2H). XYF604c [00073] 1H NMR (d6-DMSO, 300 MHz): 7.69 (d, J = 7.2 Hz, 2H), 7.54 (s, 2H) 7.22-7.47 (m, 12H) XYF564a [00074] 1H NMR (d6-DMSO, 500 MHz): 7.58 (d, J = 7.5 Hz, 2H), 7.52 (d, J = 7.5 Hz, 2H), 7.38 (s, 2H), 7.32-7.21 (m, 8H), 7.16 (t, J = 7.5 Hz, 2H), 2.53 (s, 6H) XYF564b [00075] 1H NMR (d6-DMSO, 500 MHz): 7.63 (d, J = 8.0 Hz, 2H), 7.44 (dd, J = 7.5, 1.0 Hz, 2H), 7.34 (s, 2H), 7.30-7.22 (m, 6H), 7.18 (d, J = 7.5 Hz, 2H), 7.11 (d, J = 7.5 Hz, 2H), 2.26 (s, 6H) XYF564c [00076] 1H NMR (d6-DMSO, 500 MHz): 7.49 (s, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.38 (s, 2H), 7.32-7.21 (m, 6H), 7.20 (d, J = 7.5 Hz, 2H), 7.15 (dd, J = 8.0, 1.5 Hz, 2H), 2.14 (s, 6H) XYF540 [00077] 1H NMR (d6-DMSO, 500 MHz): 7.62 (dd, J = 8.0, 2.0 Hz, 2H), 7.41 (td, J = 8.0, 2.0 Hz, 2H), 7.31-7.24 (m, 4H), 7.16 (td, J = 8.0, 1.5 Hz, 2H), 7.12-7.03 (m, 6H) XYF564-2a [00078] 1H NMR (d6-DMSO, 500 MHz): 7.26-7.23 (m, 2H), 7.20-7.05 (m, 10H), 6.96 (s, 12H), 3.86 (s, 6H)

EXAMPLE 3

Preparation of compound CLH666

[0094] ##STR00079##

[0095] The starting material G2 was purchased from a reagent company, and the synthesis of the starting material G3 was carried out in the same synthetic route as G1.

[0096] The starting materials G2 (62.7 mg, 0.192 mmol) and G3 (49.2 mg, 0.192 mmol) were dissolved in dry pyridine (0.5 mL), and then double-distilled POCl.sub.3 (21 L, 0.230 mmol) was added dropwise under N.sub.2 and reacted at 130 C. for 3 h. The reaction solution was diluted with EA, then diluted with water, adjusted to pH1 with 6 N HCl and then extracted with EA three times. The EA layer was washed with saturated brine and then saturated Na.sub.2CO.sub.3 solution. The organic phase was concentrated and subjected to silica gel column chromatography (DCM/MeOH=20/16/1) to obtain the desired compound CLH666 (23 mg, 18%, white solid). .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.53 (d, J=6.9 Hz, 1H), 7.42 (d, J=7.8 Hz, 1H), 7.16-7.37 (m, 5H), 6.83-7.16 (m, 8H), 6.61 (d, J =8.4 Hz, 1H), 3.75-4.00 (m, 2H), 3.69 (s, 3H), 3.43 (q, J=6.9 Hz, 1H), 1.22 (d, J=6.9 Hz, 3H), 1.01 (t, J=6.9 Hz, 3H).

Example 4

Preparation of compounds CLH568 and CLH582

[0097] ##STR00080##

[0098] The starting material, CLH596a (45.2 mg, 0.076 mmol), was dissolved in dry EtOAc (1 mL). 2N solution of BBr3 in DCM ((190 L, 0.3 mmol) was added dropwise under N.sub.2 and dry ice-acetone cooling, then gradually warmed to room temperature and stirred overnight. The reaction solution was diluted with water and extracted with EA three times. The EA layer was washed with brine and then saturated Na.sub.2CO.sub.3 solution. The organic phase was concentrated and purified by silica gel preparation plate (DCM/MeOH=20/16/1, developed twice) to obtain the desired compounds.

[0099] CLH568 (18.3 mg, 42.3%, white solid). .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 9.50-9.70 (br, 2H), 7.47 (d, J=6.3 Hz, 2H), 7.10-7.35 (m, 10H), 7.04 (t, J=7.8 Hz, 2H), 6.89 (J=7.8 Hz, 2H).

[0100] CLH582 (11.2 mg, 25.3%, white solid). .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.30-7.40 (m, 3H), 7.15-7.26 (m, 3H), 7.12 (d, J=7.2 Hz, 1H), 6.70-7.05 (m, 8H), 6.38 (d, J=8.1 Hz, 1H), 3.61 (s, 3H).

EXAMPLE 5

Preparation of compound CLH680

[0101] ##STR00081##

[0102] The starting material CLH736 (13 mg, 0.018 mmol) was dissolved in 4 mL of methanol, and 1 N aqueous NaOH (1 mL) was added under stirring at room temperature, and reacted at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to remove MeOH, diluted with water, and then adjusted to pH2 with 1 N HCl. The mixture was extracted with EA and washed with brine. The aqueous layer was back-extracted once and the organic phase was dried over Na.sub.2SO.sub.4, filtrated and concentrated. The obtained crude product was recrystallized from PE/DCM to give the product CLH680 (8.8 mg, 72%, white solid). .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.57 (d, J=7.5 Hz, 2H), 7.20-7.40 (m, 6H), 6.90-7.15 (m, 8H), 3.44 (q, J=7.2 Hz, 2H), 1.24 (d, J=7.2 Hz, 6H).

[0103] The following compound was synthesized in the same manner:

TABLE-US-00003 CLH638 [00082] .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.57 (d, J = 6.9 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.16-7.37 (m, 5H), 6.83-7.16 (m, 8H), 6.77 (d, J = 7.5 Hz, 1H), 3.67 (s, 3H), 3.40-3.45 (m, 1H), 1.29 (d, J = 5.7 Hz, 3H).

EXAMPLE 6

Preparation of compounds CLH600, CLH584 and CLH568a

[0104] ##STR00083##

[0105] The starting material CLH536 (100 mg, 0.186 mmol) was dissolved in MeOH (20 mL). A solution of Oxone (potassium hydrogen peroxymonosulfate, 500 mg, 1.627 mmol) in H.sub.2O was added dropwise under stirring at room temperature and reacted at room temperature overnight. The reaction solution was diluted with water, extracted with EA, washed with brine and subjected to silica gel column chromatography (DCM/MeOH=25/16/1) to obtain the products.

[0106] CLH600 (20 mg, 18%, white solid). .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 8.16 (s, 2H), 8.06 (d, J=7.2 Hz, 4H), 7.65-7.80 (m, 8H), 7.50-7.65 (m, 4H).

[0107] CLH584 (12 mg, 11%, white solid). .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 8.00-8.12 (m, 3H), 7.46-7.80 (m, 11H), 7.30-7.46 (m, 4H).

[0108] CLH568a (10 mg, 9%, white solid). .sup.1H NMR (d.sub.6-DMSO, 300 MHz): 6.60-7.90 (m, 8H), 7.56 (t, J=7.2 Hz, 2H), 7.32-7.50 (m, 7H), 7.25 (d, J=7.2 Hz, 1H).

EXAMPLE 7

Preparation of compound XYF532

[0109] ##STR00084## ##STR00085##

[0110] The starting material G1 (226 mg, 1 mmol) was dissolved in THF (4 mL), and a solution of methylmagnesium bromide in diethyl ether (3 mol/L, 0.5 mL) was added dropwise under the ice bath, then warmed to room temperature and reacted for 3 hours. The saturated ammonium chloride solution was added to quench the reaction. The mixture was extracted with EA, washed with saturated brine, dried over anhydrous sodium sulfate and dried by rotary evaporation. A solution of hydrochloric acid/ethyl acetate solution (4 mol/L, 2 mL) was added and reacted at room temperature overnight. The reaction solution was concentrated and the crude product was separated by column chromatography (PE/EA=20/110/1) to afford product G4 (135 mg, yield 60%).

[0111] The starting material G4 (135 mg, 0.60 mmol) was dissolved in carbon tetrachloride (5 mL), and NBS (139 mg, 0.78 mmol) and AIBN (10 mg) were added successively, and the mixture was heated to reflux for 5 hours, then cooled to room temperature and filtered to remove the solid. The filtrate was concentrated and separated by column chromatography to afford product G5 (82 mg, yield 45%).

[0112] The starting material G5 (100 mg, 0.33 mmol) and amine phosphite (11 mg, 0.13 mmol) were dissolved in anhydrous toluene (1.5 mL), and hexamethyldisilazane (HMDS, 110 L, 0.53 mmol) was added under argon atmosphere and heated to reflux overnight. The mixture was cooled to room temperature and dried by rotary evaporation to remove toluene. Then dichloromethane: methanol (1: 1) was added to dissolve the residue. The mixture was dried by rotary evaporation and subjected to silica gel column chromatography (DCM/MeOH=25/16/1) to give product XYF 532 (30 mg, yield 43%). .sup.1H NMR (CDCl.sub.3, 300 MHz): 7.10-7.40 (m, 18H), 2.96-3.27 (br, 4H).

EXAMPLE 8

1. Experiment Purpose

[0113] GPR84 antagonistic activity of the compounds of the invention was determined.

2. Material Source

[0114] The human GPR84 cell line was obtained by transfecting a HEK293 cell line with a plasmid encoding GPR84 and Ga16 proteins. Fluorescent dye Fluo-4AM was purchased from Invitrogen.

3. Test Principle

[0115] Intracellular Ca.sup.2+ ion is a very important second messenger of G protein-coupled receptor signaling pathway. When combined with G16 protein-coupled GPR84 and agonist, intracellular Ca.sup.2+ ion concentration can be significantly increased. Fluo-4 is a Ca.sup.2+ ion-specific fluorescent probe that binds quantitatively to Ca.sup.2+ ions and emits fluorescence. Therefore, the agonistic or antagonistic activity of the compound can be detected in a 96-well or 384-well flat-bottomed microplate using a fluorescence assay.

[0116] Detection of receptor inhibitory effect by GPR84 antagonist: After incubated with fluorescent dye Fluo-4, GPR84 cells were incubated with different concentrations of antagonistic compounds for a period of time to occupy the binding site of agonist to GPR84 (antagonistic binding site). A certain concentration of agonist (6-OAU) was added to compete with the antagonist compound for binding sites. A light source with a wavelength of 485 nm was used for excitation and changes in the fluorescence intensity of the dye caused by changes in intracellular calcium concentration at 525 nm was detected. The half-inhibitory concentration (IC.sub.50) of the compound was calculated by using GraphPad PRISM software.

4. Experiment Procedure

[0117] Preparation of HBSS: 0.4 g/L KCl (5.4 mM), 0.12 g/L Na.sub.2HPP.sub.4.12H.sub.2O (0.3 mM), 0.06 g/L KH.sub.2PO.sub.4 (0.4 mM), 0.35 g/L NaHCO.sub.3 (4.2 mM), 0.14 g/L CaCl.sub.2 (1.3 mM), 0.10 g/L MgCl.sub.2.6H.sub.2O (0.5 mM), 0.05 g/L MgSO.sub.4 (0.6 mM), 8.0 g/L NaCl (137 mM). The above ingredients were weighed and dissolved in ultrapure water. pH was adjusted to 7.4 by using hydrochloric acid or NaOH solution. The mixture was filtered and stored at 4 C. for one month.

[0118] Preparation of calcium buffer: 560 mM D-glucose (100) aqueous stock solution and 250 mM 1,2-diphenyl-4-(2-phenylsulfinyl)ethyl-3,5-pyrazolidinedione (1000) stock solution were firstly prepared. Then BSA (0.5 g), 560 mM D-glucose (1 mL) stock solution and 250 mM 1,2-diphenyl-4-(2-phenylsulfinyl)ethyl-3,5-pyrazolidinedione (100 L) stock solution were added to 100 mL HBSS to a final concentration of 0.5% BSA, 5.6 mM D-glucose, 250 M 1,2-diphenyl-4-(2-phenylsulfinyl)ethyl-3,5-pyrazolidinedione and mixed. The buffer was prepared on site.

[0119] Preparation of dyes: 3% Cremophor EL (100) stock solution in PBS and 2 mM Fluo-4 (1000) stock solution in DMSO were firstly prepared. The preparation of each milliliter of dye is as follows. 1 L of 2 mM Fluo-4 AM was mixed with 10 L of 3% Cremophor EL, then diluted with 1 mL of calcium buffer and mixed.

[0120] The cells were seeded at a density of 40,000cells/well into a 96-well cell culture plate, and cultured for 24 hours or more to a cell density of 80 to 90% for experimental detection. The culture medium in the well to be tested was removed and 40 L/well of freshly prepared dye was added and incubated in a 37-degree incubator for 40 min to 50 min.

[0121] The compound was formulated during cell incubation (this step can also be carried out in advance). The compound used as antagonist was diluted with calcium buffer freshly prepared before the experiment to 1.5 times of the final working concentration. The compound as agonist was diluted to 3 times of the final working concentration (if the compound is dissolved in DMSO, the final work concentration of DMSO shall not exceed 1%).

[0122] After the incubation step was completed, the dye was discarded, and the residue was washed with calcium buffer once and then replaced with 50 L of calcium buffer containing different concentrations of antagonist and incubated for another 10 min.

[0123] 25 L/well of calcium buffer containing a certain concentration of agonist (usually an effective concentration of agonist EC.sub.80) was added for stimulation and a light source with a wavelength of 485 nm was used for excitation and changes in the fluorescence intensity of the dye caused by changes in intracellular calcium concentration at 525 nm was detected by using a FlexStation III microplate reader.

5. Experiment Results

[0124]

TABLE-US-00004 TABLE 1 IC.sub.50 of the compound detected by the GPR84 calcium flow model Compound No. GPR84 CLH472 * CLH472a ** CLH478 *** CLH482 ** CLH508 ** CLH514 *** CLH524 ** CLH524a ** CLH528 ** CLH528a ** XYF528 ** XYF532 * CLH536 *** CLH544 ** CLH544a ** CLH548 *** CLH552 *** CLH560 *** XYF560 ** XYF604b *** XYF604c *** XYF540 *** CLH568 *** CLH572a ** CLH572b ** CLH572c ** XYF573 ** XYF573a ** XYF573b *** XYF573c *** CLH582 *** CLH584 * CLH596a *** CLH596b *** CLH596c *** CLH600 * XYF608 *** CLH616a ** CLH624 *** XYF628 * XYF628a *** XYF564b *** XYF564c *** XYF628b ** XYF628c *** XYF632 *** XYF632a *** XYF632b *** XYF632c *** CLH638 *** CLH652 ** CLH656 *** CLH656a ** XYF656 *** XYF656a ** XYF656b *** CLH666 *** CLH680 * CLH736 * XYF536c *** XYF604 ** XYF604a ** XYF564-2a *** XYF564a *** * 1-10 M; ** 0.1-1 M; *** 0.1-100 nM

[0125] All documents mentioned in the present application are incorporated herein by reference, just as each document is cited separately as a reference. In addition, it should be understood that various modifications and changes may be made by those skilled in the art after reading the above teachings of the present invention. These equivalent forms are also within the scope defined by the claims appended hereto.