COMPOUNDS HAVING AGONISTIC EFFECT AGAINST GPR84, PREPARATION METHOD FOR COMPOUNDS AND USE OF COMPOUNDS
20180237399 ยท 2018-08-23
Inventors
- Fajun NAN (Pudong, Shanghai, CN)
- Xin XIE (Pudong, Shanghai, CN)
- Yang LIU (Pudong, Shanghai, CN)
- Qing ZHANG (Pudong, Shanghai, CN)
- Linhai CHEN (Pudong, Shanghai, CN)
- Hui YANG (Pudong, Shanghai, CN)
Cpc classification
A61P29/00
HUMAN NECESSITIES
C07D309/38
CHEMISTRY; METALLURGY
C07C39/08
CHEMISTRY; METALLURGY
A61P7/00
HUMAN NECESSITIES
C07D239/54
CHEMISTRY; METALLURGY
A61K31/505
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
C07C39/19
CHEMISTRY; METALLURGY
C07D239/545
CHEMISTRY; METALLURGY
International classification
C07C39/19
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a class of compounds represented by the formula I, or pharmaceutically acceptable salts thereof, methods for their preparation, and application as small molecule tools that function as GPR84 agonists, and their use in preparing a medicament for the treatment of septicemia.
##STR00001##
Claims
1. A compound represented by formula I or a pharmaceutically acceptable salt thereof, ##STR00084## wherein, R.sub.1 is R.sub.1a, R.sub.1b or R.sub.1c; ##STR00085## each of R.sub.5a, R.sub.5b and R.sub.5c is independently methyl, isopropyl, C2-C9 alkenyl, C2-C4 alkynyl, 3-6 membered cycloalkyl, cyano, hydroxy, unsubstituted phenyl, phenyl substituted with C1-C4 alkyl, substituted phenyl substituted with C1-C3 alkoxy, or fluorophenyl; subscript n is an integer selected from 0-16; T, W and Y are each independently O, N or C; R.sub.2 is hydroxy, amino, trifluoromethyl, or C1-C3 alkyl; R.sub.3 is absent or is hydrogen, benzyl or C1-C6 alkyl; R.sub.4 is absent or is hydrogen or C1-C3 alkyl; Z is OH, NH.sub.2, O, S or C1-C6 alkylcarbonyl.
2. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein, R.sub.1 is R.sub.1a, R.sub.1b or R.sub.1c; ##STR00086## each of R.sub.5a, R.sub.5b and R.sub.5c is independently methyl, isopropyl, C2-C9 alkenyl, ethynyl, 3-4 membered cycloalkyl, cyano, hydroxy, unsubstituted phenyl, phenyl substituted with C1-C4 alkyl, methoxyphenyl, or fluorophenyl; subscript n is an integer selected from 0-9; T, W and Y are each independently N or C; R.sub.2 is hydroxy, amino, trifluoromethyl, or methyl; R.sub.3 is absent or is hydrogen or benzyl; R.sub.4 is absent or is hydrogen or methyl; Z is OH, NH.sub.2, -O, S, or C1-C6 alkylcarbonyl.
3. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein, R.sub.1 is R.sub.1a, wherein R.sub.5a is methyl, isopropyl, 3-4 membered cycloalkyl, unsubstituted phenyl, cyano, hydroxy, phenyl substituted with C1-C4 alkyl, methoxyphenyl, or fluorophenyl; R.sub.2 is hydroxy; subscript n is an integer selected from 0-14; T, W and Y are each independently O, N or C; R.sub.3 is absent or is hydrogen, benzyl or C1-C3 alkyl; R.sub.4 is absent or is hydrogen or C1-C3 alkyl; Z is OH, NH.sub.2, O, S or C1-C6 alkylcarbonyl.
4. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of the formula I has the structure of Formula II: ##STR00087## R.sub.1 is R.sub.1a, R.sub.1b or R.sub.1c; R.sub.2 is hydroxy, methyl, amino, or trifluoromethyl; W and Y are each independently N; R.sub.4 is hydrogen or C1-C3 alkyl; Z is OH or NH.sub.2.
5. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of the formula I has the structure of Formula III: ##STR00088## wherein, R.sub.1 is R.sub.1a, R.sub.1b or R.sub.1c; W is N or C.
6. A compound or pharmaceutically acceptable salt thereof according to claim 1, wherein, the compound is selected from: ##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095##
7. A pharmaceutical composition comprising a therapeutically effective amount of one or more selected from the group consisting of the compound or pharmaceutically acceptable salts thereof according to claim 1, and one or more pharmaceutically acceptable carriers.
8. A use of the compound or pharmaceutically acceptable salts thereof according to claim 1, in preparing a medicament for the treatment of septicemia.
Description
DETAILED DESCRIPTION
[0114] The following examples are provided to describe the invention in further detail. These examples are only used for illustration of the present invention without intended to limit the scope of the invention.
Preparation Examples of the Compounds
[0115] In the following Preparation Examples, NMR was measured using a Mercury-Vx 300M 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; the silica gel plate (Model No.: HSGF 254) used in TLC thin layer chromatography was produced by HuiyouSilica Gel Development Co., Ltd, Yantai, Shandong; silica gel used in the normal phase column chromatography for compound purification was produced by the branch of Ocean chemical Plant in Qingdao, Shandong, Model No.: zcx-11, 200-300 mesh.
Preparation Example 1 (Compound No.: LY214-5)
[0116] ##STR00031##
[0117] KI (11.3 mg, 0.069 mmol, 0.1 eq.) was added to a solution of compound 1 (100 mg, 0.69 mmol, 1.0 eq.) in EtOH/H.sub.2O (10 mL/5 mL), and compound 2-1 (310 mg, 0.26 mL, 3.0 eq.) was slowly added. The reaction mixture was heated at 80 C. for 6 hours. After the reaction was complete which was monitored by TLC, the residue was added dilute hydrochloric acid (1M, 10 mL), and extracted with ethyl acetate for three times. The combined organic layers were washed with brine, dried, filtered, concentrated and purified using flash column chromatography (DCM:MeOH=30:1) to give LY214-5 (white solid, 12 mg, 8%). .sup.1H NMR (DMSO-d.sub.6) 512.1 (s, 2H), 5.11 (s, 1H), 3.08 (t, J=6.0 Hz, 2H), 1.61 (m, 2H), 1.36 (m, 2H), 1.28 (m, 2H), 0.87 (t, J=6.3 Hz, 3H).
[0118] The following compound was synthesized in the same manner:
TABLE-US-00001 Cpd# Chemical structure .sup.1H NMR (300 MHz) data LY214-5
Preparation Example 2 (Compound No.: LY224-a)
[0119] ##STR00055##
[0120] Compound 3-1 (0.1 mL, 1.02 mmol, 1 eq.) was dissolved in dry DCM (5 mL), and pyridine was added (104 mg, 1.3 mmol, 1.3 eq.). The reaction mixture was cooled to 0 C., then a solution of TsCl (214 mg, 1.12 mmol, 1.1 eq.) in anhydrous DCM (5 mL) was slowly added dropwise. The reaction mixture was allowed to warm to 20 C. and stirred for 12 h monitored by TLC. Upon completion, the residue was concentrated and purified by a flash chromatography on silica gel (DCM:MeOH=30:1) to give compound 4-1 (colorless oil, 145 mg, 56%). .sup.1H NMR (300 MHz, CDCl.sub.3) 7.80 (d, J=5.1 Hz, 2H), 7.34 (d, J=8.1 Hz, 2H), 4.05 (t, J=7.2 Hz, 2H), 2.50 (m, 2H), 2.45 (s, 3H), 2.08 (m, 2H), 1.06 (t, J=7.5 Hz, 3H).
[0121] Compound 1 (67 mg, 0.47 mmol, 1.0 eq.) was dissolved in EtOH/H.sub.2O (10 mL/5 mL) and compound 4-1 (145 mg, 0.52 mmol, 1.1 eq.) was added slowly. The reaction was carried out at 80 C. for 6 h monitored by TLC. Upon completion, the mixture was added diluted hydrochloric acid (1M, 10 mL) followed by extracted with ethyl acetate (15 mL) for three times. The combined organic extracts were washed with brine, dried, filtered, concentrated and chromatographed (DCM:MeOH=30:1) to give LY224-a (white solid, 44 mg, 42%). .sup.1H NMR (300 MHz, DMSO-d.sub.6) 11.36 (s, 1H), 5.12 (s, 1H), 3.18 (t, J=6.3 Hz, 2H), 2.52-2.55 (m, 2H), 2.21-2.03 (m, 2H), 1.03 (t, J=7.5 Hz, 3H).
Preparation Example 3 (Compound No.: LY224-b)
[0122] ##STR00056##
[0123] Sodium (250 mg) was dissolved in ethanol (8 mL), then added compound 5 (500 mg, 4.9 mmol, 1 eq). After the solid was dissolved, compound 6-1 (1.1 g, 5.89 mmol, 1.2 eq.) in ethanol (5 mL) was slowly added dropwise. A white solid was precipitated. After heated to reflux for 3 hours, the reaction mixture was then cooled and filtered. The filtrate was concentrated and added 3 mL of H.sub.2O to dissolve the solid. A dilute hydrochloric acid (2M) was slowly added dropwise to adjust pH to 3-5. A white precipitation was filtered. The filter cake was added 5 mL of methanol, stirred for 1 h, filtered and dried to obtain compound LY224-b (white solid, 1.0 g, 91%). .sup.1H NMR (300 MHz, DMSO-d.sub.6) 11.64 (s, 2H), 5.02 (s, 1H), 2.52 (t, J=7.5 Hz, 2H), 1.62 (q, J=6.6 Hz 2H), 1.25 (m, 10H), 0.84 (t, J=6.6 Hz, 3H).
[0124] The following compound was synthesized in the same manner:
TABLE-US-00002 Cpd# Chemical structure .sup.1H NMR (300 MHz) data LY182
Preparation Example 4 (Compound No.: LY244)
[0125] ##STR00063##
[0126] Compound 7-1 (0.5 mL, 3.68 mmol, 1 eq.) was dissolved in dry DCM (20 mL), then added triphenylphosphine (1.2 g, 4.41 mmol, 1.2 eq.), carbon tetrabromide (1.4 g, 4.41 mmol, 1.2 eq.) and stirred at 20 C. for 12 h. Upon the reaction was completion monitored by TLC, the solvent was removed under reduced pressure, and the residue was chromatographed (petroleum ether) to afford compound 8-1 (colorless oil, 310 mg, 59%). .sup.1H NMR (300 MHz, CDCl.sub.3) 7.80 (d, J=8.1 Hz, 2H), 7.34 (d, J=8.1 Hz, 2H), 4.05 (s, 2H), 2.50 (m, 2H), 2.45 (q, J=8.1 Hz, 3H), 2.08 (m, 2H), 1.06 (t, J=7.5 Hz, 3H).
[0127] Diethyl malonate (91 mg, 0.63 mmol, 1 eq.) was dissolved in THF (10 mL), cooled to 78 C. NaH (19.7 mg, 0.82 mmol, 1.1 eq.) was added to the solution and stirred for 10 minutes. Then, compound 8-1 (100 mg, 0.69 mmol, 1.1 eq.) was slowly dripped in. The reaction was gradually warmed to 20 C. and stirred for 12 h monitored by TLC. After reaction completion, the reaction was quenched with water (5 mL), extracted with ethyl acetate (10 mL) for three times, and washed twice with water (5 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and purified by a flash chromatography on silica gel to obtain compound 9-1 (colorless oil, 52 mg, 41%). .sup.1H NMR (300 MHz, CDCl.sub.3) 7.10 (m, 4H), 4.25-4.10 (m, 4H), 3.67 (t, J=6.9 Hz, 1H), 3.17 (d, J=7.8 Hz, 2H), 2.60 (q, J=13.8 Hz, 2H), 1.27 (t, J=7.2 Hz, 3H), 1.20 (t, J=7.2, 6H).
[0128] A solution of compound 9-1 (130 mg, 0.63 mmol, 1 eq.) and NaCl (74 mg, 1.26 mmol, 2 eq.) in DMSO (2 mL) was heated at 160 C. for 3 h. The reaction mixture was added water (5 mL) and extracted three times with ethyl acetate (5 mL). The organic phase was washed three times with water (5 mL). Then, the organic phase was combined and washed with brine, dried over anhydrous sodium sulfate, and purified by a flash chromatography on silica gel to obtain compound 10-1 (colorless oil, 56 mg, 43%). .sup.1H NMR (300 MHz, CDCl.sub.3) 7.12 (m, 4H), 4.14 (q, J=14.4 Hz, 2H), 2.91 (t, J=8.4 Hz, 2H), 2.59 (t, J=8.4 Hz, 2H), 2.54 (q, J=13.8 Hz, 2H), 1.28 ((t, J=7.2 Hz, 3H), 1.22 ((t, J=7.2 Hz, 3H).
[0129] Sodium (250 mg) was dissolved in ethanol (8 mL), then added compound 2 (212 mg, 2.08 mmol, 1.1 eq.). After the solid was dissolved, compound 10-1 (390 mg, 1.89 mmol, 1 eq.) in ethanol (5 mL) was slowly added dropwise. A white solid was precipitated. After heated to reflux for 3 h, the reaction was cooled to room temperature, filtered, concentrated, dissolved with 3 mL of H.sub.2O. A dilute hydrochloric acid (2M) was slowly added dropwise to the residue to adjust pH to 3-5. A white precipitation was filtered. The filter cake was added in 5 mL of methanol, stirred for 1 h, filtered and dried to obtain compound LY244 (white solid, 147 mg, 35%). H NMR (300 MHz, DMSO) 11.50 (s, 2H), 7.12 (m, 4H), 5.06 (s, 1H), 2.95-2.88 (t, J=7.5 Hz, 2H), 2.78-2.69 (t, J=8.1 Hz, 2H), 2.60-2.53 (q, J=7.5 Hz, 2H), 1.15 (t, J=7.5 Hz, 1H).
Preparation Example 5 (Compound No.: LY237 & LY238-d)
[0130] ##STR00064##
[0131] Compound 11 (1 mL, 7.7 mmol, 1 eq.) was added in dry THF (20 mL), cooled to 0 C., then added NaH (203 mg, 8.5 mmol, 1.1 eq.), stirred for 5 minutes, added n-butyllithium (5.3 mL, 8.5 mmol, 1.6M, 1.1 eq.). Stirred for another 5 minutes, bromide (1.3 mL, 7.7 mmol, 1 eq.) was added and stirred at 0 C. for 12 h monitored by TLC. The reaction mixture turned into a yellow milky liquid. After full completion, the reaction was quenched with water (10 mL) and extracted three times with ethyl acetate (10 mL). The combined organic extracts were washed with brine, dried over anhydrous sodium sulfate, and purified by a flash chromatography on silica gel to give compound 12-1 (yellow oil, 647 mg, 34%). .sup.1H NMR (300 MHz, CDCl.sub.3) 4.19 (q, J=15 Hz, 2H), 3.42 (s, 2H), 2.53 (t, J=7.2 Hz, 2H), 1.28 (m, 14H), 0.87 (t, J=6.3 Hz, 3H). .sup.1H NMR (300 MHz, DMSO) 12.53 (s, 1H), 3.43 (s, 2H), 2.39 (t, J=7.2 Hz, 2H), 1.23 (m, 14H), 0.85 (t, J=6.3 Hz, 3H).
[0132] Compound 12-1 (647 mg, 2.67 mmol, 1 eq.) was dissolved in EtOH/H.sub.2O (10/10 mL), then added NaOH (139 mg, 3.47 mmol, 1.3 eq.). The reaction was stirred at 20 C. for 12 h. The reaction mixture was washed twice with ethyl acetate (10 mL). The aqueous layer was adjusted to acidic (pH 3-5) with 1N HCl. A white solid was precipitated, filtered and dried in vacuo to obtain compound 13-1 (white solid, 410 mg, 72%). .sup.1H NMR (300 MHz, DMSO) 12.53 (s, 1H), 3.43 (s, 2H), 2.39 (t, J=7.2 Hz, 2H), 1.23 (m, 14H), 0.85 (t, J=6.3 Hz, 3H).
[0133] Compound 13-1 (100 mg, 0.47 mmol, 1 eq.) was dissolved in anhydrous THF (10 mL), then added 1,1-carbonyldiimidazole (106 mg, 0.65 mmol, 1.4 eq.) and stirred at 20 C. for 12 h. The reaction mixture was added H.sub.2O (10 mL) and extracted three times with ethyl acetate (10 mL). The combined the organic layers were concentrated and added methanol (3 mL). The reaction was left to stand until white needle crystals were precipitated, then filtered to obtain compound 14-1 (white needle crystals, 28 mg, 16%). .sup.1H NMR (300 MHz, CDCl.sub.3) 12.30 (s, 1H), 5.91 (s, 1H), 3.07 (t, J=7.2 Hz, 2H), 2.47 t, J=7.8 Hz, 2H), 1.65 (m, 4H), 1.26 (m, 24H), 0.88 (t, J=6.7, 5.7 Hz, 6H).
[0134] Compound 14-1 (14 mg, 0.037 mmol, 1 eq.) was dissolved in 90% H.sub.2SO.sub.4 (5 mL), heated at 130 C. for 1 h. The reaction mixture was added ethyl acetate (5 mL). The phases were separated, and the organic layer was concentrated in vacuo. The crude product was purified by a flash chromatography on silica gel to give LY238-d (white solid, 6 mg, 67%). .sup.1H NMR (300 MHz, CDCl.sub.3) 5.96 (s, 1H), 5.57 (s, 1H), 5.30 (s, 1H), 2.46 (t, J=7.5 Hz, 2H), 1.62 (m, 2H), 1.26 (m, 12H), 0.87 (d, J=6.3 Hz, 3H).
[0135] A mixture of compound LY238-d (26 mg, 0.1 mmol, 1 eq.) in 30% ammonium hydroxide (5 mL) was heated to reflux at 100 C. for 14 h. A dilute hydrochloric acid (1M) was added to adjust the pH to 3-4. A white solid was precipitated, filtered, and dried to obtain compound LY237 (white solid, 20 mg, 83%). .sup.1H NMR (300 MHz, DMSO) 10.85 (s, 1H), 10.27 (s, 1H), 5.58 (s, 1H), 5.33 (s, 1H), 2.33 (t, J=7.5 Hz, 2H), 1.51 (q, J=6.6 Hz, 2H), 1.24 (m, 12H), 0.85 (t, J=6.0 Hz, 3H).
[0136] The following compound was synthesized in the same manner:
TABLE-US-00003 Cpd# Chemical structure .sup.1H NMR (300 MHz) data LY223
Preparation Example 6 (Compound No.: LY250)
[0137] ##STR00067##
[0138] 1-bromoheptane (231 mg, 1.2 mmol, 1 eq.) was slowly added dropwise under N.sub.2 atmosphere to a suspension of magnesium (58 mg, 2.4 mmol, 2 eq.) in anhydrous ether (5 mL). The reaction was refluxed at 50 C. for 45 minutes, and slowly added compound 16 (200 mg, 1.2 mmol, 1 eq.) in anhydrous ether (10 mL), then, refluxed at 50 C. for 3 h. After completion of the reaction monitored by TLC, the reaction was slowly quenched with water (5 mL) and extracted three times with ethyl acetate (10 mL). The combined organic layers were washed with brine, dried, concentrated, and chromatographed (petroleum ether:ethyl acetate=10:1) to give compound 17-1 (colorless oil, 200 mg, 60%). .sup.1H NMR (300 MHz, CDCl.sub.3) 6.51 (d, J=2.1 Hz, 2H), 6.37 (t, J=2.4 Hz, 1H), 4.64-4.54 (m, 1H), 3.78 (s, 6H), 1.57 (m, 2H), 1.26 (m, 12H), 0.85 (t, J=6.6 Hz, 3H).
[0139] Compound 17-1 (220 mg, 0.79 mmol, 1 eq.) was dissolved in dry DCM (10 mL), then added PCC (507 mg, 2.36 mmol, 3 eq.) and silica gel (880 mg), and stirred at 20 C. for 14 h monitored by TLC. After full completion, the reaction mixture was filtered, concentrated, and chromatographed (petroleumether:ethylacetate=10:1) to obtain compound 18-1 (white solid, 161 mg, 73%). .sup.1H NMR (300 MHz, CDCl.sub.3) 7.09 (d, J=2.1 Hz, 2H), 6.63 (t, J=2.1 Hz, 1H), 3.83 (s, 6H), 2.87 (t, J=7.5 Hz, 2H), 1.77-1.62 (m, 2H), 1.30 (m, 12H), 0.88 (t, J=6.6 Hz, 3H).
[0140] Compound 18-1 (157 mg, 0.56 mmol, 1 eq.) was dissolved in dry DCM (5 mL) and cooled to 78 C., then slowly added BBr.sub.3 (2M 0.85 mL, 1.68 mmol, 3 eq.) dropwise. The reaction was slowly warmed to 20 C. and stirred for 14 h monitored by TLC. The reaction was quenched by the dropwise addition of water (5 mL), and extracted three times with ethyl acetate (10 mL). The combined organic extracts were washed with brine, dried, concentrated. The residue was purified by a flash chromatography on silica gel (petroleumether:ethylacetate=3:1) to afford compound LY250 (colorless oil, 100 mg, 71%). .sup.1H NMR (300 MHz, MeOD-d.sub.4) 6.87 (d, J=2.1 Hz, 2H), 6.47 (m, 1H), 2.90 (t, J=7.2 Hz, 2H), 1.66 (m, 2H), 1.32 (m, 10H), 0.89 (t, J=6.9 Hz, 3H).
Preparation Example 7 (Compound No.: LY234)
[0141] ##STR00068##
[0142] 1-bromooctane (1 mL, 5 mmol, 1 eq.) was dissolved in toluene (20 mL), then added triphenylphosphine (1.6 g, 6 mmol, 1.2 eq.) and refluxed at 120 C. for 12 h. The reaction mixture was concentrated and diluted with n-hexane (20 mL). A white sticky solid was precipitated. The two phase was separated and the crude product 19-1 was triturated three times with petroleum ether/ethyl acetate (20 mL/10 mL) and dried in vacuo, which was used without further purification.
[0143] Compound 19-1 (460 mg, 1.01 mmol, 1.2 eq.) was dissolved in DMSO/H.sub.2O (5 mL/0.5 mL), then added compound 18 (139 mg, 0.84 mmol, 1 eq.), potassium carbonate (232 mg, 1.68 mmol, 2 eq.), refluxed at 130 C. for 12 h monitored by TLC. After then, the reaction was extracted three times with ethyl acetate (5 mL), washed three times with H.sub.2O (5 mL). The combined organic layers were washed with brine, dried, and concentrated. The residue was purified by a flash chromatography on silica gel (petroleumether:ethylacetate=50:1) to give compound 20-1 (colorless oil, 230 mg, 89%). (E/Z=1.2) E 1H NMR (300 MHz, CDCl.sub.3) 6.50 (d, J=2.1 Hz, 2H), 6.34 (t, J=2.4 Hz, 1H), 6.31 (d, J=15.6 Hz, 1H), 6.28 (m, 1H), 3.79 (s, 3H), 2.19 (q, J=13.2 Hz, 2H), 1.44 (m, 2H), 1.28 (m, 8H), 0.88 (t, J=6.6 Hz, 4H). Z .sup.1H NMR (300 MHz, CDCl.sub.3) 6.43 (d, J=2.1 Hz, 1H), 6.23 (t, J=6.4 Hz, 1H), 6.34 (d, J=11.4 Hz, 1H), 5.67 (m, 1H), 3.79 (s, 3H), 2.33 (q, J=14.1 Hz, 2H), 1.44 (m, 2H), 1.44 (m, 2H), 1.28 (m, 8H), 0.88 (t, J=6.6 Hz, 4H).
[0144] Compound 20-1 (476 mg, 1.8 mmol, 1 eq.) was dissolved in dry DCM (10 mL) and cooled to 78 C., then slowly added dropwise BBr.sub.3 (2M 2.7 mL, 5.4 mmol, 3 eq.). The reaction was slowly warmed to 20 C. and stirred for 14 h monitored by TLC. The reaction was quenched by the dropwise addition of water (10 mL), and extracted three times with ethyl acetate (15 mL). The combined organic extracts were washed with brine, dried, concentrated. The residue was purified by a flash chromatography on silica gel (petroleumether:ethylacetate=3:1) to obtain compound LY234 (a yellow oil, 320 mg, 76%). .sup.1H NMR (300 MHz, CDCl.sub.3) E 6.48-6.30 (m, 3H), 6.30-6.09 (m, 2H), 2.16 (m, 2H), 1.42 (m, 10H), 0.93 (t, J=7.5 Hz, 3H). Z 6.48-6.30 (m, 3H), 6.35 (m, 1H), 5.63 (m, 1H), 2.29 (m, 2H), 1.42 (m, 10H), 0.93 (t, J=7.5 Hz, 3H).
Preparation Example 8 (Compound No.: LY236)
[0145] ##STR00069##
[0146] Compound 19-1, 20-1 were prepared in a manner analogous to Example 7;
[0147] A mixture of compound 20-1 (100 mg, 0.38 mmol), EtOH (10 mL), Pd/C (10 mg) was stirred under an atmosphere of H.sub.2 at 20 C. for 14 h, monitored by TLC. After the reaction completion, the mixture was purged with nitrogen, and filtered. The filtrate was concentrated in vacuo and purified by a flash chromatography on silica gel (petroleumether:ethylacetate=50:1) to afford compound 21 (colorless oil, 54 mg, 54%). .sup.1H NMR (300 MHz, CDCl.sub.3) 6.35 (d, J=2.1 Hz, 2H), 6.31-6.28 (t, J=2.1 Hz, 1H), 3.78 (s, 6H), 2.54 (t, J=7.5 Hz, 2H), 1.58 (m, 4H), 1.28 (m, 6H), 0.88 (t, J=6.6 Hz, 3H).
[0148] Compound 21 (264 mg, 0.19 mmol, 1 eq.) was dissolved in dry DCM (5 mL), cooled to 78 C., then slowly added dropwise BBr.sub.3 (2M 0.27 mL, 0.54 mmol, 3 eq.). The reaction mixture was slowly warmed to 20 C. and maintained at 20 C. for 14 h monitored by TLC. The reaction was quenched by the dropwise addition of water (5 mL), and extracted three times with ethyl acetate (10 mL). The combined organic extracts were washed with brine, dried, concentrated. The residue was purified by a flash chromatography on silica gel (petroleumether:ethylacetate=3:1) to obtain compound LY236 (yellow solid, 16 mg, 70%). .sup.1H NMR (300 MHz, CDCl.sub.3) 6.24 (d, J=2.1 Hz, 2H), 6.22-6.13 (m, 1H), 4.73 (s, 2H), 2.48 (t, J=7.8 Hz, 2H), 1.56 (m, 2H), 1.26 (m, 12H), 0.88 (t, J=6.6 Hz, 3H).
Preparation Example 9 (Compound No.: LY290-b)
[0149] ##STR00070##
[0150] Na (1.2 g) was slowly dissolved in 17 mL of methanol, thereafter, added compound 23 (3.8 g, 0.05 mol, 1 eq.) in methanol (17 mL) and compound 22-2 (8.7 g, 0.05 mol, 1 eq.). A white solid was precipitated. The reaction mixture was heated to reflux for 16 h, then, cooled to 50 C., adjusted the pH to acidity with hydrochloric acid (1M, 25 mL). The white solid was gradually dissolved. The mixture was filtered to remove impurities, cooled to 0 C., and was left to stand for 12 h. The crystal was formed, filtered, washed with ice water and dried to give compound 24-1 (2.08 g, white solid, 26.3%).
[0151] Compound 24-1 (100 mg, 0.60 mmol, 1.0 eq.) was dissolved in EtOH/H.sub.2O (10 mL/5 mL), added KI (11.3 mg, 0.069 mmol, 0.1 eq.), and then, slowly added compound 25-1 (381 mg, 1.80 mmol, 3.0 eq.). The reaction was heated to 80 C. for 6 h, monitored by TLC. After the reaction was completed, the mixture was added dilute hydrochloric acid (1M, 10 mL), followed by extracted three times with ethyl acetate (15 mL). The combined organic extracts were washed with brine, dried, filtered, and concentrated. The residue was purified by a flash chromatography on silica gel (DCM:MeOH=30:1) to give compound LY290-b (white solid, 17 mg, 9.8%). .sup.1H NMR (300 MHz, DMSO) 11.31 (s, 2H), 7.26 (m, J=7.2, 2H), 7.21-7.11 (m, 3H), 3.13 (t, J=6.6, 2H), 2.59 (m, 2H), 1.71 (s, 3H), 1.69-1.60 (m, 4H).
Preparation Example 10 (Compound No.: LY274-a)
[0152] ##STR00071##
[0153] Compound 26-2 (100 mg, 0.70 mmol, 1.0 eq.) was dissolved in EtOH/H.sub.2O (10 mL/5 mL), added KI (11.6 mg, 0.07 mmol, 0.1 eq.), and then, slowly added compound 25-1 (445 mg, 2.1 mmol, 3.0 eq.). The reaction was heated at 80 C. for 6 h, monitored by TLC. After the reaction was completed, the mixture was added dilute hydrochloric acid (1M, 10 mL), followed by extracted three times with ethyl acetate (15 mL). The combined organic extracts were washed with brine, dried, filtered, and concentrated. The residue was purified by a flash chromatography on silica gel (DCM:MeOH=30:1) to give compound LY274-a (white solid, 10 mg, 5.2%). .sup.1H NMR (300 MHz, DMSO) 7.26 (m, 2H), 7.19 (m, 3H), 6.02 (s, 4H), 5.12 (s, 1H), 3.00 (t, J=6.6 Hz, 2H), 2.59 (q, J=6.6 Hz, 2H), 1.62 (m, 4H).
[0154] The following compound was synthesized in the same manner:
TABLE-US-00004 Cpd# Chemical structure .sup.1H NMR (300 MHz) data LY274-a
Preparation Example 11 (Compound No.: LY328)
[0155] ##STR00075##
[0156] Na (600 mg) was slowly dissolved in 5 mL of methanol, thereafter, added compound 23 (760 mg, 0.01 mol, 1 eq.) in methanol (5 mL) and compound 27 (2.00 g, 0.01 mol, 1 eq.). The reaction mixture was heated to reflux for 16 h, then, cooled to 50 C., adjusted the pH to acidity with hydrochloric acid (1M, 25 mL). The white solid was gradually dissolved. The mixture was filtered to remove impurities, cooled to 0 C., and was left to stand for 12 h. The crystal was formed, filtered, washed with ice water and dried to give compound 26-1 (1.00 g, white solid, 51.0%).
[0157] Compound 26-1 (100 mg, 0.50 mmol, 1.0 eq.) was dissolved in EtOH/H.sub.2O (10 mL/5 mL), added KI (11.3 mg, 0.069 mmol, 0.1 eq.), and then, slowly added compound 25-1 (318 mg, 1.50 mmol, 3.0 eq.). The reaction was heated at 80 C. for 6 h, monitored by TLC. After the reaction was completed, the mixture was added dilute hydrochloric acid (1M, 10 mL), followed by extracted three times with ethyl acetate (15 mL). The combined organic extracts were washed with brine, dried, filtered, and concentrated. The residue was purified by a flash chromatography on silica gel (DCM:MeOH=30:1) to give compound LY328 (white solid, 80 mg, 67.2%). .sup.1H NMR (300 MHz, DMSO) 13.50 (s, 1H), 7.32-7.22 (m, 2H), 7.17 (m, 3H), 6.58 (s, 1H), 3.16 (t, J=9.6 Hz, 2H), 2.60 (q, J=6.9 Hz, 2H), 1.67 (m, 4H)
Preparation Example 12 (Compound No.: LY242)
[0158] ##STR00076##
[0159] Compound LY228-6a (100 mg, 0.44 mmol, 1.0 eq.) was dissolved in toluene, added K.sub.2CO.sub.3 (120 mg, 0.88 mmol, 2.0 eq.), and then, slowly added methyl iodide (62.04 mg, 0.44 mmol, 1.0 eq.) dropwise at 0 C. The reaction was refluxed for 3 h monitored by TLC. After full completion, the mixture was concentrated under vacuum. The crude product was chromatographed (DCM:MeOH=30:1) to yield compound LY242 (white solid, 21 mg, 19.8%). .sup.1H NMR (300 MHz, DMSO) 12.24 (s, 1H), 3.30 (s, 3H), 3.09 (t, J=6.9 Hz, 2H), 1.78-1.54 (m, 2H), 1.44-1.30 (m, 2H), 1.25-1.28 (m, 4H), 1.01-0.71 (t, J=5.4 Hz, 3H).
[0160] The following compound was synthesized in the same manner:
TABLE-US-00005 Cpd# Chemical structure .sup.1H NMR (300 MHz) data LY242
Preparation Example 13 (Compound No.: LY238-c)
[0161] ##STR00079##
[0162] Compound 26-1 (2 mL, 10 mmol, 1 eq.) was dissolved in acetonitrile (20 mL), added hydrazine monohydrochloride (760 mg, 11 mmol, 1.1 eq.) and triethylamine (1.6 mL, 11 mmol, 1.1 eq.) at 0 C. Then, the mixture was stirred at room temperature for 30 minutes, added phthalic anhydride (1.5 g, 10.1 eq, 1.01 eq.), and heated to reflux for 16 h. The mixture was cooled to room temperature and concentrated. The residue was diluted with DCM (10 mL) and filtered to remove the impurities. The filtrate was washed three times with 5% ammonium hydroxide solution (10 mL) and then washed with brine (10 mL). The combined organic layers were concentrated and dried to afford compound 27-1 (yellow oil, 1.35 g, 96%). .sup.1H NMR (300 MHz, CDCl.sub.3) 2.33 (t, J=7.2 Hz, 2H), 1.72-1.59 (m, 2H), 1.45 (m, 2H), 1.29 (s, 10H), 0.87 (t, J=6.6 Hz, 3H).
[0163] Compound 27-1 (480 mg, 3.46 mmol, 1.0 eq.) was dissolved in THF (10 mL), slowly added dropwise KHMDS (10 mL, 10 mmol, 3 eq.) at 0 C., stirred for 5 minutes, and added methyl iodide (0.42 mL, 3.46 mmol, 2 eq.). After completion as indicated by TLC, the reaction was quenched with water (5 mL) and extracted three times with ethyl acetate (5 mL). The combined the organic extracts were washed with brine, dried, concentrated, and chromatographed (petroleum ether:ethyl acetate=30:1) to get compound 28-1 (yellow oil, 150 mg, 28.4%). .sup.1H NMR (300 MHz, CDCl.sub.3) 1.66-1.55 (m, 2H), 1.39 (m, 1H), 1.31 (d, J=4.5 Hz, 3H), 1.29 (s, 10H), 0.87 (t, J=6.6 Hz, 3H).
[0164] Trimethylaluminum (0.9 mL, 1.67 mmol, 1.7 eq.) was slowly added dropwise to a solution of NH.sub.4Cl (94.4 mg, 1.76 mmol, 1.8 eq.) in anhydrous toluene (10 mL) under N.sub.2 at 0 C. The mixture was stirred at room temperature. Until no methane gas emission, a solution of compound 28-1 (150 mg, 0.98 mmol, 1 eq.) in toluene was slowly added dropwise. The mixture was stirred at 80 C. for 15 h. The mixture was cooled to room temperature, added a small amount of silica gel (300 mg), stirred for 10 minutes, and filtered. The filtrate was concentrated, added HCl in MeOH (2 mL, 2N), stirred for 12 h, filtered, and concentrated to give crude product 29-1 (118 mg, yellow solid).
[0165] Na (200 mg) was dissolved in methanol (10 mL), added compound 29-1 (118 mg, 0.69 mmol, 1 eq.) and compound 22-1 (70.4 mg, 0.414 mmol, 0.6 eq.). The reaction was heated to reflux for 12 h, then, cooled to room temperature, and concentrated. A small amount of water was added to dissolve the solid, then, the pH of the mixture was adjusted to acidity with hydrochloric acid (1N). A white solid was precipitated, filtered, and chromatographed (DCM:MeOH=20:1) to give compound LY238-c (white solid, 41 mg, 26.8%). .sup.1H NMR (300 MHz, DMSO) 11.58 (s, 2H), 5.06 (s, 1H), 2.62 (m, 1H), 1.63 (m, 2H), 1.32 (m, 2H), 1.19 (m, 8H), 1.15 (d, J=6.9 Hz, 3H), 0.84 (t, J=6.9 Hz, 3H).
Preparation Example 14 (Compound No.: LY225-b)
[0166] ##STR00080##
[0167] Magnesium (118 mg, 4.92 mmol, 3 eq.) was placed in diethyl ether (10 mL). A small amount of iodine (10 mg) was added, and 1-bromooctane (3 mL, 1.64 mmol, 1 eq.) was added dropwise. After the initiation, the reaction was heated at 50 C. for 1 h and added a solution of compound 30 (300 mg, 1.64 mmol, 1 eq.) in DCM (5 mL), then, stirred at room temperature for 2 h. The reaction mixture turned into orange color. After the reaction was complete monitored by TLC, the reaction was quenched with water (5 mL), extracted three times with DCM (5 mL). The combined the organic extracts were washed with brine and dried. The residue was purified by a flash chromatography on silica gel (petroleumether:ethylacetate=30:1) to give compound 31-1. .sup.1H NMR (300 MHz, CDCl.sub.3) 2.93-2.81 (t, J=7.8 Hz, 2H), 1.88-1.70 (m, 2H), 1.43-1.15 (m, 10H), 0.88 (t, J=6.9 Hz, 3H).
[0168] Na (600 mg) was added to methanol (15 mL). After Na was dissolved, compound 31-1 (300 mg, 1.15 mmol, 1 eq.) was added and the mixture was heated to reflux for 16 h. The reaction was monitored by TLC. After the reaction was complete, the reaction mixture was cooled to room temperature and concentrated. The residue was diluted with water (5 mL) and extracted three times with ethyl acetate (5 mL). The organic extracts were combined, washed with brine, and dried. The residue was purified by a flash chromatography on silica gel (DCM:MeOH=50:1) to give compound 32-1 (white solid, 204 mg, 74.7%). .sup.1H NMR (300 MHz, CDCl.sub.3) 4.83 (s, 6H), 2.55 (t, J=7.5 Hz, 2H), 1.81-1.65 (m, 2H), 1.33 (m, 10H), 0.88 (t, J=6.9 Hz, 3H).
[0169] Compound 32-1 (100 mg, 0.395 mmol, 1 eq.) was dissolved in DCM (5 mL), then slowly added BBr.sub.3 (296 mg, 1.18 mmol, 3 eq.) dropwise at 78 C. The reaction was slowly warmed to 25 C. and stirred for 12 h monitored by TLC. After the completion, the reaction was quenched by methanol (3 mL) and concentrated. The residue was purified by a flash chromatography on silica gel (DCM:MeOH=20:1) to give compound LY225-b (white solid, 18 mg, 20.2%). .sup.1H NMR (300 MHz, DMSO) 11.19 (s, 2H), 2.38 (t, J=7.5 Hz, 2H), 1.59 (m, 2H), 1.25 (m, 10H), 0.84 (t, J=7.5 Hz, 3H).
Preparation Example 15 (Compound No.: LY240 & LY224-c)
[0170] ##STR00081##
[0171] Compound 12-2 was prepared in a manner analogous to compound 12-1; Compound 23 (350 mg, 1.54 mmol, 1.5 eq.) was added in water (0.5 mL), stirred at 70 C. until compound 23 dissolved, then, K.sub.2CO.sub.3 (213 mg, 1.54 mmol, 1.5 eq.), compound 12-2 (76 mg, 1.0 mmol, 1 eq.) was added. The reaction was heated at 105 C. in an open vessel until the solvent was completely evaporated. The reaction mixture was cooled to room temperature, added water (5 mL) to dissolve the solid. The white slurry was obtained. The pH of the mixture was adjusted to acidity with hydrochloric acid (1N). A white sticky solid was formed, and the supernatant was removed. The solid was washed with water (5 mL) for 3 times, and purified by flash chromatography on silica gel (petroleumether:ethylacetate=10:1) to afford compound LY240 (white solid, 56 mg, 23.3%). .sup.1H NMR (300 MHz, DMSO) 12.30 (s, 1H), 12.19 (s, 1H), 5.67 (s, 1H), 2.33 (t, J=8.1 Hz, 2H), 1.51 (m, 2H), 1.26 (m, 10H), 0.86 (t, J=6.3 Hz, 3H).
[0172] Compound 33 (31 mg, 0.42 mmol, 2 eq.) was dissolved in water (1 mL), then slowly added compound LY240 (50 mg, 0.21 mmol, 1 eq.) in THF/H.sub.2O (5/2 mL). The mixture was heated at 70 C. for 6 h, and then added concentrated hydrochloric acid (0.1 mL). Thereafter the reaction was carried out at 70 C. for 16 h, cooled to room temperature, extracted three times with ethyl acetate (5 mL). The combined organic extracts were washed with brine, dried and concentrated. The residue was purified by flash chromatography on silica gel (DCM:MeOH=20:1) to yield compound LY224-c (white solid, 5 mg, 10.6%). .sup.1H NMR (300 MHz, DMSO) 10.87 (s, 1H), 10.77 (s, 1H), 5.31 (s, 1H), 2.26 (t, J=7.5 Hz, 2H), 1.51 (m, 2H), 1.25 (m, 10H), 0.86 (t, J=6.6 Hz, 3H).
Preparation Example 16 (Compound No.: LY243)
[0173] ##STR00082##
[0174] Compound 34-1 (1 g, 5.6 mmol, 1 eq.) was dissolved in THF (15 mL), and then cooled to 0 C., slowly added dropwise 1,1-carbonyldiimidazole (998 mg, 6.16 mmol, 1.1 eq.) in THF (5 mL) and kept at 0 C. for 2 h. The reaction was allowed to warm to 25 C. and stirred at 25 C. for 1 h, then, was added water (5 mL), extracted three times with ethyl acetate (10 mL). The combined organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product 35-1, which are used without further purification.
[0175] n-BuLi (2.9 mL, 4.61 mmol, 1.4 eq.) was mixed with hexamethylsilane (742 mg, 4.61 mmol, 1.4 eq.) at 78 C., then stirred for 20 min at 78 C., and compound 36 (467 mg, 3.29 mmol, 1.0 eq.) was added, stirred at 78 C. for 1 h, then diethyl zinc (4.6 mL, 4.61 mmol, 1.4 eq.) was added, stirred for 20 min. The mixture was warmed to 20 C., added a solution of compound 35-1 (900 mg, 3.95 mmol) in THF (5 mL). The mixture was warmed to 10 C., stirred for 3 h, quenched with saturated NH.sub.4Cl (10 mL), and extracted with ethyl acetate (10 mL) for 3 times. The combined organic extracts were dried, filtered, concentrated, and purified by flash chromatography on silica gel to afford compound 37-1 (white solid, 321 mg, 27%).
[0176] Compound 37-1 (100 mg, 0.33 mmol, 1.0 eq.) was dissolved in ethanol (10 mL), added ammonium acetate (76 mg, 0.99 mmol, 3.0 eq.), and stirred at 25 C. for 3 h. The reaction was concentrated, and added toluene (5 mL). The mixture was refluxed at 120 C. for 3 h for removing water. The reaction was concentrated and purified by flash chromatography on silica gel (DCM:MeOH=20:1) to give compound LY243 (yellow solid, 38 mg, 48.7%). .sup.1H NMR (300 MHz, DMSO) 11.67 (s, 2H), 7.60 (d, J=8.1 Hz, 2H), 7.18 (d, J=8.1 Hz, 2H), 6.54 (s, 1H), 5.21 (s, 1H), 2.5 (t, J=7.5 Hz, 2H), 1.46-1.38 (m, 2H), 1.32-1.00 (m, 2H), 0.75 (t, J=7.5 Hz, 3H).
Preparation Example 17 (Compound No.: LY239)
[0177] ##STR00083##
[0178] Compound 38 (500 mg, 1.8 mmol, 1.0 eq.) was dissolved in ethanol (10 mL), added octylamine (387 mg, 3 mmol, 1.6 eq.) and heated at 100 C. for 16 h. The reaction mixture was concentrated, added to water (5 mL) and ethanol (5 mL). Under ice bath, a white solid was precipitated and filtered to give crude product 39-1 (white solid, 321 mg).
[0179] Na (200 mg) was added to methanol (20 mL). After Na was dissolved, compound 39-1 (700 mg, 4.1 mmol, 1 eq.) was added. The mixture was heated to reflux for 16 h, monitored by TLC. After the reaction was complete, the reaction mixture was cooled to room temperature and concentrated, added water (5 mL). The pH of the mixture was adjusted to 3-4 with 1 M hydrochloric acid. The mixture was extracted three times with ethyl acetate (5 mL). The combined organic extracts were washed with brine, dried. The residue was purified by flash chromatography on silica gel (DCM:MeOH=50:1) compound LY239 (white solid, 132 mg, 13.5%). .sup.1H NMR (300 MHz, DMSO) 10.29 (s, 2H), 6.48 (s, 1H), 4.58 (s, 1H), 3.20 (dd, J=12.9, 6.9 Hz, 2H), 1.46 (m, 2H), 1.26 (m, 10H), 0.85 (t, J=6.9 Hz, 3H).
Biological Experiment Example
[0180] Detection of Cytoplasmic Calcium Ion Concentration with Fluo-4 Fluorescent Dye Tracer Assay
1. Purpose
[0181] The GPR84 agonist activity of the compounds of the invention was tested.
2. Source of Material
[0182] The human GPR84 cell line was obtained by transfecting a plasmid encoding the GPR84 and G16 proteins in the HEK293 cell line. The fluorescent dye Fluo-4 AM was purchased from Invitrogen.
3. Principle
[0183] Intracellular Ca.sup.2+ ion is a very important second messenger of G protein-coupled receptor signaling pathway. When GPR84 coupled to G16 protein is bound to a ligand, the concentration of intracellular Ca.sup.2+ ion 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, fluorescence assay was used to detect the agonistic activity of compounds in 96-well or 384-well flat bottom microplates. The GPR84 cells were incubated with the Fluo-4 fluorescent dye and added with different concentrations of compounds for stimulation. The changes in the intracellular calcium concentration were detected by the fluorescence intensity of dyes. Fluorescence excitation was 485 nm and the detector for emission was set at 525 nm. Thereby, the concentration for 50% of maximal effect (EC.sub.50) was calculated.
4. Procedure
[0184] 1. Preparation of Hank's Balanced Salt Solution (HBSS): The ingredients, 0.4 g/L KCl (5.4 mM), 0.12 g/L Na.sub.2HPO.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), were weighed and dissolved with ultrapure water. The pH of the solution was adjusted to 7.4 with hydrochloric acid or NaOH. The solution was filtered, and stored at 4 C. for one month. [0185] 2. Preparation of Ca.sup.2+ buffer: Firstly, a 560 mM D-glucose (100) stock solution and a 250 mM Sulfinpyrazone (1000) stock solution were prepared. Then, to 100 mL of HBSS, was added BSA (0.5 g), 560 mM D-glucose stock solution (1 mL) and 250 mM Sulfinpyrazone (100 L). The final concentrations in Ca.sup.2+ buffer were 0.5% BSA, 5.6 mM D-glucose, 250 M Sulfinpyrazone. The Ca.sup.2+ buffer was mixed and used as freshly prepared. [0186] 3. Preparation of dyes solution: Firstly, a stock solution of 3% Cremophor EL (100) in PBS and a stock solution of 2 mM Fluo-4 (1000) in DMSO was prepared. Secondly, one milliliter of dye solution was prepared by mixing 1 L of 2 mM Fluo-4 AM with 10 L of 3% Cremophor EL and diluting with 1 mL of Ca.sup.2+ buffer and mixed. [0187] 4. The GPR84 cells were cultured in a 96-well plate at a starting density of 410.sup.4 cells. The cells were continually cultured for more than 24 hours so that the cell density was 80-90% for detection. [0188] 5. The culture fluid was removed from the cells to be tested. The cells were added freshly prepared dyes and incubated in a 37-degree incubator for 40 minutes to 50 minutes. [0189] 6. Preparation of dyes solution: Compounds are dissolved and diluted to 3-fold the final working concentration with freshly prepared Ca.sup.2+ buffer. If compounds are dissolved in DMSO, the final DMSO concentration should not exceed 1%. [0190] 7. After the incubation completed, the dye was removed. The cells were washed with Ca.sup.2+ buffer and then incubated with an additional 50 L of Ca.sup.2+ buffer for 5 to 10 minutes. [0191] 8. The cells were stimulated with 25 L/well of Ca.sup.2 buffer containing different concentrations of the compound. The plate was read by using the FlexStation III Multi-Mode Microplate Reader. The changes in the intracellular calcium concentration were detected by the fluorescence intensity of dyes. Fluorescence excitation was 485 nm and the detector for emission was set at 525 nm.
[0192] Taking GPR84 receptor as an example, agonist 6-OAU sampling situation is listed as below:
TABLE-US-00006 6-OAU initial concn. 6-OAU sample concn. 6-OAU final concn. 10 mM (with 100% DMSO) 30 M (with 3% DMSO) 100 M (with 1% DMSO) 1 mM (with 100% DMSO) 30 M (with 3% DMSO) 10 M (with 1% DMSO) 100 M (with 100% DMSO) 3 M (with 3% DMSO) 1 M (with 1% DMSO) 10 M (with 100% DMSO) 300 nM (with 3% DMSO) 100 nM (with 1% DMSO) 1 M (with 100% DMSO) 30 nM (with 3% DMSO) 10 nM (with 1% DMSO) 100 nM (with 100% DMSO) 3 nM (with3% DMSO) 1 nM (with 1% DMSO) 10 nM (with 100% DMSO) 0.3 nM (with 3% DMSO) 0.1 nM (with 1% DMSO) 100% DMSO 3% DMSO 1% DMSO
5. Result
[0193]
TABLE-US-00007 Compound No. EC.sub.50(M) Compound No. EC.sub.50(M) LY214-5 2.479 LY196 8.852 LY228-6a 0.2114 LY210 21.56 LY228-6b 0.6408 LY212 43.43 LY242-7 0.2311 LY248 3.856 LY256-8 0.2639 LY262-a 0.5728 LY312-12 1.164 LY276 0.4351 LY340-14 25.69 LY266-a 2.944 LY368-16 11.31 LY266-c 2.592 LY290-a 1.076 LY224-a 0.6531 LY266-b 1.991 LY262-b 0.271 LY226 0.9236 LY196b 2.105 LY182 3.669 LY210-b 0.01274 LY196 0.4542 LY238 0.04871 LY224-b 0.01135 LY244 0.08809 LY236 0.4347 LY237 0.00019 LY243 0.006099 LY224-c 0.8109 LY223 0.001339 LY238-c 0.2988 LY225-b 1.023 LY240 0.3398 LY238-d 0.003511 LY209 0.001254 LY239 0.00614 6-OAU 0.661-0.919
[0194] A series of compounds was proved to have excellent agonistic activity against GPR84. Especially activity of compound LY237 is 4500 times higher than 6-OAU, which is the best reported agonistic activity at present.