PHENYL PROPANAMIDE DERIVATIVE, AND MANUFACTURING METHOD AND PHARMACEUTICAL APPLICATION THEREOF

Abstract

The present invention provides a phenylpropanamide derivative as represented by formula (I), a manufacturing method of the derivative, application of the derivative as a -opioid receptor (KOR) agonist, and application of the derivative for manufacturing a pharmaceutical product for treating and/or preventing pain or a pain-related disease.

##STR00001##

Claims

1. A compound of formula (I): ##STR00075## or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: M is an inorganic acid or an organic acid; G is selected from the group consisting of O, NR.sup.4 and CR.sup.5R.sup.6; R.sup.1 is selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, halogen, amino, nitro, hydroxy, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, OR.sup.7, C(O)R.sup.7, C(O)OR.sup.7, S(O).sub.mR.sup.7 and NR.sup.8R.sup.9, wherein the alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each optionally substituted by one or more groups selected from the group consisting of alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.2 is selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, OR.sup.7, C(O)R.sup.7 and C(O)OR.sup.7, wherein the alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl are each optionally substituted by one or more groups selected from the group consisting of alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.3 is selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, OR.sup.7, C(O)R.sup.7 and C(O)OR.sup.7, wherein the alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl are each optionally substituted by one or more groups selected from the group consisting of alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.4 is selected from the group consisting of hydrogen, alkyl, haloalkyl, cycloalkyl, alkoxy, hydroxyalkyl, amino, alkoxycarbonyl, heterocyclyl, aryl, heteroaryl, OR.sup.7, C(O)R.sup.7, C(O)OR.sup.7, S(O).sub.mR.sup.7, NR.sup.8R.sup.9 and NHC(O)NR.sup.8R.sup.9, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each optionally substituted by one or more groups selected from the group consisting of alkyl, halogen, hydroxy, amino, alkoxycarbonyl, nitro, cyano, alkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.5 and R.sup.6 are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxyalkyl, hydroxy, amino, alkoxycarbonyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, OR.sup.7, C(O)R.sup.7, C(O)OR.sup.7, S(O).sub.mR.sup.7, NR.sup.8R.sup.9 and NHC(O)NR.sup.8R.sup.9, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each optionally substituted by one or more groups selected from the group consisting of alkyl, halogen, hydroxy, amino, alkoxycarbonyl, nitro, cyano, alkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.7 is selected from the group consisting of hydrogen, alkyl, amino, alkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each optionally substituted by one or more groups selected from the group consisting of alkyl, halogen, hydroxy, amino, nitro, cyano, alkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.8 and R.sup.9 are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxyalkyl, hydroxy, amino, alkoxycarbonyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each optionally substituted by one or more groups selected from the group consisting of alkyl, halogen, hydroxy, amino, alkoxycarbonyl, nitro, cyano, alkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; z is 0, 1, 2, 3 or 4; and m is 0, 1 or 2.

2. The compound of formula (I) according to claim 1, being a compound of formula (II): ##STR00076## or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: M, G, R.sup.2, R.sup.3 and z are as defined in claim 1.

3. The compound of formula (I) according to claim 1, being a compound of formula (III): ##STR00077## or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: M, G, R.sup.2 and z are as defined in claim 1.

4. The compound of formula (I) according to claim 1, being a compound of formula (IV): ##STR00078## or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: M, R.sup.2 and z are as defined in claim 1.

5. The compound of formula (I) according to claim 1, wherein R.sup.2 is selected from the group consisting of arylalkyl, cycloalkylalkyl and cycloalkyl, wherein the arylalkyl, cycloalkylalkyl and cycloalkyl are each optionally substituted by one or more groups selected from the group consisting of alkyl, cycloalkyl and aryl.

6. The compound of formula (I) according to claim 1, being a compound of formula (III-A): ##STR00079## or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: G is O or CR.sup.5R.sup.6; R.sup.10 is selected from the group consisting of hydrogen, alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.11 and R.sup.12 are identical or different, and each is independently selected from the group consisting of hydrogen, alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; or R.sup.11 and R.sup.12 are taken together to form a cycloalkyl; R.sup.13 is selected from the group consisting of hydrogen, alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; s is 0, 1 or 2; and R.sup.5 to R.sup.6, M and z are as defined in claim 1.

7. The compound of formula (I) according to claim 6, being a compound of formula (IV-A): ##STR00080## or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R.sup.10 to R.sup.13, M, z and s are as defined in claim 6.

8. The compound of formula (I) according to claim 6, being a compound of formula (IV-B): ##STR00081## or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R.sup.10 to R.sup.13, M, z and s are as defined in claim 6.

9. The compound of formula (I) according to claim 1, wherein z is 0 or 1.

10. A compound selected from the group consisting of: ##STR00082## ##STR00083## ##STR00084## ##STR00085##

11. A compound of formula (VI): ##STR00086## or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein: R.sup.a is an amino-protecting group; G is selected from the group consisting of O, NR.sup.4 and CR.sup.5R.sup.6; R.sup.2 is selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, OR.sup.7, C(O)R.sup.7 and C(O)OR.sup.7, wherein the alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl are each optionally substituted by one or more groups selected from the group consisting of alkyl, haloalkyl, halogen, amino, nitro, cyano, hydroxy, alkoxy, haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.4 is selected from the group consisting of hydrogen, alkyl, haloalkyl, cycloalkyl, alkoxy, hydroxyalkyl, amino, alkoxycarbonyl, heterocyclyl, aryl, heteroaryl, OR.sup.7, C(O)R.sup.7, C(O)OR.sup.7, S(O).sub.mR.sup.7, NR.sup.8R.sup.9 and NHC(O)NR.sup.8R.sup.9, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each optionally substituted by one or more groups selected from the group consisting of alkyl, halogen, hydroxy, amino, alkoxycarbonyl, nitro, cyano, alkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.5 and R.sup.6 are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxyalkyl, hydroxy, amino, alkoxycarbonyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, OR.sup.7, C(O)R.sup.7, C(O)OR.sup.7, S(O), R.sup.7, NR.sup.8R.sup.9 and NHC(O)NR.sup.8R.sup.9, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each optionally substituted by one or more groups selected from the group consisting of alkyl, halogen, hydroxy, amino, alkoxycarbonyl, nitro, cyano, alkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.7 is selected from the group consisting of hydrogen, alkyl, amino, alkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each optionally substituted by one or more groups selected from the group consisting of alkyl, halogen, hydroxy, amino, nitro, cyano, alkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R.sup.8 and R.sup.9 are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxyalkyl, hydroxy, amino, alkoxycarbonyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each optionally substituted by one or more groups selected from the group consisting of alkyl, halogen, hydroxy, amino, alkoxycarbonyl, nitro, cyano, alkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl.

12. A process for preparing the compound of formula (III) according to claim 3, comprising: ##STR00087## removing the protecting group of R.sup.a on a compound of formula (VI) under an acidic condition to obtain the compound of formula (III); wherein: M, G, z and R.sup.2 are as defined in claim 3, and R.sup.a is an amino-protecting group.

13. A pharmaceutical composition comprising a therapeutically effective amount of the compound of formula (I) according to claim 1, and one or more pharmaceutically acceptable carriers, diluents or excipients.

14.-18. (canceled)

19. A method for preventing and/or treating a opioid receptor agonist mediated and related disease, comprising administering to a subject in need thereof the pharmaceutical composition according to claim 13.

20. The method according to claim 19, wherein the opioid receptor agonist mediated and related disease is selected from the group consisting of pain, inflammation, itching, edema, hyponatremia, hypokalemia, intestinal obstruction, cough and glaucoma.

21. A method for preventing and/or treating pain and pain related diseases, comprising administering to a subject in need thereof the pharmaceutical composition according to claim 13.

22. The method according to claim 21, wherein the pain is selected from the group consisting of neuropathic pain, trunk pain, visceral pain, skin pain, arthritic pain, kidney stone pain, uterine cramp, dysmenorrhea, endometriosis, dyspepsia, post-surgical pain, post-medical treatment pain, eye pain, otitis pain, fulminant cancer pain, and GI disorder related pain.

23. A method for agonizing opioid receptor, comprising administering to a subject in need thereof the pharmaceutical composition according to claim 13.

24. The compound according to claim 1, wherein M is trifluoroacetic acid.

25. The compound according to claim 11, wherein R.sup.a is t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, allyloxycarbonyl, trichloroethoxycarbonyl, trimethylsilyloxycarbonyl, benzyloxycarbonyl, p-methylbenzenesulfonyl, p-nitrobenzenesulfonyl or tert-butyl.

Description

DESCRIPTION OF THE DRAWINGS

[0175] FIG. 1 shows the effect of the compounds of the present application on carrageenan inflammatory pain induced by carrageenan in rats.

PREFERRED EMBODIMENTS

[0176] The present invention will be further described with reference to the following examples, but the examples should not be considered as limiting the scope of the invention.

EXAMPLES

[0177] The structures of the compounds are identified by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). NMR chemical shifts (6) are given in 10.sup.6 (ppm). NMR is determined by a Bruker AVANCE-400 machine. The solvents for determination are deuterated-dimethyl sulfoxide (DMSO-d.sub.6), deuterated-chloroform (CDCl.sub.3) and deuterated-methanol (CD.sub.3OD), and the internal standard is tetramethylsilane (TMS).

[0178] MS is determined by a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, type: Finnigan LCQ advantage MAX).

[0179] Chiral high performance liquid chromatography (HPLC) analysis is determined on an LC-10A vp (Shimadzu) or SFC-analytical (Berger Instruments Inc.).

[0180] Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate is used for thin-layer silica gel chromatography (TLC). The dimension of the silica gel plate used in TLC is 0.15 mm to 0.2 mm, and the dimension of the silica gel plate used in product purification is 0.4 mm to 0.5 mm.

[0181] Yantai Huanghai 200 to 300 mesh silica gel is used as a carrier for column chromatography.

[0182] Prep Star SD-1 (Varian Instruments Inc.) or SFC-multigram (Berger Instruments Inc.) is used for chiral preparative column chromatography.

[0183] The average kinase inhibition rates and IC.sub.50 values are determined by a NovoStar ELISA (BMG Co., Germany).

[0184] The known raw materials of the present invention can be prepared by conventional synthesis methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., or Dari chemical Company, etc.

[0185] Unless otherwise stated, the reactions are carried out under nitrogen atmosphere or argon atmosphere.

[0186] The term nitrogen atmosphere or argon atmosphere means that a reaction flask is equipped with a 1 L nitrogen or argon balloon.

[0187] The term hydrogen atmosphere means that a reaction flask is equipped with a 1 L hydrogen balloon.

[0188] Pressurized hydrogenation reactions are carried out with a Parr 3916EKX hydrogenation instrument and a QL-500 hydrogen generator or HC2-SS hydrogenation instrument.

[0189] In hydrogenation reactions, the reaction system is generally vacuumed and filled with hydrogen, and the above operation is repeated three times.

[0190] CEM Discover-S 908860 type microwave reactor is used in microwave reactions.

[0191] Unless otherwise stated, the solution refers to an aqueous solution.

[0192] Unless otherwise stated, the reaction temperature in the reactions refers to room temperature, ranging from 20 C. to 30 C.

[0193] The reaction process is monitored by thin layer chromatography (TLC), and the system of developing solvent includes: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether and ethyl acetate system, D: acetone. The ratio of the volume of the solvent can be adjusted according to the polarity of the compounds.

[0194] The elution system for purification of the compounds by column chromatography and thin layer chromatography includes: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: dichloromethane and acetone system. The ratio of the volume of the solvent can be adjusted according to the polarity of the compounds, and sometimes a little alkaline reagent such as triethylamine or acidic reagent such as acetic acid can be added.

[0195] High pressure liquid chromatographic instrument used in the high performance liquid chromatography in the examples is Gilson-281, the chromatographic column is Shim-pack PREP-ODS of Shimadzu, the mobile phase used is trifluoroacetic acid buffer system, i.e., water (containing 0.05% trifluoroacetate)-acetonitrile.

[0196] Each of the compounds in the form of a trifluoroacetate salt in the examples can be obtained in a free state by the following general method: the trifluoroacetate salt thereof is dissolved in a suitable solvent (e.g., methanol, ethanol, tetrahydrofuran, acetone, etc.), and a weak base is added (such as sodium bicarbonate, sodium carbonate, potassium carbonate, etc.) to adjust the pH to neutrality, the mixture was concentrated under reduced pressure, and the residue was purified to obtain a free state.

Example 1

4-amino-14-(6R,9R,12R)-12-(4-aminobutyl)-6-benzyl-9-isobutyl-4,7,10-trioxo-1-(1-phenylcyclopropyl)-2,5,8,11-tetraazatridecan-13-oyl)piperidine-4-carboxylic acid 1

[0197] ##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052##

Step 1

4-benzyl 1-tert-butyl 4-(((benzyloxy)carbonyl)amino)piperidine-1,4-dicarboxylate 1b

[0198] 4-(((benzyloxy)carbonyl)amino)-1-(tert-butoxycarbonyl)piperidine-4-carboxylic acid 1a (1.2 g, 0.0032 mol, prepared by a known method disclosed in Bioorganic Medicinal Chemistry Letters, 2007, 7(9), 2448-2451), benzyl bromide (0.65 g, 0.0038 mol) and cesium carbonate (2.1 g, 0.0064 mol) were dissolved in 20 mL of N,N-dimethylformamide, and stirred for 12 hours at room temperature. The reaction solution was poured into water and extracted with ethyl acetate (30 mL3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with elution system B to obtain the title compound 1b (800 mg, yield: 53%).

Step 2

benzyl 4-(((benzyloxy)carbonyl)amino)piperidine-4-carboxylate hydrochloride 1c

[0199] Compound 1b (800 mg, 1.71 mmol) was dissolved in 2 mL of dichloromethane, and 2 mL of a solution of 4M hydrochloric acid in 1,4-dioxane was added. After stirring for 4 hours at room temperature, the reaction solution was concentrated under reduced pressure to obtain the crude title compound 1c (800 mg), which was used directly in the next step without purification.

Step 3

(R)-benzyl 1-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoyl)-4-(((benzyloxy)carbonyl)amino)piperidine-4-carboxylate 1e

[0200] The crude compound 1c (800 mg, 1.97 mmol) and (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoic acid 1d (926 mg, 1.97 mmol, prepared by a known method disclosed in ChemMedChem, 2015, 10(7), 1232-1239) were dissolved in 20 mL of N,N-dimethylformamide. 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (1.12 g, 3.0 mmol) and N,N-diisopropylethylamine (0.7 mL, 3.94 mmol) were added. After stirring for 12 hours at room temperature, the reaction solution was poured into 2N citric acid solution and extracted with ethyl acetate (30 mL3). The organic phases were combined, washed with saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 1e (1.6 g), which was used directly in the next step without purification.

Step 4

(R)-benzyl 1-(2-amino-6-((tert-butoxycarbonyl)amino)hexanoyl)-4-(((benzyloxy) carbonyl)amino)piperidine-4-carboxylate 1f

[0201] The crude compound 1e (1.6 g, 0.002 mol) was dissolved in 10 mL of dichloromethane, and then 10 mL of piperidine was added. After stirring for 2 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with elution system A to obtain the title compound 1f (900 mg, yield: 77%).

Step 5

(R)-benzyl 2-((R)-2-(2-chloroacetamido)-3-phenylpropionamido)-4-methylpentanoate 1i

[0202] (R)-benzyl 2-((R)-2-amino-3-phenylpropanamido)-4-methylpentanoate 1g (500 mg, 1.36 mmol, prepared by a method disclosed in the patent application US20110212882A1) and triethylamine (275 mg, 2.72 mmol) were dissolved in 10 mL of dichloromethane, and then chloroacetyl chloride (230 mg, 2 mmol) was added dropwise. After stirring for 12 hours at room temperature, the reaction solution was poured into water and washed with saturated ammonium chloride solution. The organic phase was dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title product 1i (500 mg), which was used directly in the next step without purification.

Step 6

(R)-benzyl 4-methyl-2-((R)-3-phenyl-2-(2-(((1-phenylcyclopropyl)methyl)amino) acetamido)propanamido)pentanoate 1k

[0203] The crude compound 1i (150 mg, 0.33 mmol) and (1-phenylcyclopropyl)methanamine hydrochloride 1j (74 mg, 0.4 mmol, prepared by a known method disclosed in Journal of American Chemical Society, 2015, 137(5), 2042-2046) were dissolved in 10 mL of N,N-dimethylformamide, and then potassium iodide (110 mg, 0.67 mmol) and potassium carbonate (139 mg, 1 mmol) were added. The reaction solution was warmed up to 60 C. and stirred for 5 hours, and then concentrated under reduced pressure. The resulting residue was added with water and extracted with dichloromethane (30 mL3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 1k (187 mg), which was used directly in the next step without purification.

Step 7

(9R,12R)-benzyl 9-benzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-5-(1-phenylcyclopropyl)methyl)-3-oxa-5,8,11-triazatridecan-13-oate 1l

[0204] The crude compound 1k (187 mg, 0.337 mmol) was dissolved in dichloromethane, and then di-tert-butyl dicarbonate (147 mg, 0.67 mmol) and N,N-diisopropylethylamine (130 mg, 1.01 mmol) were added. After stirring for 12 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with elution system A to obtain the title compound 1l (100 mg, yield: 45.5%).

Step 8

(9R,12R)-9-benzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-5-((1-phenylcyclopropyl)methyl)-3-oxa-5,8,11-triazatridecan-13-oic Acid 1m

[0205] Compound 1l (100 mg, 0.152 mmol) was dissolved in 10 mL of ethanol, and then palladium-carbon (100 mg, catalytic amount) was added. After completion of the addition, the reaction system was purged with hydrogen three times and stirred for 5 hours at room temperature. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 1m (86 mg), which was used directly in the next step without purification.

Step 9

benzyl 1-((9R,12R,15R)-9-benzyl-15-(4-((tert-butoxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-5-((1-phenylcyclopropyl)methyl)-3-oxa-5,8,11,14-tetraazahexadecan-16-oyl)-4-(((benzyloxy)carbonyl)amino)piperidine-4-carboxylate 1n

[0206] The crude compound 1m (86 mg, 0.152 mmol), compound 1f (91 mg, 0.152 mmol) and 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (115 mg, 0.3 mmol) were dissolved in 10 mL N,N-dimethylformamide. After stirring for 5 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with elution system A to obtain the title compound in (100 mg, yield: 57.5%).

Step 10

4-amino-1-((9R,12R,15R)-9-benzyl-15-(4-((tert-butoxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-5-((1-phenylcyclopropyl)methyl)-3-oxa-5,8,11,14-tetraazahexadecan-16-oyl)piperidine-4-carboxylic Acid 1o

[0207] Compound 1n (100 mg, 0.087 mmol) was dissolved in 10 mL of ethanol, then palladium-carbon (100 mg, catalytic amount) was added. After completion of the addition, the reaction system was purged with hydrogen three times and stirred for 12 hours at room temperature. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 1o (80 mg), which was used directly in the next step without purification.

Step 11

4-amino-1-((6R,9R,12R)-12-(4-aminobutyl)-6-benzyl-9-isobutyl-4,7,10-trioxo-1-(1-phenylcyclopropyl)-2,5,8,11-tetraazatridecan-13-oyl)piperidine-4-carboxylic acid trifluoroacetate 1p

[0208] The crude compound 1o (80 mg, 0.087 mmol) was dissolved in 10 mL of dichloromethane, then 2 mL of trifluoroacetic acid were added. After stirring for 5 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by high performance liquid chromatography to obtain the title compound 1p (10 mg, yield: 15.9%).

[0209] MS m/z (ESI): 720.4 [M+1]

[0210] .sup.1H NMR (400 MHz, CD.sub.3OD) 8.47-8.40 (m, 2H), 7.42-7.27 (m, 11H), 4.84-4.81 (m, 1H), 4.70-4.67 (m, 1H), 4.40-4.38 (m, 1H), 4.25-4.10 (m, 1H), 3.95-3.85 (m, 2H), 3.78-3.70 (m, 2H), 3.61-3.52 (m, 1H), 3.5-3.41 (m, 1H), 3.25-3.10 (m, 3H), 3.10-2.95 (m, 2H), 2.95-2.89 (m, 2H), 2.89-2.75 (m, 2H), 2.31-2.24 (m, 2H), 1.95-1.45 (m, 13H), 1.1-0.9 (m, 6H), 0.9-0.86 (m, 4H).

Step 12

4-amino-1-((6R,9R,12R)-12-(4-aminobutyl)-6-benzyl-9-isobutyl-4,7,10-trioxo-1-(1-phenylcyclopropyl)-2,5,8,11-tetraazatridecan-13-oyl)piperidine-4-carboxylic acid 1

[0211] Compound 1p (10 mg, 0.012 mmol) was dissolved in 1 mL of a mixed solution of dichloromethane and methanol (V/V=10:1), and then saturated aqueous sodium carbonate solution was added dropwise to adjust the pH to about 7. The reaction solution was stirred for 30 minutes at room temperature and left to stand and separate. The organic phase was collected, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the title compound 1 (8.6 mg, yield: 100%).

[0212] MS m/z (ESI): 720.4 [M+1]

Example 2

4-amino-1-((2R,5R,8R,14S)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazapentadecan-1-oyl)piperidine-4-carboxylic acid 2

[0213] ##STR00053## ##STR00054##

Step 1

(R)-benzyl 4-methyl-2-((R)-3-phenyl-2-(2-(((S)-2-phenylpropyl)amino)acetamido)propanamido)pentanoate 2b

[0214] Compound 1i (500 mg, 1.12 mmol) and (S)-2-phenylpropan-1-amine 2a (228 mg, 1.68 mmol, prepared by a known method disclosed in Advanced Synthesis & Catalysis, 2015, 357(18), 3875-3879) were dissolved in 10 mL of N,N-dimethylformamide, then potassium iodide (372 mg, 2.24 mmol) and potassium carbonate (309 mg, 2.24 mmol) were added. The reaction solution was warmed up to 60 C. and stirred for 12 hours. The reaction solution was cooled to room temperature, added with water, and extracted with dichloromethane (30 mL3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 2b (600 mg), which was used directly in the next step without purification.

Step 2

(9R,12R)-benzyl 9-benzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-5-((S)-2-phenylpropyl)-3-oxa-5,8,11-triazatridecan-13-oate 2c

[0215] The crude compound 2b (600 mg, 1.1 mmol) was dissolved in 10 mL of dichloromethane, and then di-tert-butyl dicarbonate (360 mg, 1.65 mmol) and triethylamine (222 mg, 2.2 mmol) were added. After stirring for 12 hours, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with elution system A to obtain the title compound 2c (380 mg, yield: 54%).

Step 3

(9R,12R)-9-benzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-5-((S)-2-phenylpropyl)-3-oxa-5,8,11-triazatridecan-13-oic Acid 2d

[0216] Compound 2c (380 mg, 0.59 mmol) was dissolved in 15 mL of methanol, and then palladium-carbon (40 mg, catalytic amount) was added. After completion of the addition, the reaction system was purged with hydrogen three times and stirred at room temperature for 12 hours. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 2d (300 mg), which was used directly in the next step without purification.

Step 4

benzyl 1-((9R,12R,15R)-9-benzyl-15-(4-((tert-butoxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-5-((S)-2-phenylpropyl)-3-oxa-5,8,11,14-tetraazahexadecan-16-oyl)-4-(((benzyloxy)carbonyl)amino)piperidine-4-carboxylate 2e

[0217] The crude compound 2d (300 mg, 0.54 mmol), compound 1f (356 mg, 0.6 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (308 mg, 0.81 mmol) and N,N-diisopropylethylamine (104 mg, 0.81 mmol) were dissolved in 10 mL of N,N-dimethylformamide. After stirring for 1.5 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with elution system A to obtain the title compound 2e (500 mg, yield: 81.8%).

Step 5

4-amino-1-((9R,12R,15R)-9-benzyl-15-(4-((tert-butoxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-5-((S)-2-phenylpropyl)-3-oxa-5,8,11,14-tetraazahexadecan-16-oyl)piperidine-4-carboxylic Acid 2f

[0218] Compound 2e (500 mg, 0.44 mmol) was dissolved in 10 mL of methanol, and then palladium-carbon (50 mg, catalytic amount) was added. After completion of the addition, the reaction system was purged with hydrogen three times and stirred for 12 hours at room temperature. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 2f (400 mg), which was used directly in the next step without purification.

Step 6

4-amino-1-((2R,5R,8R,14S)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazapentadecan-1-oyl)piperidine-4-carboxylic Acid trifluoroacetate 2g

[0219] The crude compound 2f (400 mg, 0.44 mmol) was dissolved in 5 mL of dichloromethane, and then 2 mL of a solution of 4M hydrochloric acid in 1,4-dioxane was added. After stirring for 2 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by high performance liquid chromatography to obtain the title compound 2g (150 mg, yield: 48%).

[0220] MS m/z (ESI): 708.6 [M+1]

[0221] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.40-7.17 (m, 11H), 4.89-4.82 (m, 1H), 4.79-4.74 (m, 1H), 4.40-4.39 (m, 1H), 4.22-4.15 (m, 1H), 4.01-3.95 (m, 1H), 3.85-3.60 (m, 5H), 3.49-3.36 (m, 1H), 3.21-3.09 (m, 5H), 2.96-2.92 (m, 4H), 2.27-2.25 (m, 3H), 1.83-1.45 (m, 14H), 1.34-1.33 (m, 3H), 1.01-0.92 (m, 6H)

Step 7

4-amino-1-((2R,5R,8R,14S)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazapentadecan-1-oyl)piperidine-4-carboxylic Acid 2

[0222] Compound 2g (150 mg, 0.182 mmol) was dissolved in 1 mL of a mixed solution of dichloromethane and methanol (V/V=10:1), and then saturated aqueous sodium carbonate solution was added dropwise to adjust the pH to about 7. The reaction solution was stirred for 30 minutes at room temperature and left to stand and separate. The organic phase was collected, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain title compound 2 (129 mg, yield: 100%).

[0223] MS m/z (ESI): 708.6 [M+1]

Example 3

4-amino-1-((2R,5R,8R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-14-methyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazapentadecan-1-oyl)piperidine-4-carboxylic Acid 3

[0224] ##STR00055## ##STR00056##

Step 1

(9R,12R)-benzyl 9-benzyl-12-isobutyl-2,2-dimethyl-5-(2-methyl-2-phenylpropyl)-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-oate 3b

[0225] The crude compound 1i (130 mg, 0.293 mmol) and 2-methyl-2-phenylpropan-1-amine 3a (130 mg, 0.878 mmol, prepared by a method disclosed in the patent application WO2007030582) were dissolved in 2 mL of N,N-dimethylformamide, and then potassium iodide (73 mg, 0.44 mmol) and potassium carbonate (121 mg, 0.878 mmol) were added. The reaction solution was warmed up to 80 C. and stirred for 12 hours. The reaction solution was cooled to room temperature, and then 1 mL of tetrahydrofuran and 1 mL of water were added. After stirring uniformly, di-tert-butyl dicarbonate (96 mg, 0.439 mmol) was added. The reaction solution was stirred at room temperature for 2 hours, and then concentrated under reduced pressure. The residue was added with water and extracted with ethyl acetate (30 mL3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with elution system B to obtain the title compound 3b (110 mg, yield: 57%).

Step 2

(9R,12R)-9-benzyl-12-isobutyl-2,2-dimethyl-5-(2-methyl-2-phenylpropyl)-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-oic Acid 3c

[0226] Compound 3b (110 mg, 0.162 mmol) was dissolved in methanol, and then palladium-carbon (20 mg, catalytic amount) was added. After completion of the addition, the reaction system was purged with hydrogen three times, warmed up to 30 C. and stirred for 12 hours. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 3c (74 mg), which was used directly in the next step without purification.

Step 3

methyl 1-((9R,12R,15R)-9-benzyl-15-(4-((tert-butoxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-5-(2-methyl-2-phenylpropyl)-4,7,10,13-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-oyl)-4-((tert-butoxycarbonyl)amino)piperidine-4-carboxylate 3e

[0227] The crude compound 3c (74 mg, 0.13 mmol), (R)-methyl 1-(2-amino-6-((tert-butoxycarbonyl)amino)hexanoyl)-4-((tert-butoxycarbonyl)amino)piperidine-4-carboxylate 3d (70 mg, 0.143 mmol, prepared by a method disclosed in the patent application JP5807140B1), 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (74 mg, 0.195 mmol) and N,N-diisopropylethylamine (50 mg, 0.39 mmol) were dissolved in 2 mL of N,N-dimethylformamide, and stirred at 0 C. for 2 hours. The reaction solution was concentrated under reduced pressure. The resulting residue was added with water, and extracted with ethyl acetate (30 mL3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 3e (134 mg), which was used directly in the next step without purification.

Step 4

4-amino-1-((2R,5R,8R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-14-methyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazapentadecan-1-oyl)piperidine-4-carboxylic Acid trifluoroacetate 3f

[0228] The crude compound 3e (134 mg, 0.13 mmol) was dissolved in 2 mL of a mixed solvent of tetrahydrofuran and methanol (V/V=3:1), and then 0.65 mL of 1M lithium hydroxide was added. After stirring for 2 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by high performance liquid chromatography to obtain the title compound 3f (40 mg, yield: 30%).

[0229] MS m/z (ESI): 722.6 [M+1]

[0230] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.90-7.759 (m, 2H), 7.45-7.15 (m, 10H), 4.80-4.71 (m, 1H), 4.45-4.37 (m, 1H), 4.17-4.10 (m, 1H), 4.02-3.85 (m, 2H), 3.80-3.72 (m, 3H), 3.65-3.50 m, 1H), 3.48-3.40 (m, 1H), 3.25-3.15 (m, 3H), 3.05-2.80 (m, 5H), 2.38-2.20 (m, 3H), 2.02-1.40 (m, 20H), 1.05-0.92 (m, 6H).

Step 5

4-amino-1-((2R,5R,8R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-14-methyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazapentadecan-1-oyl)piperidine-4-carboxylic Acid 3

[0231] Compound 3f (40 mg, 0.048 mmol) was dissolved in 1 mL of a mixed solution of dichloromethane and methanol (V/V=10:1). saturated aqueous sodium carbonate solution was added dropwise to adjust the pH to about 7. The reaction solution was stirred for 30 minutes at room temperature and left to stand and separate. The organic phase was collected, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the title compound 3 (34.6 mg, yield: 100%).

[0232] MS m/z (ESI): 722.6 [M+1]

Example 4

4-amino-1-((2R,5R,8R,14R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-15-methyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazahexadecan-1-oyl)piperidine-4-carboxylic Acid 4

[0233] ##STR00057## ##STR00058##

Step 1

(R)-benzyl 4-methyl-2-((R)-2-(2-(((R)-3-methyl-2-phenylbutyl)amino)acetamido)-3-phenylpropanamido)pentanoate 4b

[0234] The crude compound 1i (1 g, 2.45 mmol) and (R)-3-methyl-2-phenylbutan-1-amine 4a (500 mg, 3 mmol, prepared by a known method disclosed in Tetrahedron:Asymmetry, 2003, 14(16), 2401-2406) were dissolved in 10 mL of N,N-dimethylformamide, and then potassium iodide (1 g, 6 mmol) and potassium carbonate (1.3 g, 9.2 mmol) were added. The reaction solution was warmed up to 60 C. and stirred for 12 hours, and then concentrated under reduced pressure. The resulting residue was added with water and extracted with dichloromethane (30 mL3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 4b (500 mg), which was used directly in the next step without purification.

Step 2

(9R,12R)-benzyl 9-benzyl-12-isobutyl-2,2-dimethyl-5-((R)-3-methyl-2-phenylbutyl)-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-oate 4c

[0235] The crude compound 4b (500 mg, 0.875 mmol) was dissolved in dichloromethane, and then di-tert-butyl dicarbonate (380 mg, 1.75 mmol) and N,N-diisopropylethylamine (340 mg, 2.62 mmol) were added. After stirring for 12 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with elution system A to obtain the title compound 4c (300 mg, yield: 51.1%).

Step 3

(9R,12R)-9-benzyl-12-isobutyl-2,2-dimethyl-5-((R)-3-methyl-2-phenylbutyl)-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-oic Acid 4d

[0236] Compound 4c (300 mg, 0.447 mmol) was dissolved in 10 mL of ethanol, and then palladium-carbon (100 mg, catalytic amount) was added. After completion of the addition, the reaction system was purged with hydrogen three times and stirred for 12 hours at room temperature. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 4d (260 mg), which was used directly in the next step without purification.

Step 4

benzyl 1-((9R,12R,15R)-9-benzyl-15-(4-((tert-butoxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-5-((R)-3-methyl-2-phenylbutyl)-4,7,10,13-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-oyl)-4-(((benzyloxy)carbonyl)amino)piperidine-4-carboxylate 4e

[0237] The crude compound 4d (260 mg, 0.447 mmol), compound 1f (270 mg, 0.447 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (500 mg, 1.34 mmol) and 3 mL of triethylamine were dissolved in 10 mL of N,N-dimethylformide. After stirring for 2 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with elution system A to obtain the title compound 4e (100 mg, yield: 19.2%).

Step 5

4-amino-1-((9R,12R,15R)-9-benzyl-15-(4-((tert-butoxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-5-((R)-3-methyl-2-phenylbutyl)-4,7,10,13-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-oyl)piperidine-4-carboxylic Acid 4f

[0238] Compound 4e (100 mg, 0.086 mmol) was dissolved in 20 mL of ethanol, and the palladium-carbon (100 mg, catalytic amount) was added. After completion of the addition, the reaction system was purged with hydrogen three times and stirred for 12 hours at room temperature. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 4f (81 mg), which was used directly in the next step without purification.

Step 6

4-amino-1-((2R,5R,8R,14R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-15-methyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazahexadecan-1-oyl)piperidine-4-carboxylic Acid trifluoroacetate 4g

[0239] The crude compound 4f (81 mg, 0.086 mmol) was dissolved in 10 mL of dichloromethane, and then 3 mL of trifluroacetic acid was added. After stirring for 2 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by high performance liquid chromatography to obtain the title compound 4g (8 mg, yield: 12.7%).

[0240] MS m/z (ESI): 736.4 [M+1]

[0241] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.45-7.15 (m, 10H), 4.9-4.8 (m, 1H), 4.8-4.62 (m, 1H), 4.41-4.28 (m, 1H), 4.1-4.0 (m, 1H), 3.98-3.85 (m, 1H), 3.85-3.65 (m, 3H), 3.65-3.55 (m, 1H), 3.55-3.45 (m, 1H) 3.38-3.28 (m, 2H), 3.25-3.08 (m, 2H), 3.25-3.05 (m, 2H), 2.95-2.87 (m, 2H), 2.85-2.75 (m, 1H), 2.75-2.65 (m, 2H), 2.4-2.15 (m, 3H), 2-1.35 (m, 14H), 1.2-0.83 (m, 9H), 0.71-0.62 (d, 3H).

Step 7

4-amino-1-((2R,5R,8R,14R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-15-methyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazahexadecan-1-oyl)piperidine-4-carboxylic Acid 4

[0242] Compound 4g (8 mg, 0.009 mmol) was dissolved in 1 mL of a mixed solvent of dichloromethane and methane (V/V=10:1). Saturated sodium aqueous carbonate was added dropwised to adjust the pH to about 7. The reaction solution was stirred for 30 minutes at room temperature and left to stand and separate. The organic phase was collected, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the title compound 4 (6.9 mg, yield: 100%).

[0243] MS m/z (ESI): 736.4 [M+1]

Example 5

4-amino-1-((2R,5R,8R,14R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazapentadecan-1-oyl)piperidine-4-carboxylic Acid 5

[0244] ##STR00059## ##STR00060## ##STR00061##

Step 1

(R)-benzyl 4-methyl-2-((R)-3-phenyl-2-(2-(((R)-2-phenylpropyl)amino)acetamido)propanamido)pentanoate 5b

[0245] Compound 1i (500 mg, 1.12 mmol) and (R)-2-phenylpropan-1-amine 5a (228 mg, 1.68 mmol, prepared by a known method disclosed in Angewandte Chemie, International Edition, 2003, 42(39), 4793-4795) were dissolved in 10 mL of N,N-dimethylformamide, and then potassium iodide (372 mg, 2.24 mmol) and potassium carbonate (309 mg, 2.24 mmol) were added. The reaction solution was warmed up to 60 C. and stirred for 12 hours. The reaction solution was cooled to room temperature, added with water, and extracted with dichloromethane (30 mL3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 5b (600 mg), which was used directly in the next step without purification.

Step 2

(9R,12R)-benzyl 9-benzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-5-((R)-2-phenylpropyl)-3-oxa-5,8,11-triazatridecan-13-oate 5c

[0246] The crude compound 5b (600 mg, 1.1 mmol) was dissolved in 20 mL of dichloromethane, and then di-tert-butyl dicarbonate (361 mg, 1.66 mmol) and triethylamine (222 mg, 2.2 mmol) were added. After stirring for 12 hours at room temperature, the reaction solution was concentrated under pressure. The resulting residue was purified by thin layer chromatography with elution system A to obtain the title compound 5c (580 mg, yield: 82%).

Step 3

(9R,12R)-9-benzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-5-((R)-2-phenylpropyl)-3-oxa-5,8,11-triazatridecan-13-oic Acid 5d

[0247] Compound 5c (580 mg, 0.9 mmol) was dissolved in 10 mL of methanol, and then palladium-carbon (60 mg, catalytic amount) was added. After completion of the addition, the reaction system was purged with hydrogen three times and stirred for 12 hours at room temperature. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 5d (500 mg), which was used directly in next step without purification.

Step 4

benzyl 1-((9R,12R,15R)-9-benzyl-15-(4-((tert-butoxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-5-((R)-2-phenylpropyl)-3-oxa-5,8,11,14-tetraazahexadecan-16-oyl)-4-(((benzyloxy)carbonyl)amino)piperidine-4-carboxylate 5e

[0248] The crude compound 5d (365 mg, 0.66 mmol), compound 1f (393 mg, 0.66 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (376 mg, 0.99 mmol) and N,N-diisopropylethylamine (0.16 mL, 0.99 mmol) were dissolved in 10 mL of N,N-dimethylformamide. After stirring for 2 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with elution system A to obtain the title compound 5e (170 mg, yield: 23%).

Step 5

4-amino-1-((9R,12R,15R)-9-benzyl-15-(4-((tert-butoxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-5-((R)-2-phenylpropyl)-3-oxa-5,8,11,14-tetraazahexadecan-16-oyl)piperidine-4-carboxylic Acid 5f

[0249] Compound 5e (80 mg, 0.0706 mmol) was dissolved in 10 mL of methanol, then palladium-carbon (10 mg, catalytic amount) was added. After completion of the addition, the reaction system was purged with hydrogen three times and stirred for 12 hours at room temperature. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 5f (60 mg), which was used directly in the next step without purification.

Step 6

4-amino-1-((2R,5R,8R,14R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazapentadecan-1-oyl)piperidine-4-carboxylic Acid trifluoroacetate 5g

[0250] The crude product 5f (60 mg, 0.066 mmol) was dissolved in 2 mL of dichloromethane, and then 1 mL of a solution of 4M hydrochloric acid in 1,4-dioxane was added. After stirring for 2 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by high performance liquid chromatography to obtain the title compound 5g (30 mg, yield: 55.6%).

[0251] MS m/z (ESI): 708.6 [M+1]

Step 7

4-amino-1-((2R,5R,8R,14R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazapentadecan-1-oyl)piperidine-4-carboxylic Acid 5

[0252] Compound 5g (30 mg, 0.028 mmol) was dissolved by 5 mL of a mixed solvent of methanol/water, and then sodium bicarbonate solid (10 mg) was added to adjust the pH to 7. The reaction solution was stirred for 30 minutes, and then concentrated under reduced pressure. The resulting residue was added with 10 mL of dichloromethane, stirred for 30 minutes, and filtered. The filter cake was rinsed with 10 mL of dichloromethane, and the filtrate was concentrated under reduced pressure to obtain the title compound 5 (17 mg, yield: 85.9%).

[0253] MS m/z (ESI): 708.6 [M+1]

[0254] .sup.1H NMR (400 MHz, CD.sub.3OD): 7.33-7.19 (m, 10H), 4.90-4.84 (m, 2H), 4.64-4.61 (m, 2H), 4.42-4.39 (m, 1H), 3.86-3.74 (m, 5H), 3.20-3.12 (m, 4H), 2.94-2.84 (m, 4H), 2.61-2.54 (m, 2H), 2.20-2.15 (m, 3H), 1.79-1.70 (m, 2H), 1.68-1.60 (m, 8H), 1.45-1.40 (m, 3H), 1.30-1.20 (m, 5H), 0.99-0.76 (m, 6H).

Example 6

(R)N((R)-6-amino-1-morpholino-1-oxohexan-2-yl)-4-methyl-2-((R)-3-phenyl-2-(2-(((1S,2R)-2-phenylcyclopropyl)amino)acetamido)propanamido)pentanamide 6

[0255] ##STR00062## ##STR00063## ##STR00064##

Step 1

(R)-benzyl tert-butyl (6-morpholino-6-oxohexane-1,5-diyl)dicarbamate 6b

[0256] (R)-2-(((benzyloxy)carbonyl)amino)-6-((tert-butoxycarbonyl)amino)hexanoic acid 6a (1.14 g, 3 mmol, prepared by a known method disclosed in African Journal of Pure and Applied Chemistry, 2009, 3(6), 108-115), morpholino (0.31 mL, 3.6 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (1.73 g, 4.5 mmol) and N,N-diisopropylethylamine (0.8 mL, 4.5 mmol) were dissolved in 10 mL of N,N-dimethylformamide, and stirred for 2 hours at room temperature. The reaction solution was added with 50 mL ethyl acetate, washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution and saturated sodium chloride solution successively, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 6b (1.3 g), which was used directly in the next step without purification.

Step 2

(R)-tert-butyl (5-amino-6-morpholino-6-oxohexyl)carbamate 6c

[0257] The crude compound 6b (1.3 g, 2.9 mmol) was dissolved in 15 mL of methanol, and then palladium-carbon (350 mg, catalytic amount) was added. After completion of the addition, the reaction system was purged with hydrogen three times and stirred for 12 hours at room temperature. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 6c (914 mg), which was used directly in the next step without purification.

Step 3

(R)-benzyl 4-methyl-2-((R)-3-phenyl-2-(2-(((1S,2R)-2-phenylcyclopropyl)amino)acetamido)propanamido)pentanoate 6e

[0258] The crude compound 1i (300 mg, 0.675 mmol) and (1S,2S)-2-phenylcyclopropanamine 6d (120 mg, 0.68 mmol, prepared by a method disclosed in the patent application US20060116370A1) were dissolved in 10 mL of N,N-dimethylformamide, and then potassium iodide (560 mg, 3.375 mmol) and potassium carbonate (465 mg, 3.375 mmol) were added. The reaction solution was warmed up to 60 C. and stirred for 5 hours, and then concentrated under reduced pressure. The resulting residue was added with water and extracted with dichloromethane (30 mL3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the title compound 6e (200 mg), which was used directly in the next step without purification.

Step 4

(9R,12R)-benzyl 9-benzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-5-((1S,2R)-2-phenylcyclopropyl)-3-oxa-5,8,11-triazatridecan-13-oate 6f

[0259] The crude compound 6e (200 mg, 0.35 mmol) was dissolved in dichloromethane, and then di-tert-butyl dicarbonate (100 mg, 0.525 mmol) and triethylamine (110 mg, 1.05 mmol) were added. After stirring for 12 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with elution system A to obtain the title compound 6f (140 mg, yield: 62.5%).

Step 5

(9R,12R)-9-benzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-5-((1S,2R)-2-phenylcyclopropyl)-3-oxa-5,8,11-triazatridecan-13-oic Acid 6g

[0260] Compound 6f (140 mg, 0.218 mmol) was dissolved in 4.5 mL of a mixed solvent of tetrahydrofuran, methanol and water (V/V/V=4:4:1), and then lithium hydroxide monohydrate (55 mg, 1.31 mmol) was added. After stirring for 12 hours at room temperature, the reaction solution was concentrated under reduced pressure to remove methanol and tetrahydrofuran solvents. Water was added, and 1M hydrochloric acid was added dropwise to adjust the pH to 6. The reaction solution was extracted with dichloromethane (30 mL3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 6g (130 mg), which was used directly in next step without purification.

Step 6

tert-butyl ((10R,13R,16R)-16-phenyl-13-isobutyl-2,2-dimethyl-10-(morpholine-4-carbonyl)-4,12,15,18-tetraoxo-3-oxa-5,11,14,17-tetraazanonadecan-19-yl)((1S,2R)-2-phenylcyclopropyl)carbamate 6h

[0261] The crude compound 6g (130 mg, 0.218 mmol), the crude compound 6c (85 mg, 0.26 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (125 mg, 0.327 mmol) and N,N-diisopropylethylamine (85 mg, 0.654 mmol) were dissolved in 5 mL of N,N-dimethylformamide, and stirred for 12 hours at room temperature. The reaction solution was added with 30 mL of acetyl acetate, washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution and saturated sodium chloride solution successively, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with elution system A to obtain the title compound 6h (130 mg, yield: 70%).

Step 7

(R)N((R)-6-amino-1-morpholino-1-oxohexan-2-yl)-4-methyl-2-((R)-3-phenyl-2-(2-(((1S,2R)-2-phenylcyclopropyl)amino)acetamido)propanamido)pentanamide 6

[0262] Compound 6h (60 mg, 0.071 mmol) was dissolved in 3 mL of dichloromethane, and then 0.8 mL of a solution of 4M hydrochloric acid in 1,4-dioxane was added. After stirring for 2 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was dissolved in a mixed solvent of methanol and water (V:V=20:1). Sodium carbonate was added to adjust the pH to greater than 8. The solution was concentrated under reduced pressure. The resulting residue was added with 10 mL of dichloromethane, stirred for 10 min, and filtered. The filtrate was concentrated under reduced pressure to obtain the title compound 6 (19 mg, yield: 43.5%).

[0263] MS m/z (ESI): 649.3 [M+1]

[0264] 1H NMR (400 MHz, CD3OD) 7.26-7.23 (m, 10H), 4.95-4.93 (m, 1H), 4.80-4.78 (m, 1H), 4.70-4.68 (m, 1H), 3.66-3.60 (m, 8H), 3.32-3.30 (m, 6H), 3.28-3.26 (m, 1H), 3.18-3.16 (m, 1H), 2.94-2.91 (m, 1H), 2.65-2.63 (m, 1H), 2.26-2.23 (m, 1H), 1.71-1.68 (m, 5H), 1.48-1.42 (m, 6H), 0.95-0.93 (dd, 6H).

Example 7

4-amino-1-((2R,5R,8R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazahexadecan-1-oyl)piperidine-4-carboxylic Acid 7

[0265] ##STR00065## ##STR00066##

Step 1

(9R,12R)-9-benzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-5-(2-phenylbutyl)-3-oxa-5,8,11-triazatridecan-13-oic Acid 7a

[0266] Compound 11 (300 mg, 0.458 mmol) was dissolved in 10 mL of methanol, and then a catalytic amount of palladium-carbon was added. After completion of the addition, the reaction system was purged with hydrogen three times and stirred for 12 hours at room temperature. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 7a (189 mg), which was used directly in the next step without purification.

Step 2

benzyl 1-((9R,12R,15R)-9-benzyl-15-(4-((tert-butoxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-5-(2-phenylbutyl)-3-oxa-5,8,11,14-tetraazahexadecan-16-oyl)-4-(((benzyloxy)carbonyl)amino)piperidine-4-carboxylate 7b

[0267] The crude compound 7a (189 mg, 0.34 mmol), compound 1f (200 mg, 0.34 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (194 mg, 0.51 mmol) and triethylamine (67 mg, 0.68 mmol) were dissolved in 10 mL of N,N-dimethylformamide. After stirring for 12 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with elution system A to obtain the title compound 7b (80 mg, yield: 20%).

Step 3

4-amino-1-((9R,12R,15R)-9-benzyl-15-(4-((tert-butoxycarbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-5-(2-phenylbutyl)-3-oxa-5,8,11,14-tetraazahexadecan-16-oyl)piperidine-4-carboxylic Acid 7c

[0268] Compound 7b (80 mg, 0.07 mmol) was dissolved in 10 mL of methanol, and then palladium-carbon (10 mg, catalytic amount) was added. After completion of the addition, the reaction system was purged with hydrogen three times and stirred for 12 hours at room temperature. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 7c (50 mg), which was used directly in the next step without purification.

Step 4

4-amino-1-((2R,5R,8R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazahexadecan-1-oyl)piperidine-4-carboxylic acid trifluoroacetate 7d

[0269] The crude compound 7c (50 mg, 0.054 mmol) was dissolved in 10 mL of dichloromethane, and then 2 mL of a solution of 4M hydrochloric acid in 1,4-dioxane was added. After stirring for 1 hour at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by high performance liquid column chromatography to obtain the title compound 7d (10 mg, yield: 25.6%).

[0270] MS m/z (ESI): 722.6 [M+1]

[0271] .sup.1H NMR (400 MHz, CD.sub.3OD) 7.42-7.25 (m, 10H), 4.84-4.69 (m, 3H), 4.39-4.38 (m, 2H), 3.90-3.73 (m, 8H), 3.22-3.19 (m, 4H), 2.94-2.67 (m, 5H), 2.24-2.19 (m, 3H), 1.79-1.58 (m, 15H), 0.99-0.93 (m, 6H), 0.78 (t, 3H)

Step 5

4-amino-1-((2R,5R,8R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazahexadecan-1-oyl)piperidine-4-carboxylic Acid 7

[0272] Compound 7d (10 mg, 0.012 mmol) was dissolved in 1 mL of a mixed solvent of dichloromethane and methanol (V/V=10:1), and then saturated aqueous sodium carbonate solution was added to adjust the pH to about 7. The reaction solution was stirred for 30 minutes at room temperature and left to stand and separate. The organic phase was collected, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the title compound 7 (8.6 mg, yield: 100%).

[0273] MS m/z (ESI): 722.6 [M+1]

Example 8

4-amino-1-((2R,5R,8R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazatetradecan-1-oyl)piperidine-4-carboxylic Acid 8

[0274] ##STR00067## ##STR00068## ##STR00069##

Step 1

(R)-methyl 2-amino-6-(((benzyloxy)carbonyl)amino)hexanoate hydrochloride 8b

[0275] 1.3 mL of dichlorosulfoxide was dissolved in 20 mL of methanol, and cooled to 0 C. in a ice bath. (R)-2-amino-6-(((benzyloxy)carbonyl)amino)hexanoic acid 8a (2 g, 7.1 mmol) was then added. After stirring for 12 hours at room temperate, the reaction solution was concentrated under reduced pressure to obtain the crude title compound 8b (2.09 g), which was used directly in the next step without purification.

Step 2

(9R,12R)-benzyl 9-benzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-5-phenylethyl-3-oxa-5,8,11-triazatridecan-13-oate 8d

[0276] 2-((tert-butoxycarbonyl)(phenylethyl)amino)acetic acid 8c (332 mg, 1.19 mmol, prepared by a method disclosed in the patent U.S. Pat. No. 6,245,746B1) and compound 1g (439 mg, 1.19 mmol) were dissolved in 6.6 mL of N,N-dimethylformamide, and then 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (679 mg, 1.785 mmol) and N,N-diisopropylethylamine (615 mg, 4.76 mmol) were added. The solution was stirred at 0 C. for 2 hours, and then concentrated under reduced pressure. The resulting residue was added with water and extracted with ethyl acetate (30 mL3). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with elution system B to obtain the title compound 8d (410 mg, yield: 55%).

Step 3

(9R,12R)-9-benzyl-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-5-phenylethyl-3-oxa-5,8,11-triazatridecan-13-oic Acid 8e

[0277] Compound 8d (410 mg, 0.65 mmol) was dissolved in 20 mL of methanol, and then palladium-carbon (60 mg, catalytic amount) was added. After completion of the addition, the reaction system was purged with hydrogen three times and stirred for 12 hours at room temperature. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 8e (313 mg), which was used directly in the next step without purification

Step 4

(9R,12R,15R)-methyl 9-benzyl-15-(4-(((benzyloxy)carbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-5-phenyl ethyl-3-oxa-5,8,11,14-tetraazahexadecan-16-oate

[0278] The crude compound 8e (100 mg, 0.125 mmol) and the crude compound 8b (85 mg, 0.231 mmol) were dissolved in 5 mL of N,N-dimethylformamide, and then 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (105 mg, 0.277 mmol) and N,N-diisopropylethylamine (72 mg, 0.555 mmol) were added. After stirring for 2 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was added with water and extracted with ethyl acetate (30 mL3). The organic phases were combined, washed with saturated ammonium chloride solution (30 mL3), saturated sodium bicarbonate solution (30 mL3) and saturated sodium chloride solution (30 mL3) successively, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 8f (140 mg), which was used directly in the next step without purification.

Step 5

(9R,12R,15R)-9-benzyl-15-(4-(((benzyloxy)carbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-5-phenylethyl-3-oxa-5,8,11,14-tetraazahexadecan-16-oic Acid 8g

[0279] The crude compound 8f (140 mg, 0.17 mmol) was dissolved in 7 mL of a mixed solvent of tetrahydrofuran, methanol and water (V/V/V=3:3:1), and then lithium hydroxide monohydrate (40 mg, 0.85 mmol) was added. After stirring for 0.5 hour at room temperature, the reaction solution was concentrated under reduced pressure to remove methanol and tetrahydrofuran solvents. Water was added, then 1M hydrochloric acid was added dropwise to adjust the pH to 6. The reaction solution was extracted with dichloromethane (30 mL3). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 8g (220 mg), which was used directly in the next step without purification.

Step 6

benzyl 1-((9R,12R,15R)-9-benzyl-15-(4-(((benzyloxy)carbonyl)amino)butyl)-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-5-phenyl ethyl-3-oxa-5,8,11,14-tetraazahexadecan-16-oyl)-4-(((benzyloxy)carbonyl)amino)piperidine-4-carboxylate 8h

[0280] The crude compound 8g (220 mg, 0.17 mmol) and the crude compound 1c (130 mg, 0.26 mmol) were dissolved in 5 mL of N,N-dimethylformamide, and then 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (100 mg, 0.26 mmol) and N,N-diisopropylethylamine (66 mg, 0.51 mmol) were added. After stirring for 3 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was added with water and extracted with ethyl acetate (30 mL3). The organic phases were combined, washed with saturated ammonium chloride solution (30 mL3), saturated sodium bicarbonate solution (30 mL3) and saturated sodium chloride solution (30 mL3) successively, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with elution system A to obtain the title compound 8h (170 mg, yield: 87%).

Step 7

1-((9R,12R,15R)-15-(4-aminobutyl)-9-benzyl-12-isobutyl-2,2-dimethyl-4,7,10,13-tetraoxo-5-phenylethyl-3-oxa-5,8,11,14-tetraazahexadecan-16-oyl)-4-(carboxyamino)piperidine-4-carboxylic Acid 8i

[0281] Compound 8h (170 mg, 0.147 mmol) was dissolved in 5 mL of methanol, then palladium-carbon (50 mg, catalytic amount) was added. After completion of the addition, the reaction system was purged with hydrogen three times and stirred for 12 hours at room temperature. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 8i (140 mg), which was used directly in the next step without purification.

Step 8

4-amino-1-(2R,5R,8R)-2-(4-aminobutyl)-8-benzyl-5-isobutyl-4,7,10-trioxo-14-phenyl-3,6,9,12-tetraazatetradecan-1-oyl)piperidine-4-carboxylic Acid 8

[0282] The crude compound 8i (140 mg, 0.167 mmol) was dissolved in 3 mL of dichloromethane, and then 0.5 mL of a solution of 4M hydrochloric acid in 1,4-dioxane was added. After stirring for 1 hour at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was dissolved in a mixed solvent of methanol and water (V/V=20:1), and then sodium carbonate was added to adjust the pH to greater than 8. The reaction solution was concentrated under reduced pressure. The resulting residue was added with 10 mL of dichloromethane, stirred for 10 minutes and filtered. The filtrate was concentrated under reduced pressure to obtain the title compound 8 (19 mg, yield: 17.4%).

[0283] MS m/z (ESI): 694.4 [M+1]

[0284] .sup.1H NMR (400 MHz, CD3OD) 7.32-7.01 (m, 10H), 4.68-4.66 (m, 1H), 4.44-4.42 (m, 1H), 3.75-3.65 (m, 6H), 3.35 (s, 2H), 3.32-3.28 (m, 6H), 3.20-3.18 (m, 2H), 3.00 (s, 2H), 2.70-2.58 (m, 5H), 2.10-1.98 (m, 3H), 1.55-1.50 (m, 6H), 0.95-0.93 (dd, 6H).

Example 9

(R)N((R)-6-amino-1-(4-(3-methylureido)piperidin-1-yl)-1-oxohexan-2-yl)-4-methyl-2-((R)-3-phenyl-2-(2-(((R)-2-phenylpropyl)amino)acetamido)propanamido)pentanamide 9

[0285] ##STR00070## ##STR00071##

Step 1

tert-butyl 4-(3-methylureido)piperidine-1-carboxylate 9c

[0286] Compound 9a (8.11 g, 40 mmol, prepared by a method disclosed in the patent application WO2006115353) was dissolved in 130 mL of dichloromethane, and then N,N-diisopropylethylamine (15.51 g, 120 mmol) was added. After cooling to 0 C., the reaction solution was added with 9b (3.74 g, 40 mmol) and stirred for 2 hours at room temperature. The reaction solution was added with 200 mL of saturated sodium bicarbonate solution and extracted with dichloromethane (200 mL3). The organic phases were combined, washed with saturated sodium chloride solution (200 mL), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 9c (9.3 g), which was used directly in next step without purification.

Step 2

1-methyl-3-(piperidin-4-yl)urea hydrochloride 9d

[0287] The crude compound 9c (1 g, 4 mmol) was dissolved in 10 mL of dichloromethane, then 2 mL of a solution of 4M hydrochloric acid in 1,4-dioxane was added. After stirring for 2 hours, the reaction solution was concentrated under reduced pressure to obtain the crude title compound 9d (1 g, white solid), which was used directly in the next step without purification.

Step 3

(R)-(9H-fluoren-9-yl)methyl tert-butyl (6-(4-(3-methylureido)piperidin-1-yl)-6-oxohexane-1,5-diyl)dicarbamate 9e

[0288] The crude compound 9d (1 g, 5.16 mmol) and compound 1d (2.42 g, 5.16 mmol) were dissolved in 20 mL of N,N-dimethylformamide, and then 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (2.94 g, 7.74 mmol) and triethylamine (1.03 g, 10.32 mmol) were added. After stirring for 4 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with elution system A to obtain the title compound 9e (1 g, yield: 32%).

Step 4

(R)-tert-butyl (5-amino-6-(4-(3-methylureido)piperidin-1-yl)-6-oxohexyl)carbamate 9f

[0289] Compound 9e (304 mg, 0.5 mmol) was dissolved in 2 mL of N,N-dimethylformamide, and then 6 mL of triethylamine was added. The reaction solution was stirred for 12 hours, and then used directly in next step without purification.

Step 5

tert-butyl ((10R,13R,16R)-16-benzyl-13-isobutyl-2,2-dimethyl-10-(4-(3-methylureido)piperidine-1-carbonyl)-4,12,15,18-tetraoxo-3-oxa-5,11,14,17-tetraazanonadecan-19-yl)((R)-2-phenylpropyl)carbamate 9g

[0290] The crude compound 9f (193 mg, 0.5 mmol), the crude compound 5d (277 mg, 0.5 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (380 mg, 1 mmol) and N,N-diisopropylethylamine (129 mg, 1 mmol) were dissolved in 30 mL of N,N-dimethylformamide, and stirred for 12 hours at room temperature. The reaction solution was added with 10 mL of saturated citric acid solution and 20 mL of water, and extracted with ethyl acetate (30 mL3). The organic phases were combined, washed with saturated sodium bicarbonate solution (20 mL) and saturated sodium chloride solution (20 mL) successively, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 9g (500 mg), which was used directly in next step without purification.

Step 6

(R)N((R)-6-amino-1-(4-(3-methylureido)piperidin-1-yl)-1-oxohexan-2-yl)-4-methyl-2-((R)-3-phenyl-2-(2-(((R)-2-phenylpropyl)amino)acetamido)propanamido)pentanamide trifluoroacetate 9h

[0291] The crude compound 9g (184 mg, 0.2 mmol) was dissolved in 10 mL of dichloromethane, and then 2 mL of a solution of 4M hydrochloric acid in 1,4-dioxane was added. After stirring for 12 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by high performance liquid chromatography to obtain the title compound 9h (20 mg, yield: 14%).

[0292] MS m/z (ESI): 721.6 [M+1]

[0293] .sup.1H NMR (400 MHz, CD.sub.3OD) 8.37-8.25 (m, 1H), 7.73-7.65 (m, 1H), 7.41-7.37 (m, 2H), 7.39-7.29 (m, 7H), 7.18-7.16 (m, 2H), 4.93-4.90 (m, 1H), 4.83-4.82 (m, 1H), 4.54-4.50 (m, 2H), 4.00-3.92 (m, 1H), 3.88-3.60 (m, 6H), 3.15-3.08 (m, 5H), 3.05-2.98 (m, 4H), 2.70 (s, 3H), 2.15-1.88 (m, 3H), 1.79-1.61 (m, 15H), 1.01-0.96 (m, 6H).

Step 7

(R)N((R)-6-amino-1-(4-(3-methylureido)piperidin-1-yl)-1-oxohexan-2-yl)-4-methyl-2-((R)-3-phenyl-2-(2-(((R)-2-phenylpropyl)amino)acetamido)propanamido)pentanamide 9

[0294] Compound 9h (20 mg, 0.024 mmol) was dissolved in 1 mL of a mixed solvent of dichloromethane and methanol (V/V=10:1), and then saturated aqueous sodium carbonate solution was added dropwise to adjust the pH to about 7. The reaction solution was stirred for 30 minutes at room temperature and left to stand and separate. The organic phase was collected, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the title compound 9 (17 mg, yield: 100%).

[0295] MS m/z (ESI): 721.6 [M+1]

Example 10

(R)N((R)-6-amino-1-morpholino-1-oxohexan-2-yl)-2-((R)-2-(2-((2,3-dihydro-1H-inden-2-yl)amino)acetamido)-3-phenylpropanamido)-4-methylpentanamide 10

[0296] ##STR00072## ##STR00073## ##STR00074##

Step 1

(R)-(9H-fluoren-9-yl)methyl (6-((1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)amino)-1-morpholino-1-oxohexan-2-yl)carbamate 10b

[0297] (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)amino)hexanoic acid 10a (1.06 g, 2 mmol, purchased from Accela ChemBio Inc.) and morpholine (200 mg, 2.4 mmol) were dissolved in 10 mL of N,N-dimethylformamide, and then 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (1.51 g, 4 mmol) and triethylamine (400 mg, 4 mmol) were added. After stirring for 3 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was added with 5 mL of saturated citric acid solution and 50 mL of water, and extracted with ethyl acetate (100 mL3). The organic phases were combined, washed with water (100 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 10b (1.3 g), which was used directly in the next step without purification.

Step 2

(R)-2-(1-((5-amino-6-morpholino-6-oxohexyl)amino)ethylidene)-5,5-dimethylcyclohexane-1,3-dione 10c

[0298] The crude compound 10b (1.3 g, 2 mmol) was dissolved in 5 mL of dichloromethane, and then 2 mL of piperidine was added. After stirring for 12 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with elution system A to obtain the title compound 10c (500 mg, yield: 75%).

Step 3

(R)-benzyl 2-((R)-2-(2-((2,3-dihydro-1H-inden-2-yl)amino)acetamido)-3-phenylpropanamido)-4-methylpentanoate 10e

[0299] The crude compound 1i (222 mg, 0.5 mmol) and 2,3-dihydro-1H-inden-2-amine hydrochloride 10d (127 mg, 0.4 mmol, prepared by a known method disclosed in Tetrahedron, 2005, 61(28), 6801-6807) were dissolved in 5 mL of N,N-dimethylformamide, and then potassium iodide (415 mg, 2.5 mmol) and potassium carbonate (345, 2.5 mmol) were added. The reaction solution was warmed up to 60 C. and stirred for 12 hours, then concentrated under reduced pressure. The resulting residue was purified with thin layer chromatography with elution system A to obtain the title compound 10e (300 mg, yield: 100%).

Step 4

(9R,12R)-benzyl 9-benzyl-5-(2,3-dihydro-1H-inden-2-yl)-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-oate 10f

[0300] Compound 10e (300 mg, 0.55 mmol) was dissolved in 10 mL of dichloromethane, then di-tert-butyl dicarbonate (181 mg, 083 mmol), and N,N-diisopropylethylamine (0.3 mL, 1.65 mmol) were added. After stirring for 12 hours at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified with thin layer chromatography with elution system A to obtain the title compound 10f (170 mg, yield: 48%).

Step 5

(9R,12R)-9-benzyl-5-(2,3-dihydro-1H-inden-2-yl)-12-isobutyl-2,2-dimethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-oic Acid 10g

[0301] Compound 10f (170 mg, 0.26 mmol) was dissolved in 10 mL of methanol, and then palladium-carbon (50 mg, 10%) was added. The reaction system was purged with hydrogen three times and stirred for 12 hours at room temperature. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude title compound 10g (112 mg), which was used directly in next step without purification.

Step 6

tert-butyl ((4R,7R,10R)-4-benzyl-16-(4,4-dimethyl-2,6-dioxocyclohexylidene)-7-isobutyl-10-(morpholine-4-carbonyl)-2,5,8-trioxo-3,6,9,15-tetraazaheptadecyl)(2,3-dihydro-1H-inden-2-yl)carbamate 10h

[0302] The crude compound 10g (112 mg, 0.2 mmol), compound 10c (78 mg, 0.2 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (114 mg, 0.3 mmol) and N,N-diisopropylethylamine (0.1 mL, 0.6 mmol) were dissolved in 15 mL of N,N-dimethylformamide and stirred for 2 hours at room temperature. The reaction solution was concentrated under reduced pressure. The resulting residue was added with 100 mL of ethyl acetate, washed with water (50 mL3) and saturated ammonium chloride solution (50 mL3) successively, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the crude title compound 10h (183 mg), which was used directly in next step without purification.

Step 7

tert-butyl (2-(((R)-1-(((R)-1-(((R)-6-amino-1-morpholino-1-oxohexan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)(2,3-dihydro-1H-inden-2-yl)carbamate 10i

[0303] The crude compound 10h (183 mg, 0.2 mmol) was dissolved in 10 mL of methanol, and then 0.5 mL of hydrazine hydrate was added. After stirring at room temperature for 1 hour, the reaction solution was concentrated under reduced pressure to obtain the crude title compound 10i (150 mg), which was used directly in next step without purification.

Step 8

(R)N((R)-6-amino-1-morpholino-1-oxohexan-2-yl)-2-((R)-2-(2-((2,3-dihydro-1H-inden-2-yl)amino)acetamido)-3-phenylpropanamido)-4-methylpentanamide trifluoroacetate 10j

[0304] The crude compound 10i (150 mg, 1.31 mmol) was dissolved in 5 mL of dichloromethane, and then 1 mL of trifluoroacetic acid was added. After stirring for 1 hour at room temperature, the reaction solution was concentrated under reduced pressure. The resulting residue was purified by high performance liquid chromatography to obtain the title compound 10j (5 mg, yield: 4%).

[0305] MS m/z (ESI): 649.4 [M+1]

[0306] .sup.1H NMR (400 MHz, DMSO-d.sub.6) 8.36 (d, 1H), 7.88 (d, 2H), 7.21-7.33 (m, 9H), 4.61-4.64 (m, 1H), 4.36-4.47 (m, 1H), 4.02-4.15 (m, 2H), 3.60-3.70 (m, 3H), 3.45-3.59 (m, 5H), 3.35-3.44 (m, 3H), 2.90-3.25 (m, 9H), 1.55-1.80 (m, 6H), 1.30-1.45 (m, 4H), 0.98 (d, 3H), 0.92 (d, 3H).

Step 9

(R)N((R)-6-amino-1-morpholino-1-oxohexan-2-yl)-2-((R)-2-(2-((2,3-dihydro-1H-inden-2-yl)amino)acetamido)-3-phenylpropanamido)-4-methylpentanamide 10

[0307] Compound 10j (5 mg, 0.0057 mmol) was dissolved in 2 mL of a mixed solvent of dichloromethane and methanol (V/V=10:1), and then saturated sodium carbonate was added dropwised to adjust the pH to about 7. The reaction solution was stirred for 30 minutes at room temperature and left to stand and separate. The organic phase was collected, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the title compound 10 (4 mg, yield: 100%).

[0308] MS m/z (ESI): 649.4 [M+1]

Biological Assay

[0309] The present invention will be further described with reference to the following test examples, but the examples should not be considered as limiting the scope of the invention.

Test Example 1

[0310] 1. Experimental Objective

[0311] The objective of this experiment is to determine the agonistic effect of the compounds of the present invention on human KOR (h-KOR) receptors, and to evaluate the in vitro activity of the compounds according to the values of EC.sub.50.

[0312] 2. h-KOR Activity Test

[0313] 2.1 Experimental Objective

[0314] The compounds of the present invention can activate h-KOR receptor, thereby reducing intracellular cAMP levels. The second messenger cAMP enters the nucleus and binds to the CRE of the DNA, thereby initiating the expression of the downstream luciferase. Luciferase reacts with its substrate to emit fluorescence, and the measured fluorescence signals reflect the agonistic activity of the compounds.

[0315] 2.2 Experimental Method

[0316] The activity of the test example compounds on agonizing h-KOR and affecting downstream cAMP levels was tested by the following method.

[0317] 2.1.1 Experimental Materials and Instruments

TABLE-US-00003 Reagent name Supply company Item number HEK293 cell line Cell bank of the typical GNHu43 culture preservation Committee of Chinese Academy of Sciences DMSO Shanghai Titanchem G75927B DMEM high glucose Thermo HyCLone SH30243018 medium Fetal bovine serum (FBS) Gibco 10099-141 CRE/pGL4.29 Promega E8471 KOR-1/pcDNA3.1(+) GENEWIZ Biological Synthesis Technology Co., Ltd ONE-Glo Luciferase Promega E6110 Assay System

[0318] 2.2.2 Experimental Procedure

[0319] 1) Obtaining HEK293/KOR/CRE Monoclonal Cell Lines

[0320] KOR/pcDNA3.1 (+) and CRE/pGL4.29 were transferred into HEK293 cells. G418 and hygromycin were added into the culture medium, and HEK293/KOR/CRE monoclonal cell lines were screened in a 96-well cell culture plate.

[0321] 2) Agonistic Effect of Example Compounds on h-KOR

[0322] HEK293/h-KOR/CRE monoclonal cells were cultured in a DMEM/high glucose medium (10% FBS, 1 mg/ml G418, 200 g/ml hygromycin, mixed uniformly), and passaged every 3 days. On the day of the experiment, a cell suspension was prepared with a fresh cell medium, added to a 96 well plate (BD, #356692) with 20,000 cells/well, and incubated in 5% CO.sub.2 at 37 C. On the second day, the compound was dissolved in pure DMSO at a concentration of 20 mM, then formulated with DMSO to a first concentration of 200 nM and diluted in 3 fold concentration gradient to 8 concentrations. 90 l of DMSO were added to blank and control wells. The compound solution was diluted 20-fold with DMEM/hyperglucose (SH30243.01B, Hyclone) medium containing 10 l, M Forskolin. The cell culture plates inoculated on the first day were taken out, and 10 l of the diluted drug or the control (0.5% DMSO) was added to each well. The plate was gently shaken, and placed at 37 C. for 4 hours. In a 96-well cell culture plate, 100 l of luciferase assay solution (Promega, # E6110) was added to each well. The plate was placed for 5 minutes at room temperature. The chemiluminescence value was measured using Victor 3.0. The EC.sub.50 values of the compounds were calculated using Graphpad Prism software based on each concentration of the compound and the corresponding signal value.

[0323] 2.3 Test Results

[0324] The activity of the compounds of the present invention on agonizing h-KOR and affecting downstream cAMP levels was determined by the above test, and the EC.sub.50 values are shown in Table 1.1.

TABLE-US-00004 TABLE 1.1 EC.sub.50 of the compounds of the present invention on agonizing h-KOR receptor and affecting cAMP levels Example No. EC.sub.50(pM) 1 56 2 15 3 4 4 9 5 1 6 79 7 3 8 13 9 0.5 10 >1000

[0325] Conclusion: The compounds of the present invention have significant agonistic effects on the h-KOR receptor. In particular, when the substituent on the amino group of glycine is a substituted or unsubstituted ethylene group, the compound has an unexpected effect.

Pharmacokinetics Evaluation

Test Example 2. Pharmacokinetics Assay of the Compounds of Examples 2, 5 and 8 of the Present Invention in Rats

[0326] 1. Abstract

[0327] Rats were used as test animals. The drug concentration in plasma at different time points was determined by LC/MS/MS after intravenous administration of the compounds of Examples 2, 5 and 8 to the rats. The pharmacokinetic behavior of the compounds of the present invention was studied and evaluated in SD rats.

[0328] 2. Protocol

[0329] 2.1 Test Compounds

[0330] Compounds of Examples 2, 5 and 8

[0331] 2.2 Test animals

[0332] 12 Sprague-Dawley (SD) rats, half male and half female, were purchased from SINO-BRITISH SIPPR/BK LAB. ANIMAL LTD., CO, with License No.: SOCK (Shanghai) 2008-0016.

[0333] 2.3 Preparation of the Test Compounds

[0334] The appropriate amount of the test compounds was weighed, and added with 5% 5% DMSO+5% PEG400+90% normal saline successively.

[0335] 2.4 Administration

[0336] After an overnight fast, 12 SD rats, half male and half female, were divided into 3 groups equally, and administered intravenously at a dose of an administration volume of 5 mL/kg.

[0337] 3. Process

[0338] In the venous group, blood (0.2 mL) was taken from the orbital sinus before administration and 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 11.0 and 24.0 hours after administration. The samples were stored in heparinized tubes, and centrifuged for 10 minutes at 3,500 rpm to separate the blood plasma. The plasma samples were stored at 20 C.

[0339] The concentration of the test compounds in SD rat plasma after intravenous administration was determined by LC-MS/MS.

[0340] 4. Results of Pharmacokinetic Parameters in SD Rats

[0341] Pharmacokinetic parameters of the compounds of Examples 2, 5 and 8 of the present invention are shown below.

TABLE-US-00005 Pharmacokinetics Parameters (1 mg/kg) Mean Apparent Area Under Residence Distribution Curve Half-Life Time Clearance Volume Example AUC t.sub.1/2 MRT CL Vz No. (ng/mL*h) (h) (h) (ml/minute/kg) (ml/kg) 2 2563 427 0.488 0.026 0.525 0.099 6.66 1.28 280 46 5 1697 363 0.469 0.092 0.557 0.124 10.1 1.8 410 112 8 1522 436 0.502 0.038 0.566 0.112 11.6 3.3 499 111

[0342] Conclusion: The compounds of the present invention have good pharmacokinetic properties in rats.

Test Example 3. Pharmacokinetics Assay of the Compound of Example 5 of the Present Invention in Dogs

[0343] 1. Abstract

[0344] Beagle dogs were used as test animals. The drug concentration in plasma at different time points was determined by LC/MS/MS after intravenous administration of the compound of Example 5 to the Beagle dogs. The pharmacokinetic behavior of the compound of the present invention was studied and evaluated in Beagle dogs.

[0345] 2. Protocol

[0346] 2.1 Test Compound

[0347] Compound of Example 5

[0348] 2.2 Test animals

[0349] 3 Beagle dogs in one group, male, were purchased from Medicilon Pharmaceutical Technology (Shanghai) Co., Ltd.

[0350] 2.3 Preparation of the Test Compound

[0351] The appropriate amount of the test compound was weighed, and added with 100% normal saline.

[0352] 2.4 Administration

[0353] After an overnight fast, 3 Beagle dogs in one group, male, were administered intravenously at a dose of an administration volume of 2 mL/kg.

[0354] 3. Process

[0355] In the venous group, blood (1 mL) was taken from the jugular vein before administration and 5 minutes, 15 minutes, 0.5, 1.0, 2.0, 4.0, 8.0, 12.0 and 24.0 hours after administration. The samples were stored in heparinized tubes, and centrifuged for 10 minutes at 3,500 rpm to separate the blood plasma. The plasma samples were stored at 80 C.

[0356] The concentration of the test compound in Beagle dog plasma after intravenous administration was determined by LC-MS/MS.

[0357] 4. Results of Pharmacokinetic Parameters in Beagle Dogs

[0358] Pharmacokinetic parameters of the compound of Example 5 of the present invention are shown below.

TABLE-US-00006 Pharmacokinetics Parameters (0.3 mg/kg) Mean Apparent Area Under Residence Distribution Curve Half-Life Time Clearance Volume Example AUC t.sub.1/2 MRT CL Vz No. (ng/mL*h) (h) (h) (ml/minute/kg) (ml/kg) 5 1975 165 1.34 0.04 1.43 0.08 2.54 0.21 296 19

[0359] Conclusion: The present compound of the invention has good pharmacokinetic properties in Beagle dogs.

Test Example 4. Experimental Report of KOR Agonist in the Treatment of Carrageenan-Induced Inflammatory Pain in Rats

[0360] 1. Experimental Objective

[0361] A carrageenan inflammatory pain model in rats was established to evaluate the therapeutic effect of KOR agonists on inflammatory pain in rats.

[0362] 2. Experimental Method and Experimental Materials

[0363] 2.1. Test Animals and Feeding Conditions

[0364] Male Wistar rats were purchased from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China, Certificate No. 2015000513408, License No. SCXK (Shanghai) 2012-0002). The rats were 150-180 g, and fed at 5/cage, in a condition of 12/12 hours light/dark cycle adjustment, constant temperature of 231 C., humidity of 5060%, and free access to food and water. After purchase, the animals adapted to this condition for 7 days before the experiment was started.

[0365] 2.2. Test Compound

[0366] Compound of Example 5;

[0367] -Carrageenan, Batch No. BCBP8978V, purchased from sigma.

[0368] 0.9% Sodium chloride solution (500 mL, 4.5 g)

[0369] 1% -Carrageenan was placed in physiological saline, and stirred overnight to form a jelly-like suspension.

[0370] The compound dose was calculated on bases.

[0371] 2.3. Experimental Design and Experimental Method

[0372] 2.3.1 Animal Grouping

[0373] After adaptive feeding, the rats were grouped as follows:

TABLE-US-00007 Groups of Inflammatory Pain Model n Molding Method Administration Method Blank control 8 0.9% NS 0.9% NS (i.v., once) group (s.c., 0.1 ml/rat, once) Model group 8 1% -Carrageenan 0.9% NS (i.v., once) (s.c., 0.1 ml/rat, once) Example 5 8 1% -Carrageenan Example 5 (0.1, 0.3 group (0.1 and (s.c., 0.1 ml/rat, once) mg/kg i.v., once) 0.3 mg/kg) Note: NS: normal saline used in the preparation of carrageenan solution; i.v.: intravenous injection; s.c.: subcutaneous injection.

[0374] 2.3.2. Experiment Method.sup.[1] [2]:

[0375] The experimental method was modified in accordance with the method of Document 1 (Kazunari Nakao et al.). Before inflammatory pain experiment, rats were randomly divided into the following groups according to body weight: blank control group, model group, Example 5-0.1 mg/kg group, and Example 5-0.3 mg/kg group. There were 8 rats in each group. Inflammatory pain model was made in Wistar rat footpads that were subcutaneously injected with 1% carrageenan (100 l). After 4 hours, the rats were subjected to a plantar tenderness test to evaluate the mechanical pain threshold. Single tail vein administration of the drug (1 ml/kg) was carried out 30 minutes before detection, and the control group and the model group were given corresponding solvents.

[0376] Note: Documents 1, CJ-023,423, a Novel, Potent and Selective Prostaglandin KOR Receptor Antagonist with Anti-hyperalgesic Properties. The Journal of Pharmacology and Experimental Therapeutics, 2007, 322(2):686-694.

[0377] 2.4 Experimental Apparatus

[0378] Electronic Von Frey: UGO BASILE, type 38450.

[0379] 2.5 Data Representation and Statistical Processing

[0380] The experimental data were expressed as meanstandard deviation (S.D.). Statistical comparisons were performed using the excel software t test. The data between the model group and the control group were analyzed and compared to determine whether there was a significant statistical significance. *P<0.05 indicates that there is a significant difference between the model group and the control group, ** P<0.01 indicates that there is a high significant difference between the model group and the control group, #P<0.05 indicates that there is a significant difference between the model group and the control group, ##P<0.01 indicates that there is a high significant difference between the model group and the administration group.

[0381] 3. Results: Effect of the Compound of the Present Invention on Carrageenan-Induced Carrageenan Inflammatory Pain in Rats

[0382] The experimental results in rats showed that the threshold of tenderness in the blank control group was about 20 g, and the threshold of tenderness in the model group was 7.6 g. Compared with the blank control group, the threshold of tenderness in the model group was significantly decreased (P<0.01). Compared with the model group, all drugs could significantly increase the tenderness threshold of inflammatory rats (P<0.01). The threshold of tenderness of Example 5-0.1 mg/kg and Example 5-0.3 mg/kg were 13.7 g and 23.2 g, respectively. The increases were 79.5% and 204.5% respectively, with significant dose dependency. (see FIG. 1).

[0383] 4. Discussion

[0384] -Carrageenan is a colloidal substance extracted from aquatic plant, and has an allergic stimulating effect. Carrageenan alone can induce inflammation and cause pain. In this experiment, the model of carrageenan inflammatory pain was established to observe the changes of the threshold of tenderness after KOR agonist administration in rats, and to evaluate the analgesic effect of the drug on subacute inflammatory pain and its intensity. The experiment used an electronic tactile measuring instrument to measure the response of the rat to tenderness. The electronic tactile measuring instrument (e-VF) was designed using Ugo Basile original design to evaluate rat and mouse allergies and allodynia. The instrument automatically records the stimulus time and stimulation intensity of the animals. The unique prism design makes it easy to observe the plantar area of the test animals during the experiment. During the detection, the instrument can sense the test animal to retract the test claw, or it can be judged by the foot switch. More focused positioning is more suitable for local pain and neuropathic pain measurement.

[0385] 5. Conclusion

[0386] The test compound could improve inflammatory pain in rats in a dose-dependent manner.