Polypeptide compound, pharmaceutical composition, preparation method and application thereof

Abstract

The present invention discloses a polypeptide compound, a pharmaceutical composition, and a preparation method and use thereof. The structural formula of the polypeptide compound is shown in general formula (I): ##STR00001## Such polypeptide compounds as κ-opioid receptor agonists have the advantages of better activity and the potential to become clinical candidate compounds.

Claims

1. A polypeptide compound selected from the group consisting of ##STR00025## ##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032## or pharmaceutically acceptable salts thereof.

2. A pharmaceutical composition comprising the polypeptide compound or pharmaceutically acceptable salts thereof according to claim 1.

Description

DETAILED DESCRIPTION

(1) In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with examples. The exemplary embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

Example 1

Preparation of Polypeptide Compound TM-1

(2) Step 3-1: A solution of Int-1 (0.545 g, 0.622 mmol), HOBT (0.201 g, 1.492 mmol), HBTU (0.566 g, 1.492 mmol), and DIEA (0.320 g, 2.488 mmol) in DCM (15 mL) were stirred at room temperature for 0.5 h, and then crude Int-4 was added. The mixture was reacted at room temperature for 2 h. The reaction solution was washed with saturated ammonium chloride solution, water and saturated brine, and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was purified by Prep-HPLC to obtain an intermediate compound 1-1;

(3) Step 3-2: TFA (1 mL) was added dropwise to a solution of 1-1 (500 mg) in DCM (2 mL), and the mixture was stirred at room temperature for 1 h. After concentrating and drying, the crude product was purified by Prep-HPLC to obtain a trifluoroacetate salt of polypeptide compound TM-1.

(4) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-1 are as follows:

(5) ESI-MS (m/z): 665.4 (M+H+)

(6) 1H NMR (400 MHz, DMSO-d6+D2O): δ 7.28-7.21 (m, 10H), 4.70-4.59 (m, 2H), 4.42-4.36 (m, 1H), 4.18-4.11 (m, 1H), 3.98 (s, 1H), 3.13-2.87 (m, 4H), 2.86-2.59 (m, 4H), 1.74-1.09 (m, 14H), 1.00-0.91 (m, 1H), 0.89-0.84 (m, 6H).

(7) The preparation process of Int-1 is as follows:

(8) Step 1-1: Synthesis of Intermediate Int-1-1: 2-CTC Resin (degree of substitution was 0.993 mmol/g, 2.000 g) was swelled using DCM (20 mL) at room temperature for 15 min, and the solvent was removed. The mixed solution of Fmoc-D-Lys(Boc)-OH (1.120 g, 2.4 mmol) and DIEA (0.516 g, 4.0 mmol) in DCM (15 mL) was added to the swollen resin and the mixture was reacted at room temperature for 2 h; then methanol (2 mL) and DIEA (1 mL) were added to continue the reaction for 0.5 h. The solvent was removed until dryness and the resulting mixture was washed with DCM (30 mL) for three times, and finally washed with DMF (30 mL) for three times, and after that the resin was directly put into the next reaction;

(9) Step 1-2: Piperidine/DMF (V/V=1/4, 20 mL) was added to the product obtained in step 1-1, and the mixture was reacted for 10 min at room temperature, and then removed of solvent until dryness. Piperidine/DMF (V/V=1/4, 20 mL) was added again, and the mixture was reacted for 10 min at room temperature, and then removed of solvent until dryness, and washed with DMF (30 mL) for 5 times. After the last washing, the pH of the waste liquid was tested to be neutral; Fmoc-D-Leu-OH (1.809 g, 4.0 mmol), HOBT (0.543 g, 4.0 mmol) and HBTU (1.521 g, 4.0 mmol) were added to DMF (20 mL) under ice bath conditions for activation for 10 min, and then DIEA (0.780 g, 6 mmol) was added and the mixture was reacted for 5 min. Finally, the activation solution was added to the resin and the mixture was reacted at room temperature for 2 h. The resin was treated with 5% ninhydrin solution (heated at 100° C. for 10 min) and the color did not change. The solution was removed until dryness and washed with DMF (30 mL) for 5 times. After the last washing, the pH of the waste liquid was tested to be neutral. After removing solvent until dryness, the resulting mixture was directly used in the next reaction;

(10) Step 1-3: Piperidine/DMF (V/V=1/4, 20 mL) was added to the product obtained in step 1-2, and the mixture was reacted for 10 min at room temperature, and then removed of solvent until dryness. Piperidine/DMF (V/V=1/4, 20 mL) was added again, and the mixture was reacted for 10 min at room temperature, and then removed of solvent until dryness, and washed with DMF (30 mL) for 5 times. After the last washing, the pH of the waste liquid was tested to be neutral; Fmoc-D-Phe-OH (1.547 g, 4.0 mmol), HOBT (0.543 g, 4.0 mmol) and HBTU (1.521 g, 4.0 mmol) were added to DMF (20 mL) under ice bath conditions for activation for 10 min, and then DIEA (0.780 g, 6 mmol) was added and the mixture was reacted for 5 min. Finally, the activation solution was added to the resin and the mixture was reacted at room temperature for 2 h. The resin was treated with 5% ninhydrin solution (heated at 100° C. for 10 min) and the color did not change. The solution was removed until dryness and washed with DMF (30 mL) for 5 times. After the last washing, the pH of the waste liquid was tested to be neutral. After removing solvent until dryness, the resulting mixture was directly used in the next reaction;

(11) Step 1-4: Synthesis of Intermediate Int-1-4: Piperidine/DMF (V/V=1/4, 20 mL) was added to the product obtained in step 1-3, and the mixture was reacted for 10 min at room temperature, and then removed of solvent until dryness. Piperidine/DMF (V/V=1/4, 20 mL) was added again, and the mixture was reacted for 10 min at room temperature, and then removed of solvent until dryness, and washed with DMF (30 mL) for 5 times. After the last washing, the pH of the waste liquid was tested to be neutral; Boc-D-Phe-OH (1.547 g, 4.0 mmol), HOBT (0.543 g, 4.0 mmol) and HBTU (1.521 g, 4.0 mmol) were added to DMF (20 mL) under ice bath conditions for activation for 10 min, and then DIEA (0.780 g, 6 mmol) was added and the mixture was reacted for 5 min. Finally, the activation solution was added to the resin and the mixture was reacted at room temperature for 2 h. The resin was treated with 5% ninhydrin solution (heated at 100° C. for 10 min) and the color did not change. The solution was removed until dryness and washed with DMF (30 mL) for 5 times. After the last washing, the pH of the waste liquid was tested to be neutral. After removing solvent until dryness, the resulting mixture was directly used in the next reaction;

(12) Step 1-5: Int-1-4 (4.2 g) was added to trifluoroethanol/DCM (50 mL, V=/V=1/4) at room temperature, and the mixture was reacted for 2 h at room temperature. The resulting mixture was filtered with suction, and washed twice with DCM (30 mL), and the organic phase was concentrated to about 5 mL. The concentrated solution was added dropwise to 100 mL of methyl tert-butyl ether with stirring and the mixture was settled to obtain 2.10 g of product, which was detected by LCMS as target product Int-1.

(13) ESI-MS (m/z): 754.4 (M+H.sup.+)

(14) The preparation process of Int-4 is as follows:

(15) TFA (1 mL) was added dropwise to a solution of 1-N-tert-butoxycarbonylpiperidine-4-boronic acid pinacol ester (0.045 g, 1.1 eq) in DCM (2 mL), and the mixture was stirred at room temperature for 0.5 h. The mixture was concentrated to dryness under reduced pressure, and the obtained transparent oil was directly used for the next reaction.

(16) ESI-MS (m/z): 212.3 (M+H.sup.+)

Example 2

Preparation of Polypeptide Compound TM-2

(17) This example was based on example 1 except that:

(18) The amount of Int-1 was 0.093 g; Int-4 was replaced with pyrrole-3-boronic acid pinacol ester; the synthesis of pyrrole-3-boronic acid pinacol ester was similar to Int-4, and was achieved by removing the protective group of N-tert-butoxycarbonyl-pyrrole-3-boronic acid pinacol ester under acidic conditions. The obtained crude product was purified by Prep-HPLC to obtain two isomers of the trifluoroacetate salt of the target compound TM-2, TM-2A (10.5 mg) and TM-2B (12.2 mg). The structures were identified as (R)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-3-pyrrolidineboronic acid and (S)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-3-pyrrolidineboronic acid, respectively.

(19) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-2A were as follows:

(20) ESI-MS (m/z): 651.4 (M+H.sup.+)

(21) 1H NMR (400 MHz, DMSO): δ8.76-8.73 (m, 1H), 8.38-8.16 (m, 2H), 8.02 (s, 3H), 7.70 (s, 3H), 7.39-7.13 (m, 10H), 4.69-4.64 (m, 1H), 4.52-4.43 (m, 2H), 4.02 (s, 1H), 3.44-3.25 (m, 3H), 3.14-3.02 (m, 3H), 2.96-2.90 (m, 1H), 2.87-2.66 (m, 3H), 2.01-1.92 (m, 1H), 1.71-1.29 (m, 11H), 0.92-0.87 (m, 6H).

(22) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-2B were as follows:

(23) ESI-MS (m/z): 651.4 (M+H.sup.+)

(24) 1H NMR (400 MHz, DMSO): δ8.76-8.70 (m, 1H), 8.39-8.21 (m, 2H), 8.03 (s, 3H), 7.70 (s, 3H), 7.41-7.15 (m, 10H), 4.69-4.66 (m, 1H), 4.55-4.44 (m, 2H), 4.05 (s, 1H), 3.48-3.30 (m, 3H), 3.18-3.02 (m, 3H), 3.01-2.90 (m, 1H), 2.92-2.71 (m, 3H), 2.03-1.97 (m, 1H), 1.70-1.20 (m, 11H), 0.90-0.85 (m, 6H).

Example 3

Preparation of Polypeptide Compound TM-3

(25) This example was based on example 1 except that:

(26) The amount of Int-1 was 0.090 g; Int-4 was replaced with 2,5-dihydro-1H-pyrrole-3-boronic acid pinacol ester; the synthesis of 2,5-dihydro-1H-pyrrole-3-boronic acid pinacol ester was similar to Int-4, and was achieved by removing the protective group of N-tert-butoxycarbonyl-2,5-dihydro-1H-pyrrole-3-boronic acid pinacol ester under acidic conditions. The obtained crude product was purified by Prep-HPLC to obtain 13.3 mg of the trifluoroacetate salt of the target compound TM-3.

(27) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-3 were as follows:

(28) ESI-MS (m/z): 649.4 (M+H.sup.+)

(29) 1H NMR (400 MHz, CD.sub.3OD): δ 7.44-7.12 (m, 10H), 6.58-6.34 (m, 1H), 4.77-4.52 (m, 3H), 4.48-4.25 (m, 4H), 4.11-4.06 (m, 1H), 3.28-3.18 (m, 1H), 3.06-2.89 (m, 4H), 1.88-1.80 (m, 1H), 1.79-1.38 (m, 8H), 1.02-0.91 (m, 6H).

Example 4

Preparation of Polypeptide Compound TM-4

(30) This example was based on example 1 except that:

(31) The amount of Int-1 was 0.057 g; Int-4 was replaced with aminomethylphenylboronic acid pinacol ester. The amount of aminomethylphenylboronic acid pinacol ester was 18 mg. The obtained crude product was purified by Prep-HPLC to obtain 20.1 mg of the trifluoroacetate salt of the target compound TM-4.

(32) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-4 were as follows:

(33) ESI-MS (m/z): 687.4 (M+H.sup.+)

(34) 1H NMR (400 MHz, DMSO): δ 8.77-8.72 (m, 1H), 8.49-8.33 (m, 2H), 8.11-7.95 (m, 5H), 7.66 (d, J=8.8 Hz, 4H), 7.40-7.06 (m, 12H), 4.69-4.64 (m, 1H), 4.49-4.35 (m, 1H), 4.34-4.23 (m, 3H), 4.01 (s, 1H), 3.19-3.03 (m, 2H), 2.98-2.87 (m, 1H), 2.86-2.69 (m, 3H), 1.78-1.45 (m, 7H), 1.36-1.25 (m, 2H), 0.97- 0.74 (m, 6H).

Example 5

Preparation of Polypeptide Compound TM-5

(35) This example was based on example 1 except that:

(36) The amount of Int-1 was 0.075 g; Int-4 was replaced with 3,6-dihydro-2H-pyridine-5-boronic acid pinacol ester; the synthesis of 3,6-dihydro-2H-pyridine-5-boronic acid pinacol ester was similar to Int-4, and was achieved by removing the protective group of N-tert-butoxycarbonyl-3,6-dihydro-2H-pyridine-5-boronic acid pinacol ester under acidic conditions. The obtained crude product was purified by Prep-HPLC to obtain 22.0 mg of the trifluoroacetate salt of the target compound TM-5.

(37) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-5 were as follows:

(38) ESI-MS (m/z): 663.4 (M+H.sup.+)

(39) 1H NMR (400 MHz, MeOD): δ 7.52-7.15 (m, 10H), 6.59-6.40 (m, 1H), 4.79-4.64 (m, 1H), 4.42 (dt, J=10.2, 6.1 Hz, 1H), 4.30-3.94 (m, 3H), 3.85-3.53 (m, 2H), 3.32-3.12 (m, 2H), 3.04-2.91 (m, 4H), 2.27 (s, 2H), 1.97-1.55 (m, 7H), 1.54-1.32 (m, 2H), 1.00-0.95 (m, 6H).

Example 6

Preparation of Polypeptide Compound TM-6

(40) This example was based on example 1 except that:

(41) The amount of Int-1 was 0.075 g; Int-4 was replaced with p-aminophenylboronic acid pinacol ester. The amount of p-aminophenylboronic acid pinacol ester was 22 mg. The obtained crude product was purified by Prep-HPLC to obtain 3.0 mg of the trifluoroacetate salt of the target compound TM-6.

(42) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-6 were as follows:

(43) ESI-MS (m/z): 755.4 (M+H.sup.+)

(44) 1H NMR (400 MHz, CD.sub.3OD): δ 7.79-7.47 (m, 4H), 7.44-7.12 (m, 10H), 4.79-4.66 (m, 1H), 4.58-4.35 (m, 2H), 4.09-4.06 (m, 1H), 3.05-2.90 (m, 4H), 2.09-1.89 (m, 1H), 1.88-1.61 (m, 6H), 1.61-1.45 (m, 2H), 1.41-1.34 (m, 2H), 1.06-0.92 (m, 6H).

Example 7

Preparation of Polypeptide Compound TM-7

(45) This example was based on example 1 except that:

(46) The amount of Int-1 was 0.190 g; Int-4 was replaced with Int-2. The obtained crude product was purified by Prep-HPLC to obtain 33.0 mg of the trifluoroacetate salt of the target compound TM-7.

(47) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-7 were as follows:

(48) ESI-MS (m/z): 705.4 (M+H.sup.+)

(49) 1H NMR (400 MHz, DMSO-d6): δ 8.58 (dd, J=160.0, 8.2 Hz, 1H), 8.18-7.84 (m, 3H), 7.69 (s, 2H), 7.43-7.14 (m, 10H), 4.83-4.40 (m, 2H), 4.40-4.20 (m, 1H), 4.19-3.66 (m, 2H), 3.18-3.03 (m, 3H), 2.96-2.60 (m, 5H), 1.87-1.58 (m, 6H), 1.57-1.41 (m, 6H), 1.41-1.22 (m, 10H), 0.92-0.84 (m, 6H).

(50) The preparation process of Int-2 is as follows:

(51) Step 2-2-1: DIC (0.070 g, 0.55 mmol), DMAP (0.006, 0.05 mmol), N-hydroxyphthalimide (0.098 g, 0.6 mmol) were added to a solution of Int-2-1 (0.135 g, 0.5 mmol) in DCM (15 mL) at room temperature and the mixture was stirred at room temperature for 2 h. The reaction solution was diluted with 20 mL DCM and respectively washed with 1N HCl and water twice. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a white solid Int-2-2 (0.120 g), which was directly used in the next reaction. Int-2-1 was 7-(tert-butoxycarbonyl)-7-azaspiro[3.5]nonane-2-carboxylic acid;

(52) Step 2-2-2: Reaction flask A: Under the protection of nitrogen at 0° C., MeLi (1.6M, 1 mL) was slowly added to a solution of bis(pinacolato)diboron (0.420 g, 1.65 mmol) in anhydrous THF (7 mL). After addition, the mixture was continued to be stirred at 0° C. for 0.5 h, and then at room temperature for 0.5 h.

(53) Reaction flask B: Int-2-2 (0.12 g) and magnesium bromide diethyl ether complex (0.129 g) were dissolved in anhydrous THF (3 mL) under nitrogen protection at 0° C., and then nickel chloride hexahydrate (0.012 g), 4,4′-dimethoxy-2,2′-bipyridine (0.014 g) and anhydrous THF (3 mL) were added with stirring for 0.5 h until the reaction system turned pale green.

(54) The solution in reaction flask A was one-time added to reaction flask B at 0° C., and the reaction system turns brown. The mixture was stirred at 0° C. for 1 h, and then warmed to room temperature and stirred for 1 h. TLC was used to monitor the completion of the Int-2-2 reaction. The reaction mixture was added to 20 mL of saturated ammonium chloride solution, and the mixture was stirred for 10 min. 25 mL of ethyl acetate was added for extraction, and the aqueous phase was extracted with 15 mL of ethyl acetate; the combined organic phase was washed with saturated sodium chloride solution twice, dried over anhydrous sodium sulfate, filtered and concentrated to obtain 0.550 g crude product; the crude product was purified by column chromatography to obtain 0.11 g of transparent oily substance, ESI-MS (m/z): 352.3 (M+H+);

(55) Step 2-2-3: Int-2-3 (0.11 g, 0.313 mmol) was added to DCM (4 mL), and then TFA (2 mL) was added. The mixture was stirred at room temperature for 0.5 h and then concentrated to dryness to obtain crude product oil (Int-2), which was directly used in the next reaction, ESI-MS (m/z): 252.3 (M+H+).

Example 8

Preparation of Polypeptide Compound TM-8

(56) This example was based on example 1 except that:

(57) The amount of Int-1 was 0.220 g; Int-4 was replaced with Int-3. The obtained crude product was purified by Prep-HPLC to obtain 6.5 mg of the trifluoroacetate salt of the target compound TM-8.

(58) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-8 were as follows:

(59) ESI-MS (m/z): 680.4 (M+H+)

(60) 1H NMR (400 MHz, DMSO-d6+D2O): δ 7.41-7.22 (m, 10H), 4.79-4.69 (m, 1H), 4.44-4.42 (m, 1H), 4.11-4.09 (m, 2H), 3.95-3.57 (m, 4H), 2.99-2.92 (m, 4H), 2.20-2.00 (m, 2H), 1.85-1.33 (m, 13H), 1.02-0.96 (m, 6H).

(61) The preparation process of Int-3 is as follows:

(62) Step 2-3-1: DIC (0.3 g, 1.1 eq), DMAP (0.027 g, 0.1 eq), N-hydroxyphthalimide (0.42 g, 1.2 eq) were added to a solution of Int-3-1 (1.0 g, 1 eq) in DCM (15 mL) at room temperature and the mixture was stirred at room temperature for 2 h. The reaction solution was diluted with 20 mL DCM and washed with 1N HCl and water twice, respectively. The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a white solid Int-3-2 (1.29 g), which was directly used in the next reaction. Int-3-1 was 4-(tert-butoxycarbonylamino)-1-fluorenylmethoxycarbonylpiperidine-4-carboxylic acid;

(63) ESI-MS (m/z): 612.2 (M+H.sup.+).

(64) Step 2-3-2: Reaction flask A: Under the protection of nitrogen at 0° C., MeLi (1.6M, 4 ml, 3 eq) was slowly added to a solution of bis(pinacolato)diboron (1.798 g, 3.3 eq) in anhydrous THF (7 mL). After addition, the mixture was continued to be stirred at 0° C. for 0.5 h, and then at room temperature for 0.5 h. Reaction flask B: Int-3-2 (1.29 g) and magnesium bromide diethyl ether complex (0.545 g, 1 eq) were dissolved in anhydrous THF (3 mL) under nitrogen protection at 0° C., and then nickel chloride hexahydrate (0.05 g, 0.1 eq), 4,4′-dimethoxy-2,2′-bipyridine (0.06 g, 0.13 eq) and anhydrous THF (7 mL) were added with stirring for 0.5 h until the reaction system turned pale green.

(65) The solution in reaction flask A was one-time added to reaction flask B at 0° C., and the reaction system turned brown. The mixture was stirred at 0° C. for 1 h, and then warmed to room temperature and stirred for 1 h. TLC was used to monitor the completion of the Int-3-2 reaction. The reaction mixture was added to 50 mL of saturated ammonium chloride solution, and the mixture was stirred for 10 min. 25 mL of ethyl acetate was added for extraction, and the aqueous phase was extracted with 25 mL of ethyl acetate; the combined organic phase was washed with saturated sodium chloride solution twice, dried over anhydrous sodium sulfate, filtered and concentrated to obtain 3.00 g crude product; the crude product was purified by column chromatography to obtain 0.650 g of transparent oily substance, (yield: 55%)

(66) ESI-MS (m/z): 549.3 (M+H.sup.+)

(67) Step 2-3-3: Int-3-3 (0.08 g, 0.146 mmol) and methylamine methanol solution (4M, 4 mL) were stirred at room temperature for 0.5 h, and concentrated to dryness under reduced pressure. The crude product oil obtained was Int-3, which was directly used into the next reaction; ESI-MS (m/z): 327.2 (M+H.sup.+).

Example 9

Preparation of Polypeptide Compound TM-9

(68) This example was based on example 1 except that: Int-4 was replaced with pyrrole-2-boronic acid pinacol ester. The obtained crude product was purified by Prep-HPLC to obtain two isomers TM-9A (58.1 mg) and TM-9B (60.2 mg) of the trifluoroacetate salt of the target compound TM-9. The structures were identified as (R)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-2-pyrrolidineboronic acid and (S)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-2-pyrrolidineboronic acid.

(69) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-9A were as follows:

(70) ESI-MS (m/z): 651.5 (M+H.sup.+)

(71) 1H NMR (400 MHz, DMSO): δ 8.87-8.75 (m, 1H), 8.39-8.16 (m, 2H), 8.05 (s, 3H), 7.72 (s, 3H), 7.41-7.13 (m, 10H), 4.68-4.65 (m, 2H), 4.52-4.40 (m, 1H), 4.08-4.02 (m, 1H), 3.74-3.55 (m, 2H), 3.34-3.22 (m, 2H), 3.06-2.80 (m, 5H), 2.25-1.26 (m, 7H), 0.92-0.87 (m, 6H).

(72) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-9B were as follows:

(73) ESI-MS (m/z): 651.5 (M+H.sup.+)

(74) 1H NMR (400 MHz, DMSO): δ 8.88-8.75 (m, 1H), 8.42-8.16 (m, 2H), 8.05 (s, 3H), 7.75 (s, 3H), 7.4-7.10 (m, 10H), 4.69-4.64 (m, 2H), 4.50-4.41 (m, 1H), 4.09-4.03 (m, 1H), 3.75-3.55 (m, 2H), 3.38-3.22 (m, 2H), 3.09-2.80 (m, 5H), 2.20-1.20 (m, 7H), 0.93-0.89 (m, 6H).

Example 10

Preparation of Two Isomers TM-10A and TM-10B of Polypeptide Compound TM-10

(75) Anhydrous magnesium sulfate (10 eq) and pinacol (5 eq) were added to a solution of TM-2A (20 mg) or TM-2B (20 mg) in THF, and the mixture was stirred overnight at room temperature. After filtration and concentration, the crude product was purified by Prep-HPLC to obtain two isomers of the target compound, TM-10A (5.1 mg) and TM-10B (5.7 mg). The structures were identified as (R)-1-(D)-Phe-D-Phe-D-Leu-D-Lys)-3-pyrrolidine borate pinacol ester and (S)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-3-pyrrolidine borate pinacol ester, respectively.

(76) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-10A were as follows:

(77) ESI-MS (m/z): 733.5 (M+H.sup.+)

(78) 1H NMR (400 MHz, DMSO): δ8.76-8.73 (m, 1H), 8.38-8.16 (m, 2H), 8.02 (s, 3H), 7.70 (s, 3H), 7.39-7.13 (m, 10H), 4.69-4.64 (m, 1H), 4.52-4.43 (m, 2H), 4.02 (s, 1H), 3.44-3.25 (m, 3H), 3.14-3.02 (m, 3H), 2.96-2.90 (m, 1H), 2.87-2.66 (m, 3H), 2.01-1.92 (m, 1H), 1.71-1.29 (m, 11H), 1.20 (s, 12H), 0.92-0.87 (m, 6H).

(79) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-10B were as follows:

(80) ESI-MS (m/z): 733.5 (M+H.sup.+)

(81) 1H NMR (400 MHz, DMSO): δ8.76-8.70 (m, 1H), 8.39-8.21 (m, 2H), 8.03 (s, 3H), 7.70 (s, 3H), 7.41-7.15 (m, 10H), 4.69-4.66 (m, 1H), 4.55-4.44 (m, 2H), 4.05 (s, 1H), 3.48-3.30 (m, 3H), 3.18-3.02 (m, 3H), 3.01-2.90 (m, 1H), 2.92-2.71 (m, 3H), 2.03-1.97 (m, 1H), 1.70-1.20 (m, 11H), 1.20 (s, 12H), 0.90-0.85 (m, 6H).

Example 11

Preparation of Two Isomers TM-11A and TM-11B of Polypeptide Compound TM-11

(82) The method in this example was similar to that of example 10 except that pinacol was replaced with citric acid. After purified by Prep-HPLC, two isomers of the target compound, TM-11A (7.2 mg) and TM-11B (9.1 mg), were obtained, and their structures were identified as (R)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-3-pyrrolidineboronic acid citrate and (S)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-3-pyrrolidineboronic acid citrate, respectively.

(83) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-11A were as follows:

(84) ESI-MS (m/z): 807.5 (M+H.sup.+)

(85) 1H NMR (400 MHz, DMSO): δ 13.5 (s, 2H), 8.76-8.73 (m, 1H), 8.38-8.16 (m, 2H), 8.02 (s, 2H), 7.70 (s, 2H), 7.39-7.13 (m, 10H), 4.69-4.64 (m, 1H), 4.52-4.43 (m, 2H), 4.02 (s, 1H), 3.44-3.25 (m, 3H), 3.14-3.02 (m, 3H), 2.96-2.90 (m, 1H), 2.87-2.66 (m, 3H), 2.61 (s, 4H), 2.01-1.92 (m, 1H), 1.71-1.29 (m, 11H), 1.20 (s, 12H), 0.92-0.87 (m, 6H).

(86) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-11B were as follows:

(87) ESI-MS (m/z): 807.5 (M+H.sup.+)

(88) 1H NMR (400 MHz, DMSO): δ 13.5 (s, 2H), 8.76-8.70 (m, 1H), 8.39-8.21 (m, 2H), 8.03 (s, 2H), 7.70 (s, 2H), 7.41-7.15 (m, 10H), 4.69-4.66 (m, 1H), 4.55-4.44 (m, 2H), 4.05 (s, 1H), 3.48-3.30 (m, 3H), 3.18-3.02 (m, 3H), 3.01-2.90 (m, 1H), 2.92-2.71 (m, 3H), 2.61 (s, 4H), 2.03-1.97 (m, 1H), 1.70-1.20 (m, 11H), 1.20 (s, 12H), 0.90-0.85 (m, 6H).

Example 12

Preparation of Two Isomers TM-12A and TM-12B of Polypeptide Compound TM-12

(89) This example was based on example 1 except that: Int-4 was replaced with piperidine-3-boronic acid pinacol ester. The obtained crude product was purified by Prep-HPLC to obtain two isomers TM-12A (15.1 mg) and TM-12B (20.2 mg) of the trifluoroacetate salt of the target compound TM-12. The structures were identified as (R)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-3-piperidine boronic acid and (S)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-3-piperidine boronic acid, respectively.

(90) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-12A were as follows:

(91) ESI-MS (m/z): 665.4 (M+H.sup.+)

(92) .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 7.28-7.21 (m, 10H), 4.71-4.59 (m, 2H), 4.45-4.40 (m, 1H), 4.18-4.10 (m, 1H), 4.01-3.90 (m, 1H), 3.13-2.87 (m, 4H), 2.86-2.59 (m, 4H), 1.74-1.11 (m, 13H), 1.02-0.90 (m, 1H), 0.90-0.84 (m, 6H).

(93) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-12B were as follows:

(94) ESI-MS (m/z): 665.4 (M+H.sup.+)

(95) .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 7.33-7.25 (m, 10H), 4.72-4.59 (m, 2H), 4.43-4.36 (m, 1H), 4.19-4.13 (m, 1H), 4.11-3.92 (s, 1H), 3.12-2.87 (m, 4H), 2.80-2.51 (m, 4H), 1.70-1.03 (m, 13H), 1.05-0.90 (m, 1H), 0.88-0.84 (m, 6H).

Example 13

Preparation of Two Isomers TM-13A and TM-13B of Polypeptide Compound TM-13

(96) This example was based on example 1 except that: Int-4 was replaced with piperidine-2-boronic acid pinacol ester. The obtained crude product was purified by Prep-HPLC to obtain two isomers TM-13A (12.2 mg) and TM-13B (10.0 mg) of the trifluoroacetate salt of the target compound TM-13. The structures were identified as (R)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-2-piperidine boronic acid and (S)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-2-piperidine boronic acid, respectively.

(97) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-13A were as follows:

(98) ESI-MS (m/z): 665.4 (M+H.sup.+)

(99) .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 7.32-7.20 (m, 10H), 4.70-4.58 (m, 2H), 4.48-4.42 (m, 1H), 4.18-4.07 (m, 1H), 4.00-3.92 (m, 1H), 3.13-2.83 (m, 4H), 2.89-2.61 (m, 4H), 1.77-1.13 (m, 13H), 1.00-0.81 (m, 1H), 0.90-0.84 (m, 6H).

(100) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-13B were as follows:

(101) ESI-MS (m/z): 665.4 (M+H.sup.+)

(102) .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 7.30-7.20 (m, 10H), 4.72-4.58 (m, 2H), 4.44-4.42 (m, 1H), 4.16-4.05 (m, 1H), 4.01-3.93 (m, 1H), 3.15-2.83 (m, 4H), 2.92-2.60 (m, 4H), 1.78-1.15 (m, 13H), 1.05-0.80 (m, 1H), 0.97-0.85 (m, 6H).

Example 14

Preparation of Polypeptide Compound TM-14

(103) This example was based on example 1 except that: Int-4 was replaced with 1-tert-butoxycarbonyl-3,6-dihydro-2H-pyridine-5-boronic acid pinacol ester. The obtained crude product was purified by Prep-HPLC to obtain 15.0 mg of trifluoroacetate salt of the target compound TM-14.

(104) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-14 were as follows:

(105) ESI-MS (m/z): 663.4 (M+H.sup.+)

(106) 1H NMR (400 MHz, d6-DMSO): δ 8.77-8.70 (m, 1H), 8.38-8.29 (m, 1H), 8.20-8.00 (m, 3H), 7.75-7.50 (brs, 4H), 7.25-7.06 (m, 10H), 6.30-6.28 (m, 1H), 4.79-4.60 (m, 2H), 4.47-4.31 (m, 1H), 4.150-3.90 (m, 3H), 3.85-3.53 (m, 2H), 3.22-3.01 (m, 3H), 2.84-2.71 (m, 3H), 2.27-2.15 (m, 2H), 1.67-1.35 (m, 7H), 1.34-1.22 (m, 3H), 0.90-0.77 (m, 6H).

Example 15

Preparation of Polypeptide Compound TM-15

(107) This example was based on example 1 except that: Int-4 was replaced with tert-butyl 4-[(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methylene]piperidine-1-carboxylate. The obtained crude product was purified by Prep-HPLC to obtain 22.6 mg of the trifluoroacetate salt of the target compound TM-15.

(108) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-15 were as follows:

(109) ESI-MS (m/z): 677.4 (M+H.sup.+)

(110) 1H NMR (400 MHz, CD3OD): δ 7.40-7.20 (m, 10H), 5.38-5.29 (m, 1H), 4.70-4.60 (m, 1H), 4.15-4.00 (m, 1H), 3.85-3.66 (m, 2H), 3.50-3.28 (m, 3H), 3.25-3.10 (m, 2H), 3.00-2.81 (m, 4H), 2.75-2.25 (m, 4H), 1.90-1.70 (m, 1H), 1.70-1.63 (m, 6H), 1.52-1.31 (m, 2H), 1.30-1.21 (m, 1H), 0.99-0.82 (m, 6H).

Example 16

Preparation of Two Isomers TM-16A and TM-16B of Polypeptide Compound TM-16

(111) The method in this example was similar to that of example 10 except that TM-2A or TM-2B was replaced with TM-12. After filtration and concentration, the crude product was purified by Prep-HPLC to obtain two isomers, TM-16A (8.1 mg) and TM-16B (12.2 mg), respectively. The structures were identified as (R)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-3-piperidine boronic acid pinacol ester and (S)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-3-piperidine boronic acid pinacol ester, respectively.

(112) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-16A were as follows:

(113) ESI-MS (m/z): 747.4 (M+H.sup.+)

(114) .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 7.29-7.21 (m, 10H), 4.71-4.58 (m, 2H), 4.46-4.40 (m, 1H), 4.18-4.12 (m, 1H), 4.01-3.93 (m, 1H), 3.13-2.88 (m, 4H), 2.86-2.61 (m, 4H), 1.77-1.11 (m, 13H), 1.22 (s, 12H), 1.02-0.88 (m, 1H), 0.90-0.84 (m, 6H).

(115) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-16B were as follows:

(116) ESI-MS (m/z): 747.4 (M+H.sup.+)

(117) .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 7.35-7.25 (m, 10H), 4.72-4.59 (m, 2H), 4.43-4.36 (m, 1H), 4.19-4.13 (m, 1H), 4.11-3.92 (s, 1H), 3.13-2.87 (m, 4H), 2.83-2.51 (m, 4H), 1.75-1.03 (m, 13H), 1.21 (s, 12H), 1.05-0.90 (m, 1H), 0.88-0.84 (m, 6H).

Example 17

Preparation of Polypeptide Compound TM-17

(118) The synthesis method of this example made reference to example 10. After filtration and concentration, the crude product was purified by Prep-HPLC to obtain the target product TM-17 (8.1 mg).

(119) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-17 were as follows:

(120) ESI-MS (m/z): 745.5 (M+H.sup.+)

(121) 1H NMR (400 MHz, d6-DMSO): δ 8.78-8.60 (m, 1H), 8.48-8.29 (m, 1H), 8.20-8.00 (m, 3H), 7.75-7.52 (brs, 4H), 7.28-7.07 (m, 10H), 6.32-6.28 (m, 1H), 4.82-4.61 (m, 2H), 4.48-4.32 (m, 1H), 4.150-3.92 (m, 3H), 3.86-3.56 (m, 2H), 3.23-3.02 (m, 3H), 2.84-2.75 (m, 3H), 2.32-2.17 (m, 2H), 1.69-1.36 (m, 7H), 1.35-1.22 (m, 3H), 0.92-0.78 (m, 6H).

Example 18

Preparation of Polypeptide Compound TM-18

(122) This example was based on example 7 except that: Int-2-1 was replaced with 1-(tert-butoxycarbonyl)azetidine-3-carboxylic acid. The obtained crude product was purified by Prep-HPLC to obtain 12.1 mg of the trifluoroacetate salt of the target compound TM-18.

(123) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-18 were as follows:

(124) ESI-MS (m/z): 637.5 (M+H.sup.+)

(125) 1H NMR (400 MHz, DMSO-d6): δ 8.58 (m, 3H), 8.18-7.86 (m, 3H), 7.72 (s, 3H), 7.53-7.16 (m, 10H), 4.86-4.41 (m, 2H), 4.42-4.25 (m, 1H), 4.21-3.76 (m, 2H), 3.28-3.05 (m, 3H), 2.92-2.62 (m, 5H), 1.89-1.68 (m, 6H), 1.52-1.40 (m, 6H), 1.40-1.20 (m, 10H), 0.91-0.7 (m, 6H).

Example 19

Preparation of Two Isomers TM-19A and TM-19B of Polypeptide Compound TM-19

(126) The synthesis method of this example made reference to example 2 except that the protective amino acid reagent N-tert-butoxycarbonyl-D-phenylalanine in the synthesis of the corresponding polypeptide intermediate was replaced with (S)-2-benzyl-3-N-tert-butoxycarbonylaminopropionic acid. The obtained crude product was purified by Prep-HPLC to obtain the two isomers of trifluoroacetate salt of the target compound TM-19, TM-19A (9.5 mg) and TM-19B (8.0 mg). The structures were identified as (R)-1-((S-2-benzylaminopropionic acid)-D-Phe-D-Leu-D-Lys)-3-pyrrolidineboronic acid and (S)-1-((S-2-benzylaminopropionic acid)-D-Phe-D-Leu-D-Lys)-3-pyrrolidineboronic acid, respectively.

(127) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-19A were as follows:

(128) ESI-MS (m/z): 665.5 (M+H.sup.+)

(129) 1H NMR (400 MHz, DMSO): δ 8.86-8.73 (m, 1H), 8.48-8.20 (m, 2H), 8.05 (brs, 3H), 7.80 (s, 3H), 7.38-7.10 (m, 10H), 4.52-4.43 (m, 2H), 4.02 (s, 1H), 3.66-3.60 (m, 2H), 3.44-3.25 (m, 3H), 3.14-3.02 (m, 3H), 2.97-2.90 (m, 1H), 2.88-2.66 (m, 3H), 2.77-2.73 (m, 1H), 2.05-1.92 (m, 1H), 1.70-1.28 (m, 11H), 0.92-0.89 (m, 6H).

(130) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-19B were as follows:

(131) ESI-MS (m/z): 665.5 (M+H.sup.+)

(132) 1H NMR (400 MHz, DMSO): δ 8.86-8.76 (m, 1H), 8.49-8.20 (m, 2H), 8.10 (brs, 3H), 7.90 (s, 3H), 7.45-7.01 (m, 10H), 4.53-4.42 (m, 2H), 4.05 (s, 1H), 3.69-3.60 (m, 2H), 3.47-3.25 (m, 3H), 3.11-3.00 (m, 3H), 2.92-2.84 (m, 1H), 2.84-2.66 (m, 3H), 2.79-2.73 (m, 1H), 2.01-1.92 (m, 1H), 1.73-1.29 (m, 11H), 0.91-0.89 (m, 6H).

Example 20

Preparation of Two Isomers TM-20A and TM-20B of Polypeptide Compound TM-20

(133) The synthesis method of this example made reference to example 2 except that the protective amino acid reagent N-tert-butoxycarbonyl-D-phenylalanine in the synthesis of the corresponding polypeptide intermediate was replaced with (R)-N-tert-butoxycarbonyl-N-(2-phenylpropyl)alanine. The obtained crude product was purified by Prep-HPLC to obtain the two isomers of trifluoroacetate salt of the target compound TM-20, TM-20A (21.2 mg) and TM-20B (15.0 mg). The structures were identified as (R)-1-((R-2-phenylpropyl-Gly)-D-Phe-D-Leu-D-Lys)-3-pyrrolidineboronic acid and (S)-1-((R-2-phenylpropyl-Gly)-D-Phe-D-Leu-D-Lys)-3-pyrrolidineboronic acid.

(134) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-20A were as follows:

(135) ESI-MS (m/z): 679.4 (M+H.sup.+)

(136) 1H NMR (400 MHz, DMSO): δ 8.86-8.73 (m, 1H), 8.48-8.20 (m, 2H), 8.05 (brs, 3H), 7.80 (s, 3H), 7.38-7.10 (m, 10H), 4.52-4.43 (m, 2H), 4.38-4.16 (m, 2H), 4.02 (s, 1H), 3.66-3.60 (m, 2H), 3.44-3.25 (m, 3H), 3.14-3.02 (m, 3H), 2.97-2.80 (m, 3H), 2.88-2.66 (m, 1H), 2.77-2.73 (m, 1H), 2.05-1.92 (m, 1H), 1.70-1.28 (m, 14H), 0.92-0.89 (m, 6H).

(137) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-20B were as follows:

(138) ESI-MS (m/z): 679.4 (M+H.sup.+)

(139) 1H NMR (400 MHz, DMSO): δ 9.06-8.80 (m, 1H), 8.58-8.30 (m, 2H), 8.00 (brs, 3H), 7.90 (brs, 3H), 7.48-7.20 (m, 10H), 4.50-4.45 (m, 2H), 4.39-4.26 (m, 2H), 4.05 (s, 1H), 3.68-3.60 (m, 2H), 3.49-3.22 (m, 3H), 3.17-3.02 (m, 3H), 2.99-2.83 (m, 3H), 2.88-2.60 (m, 1H), 2.81-2.72 (m, 1H), 2.01-1.90 (m, 1H), 1.75-1.28 (m, 14H), 0.96-0.89 (m, 6H).

Example 21

Preparation of Two Isomers TM-21A and TM-21B of Polypeptide Compound TM-21

(140) The synthesis method of this example made reference to example 2 except that the protective amino acid reagent N-fluorenylmethyloxycarbonyl-D-leucine in the synthesis of the corresponding polypeptide intermediate was replaced with N-fluorenylmethyloxycarbonyl-D-norleucine. The obtained crude product was purified by Prep-HPLC to obtain the two isomers of trifluoroacetate salt of the target compound TM-21, TM-21A (7.2 mg) and TM-21B (8.5 mg). The structures were identified as (R)-1-(D-Phe-D-Phe-D-Nle-D-Lys)-3-pyrrolidineboronic acid and (S)-1-(D-Phe-D-Phe-D-Nle-D-Lys)-3-pyrrolidineboronic acid, respectively.

(141) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-21A were as follows:

(142) ESI-MS (m/z): 651.4 (M+H.sup.+)

(143) 1H NMR (400 MHz, DMSO): δ 8.86-8.75 (m, 1H), 8.39-8.12 (m, 2H), 8.00 (s, 3H), 7.87 (s, 3H), 7.41-7.13 (m, 10H), 4.72-4.62 (m, 1H), 4.55-4.41 (m, 2H), 4.03 (s, 1H), 3.48-3.22 (m, 3H), 3.19-3.00 (m, 3H), 2.91-2.83 (m, 1H), 2.88-2.67 (m, 3H), 2.00-1.92 (m, 1H), 1.71-1.20 (m, 14H), 0.90-0.89 (m, 3H).

(144) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-21B were as follows:

(145) ESI-MS (m/z): 651.4 (M+H.sup.+)

(146) 1H NMR (400 MHz, DMSO): δ 8.86-8.76 (m, 1H), 8.34-8.10 (m, 2H), 8.01 (s, 3H), 7.88 (s, 3H), 7.42-7.10 (m, 10H), 4.73-4.62 (m, 1H), 4.56-4.40 (m, 2H), 4.02 (s, 1H), 3.49-3.22 (m, 3H), 3.23-3.00 (m, 3H), 2.95-2.82 (m, 1H), 2.89-2.61 (m, 3H), 2.01-1.90 (m, 1H), 1.72-1.17 (m, 14H), 0.93-0.86 (m, 3H).

Example 22

Preparation of Two Isomers TM-22A and TM-22B of Polypeptide Compound TM-22

(147) The synthesis method of this example made reference to example 2 except that the protective amino acid reagent Fmoc-D-lysine in the synthesis of the corresponding polypeptide intermediate was replaced with N2-(9-fluorenylmethoxycarbonyl)-N6-butoxycarbonyl-N6-(2-(2-methoxyethoxy)ethyl)-D-lysine (referring to patent WO2018059331). The obtained crude product was purified by Prep-HPLC to obtain the two enantiomers of trifluoroacetate salt of the target compound TM-22, TM-22A (13.9 mg) and TM-22B (17.1 mg). The structures were identified as (R)-1-(N2-D-Phe-D-Phe-D-Leu-N6-(2-(2-methoxyethoxy)ethyl)-D-Lys)-3-pyrrolidineboronic acid and (S)-1-(N2-D-Phe-D-Phe-D-Leu-N6-(2-(2-methoxyethoxy)ethyl)-D-Lys)-3-pyrrolidineboronic acid, respectively.

(148) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-22A were as follows:

(149) ESI-MS (m/z): 753.5 (M+H.sup.+)

(150) 1H NMR (400 MHz, DMSO): δ 8.77-8.74 (m, 1H), 8.39-8.16 (m, 2H), 8.03 (brs, 2H), 7.72 (brs, 3H), 7.43-7.13 (m, 10H), 4.67-4.66 (m, 1H), 4.52-4.40 (m, 2H), 4.02 (s, 1H), 3.58-3.35 (m, 9H), 3.30 (s, 3H), 3.14-3.02 (m, 3H), 2.96-2.90 (m, 1H), 2.88-2.66 (m, 5H), 2.01-1.92 (m, 1H), 1.71-1.29 (m, 11H), 0.92-0.87 (m, 6H).

(151) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-22B were as follows:

(152) ESI-MS (m/z): 753.5 (M+H.sup.+)

(153) 1H NMR (400 MHz, DMSO): δ 8.81-8.75 (m, 1H), 8.38-8.13 (m, 2H), 8.05 (brs, 2H), 7.70 (brs, 3H), 7.49-7.12 (m, 10H), 4.63-4.61 (m, 1H), 4.57-4.41 (m, 2H), 4.03 (s, 1H), 3.50-3.30 (m, 9H), 3.28 (s, 3H), 3.15-3.02 (m, 3H), 2.94-2.90 (m, 1H), 2.85-2.66 (m, 5H), 2.04-1.92 (m, 1H), 1.71-1.29 (m, 11H), 0.92-0.88 (m, 6H).

Example 23

Preparation of Polypeptide Compound TM-23

(154) This example was based on example 1 except that: Int-4 was replaced with 1H-indazole-5-boronic acid pinacol ester. The obtained crude product was purified by Prep-HPLC to obtain 10.5 mg of the trifluoroacetate salt of the target compound TM-23.

(155) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-23 were as follows:

(156) ESI-MS (m/z): 698.4 (M+H.sup.+)

(157) 1H NMR (400 MHz, DMSO): δ 8.70-8.42 (m, 2H), 8.19-8.02 (m, 1H), 7.65 (m, 1H), 7.44-7.03 (m, 12H), 4.68-4.60 (m, 1H), 4.53-4.35 (m, 1H), 4.37-4.23 (m, 3H), 4.05 (s, 1H), 3.18-3.06 (m, 2H), 2.97-2.83 (m, 1H), 2.80-2.60 (m, 3H), 1.79-1.43 (m, 7H), 1.34-1.22 (m, 2H), 0.98-0.74 (m, 6H).

Example 24

Preparation of Polypeptide Compound TM-24

(158) This example was based on example 7 except that: Int-2-1 was replaced with (1S,4R)-7-(tert-butoxycarbonyl)-7-azabicyclo[2.2.1]heptane-2-carboxylic acid. The obtained crude product was purified by Prep-HPLC to obtain 17.7 mg of the trifluoroacetate salt of the target compound TM-24.

(159) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-24 were as follows:

(160) ESI-MS (m/z): 677.5 (M+H.sup.+)

(161) 1H NMR (400 MHz, DMSO-d6): δ 8.59 (m, 3H), 8.17-7.80 (m, 3H), 7.71 (s, 3H), 7.55-7.16 (m, 10H), 4.82-4.40 (m, 1H), 4.49-4.24 (m, 1H), 4.20-3.76 (m, 3H), 3.20-3.05 (m, 3H), 2.92-2.61 (m, 5H), 1.95-1.69 (m, 8H), 1.50-1.40 (m, 8H), 1.42-1.20 (m, 10H), 0.93-0.71 (m, 6H).

Example 25

Preparation of Polypeptide Compound TM-25

(162) This example was based on example 1 except that: Int-4 was replaced with 1-amino-4-cyclohexylboronic acid pinacol ester. The obtained crude product was purified by Prep-HPLC to obtain 11.2 mg of the trifluoroacetate salt of the target compound TM-25.

(163) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-25 were as follows:

(164) ESI-MS (m/z): 679.4 (M+H.sup.+)

(165) .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 7.29-7.20 (m, 10H), 4.74-4.58 (m, 2H), 4.45-4.33 (m, 1H), 4.18-4.10 (m, 1H), 3.95 (s, 1H), 3.16-2.86 (m, 1H), 2.86-2.59 (m, 4H), 1.75-1.12 (m, 18H), 1.00-0.90 (m, 1H), 0.90-0.84 (m, 6H).

Example 26

Preparation of Polypeptide Compound TM-26

(166) This example was based on example 1 except that: Int-4 was replaced with 2-aminoethylboronic acid pinacol ester. The obtained crude product was purified by Prep-HPLC to obtain 11.2 mg of the trifluoroacetate salt of the target compound TM-26.

(167) ESI-MS (m/z): 607.4 (M−18+H.sup.+)

(168) 1H NMR (400 MHz, DMSO) δ 8.79-8.75 (m, 1H), 8.40-8.38 (m, 1H), 8.03 (s, 3H), 7.852-7.73 (m, 4H), 7.61 (s, 2H), 7.32-7.20 (m, 10H), 4.69-4.63 (m, 1H), 4.39-4.32 (m, 1H), 4.20-4.18 (m, 1H), 4.01 (s, 1H), 3.18-3.01 (m, 4H), 2.95-2.90 (m, 1H), 2.86-2.70 (m, 3H), 1.71-1.58 (m, 2H), 1.56-1.43 (m, 5H), 1.31-1.25 (m, 2H), 0.99-0.87 (m, 6H), 0.84-0.79 (m, 2H).

Example 27

Preparation of Polypeptide Compound TM-27

(169) This example was based on example 1 except that: Int-4 was replaced with 3-azabicyclo[3.1.0]hexyl-1-boronic acid pinacol ester (the synthesis of this fragment made reference to Org. Lett. 2017, 19, 9, 2450-2453). The obtained crude product was purified by Prep-HPLC to obtain trifluoroacetate salt of two isomers TM-27A (6.1 mg) and TM-27B (5.0 mg) of the target compound TM-13. The structures were identified as ((R)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-3-azabicyclo[3.1.0]hexylboronic acid and ((S)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-3-azabicyclo[3.1.0]hexylboronic acid, respectively.

(170) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-27A were as follows:

(171) ESI-MS (m/z): 663.4 (M+H.sup.+)

(172) 1H NMR (400 MHz, DMSO): δ 8.73-8.75 (m, 1H), 8.39-8.12 (m, 2H), 8.02 (s, 3H), 7.76 (s, 3H), 7.38-7.13 (m, 10H), 4.66-4.60 (m, 1H), 4.52-4.41 (m, 2H), 4.04 (s, 1H), 3.41-3.25 (m, 3H), 3.14-3.00 (m, 3H), 2.98-2.90 (m, 1H), 2.82-2.60 (m, 3H), 2.00-1.92 (m, 1H), 1.70-1.29 (m, 12H), 0.91-0.85 (m, 6H).

(173) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-27B were as follows:

(174) ESI-MS (m/z): 663.4 (M+H.sup.+)

(175) 1H NMR (400 MHz, DMSO): δ 8.81-8.72 (m, 1H), 8.38-8.10 (m, 2H), 8.00 (s, 3H), 7.79 (s, 3H), 7.39-7.12 (m, 10H), 4.66-4.61 (m, 1H), 4.52-4.43 (m, 2H), 4.05 (s, 1H), 3.42-3.22 (m, 3H), 3.14-3.01 (m, 3H), 3.01-2.90 (m, 1H), 2.85-2.60 (m, 3H), 2.03-1.91 (m, 1H), 1.77-1.29 (m, 12H), 0.91-0.86 (m, 6H).

Example 28

Preparation of Polypeptide Compound TM-28

(176) This example was based on example 7 except that: Int-2-1 was replaced with 1-tert-butyloxycarbonyl-4-methyl-4-piperidinecarboxylic acid. The obtained crude product was purified by Prep-HPLC to obtain 23.1 mg of the trifluoroacetate salt of the target compound TM-28.

(177) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-28 were as follows:

(178) ESI-MS (m/z): 679.5 (M+H.sup.+)

(179) .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 7.31-7.20 (m, 10H), 4.75-4.59 (m, 2H), 4.46-4.36 (m, 1H), 4.18-4.11 (m, 1H), 3.98 (s, 1H), 3.13-2.87 (m, 4H), 2.86-2.59 (m, 4H), 1.74-1.09 (m, 14H), 1.53 (s, 3H), 0.89-0.84 (m, 6H).

Example 29

Preparation of Polypeptide Compound TM-29

(180) The method in this example was similar to that of example 10 except that TM-2A or TM-2B was replaced with TM-1, and pinacol was replaced with isopropanol. After purified by Prep-HPLC, the trifluoroacetate salt of the target compound TM-29 (9.0 mg) was obtained.

(181) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-29 were as follows:

(182) ESI-MS (m/z): 749.5 (M+H.sup.+)

(183) .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 7.28-7.21 (m, 10H), 4.70-4.59 (m, 2H), 4.42-4.36 (m, 1H), 4.18-4.11 (m, 1H), 3.98 (s, 1H), 3.82-3.77 (m, 2H), 3.13-2.87 (m, 4H), 2.86-2.59 (m, 4H), 1.74-1.09 (m, 14H), 1.00-0.91 (m, 13H), 0.89-0.84 (m, 6H).

Example 30

Preparation of Polypeptide Compound TM-30

(184) This example was based on example 7 except that: Int-2-1 was replaced with 1-(tert-butoxycarbonyl)indoline-3-carboxylic acid. The obtained crude product was purified by Prep-HPLC to obtain trifluoroacetate salt of the two isomers TM-30A (7.2 mg) and TM-30B (9.0 mg) of the target compound TM-30. The structures were identified as (R)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-3-indoline boronic acid and (S)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-3-indoline boronic acid.

(185) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-30A were as follows:

(186) ESI-MS (m/z): 699.4 (M+H.sup.+)

(187) 1H NMR (400 MHz, DMSO+D.sub.2O): δ 7.67-7.00 (m, 14H), 4.69-4.64 (m, 1H), 4.49-4.35 (m, 1H), 4.34-4.23 (m, 3H), 4.01 (s, 1H), 2.98-2.87 (m, 1H), 2.86-2.69 (m, 3H), 2.39-2.23 (m, 2H), 1.78-1.45 (m, 7H), 1.36-1.25 (m, 3H), 0.97-0.74 (m, 6H).

(188) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-30B were as follows:

(189) ESI-MS (m/z): 699.4 (M+H.sup.+)

(190) 1H NMR (400 MHz, DMSO+D.sub.2O): δ 7.67-7.03 (m, 14H), 4.70-4.65 (m, 1H), 4.49-4.33 (m, 1H), 4.34-4.20 (m, 3H), 4.00 (s, 1H), 2.99-2.81 (m, 1H), 2.86-2.68 (m, 3H), 2.34-2.20 (m, 2H), 1.80-1.40 (m, 7H), 1.36-1.25 (m, 3H), 0.99-0.75 (m, 6H).

Example 31

Preparation of Polypeptide Compound TM-31

(191) This example was based on example 7 except that: Int-2-1 was replaced with 1-tert-butoxycarbonyl-azepane-4-carboxylic acid. The obtained crude product was purified by Prep-HPLC to obtain trifluoroacetate salt of the two isomers TM-31A (18.8 mg) and TM-31B (12.0 mg) of the target compound TM-31. The structures were identified as (R)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-4-azacycloheptylboronic acid and (S)-1-(D-Phe-D-Phe-D-Leu-D-Lys)-4-azacycloheptylboronic acid.

(192) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-31A were as follows:

(193) ESI-MS (m/z): 679.5 (M+H.sup.+)

(194) .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 7.32-7.21 (m, 10H), 4.80-4.55 (m, 2H), 4.40-4.36 (m, 1H), 4.18-4.10 (m, 1H), 3.92 (s, 1H), 3.23-2.67 (m, 4H), 2.76-2.58 (m, 4H), 1.79-1.02 (m, 16H), 1.00-0.90 (m, 1H), 0.88-0.84 (m, 6H).

(195) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-31B were as follows:

(196) ESI-MS (m/z): 679.5 (M+H.sup.+)

(197) .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 7.33-7.22 (m, 10H), 4.84-4.50 (m, 2H), 4.41-4.36 (m, 1H), 4.21-4.12 (m, 1H), 3.93 (s, 1H), 3.26-2.67 (m, 4H), 2.72-2.58 (m, 4H), 1.78-1.00 (m, 16H), 1.07-0.91 (m, 1H), 0.89-0.85 (m, 6H).

Example 32

Preparation of Polypeptide Compound TM-32

(198) This example was based on example 1 except that: Int-4 was replaced with Int-5. The obtained crude product was purified by Prep-HPLC to obtain 22.3 mg of trifluoroacetate salt of the target compound TM-32.

(199) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-32 were as follows:

(200) ESI-MS (m/z): 768.5 (M+H.sup.+)

(201) 1H NMR (400 MHz, DMSO-d6+D2O): δ 7.29-7.18 (m, 10H), 4.70-4.57 (m, 2H), 4.40-4.33 (m, 1H), 4.22-4.10 (m, 1H), 3.95 (s, 1H), 3.20-2.85 (m, 6H), 2.86-2.44 (m, 5H), 1.74-1.09 (m, 14H), 1.10-0.93 (m, 2H), 0.87-0.80 (m, 6H).

(202) Among them, the preparation process of Int-5 was as follows:

(203) A solution of Int-5-1, HATU and DIEA in DMF was stirred at room temperature for 30 minutes, and then 2-aminoethylboronic acid pinacol ester was added. The mixture was reacted at room temperature for 30 minutes, and then the reaction solution was poured into water, and ethyl acetate was added to extract the solution for three times. The organic phase was dried with anhydrous sodium sulfate, filtered and concentrated to obtain the intermediate compound Int-5-2;

(204) ESI-MS (m/z): 383.3 (M+H+)

(205) TFA was added dropwise to the solution of Int-5-2 in DCM and the mixture was stirred at room temperature for 30 minutes. The mixture was concentrated to dryness under reduced pressure, and the target compound Int-5 was obtained as a transparent oil, which was directly used in the next reaction.

(206) ESI-MS (m/z): 283.3 (M+H+)

Example 33

Preparation of Polypeptide Compound TM-33

(207) This example was based on example 7 except that: Int-2-1 was replaced with 5-tert-butoxycarbonyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carboxylic acid. The obtained crude product was purified by Prep-HPLC to obtain 22.1 mg of trifluoroacetate salt of the target compound TM-33.

(208) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-33 were as follows:

(209) ESI-MS (m/z): 719.4 (M+H.sup.+)

(210) .sup.1H NMR (400 MHz, DMSO-d.sub.6+D.sub.2O): δ 7.32-7.11 (m, 11H), 4.80-4.55 (m, 4H), 4.40-4.36 (m, 1H), 4.18-4.10 (m, 1H), 3.92 (s, 1H), 3.23-2.67 (m, 6H), 2.76-2.58 (m, 4H), 1.79-1.02 (m, 12H), 0.88-0.84 (m, 6H).

Example 34

Preparation of Polypeptide Compound TM-34

(211) This example was based on example 7 except that: Int-2-1 was replaced with 1-tert-butoxycarbonyl-4-hydroxy-4-carboxypiperidine. The obtained crude product was purified by Prep-HPLC to obtain 9.5 mg of trifluoroacetate salt of the target compound TM-34.

(212) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-34 were as follows:

(213) ESI-MS (m/z): 681.4 (M+H.sup.+)

(214) 1H NMR (400 MHz, DMSO-d6+D2O): δ7.45-7.22 (m, 10H), 4.72-4.61 (m, 1H), 4.47-4.42 (m, 1H), 4.12-4.06 (m, 2H), 3.99-3.59 (m, 4H), 2.97-2.91 (m, 4H), 2.22-2.06 (m, 2H), 1.83-1.30 (m, 13H), 1.00-0.96 (m, 6H).

Example 35

Preparation of Polypeptide Compound TM-35

(215) The synthesis method of this example made reference to example 1 except that the protective amino acid reagent N-tert-butoxycarbonyl-D-lysine in the synthesis of the corresponding polypeptide intermediate was replaced with N-fluorenylmethoxycarbonyl-R-4-tert-butoxycarbonylaminophenylglycine. The obtained crude product was purified by Prep-HPLC to obtain trifluoroacetate salt of the target compound TM-35 (10.2 mg).

(216) The mass spectrum and nuclear magnetic resonance characterization of the prepared TM-35 were as follows:

(217) ESI-MS (m/z): 685.4 (M+H.sup.+)

(218) 1H NMR (400 MHz, DMSO-d6+D2O): δ7.29-7.21 (m, 14H), 4.77-4.58 (m, 2H), 4.46-4.32 (m, 1H), 4.19-4.12 (m, 1H), 3.99 (s, 1H), 3.12-2.87 (m, 2H), 2.82-2.56 (m, 4H), 1.74-1.09 (m, 8H), 1.01-0.92 (m, 1H), 0.86-0.80 (m, 6H).

Example 36

Preparation of Polypeptide Compound TM-36

(219) The synthesis method of this example made reference to example 1 except that the protective amino acid reagent N-tert-butoxycarbonyl-D-phenylalanine in the synthesis of the corresponding polypeptide intermediate was replaced with N-tert-butoxycarbonyl-D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. The obtained crude product was purified by Prep-HPLC to obtain trifluoroacetate salt of the target compound TM-36 (13.5 mg).

(220) ESI-MS (m/z): 697.3 (M+H+)

(221) 1H NMR (400 MHz, DMSO-d6+D2O): δ7.30-7.23 (m, 9H), 4.73-4.57 (m, 2H), 4.46-4.39 (m, 1H), 4.22-4.10 (m, 3H), 3.95 (s, 1H), 3.10-2.86 (m, 4H), 2.80-2.54 (m, 4H), 1.74-1.05 (m, 14H), 1.00-0.92 (m, 1H), 0.87-0.81 (m, 6H).

(222) The solvent used for swelling CTC resin, the solvent used for washing the resin and the solvent used in the condensation condition in the present invention include but not limited to one or more solvents selected from DCM, THF, DMF, DMA, NMP, DMSO; the amino acid protecting group and side chain protecting group, in addition to fluorenylmethoxycarbonyl and tert-butoxycarbonyl, includes benzyloxycarbonyl, methoxycarbonyl, ethoxycarbonyl, trifluoroacetyl, p-methoxybenzyl, allyloxycarbonyl and the like; the condensation agent includes but not limited to HATU, HBTU, HCTU, EDCI, PyBOP, CDI, HOBT and the like.

(223) The preparation process of the tetrapeptide intermediate Int-1 was as follows:

(224) ##STR00018## ##STR00019##

(225) The general synthesis scheme used in the preparation of boronic acid intermediates Int-2, Int-3 and Int-4 was as follows:

(226) ##STR00020## ##STR00021##

(227) The preparation scheme of boronic acid intermediates Int-5 was as follows:

(228) ##STR00022##

(229) The general synthesis scheme used in the preparation of target compounds TM-1 to TM-36 was shown as follows:

(230) ##STR00023##

(231) The general synthesis scheme used in the preparation of target compounds TM-10, 11, 16, 17 and 29 was shown as follows:

(232) ##STR00024##

(233) The meanings of the abbreviations of the materials used in the present invention were shown in Table 1:

(234) TABLE-US-00001 English abbreviation Full name Boc tert-butyloxycarbonyl Fmoc fluorenylmethoxycarbonyl DIEA diisopropylethylamine HBTU O-(benzotriazol-1-yl)-N,N,N,N- tetramethyluroniumhexafluorophosphate HOBt 1-hydroxybenzotriazole DIC N,N′-diisopropylcarbodiimide DCM dichloromethane 2-CTC Resin 2-chlorotritylchloride resin DMF N,N-dimethylformamide LCMS liquid chromatography-mass spectrometer hKOR Human κ-opioid receptor DOR δ-opioid receptor MOR μ-opioid receptor

(235) Biological evaluation of some polypeptide derivatives prepared in the above examples were carried out.

(236) 1. Agonistic activity and selectivity of the κ-opioid receptors

(237) Forskolin can stimulate the release of cAMP from HEK293 cells with high expression of human κ (or μ, or δ)-opioid receptors. The κ-opioid receptor agonists can inhibit the release of cAMP from the HEK293 cells with high expression of human κ-opioid receptors stimulated by Forskolin, but do not affect the release of cAMP from the HEK293 cells with high expression of human μ (or δ)-opioid receptors stimulated by Forskolin. The efficacy of the compounds of the invention as κ-opioid receptor agonists was determined by measuring the ability of the compounds of Examples to inhibit adenylate cyclase activity.

(238) Cell culture: The HEK293 cell line highly expressing human κ (or μ, or δ)-opioid receptor were cultured in DMEM medium containing 10% FBS.

(239) Stimulation: the test compound was 4-fold diluted in a gradient manner to obtain 10 concentrations, and 50 nl of each was transferred to a 384-well plate, and then 10 nl Forskolin was added; the cells were digested, re-suspended, and counted; and then 10 μl of cell suspension (5×10.sup.5 cell/mL) was added, and the cells were mixed gently, and incubated at 23° C. for 60 minutes.

(240) Detection: cAMP Assay Kit (Cisbio) was used, cAMPD2 and Anti-cAMP conjugate were added, and the resultant mixture was incubated for 1 h at room temperature. The plate was read using envision (Perkin Elmer) and EC.sub.50 was obtained by means of fitting with a four-parameter equation.

(241) Experimental results: As shown in Table 2, the agonistic activity (EC.sub.50) of all the tested compounds are below the nM level, and they have excellent selectivity for the κ-opioid receptor.

(242) TABLE-US-00002 TABLE 2 Agonistic activity and selectivity of the compounds on κ-opioid receptors (EC.sub.50) KOR DOR MOR Compounds (EC.sub.50, nM) (EC.sub.50, nM) (EC.sub.50, nM) TM-1 0.0984 >10000 >10000 TM-2A 0.0331 >10000 >10000 TM-3 0.0547 >10000 >10000 TM-5 0.0270 >10000 >10000 TM-7 0.0087 >10000 >10000 TM-8 0.0846 >10000 >10000 TM-9A 0.0012 >10000 >10000 TM-13A 0.0946 >10000 >10000 TM-22B 0.0095 >10000 >10000 TM-26 0.0082 >10000 >10000

(243) 2. Inhibition of Cytochrome P.sub.450 Oxidase

(244) The human liver microsomes (0.253 mg/mL protein) containing cytochrome P.sub.450, test compounds (0.05-50 μM), CYPs substrates (10 μM p-acetaminophen, 5 μM diclofenac, 30 μM mephenytoin, 5 μM dextromethorphan hydrobromide, 2 μM midazolam), 1.0 mM NADP were incubated at 37° C. for 10 minutes. Naflavone, sulfafenpyrazole, N-3-benzylnivan, quinidine, and ketoconazole were used as reference inhibitors. The results are shown in Table 3. The IC.sub.50 of the test compounds are all greater than 50 μM.

(245) TABLE-US-00003 TABLE 3 Inhibitory activity (IC.sub.50) of the compounds on cytochrome P.sub.450 CYP isoenzyme CYPs 1A2 2D6 3A4 2C9 2C19 Compounds (μM) (μM) (μM) (μM) (μM) TM-2A >50 >50 >50 >50 >50 TM-3 >50 >50 >50 >50 >50 TM-8 >50 >50 >50 >50 >50 TM-9A >50 >50 >50 >50 >50 TM-22B >50 >50 >50 >50 >50 TM-26 >50 >50 >50 >50 >50

(246) 3. Membrane Permeability of the Compounds

(247) The Caco-2 cell line is a human colon adenocarcinoma cell line that differentiates in culture and is used to model the epithelial lining of the human small intestine. Compounds of the present invention were tested in a membrane permeability assay using Caco-2 cell membrane layer in a standard assay. The apparent permeability coefficient (Papp) can be determined in the apical-to-basolateral (A-B) direction across cell monolayers cultured on 96-well polycarbonate membrane filters. The compound was maintained at pH 7.4 on the acceptor side at a concentration of 5 μM, and the test plate was gently shaken and incubated at 37° C. for 120 minutes. Samples were taken at time zero from the donor side and at the end of the incubation period from both the donor and acceptor sides. Samples were analyzed by HPLC-MS/MS. The Papp value (expressed as 10.sup.6 cm/sec) was then calculated based on the appearance rate of compound on the recipient side. Papp can be calculated by the following equation:
Papp=(VA×[drug].sub.acceptor)/(Area×Time×[drug].sub.initial, donor)
wherein Papp is the apparent permeability; VA is the volume of the acceptor side, Area is the surface area of the membrane, [drug].sub.initial, donor is the concentration on the donor side at time zero, [drug].sub.acceptor is the concentration of the compounds on the recipient side at the end of the incubation period, Time is the total incubation time.

(248) TABLE-US-00004 TABLE 4 membrane permeability of the compounds Mean permeability (Papp) Compounds (10.sup.−6, cm/s) TM-2A <0.05 TM-3 <0.20 TM-8 <0.10 TM-9A <0.05 TM-22B <0.10 TM-26 <0.10

(249) 4. Acetic Acid Writhing Test in Mice

(250) The analgesic effect of the compound of the invention was evaluated by measuring the analgesic ED.sub.50 of the compounds of Examples in the acetic acid writhing test in mice.

(251) The ICR male mice (18-25 g) were randomly separated into the drug group and the model group. 15 minutes after IV administration, 0.6% (v/v) acetic acid solution was intraperitoneally injected at 10 mL/kg. The number of writhes of the mice within 15 min was counted. The inhibition rate on the writhes was calculated based on the number of writhes. The ED.sub.50 of the compound to be tested was calculated based on inhibition rate according to a four-parameter equation.

(252) TABLE-US-00005 ED.sub.50 Compounds (μg/kg) TM-2A 0.679 TM-9A 0.377

(253) The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.