CN111647041B - Endomorphin analogues and uses thereof - Google Patents

Abstract

Endomorphin analogs and uses thereof, the compounds of the invention are mu-opioid receptor antagonists, useful in the treatment of withdrawal symptoms of opioid addiction, or in the relief of respiratory depression and gastrointestinal side effects due to opioid drugs; in addition, the compounds of the present invention contain 2, 6-dimethyltyrosine (Dmt) having antioxidant properties and are useful for the prevention and treatment of diseases associated with free radicals, such as: neurodegenerative diseases, diabetic complications, cerebral apoplexy, myocardial infarction, intervention therapy of coronary heart disease, inflammation, glaucoma, cataract, etc.

Description

Endomorphin analogs and their applications

technical field

The invention belongs to the field of pharmacy, and in particular relates to a class of endomorphin analogs and applications thereof.

Background technique

Morphine, fentanyl, oxycodone, and pethidine are commonly used opioid analgesics in clinical practice, and morphine is the gold standard for analgesics. Morphine is mostly used for severe pain caused by trauma, surgery, burns, cancer, etc. It is also used for angina pectoris caused by myocardial infarction. Although other types of analgesic drugs have been developed, opioid drugs represented by morphine still have an irreplaceable position in the treatment of moderate and severe pain.

Opioids such as morphine exert analgesic effects by acting on opioid receptors. Opioid receptors are widely present in the brain and gastrointestinal tract. Traditionally, opioid receptors can be divided into three types: μ, δ, and κ. Exogenous or endogenous opioids, when they bind to opioid receptors in different parts, will produce different physiological effects. For example, when it binds to the opioid receptors of pain-sensing nerve endings, it produces analgesic effect; when it binds to the opioid receptors of the respiratory center, it will reduce the response of the respiratory center to _CO2 , resulting in respiratory depression; it binds to the gastrointestinal tract opioid receptors Opioids can also cause smooth muscle spasm, leading to gastric colic, biliary colic, or bronchial asthma, polar respiratory syndrome, etc.

Acute or excessive administration of opioids such as morphine can cause respiratory depression, nausea, vomiting, constipation and other gastrointestinal side effects. Long-term administration can lead to side effects such as tolerance and addiction. break symptoms.

Opioid receptor antagonists are a class of substances that have opioid receptor binding function but do not have opioid receptor activation, and antagonize the physiological effects of morphine and other opioids by competitively binding to the opioid receptor binding pocket. Opioid receptor antagonists are not only of great value in scientific research, clinically, opioid receptor antagonists are used to suppress the withdrawal symptoms of morphine drug addiction, for respiratory depression caused by opioid overdose, and for relieving Gastrointestinal side effects caused by opioids, for acute alcohol poisoning. Recent studies have shown that opioid receptor antagonists can also be used to treat respiratory failure caused by organophosphine pesticides, carbon monoxide poisoning, neonatal hypoxic-ischemic encephalopathy, heart failure, cardiac arrest first aid, edematous pancreatitis, etc. Studies have also shown that opioid receptors are related to the occurrence, development, and migration of tumors, and opioid receptor antagonists can significantly inhibit tumor growth (Shandong Chemical Industry, 2018, 47, 72-73; Chongqing Medicine, 2012, 41, 2772- 74). With the deepening of research, the clinical application of opioid receptor antagonists will become more and more extensive.

Endomorphin (H-Tyr-Pro-Phe/Trp-Phe-NH ₂ ) is an endogenous opioid receptor agonist discovered in 1997, replacing the first tyrosine (Tyr) with its analog 2, 6-dimethyltyrosine (Dmt), or replace the aromatic amino acid at position 3 with another aromatic amino acid, such as 2,6-dimethylphenylalanine (Dmp), 2,4,6-trimethyl Phenylalanine (Tmp) can improve the biological activity of endomorphin, or induce new pharmacological properties (Bioorg MedChem.2014,22(7):2208-19). The compound of the present invention introduces a basic group into the ring of proline, and such compound exhibits μ-receptor antagonistic activity.

Contents of the invention

Technical problem to be solved: the present invention provides a class of endomorphin analogs and applications thereof. This type of compound is a μ-receptor antagonist, which can be used to treat withdrawal symptoms, respiratory depression, and gastrointestinal side effects caused by opioids ; In addition, the compounds of the present invention have antioxidant properties and can be used for the prevention and treatment of diseases related to free radicals, such as: neurodegenerative diseases, diabetic complications, stroke, myocardial infarction, interventional treatment of coronary heart disease, inflammation, glaucoma, cataract etc.

Technical solution: endomorphin analogues, conforming to the following general structural formula:

Wherein, R ₁ =-(CH ₂ ) _n NH ₂ , n=0-4; or -(CH ₂ ) _n NHC(=NH)NH ₂ , n=0-4; R ₂ =natural or unnatural aroma Amino acid side chain; R ₃ =-(CH ₂ ) _n NH ₂ , n=1-4; or -(CH ₂ ) _n NHC(=NH)NH ₂ , n=1-4.

The above-mentioned natural or unnatural aromatic amino acid side chain is benzyl, 2', 4', 6'-trimethylbenzyl, 2', 6'-trimethylbenzyl, indolyl or 1-naphthyl.

The preferred structural formula of the above-mentioned endomorphin analogs is as follows:

Application of the above-mentioned endomorphin analogs in the preparation of drugs for preventing or treating withdrawal symptoms of opioid addiction.

Application of the above-mentioned endomorphin analogs in the preparation of drugs for alleviating respiratory depression caused by opioids.

Application of the above-mentioned endomorphin analogs in the preparation of drugs for neurodegenerative diseases, diabetes complications, cerebral apoplexy, myocardial infarction, interventional treatment of coronary heart disease, inflammation, glaucoma or cataract, etc.

Beneficial effects: Naloxone is a commonly used opioid receptor antagonist. Clinically, although it can effectively treat respiratory depression caused by overdose of opioids, its shortcoming is that its effective time is too short, and it often needs multiple administrations. Medicine (JAMA, 2018; 320(2):129-130). Although the endomorphin analog of the present invention is a tetrapeptide compound, it contains three unnatural amino acids and has good anti-enzymolysis effect. In addition, continuous or excessive use of opioids such as morphine or fentanyl can lead to excessive production of reactive oxygen species (ROS) or reactive nitrogen species (RNS), resulting in an imbalance of the redox system in the body (LifeSci, 2020 246: 117400; Front Pharmacol,2020,11:82), oxidative stress is related to side effects such as addiction and tolerance of opioids. The compounds of the present invention contain 2,6-dimethyltyrosine residues with antioxidant activity (Bioorg Med Chem Lett, 2016, 26, 3629-3631), therefore, the compounds of the present invention, in addition to directly antagonizing opioid receptors In addition to the stimulant effect, it can also improve the imbalance of redox in the body and improve the side effects.

Description of drawings

Figure 1 is a schematic diagram of the synthesis of N-Fmoc-2(Boc-aminoethyl)-Pro-OH(14); reagents and conditions: (a) Cbz-Cl, THF/NaHCO ₃ , room temperature, 12h; (b) SOCl ₂ , MeOH , reflux, 12h; (c) TCCA, TEMPO, DCM, 0℃, 2h; (d) benzyl 2-(diethoxyphosphoryl) acetate, 60% NaH, THF, -20℃, 1h; (e) Pd/C, H ₂ (4atm), MeOH, 12h; (f) NMM, IBCF, THF, 0°C, 10min, NaBH ₄ , 0°C, 20min; (g) TsCl, TEA, DMAP, DCM, 0°C to room temperature, 12h; ( h) NaN ₃ , DMF, 65°C, 12h; (i) PPh ₃ , H ₂ O, THF, reflux, 12h; (j) (Boc) ₂ O, TEA, H ₂ O/1,4-dioxane, 0 ℃ to room temperature, 12h; (k) LiOH, THF/H ₂ O, room temperature, 12h. (l) Fmoc-OSu, NaHCO ₃ , acetone/water, room temperature 12h.

Figure 2 is a schematic diagram of the synthesis of cis-4-(2-BocNH-Et)-N ^a -Fmoc-Pro-OH (18); reagents and conditions: (a) N,N'-Di-Boc-N"-trifluoromethanesulfonyl -Guanidine, DIPEA, DCM, 0℃ to room temperature, 12h; (b) LiOH, THF/H ₂ O, room temperature, 12h; (c) Pd/C, H ₂ (4atm), MeOH, 12h; (d) Fmoc -OSu, NaHCO ₃ , acetone/water, room temperature 12h.

Figure 3 is a schematic diagram of the synthesis of N-Fmoc-2-(Boc-amino)-Pro-OH (24), reagents and conditions: (a) MsCl, pyridine, 0°C to room temperature, 12h; (b) NaN ₃ , DMF , 65℃, 12h; (c) TFA, DCM, 2h; (d) Fmoc-Osu, 6% NaHCO ₃ /1,4-dioxane, room temperature, 12h; (e) Pd/C, H ₂ (8atm), Room temperature, 3d; (f) (Boc) ₂ O, TEA, H ₂ O/1,4-dioxane, 0℃ to room temperature, 12h.

Fig. 4 is trans-4-BocNH-N ^a -Fmoc-Pro-OH (32) and trans-4-(diBoc-gunidino)-N ^a -Fmoc-Pro-OH (35) synthetic schematic; Reagents and conditions: ( a) Cbz-Cl, THF/NaHCO ₃ , room temperature, 12h; (b) BnBr, TEA, THF, 0°C to room temperature, 12h; (c) TsCl, TEA, DMAP, DCM, 0°C to room temperature, 12h; ( d) NaN ₃ , DMF, 65℃, 12h; (e) PPh ₃ , H ₂ O, THF, reflux, 12h; (f) (Boc) ₂ O, TEA, H ₂ O/1,4-dioxane, 0 ℃ to room temperature, 12h; (g) Pd/C, H ₂ , room temperature, 12h; (h) Fmoc-Osu, 6%NaHCO ₃ /1,4-dioxane, room temperature, 12h; (i) N,N'- Di-Boc-N"-trifluoromethanesulfonyl-guanidine, DIPEA, DCM, 0℃ to room temperature, 12h.

Detailed ways

The following examples allow those skilled in the art to fully understand the present invention, but do not limit the present invention in any way. The synthesis of the proline analogs conveniently used for solid-phase peptide synthesis containing amino or guanidine groups on the ring involved in the present invention can be shown in Figures 1-4.

The synthesis of embodiment 1 anti-N-benzyloxycarbonyl-4-hydroxyl-proline (1)

4-Hydroxyproline (10g, 76.4mmol) was dissolved in a mixed solution of saturated NaHCO ₃ (376mL) and THF (376mL), and under ice-bath conditions, benzyl chloroformate (15.4g, 91.6mmol) was added dropwise THF (20 mL) solution was removed from the ice bath and stirred overnight. After the reaction was complete, the pH was adjusted to 3 with 3 mol/L hydrochloric acid, extracted with EA, and the organic layer was collected and dried over anhydrous Na ₂ SO ₄ . Rotary evaporation gave 25 g of colorless oily liquid. It was directly used for the next reaction without purification.

The synthesis of embodiment 2 trans-N-benzyloxycarbonyl-4-hydroxyl-proline methyl ester (2)

Under ice-bath conditions, add SOCl ₂ (12.5mL, 164mmol) to 300mL of methanol solution, dissolve compound 1 (25g, 94mmol) in a small amount of methanol (15mL), add to the above system, stir for 10min, remove the ice bath, reflux, overnight. After the reaction stopped, spin dry. Silica gel column chromatography (PE:EA=3:1-1:1) gave 22.5 g of a colorless oily substance, with a yield of 85.7%.

Example 3 Synthesis of N-benzyloxycarbonyl-4-oxo-proline methyl ester (3)

Compound 2 (10g, 35.8mmol) was dissolved in dichloromethane (200mL), trichloroisocyanic acid (6.3g, 27.3mmol) was added in batches, and stirred for 15min; at 0°C, TEMPO (0.58g, 3.7mmol) was added in batches ), stirred for 2h. After the reaction is complete, wash with saturated NaHCO ₃ , 10% citric acid, water, and saturated NaCl, and dry over anhydrous Na ₂ SO ₄ . After rotary evaporation, the crude product was subjected to silica gel column chromatography (PE:EA=3:1-1:1) to obtain 7.45 g of light yellow oil with a yield of 75.1%. ¹ H NMR (400MHz, CDCl ₃ ) δ7.35–7.32(m,5H),5.24–5.10(m,2H),4.87–4.81(m,1H),3.98–3.90(m,2H),3.76–3.62 (m,3H),2.95–2.89(m,1H),2.63–2.57(m,1H).

Example 4 Synthesis of N-benzyloxycarbonyl-4-(benzyloxycarbonylvinyl)-proline methyl ester (4)

2-(Diethoxyphosphoryl) benzyl acetate (5.67g, 19.8mmol) was dissolved in 30mL THF solution, at -20°C, 60% NaH (0.79g, 19.8mmol) was added, stirred for 30min; then added Compound 3 (5g, 18mmol), continued to react at this temperature for 40min. After the reaction was complete, it was quenched with ice water, extracted 4 times with EA, washed with saturated NaCl, and dried over anhydrous Na ₂ SO ₄ . Silica gel column chromatography (PE:EA=4:1) gave colorless oily substance 4a (2.1g, 28.5%) and colorless oily substance 4b (4.1g, 55.5%). 4a: ESI-MS: m/z 432.2[M+Na] ⁺ ; ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.36–7.28 (m, 5H), 6.04 (d, J=12Hz, 1H), 5.13–5.01(m,4H),4.67–4.61(m,1H),4.35–4.20(m,2H),3.65–3.58(m,3H),3.45–3.22(m,2H).4b:ESI-MS : m/z 432.2[M+Na] ⁺ ; ¹ H NMR (400MHz, CDCl ₃ ) δ7.38–7.31(m,5H),5.87(s,1H),5.22–5.08(m,4H),4.65– 4.57(m,3H),3.73–3.60(m,3H),3.15–3.11(m,1H),2.86–2.81(m,1H).

Synthesis of cis-4-carboxymethyl-proline methyl ester (5) in embodiment 5

Compound 4b (3.4 g, 8.3 mmol) was dissolved in 50 mL of methanol, 0.34 g of 10% Pd/C was added, and stirred overnight under 4 atmospheres of hydrogen. After the reaction stopped, it was suction filtered and spin-dried. It was directly used for the next reaction without purification.

Synthesis of embodiment 6 cis-N-benzyloxycarbonyl-4-carboxymethyl-proline methyl ester (6)

According to the method described in Example 1, compound 5 and benzyl chloroformate (2.16 g, 12.7 mmol) were used as starting materials. Silica gel column chromatography (DCM:MeOH=30:1) gave 2.3 g of a light yellow oily substance, with a yield of 86.2%. ESI MS: m/z 320.1[MH] ^– ; ¹ HNMR (400MHz, CDCl ₃ ) δ7.36–7.28(m,5H),5.19–5.00(m,2H),4.38–4.32(m,1H),3.93 –3.88(m,1H),3.74–3.54(m,3H),3.21–3.09(m,1H),2.61–2.44(m,4H),1.73–1.63(m,1H).

Example 7 Synthesis of cis-N-benzyloxycarbonyl-4-hydroxyethyl-proline methyl ester (7)

Compound 6 (1.98g, 6.2mmol), N-methylmorpholine (0.69mL, 6.2mmol) were dissolved in THF (20mL), and isobutyl chloroformate (0.77mL, 6.2mmol) was added dropwise at 0°C, Stir for 10 min. After the reaction was complete, filter, the filter cake was washed twice with THF, and the filtrate was collected and transferred to a two-necked bottle. At 0°C, under the protection of argon, an aqueous solution of sodium borohydride (0.37 g, 9.3 mmol) was added dropwise through a syringe, and stirring was continued at this temperature for 20 min. After the reaction was complete, it was washed with water, extracted with EA, and the organic phase was collected and dried over anhydrous Na ₂ SO ₄ . Silica gel column chromatography (PE:EA=1:1) gave 1.7 g of a light yellow oily substance, with a yield of 90%. ESI-MS: m/z 330.1[M+Na] ⁺ ; ¹ H NMR (400MHz, CDCl ₃ ) δ7.34–7.26(m,5H),5.18–4.98(m,2H),4.43–4.25(m, 1H),3.90–3.79(m,1H),3.73–3.53(m,5H),3.13–3.01(m,1H),2.65(s,1H),2.49–2.43(m,1H),2.33–2.12( m,1H),1.66–1.54(m,3H).

Example 8 Synthesis of cis-N-benzyloxycarbonyl-4-[(p-toluenesulfonyloxy)ethyl]-proline methyl ester (8)

Compound 7 (1.7g, 5.5mmol) was dissolved in anhydrous DCM (30mL), then TEA (1.15mL, 8.3mmol), DMAP (201mg, 1.65mmol) were added, and p-toluenesulfonyl chloride was added in batches at 0°C ( 1.25g, 6.6mmol), stirred overnight. After the reaction is complete, wash with water and saturated NaCl twice, collect the organic phase, and dry over anhydrous Na ₂ SO ₄ . Silica gel column chromatography (PE:EA=4:1) gave 2 g of a colorless oily substance, with a yield of 80.3%. ESI-MS: m/z 484.1[M+Na] ⁺ ; ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.79 (dd, J=8.2, 6.1Hz, 2H), 7.46 (dd, J=11.0, 8.1Hz,2H),7.36–7.26(m,5H),5.09–4.93(m,2H),4.26–4.15(m,1H),4.03–4.00(m,2H),3.66–3.52(m,4H) ,2.95–2.86(m,1H),2.40(d,J＝7.7Hz,3H),2.32–2.12(m,2H),1.72–1.64(m,2H).1.44–1.38(m,1H)

Example 9 Synthesis of cis-N-benzyloxycarbonyl-4-azidoethyl-proline methyl ester (9)

Compound 8 (2g, 5.5mmol) was dissolved in DMF (30mL), sodium azide (1.43g, 22mmol) was added, heated to 65°C, and reacted overnight. After the reaction was complete, ice water was added, extracted 4 times with EA, washed twice with water and saturated NaCl respectively, and dried. It was directly used for the next reaction without purification. ESI-MS: m/z 355.1[M+Na] ⁺ ; ¹ H NMR (400MHz, CDCl ₃ ) δ7.36–7.28(m,5H),5.19–4.99(m,2H),4.46–4.27(m, 1H),3.92–3.79(m,1H),3.75–3.54(m,3H),3.32–3.28(m,2H),3.14–3.02(m,1H),2.52–2.44(m,1H),2.32– 2.13(m,1H),1.72–1.56(m,3H).

Example 10 Synthesis of cis-N-benzyloxycarbonyl-4-aminoethyl-proline methyl ester (10)

Compound 9 (1.1g, 3.2mmol) was dissolved in THF, triphenylphosphine (1.68g, 6.4mmol) and water (115μL, 6.4mmol) were added, and refluxed overnight. After the reaction was complete, ice water was added, extracted with EA three times, washed with water and saturated NaCl twice, and dried over anhydrous Na ₂ SO ₄ . Silica gel column chromatography (DCM:MeOH:TEA=10:1:0.1) gave 840 mg of a yellow oil with a yield of 85.7%. ESI-MS: m/z[M+H] ⁺ 307.1; ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.40–7.26(m,5H), 5.10–4.93(m,2H), 4.30–4.19( m,1H),3.73–3.70(m,1H),3.64–3.53(m,3H),3.00–2.88(m,1H),2.56–2.38(m,4H),2.30–1.99(m,2H), 1.50–1.34(m,3H).

Example 11 Synthesis of cis-N-benzyloxycarbonyl-4-[(tert-butoxycarbonylamino) ethyl]-proline methyl ester (11)

Compound 10 (830mg, 2.7mmol) was dissolved in a mixed solution of water (5mL) and TEA (0.75mL, 5.42mmol), and (Boc) ₂ O (650mg, 2.98mmol) of 1,4- A solution of dioxane (10 mL) was stirred overnight. After the reaction was complete, it was concentrated, adjusted to pH 2 with 10% citric acid, extracted 4 times with EA, washed twice with water and saturated NaCl, and dried over anhydrous Na ₂ SO ₄ . Silica gel column chromatography (PE:EA=2:1) gave a colorless oil (1.04 g), yield 95%. ESI-MS: m/z429.2[M+Na] ⁺ ; ¹ H NMR (400MHz, CDCl ₃ ) δ7.32–7.25(m,5H),5.15–4.96(m,2H),4.70(s,1H ),4.30–4.23(m,1H),3.87–3.73(m,1H),3.71–3.50(m,3H),3.09–3.00(m,3H),2.46–2.41(m,1H),2.21–2.10 (m,1H),1.59–1.52(m,3H),1.39(d,J=5.1Hz,9H).

Example 12 Synthesis of cis-N-benzyloxycarbonyl-4-[(tert-butoxycarbonylamino)ethyl]-proline (12)

Compound 11 (1.04g, 2.56mmol) was dissolved in a mixed solution of THF (30mL) and water (10mL), anhydrous LiOH (202mg, 8.42mmol) was added, and stirred overnight. After the reaction is complete, concentrate, add water, adjust the pH to 3-4 with 3mol/L hydrochloric acid, extract 3 times with EA, and dry over anhydrous Na ₂ SO ₄ . Concentration gave 800mg of light yellow oily liquid. ESI-MS: m/z [M+H] ⁺ 293.1; [M+Na] ⁺ 415.2; ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.37–7.27 (m, 5H), 6.82 (q, J ＝5.3Hz,1H),5.09–5.01(m,2H),4.23–4.05(m,2H),3.76–3.70(m,1H),2.92–2.85(m,3H),2.48–2.38(m,1H ),2.15–2.03(m,1H),1.49–1.42(m,3H),1.36(d,J＝8.4Hz,9H).

Example 13 Synthesis of cis-4-[(tert-butoxycarbonylamino)ethyl]-proline (13)

Compound 12 (800 mg, 2.04 mmol) was dissolved in MeOH (30 mL), 10% Pd/C (0.1 g), and stirred overnight under H ₂ atmosphere. After the reaction is complete, concentrate. 296 mg of white solid was obtained, which was directly put into the next reaction without purification.

Example 14 Synthesis of cis-N-fluorenylmethoxycarbonyl-4-[(tert-butoxycarbonylamino)ethyl]-proline (14)

Compound 13 (296 mg, 1.14 mmol) and NaHCO ₃ (95.7 mg, 1.14 mmol) were dissolved in a mixed solvent of water and acetone (10 mL:10 mL). At 0°C, fluorenylmoxycarbonyl succinimide (301 mg, 1.25 mmol) was added, and stirred overnight at room temperature. After the reaction was complete, adjust the pH to 3 with 3 mol/L hydrochloric acid, extract 4 times with EA, wash with water and saturated NaCl twice, and dry over anhydrous Na ₂ SO ₄ . Silica gel column chromatography (DCM:MeOH:CH ₃ CO ₂ H=40:1:0.4) gave 590 mg of white solid powder with a yield of 45%. ESI-MS: m/z[MH] ^– 479.2; ¹ H NMR (400MHz, CDCl ₃ ) δ7.77–7.70(m,2H),7.60–7.56(m,2H),7.42–7.26(m,4H) ,4.61–4.24(m,4H),3.85–3.73(m,1H),3.16–3.07(m,3H),2.47–2.19(m,2H),1.85–1.82(m,1H),1.63–1.58( m,3H), 1.44(s,9H).

Example 15 cis-N-benzyloxycarbonyl-4-[(bis-tert-butoxycarbonylguanidino)ethyl]-proline methyl ester (15)

1,3-Di-tert-butoxycarbonyl-2-(trifluoromethylsulfonyl)guanidine (3.2 g, 10.4 mmol) and DIPEA (1.81 mL, 10.4 mmol) were dissolved in 40 mL of anhydrous DCM and added at 0 °C Compound 10 (2.89 g, 9.45 mmol). Stir at room temperature overnight, after the reaction is complete, concentrate, silica gel column chromatography (PE:EA=5:1) to obtain 3.24 g of yellow oily liquid, yield 62.5%. ESI-MS: m/z[M+Na] ⁺ 571.3; ¹ H NMR (400MHz, DMSO-d ₆ ) δ11.49 (s, 1H), 8.34 (q, J=5.5Hz, 1H), 7.38–7.26 (m,5H),5.11–4.93(m,2H),4.38–4.18(m,1H),3.81–3.73(m,1H),3.63–3.53(m,3H),3.31–3.27(m,2H) ,3.03–2.90(m,1H),2.55–2.47(m,1H),2.21–2.12(m,1H),1.61–1.55(m,3H),1.46(d,J＝5.6Hz,9H),1.38 (d,J=3.9Hz,9H).

Example 16 Synthesis of cis-4-[(bis-tert-butoxycarbonylguanidine) ethyl]-proline methyl ester (16)

Compound 15 (1.85g, 3.37mmol) was dissolved in a mixed solution of THF (40mL) and water (13mL), anhydrous LiOH (242mg, 10.1mmol) was added, and stirred overnight. After the reaction is complete, concentrate, add water, adjust the pH to 3-4 with 3mol/L hydrochloric acid, extract 3 times with EA, and dry over anhydrous Na ₂ SO ₄ . After concentration, silica gel column chromatography (DCM:MeOH=20:1) gave 1.53 g of a colorless oily liquid with a yield of 85%. ESI-MS: m/z[M+H] ⁺ 535.3, [M+Na] ⁺ 557.3; ¹ H NMR (400MHz, DMSO-d ₆ ) δ11.52(s, 1H), 8.34(s, 1H), 7.37–7.28(m,5H),5.07–4.99(m,2H),4.21–4.08(m,1H),3.80–3.75(m,1H),3.30(s,2H),2.98–2.89(m,1H ),2.53–2.48(m,1H),2.16–2.12(m,1H),1.62–1.49(m,3H),1.46(d,J＝5.9Hz,9H),1.38(d,J＝4.4Hz, 9H).

Example 17 Synthesis of cis-4-[(bis-tert-butoxycarbonylguanidine) ethyl]-proline (17)

Compound 16 (1.53g, 2.86mmol) was dissolved in 50mL of methanol, 0.15g of 10% Pd/C was added, stirred overnight at room temperature under hydrogen atmosphere, after the reaction was complete, filtered, and the filtrate was concentrated to obtain 1.1g of crude product. It was directly used for the next reaction without purification.

Example 18 Synthesis of cis-N-fluorenylmethoxycarbonyl-4-[(bis-tert-butoxycarbonylguanidine)ethyl]-proline (18)

According to the method described in Example 14, compound 17 (1.1 g, 2.7 mmol), NaHCO ₃ (0.23 g, 2.7 mmol), and fluorenyl moxycarbonyl succinimide (713 mg, 2.97 mmol) were used as starting materials. Silica gel column chromatography (DCM:MeOH:CH ₃ COOH=30:1:0.1) gave 1.14 g of a white solid with a yield of 64.2%. ESI-MS: m/z[MH] ^- 621.3; ¹ H NMR (400MHz, DMSO-d ₆ ) δ12.51(s,1H),11.52(s,1H),8.36(s,1H),7.91–7.87 (m,2H),7.66–7.63(m,2H),7.43–7.29(m,4H),4.36–4.01(m,4H),3.80–3.63(m,1H),3.31(s,2H),3.03 –2.90(m,1H),2.63–2.46(m,1H),2.17–2.05(m,1H),1.61–1.54(m,2H),1.47–1.39(m,19H).

Example 19 Synthesis of N-Boc-4-(trans-OTs)-Pro-OBn(19)

Add N-Boc-4-(trans-OH)-Pro-OBn (10g, 31.1mmol) to 100mL pyridine, after dissolving, add methanesulfonyl chloride (4.9mL, 63.3mmol) dropwise in ice bath, remove the ice Bath, react for 24h. Under ice-salt bath conditions, 18.2 mL of 10% water/pyridine solution was added dropwise, stirred for 10 min, and concentrated. Pour into ice water until no white flocs precipitated, extracted 4 times with ethyl acetate, discarded the aqueous layer, washed the organic layer 3 times with 5wt.% NaHCO ₃ , washed 2 times with saturated brine, and dried over anhydrous sodium sulfate. Silica gel column chromatography (PE:EA=3:1-1:1) gave 9.1 g of a light yellow oily substance with a yield of 69.6%. MS (ESI) calcd for _C18H25NO7S [M+Na] ⁺ : 422.14; _found : _m /z 422.10.

Example 20 Synthesis of N-Boc-4-(cis-N ₃ )-Pro-OBn(20)

After compound 19 (13g, 32.58mmol) was dissolved in 300mL DMF, sodium azide (9.1g, 140mmol) was added and reacted at 55°C for 16h. Pour ice water into the reaction solution until the white flocs no longer precipitate. Extracted 4 times with ethyl acetate, discarded the aqueous layer, washed the organic layer 3 times with water, washed 2 times with saturated brine, and dried over anhydrous sodium sulfate. Silica gel column chromatography (PE:EA=5:1～1:1) gave 7 g of a light yellow oily substance with a yield of 57%.

Example 21 Synthesis of H-4-(cis-N ₃ )-Pro-OBn(21)

Add 60 mL of a mixed solvent of trifluoroacetic acid and dichloromethane (1:1) to compound 20 (7 g, 21.22 mmol), and stir for 2 h. Add 5wt.% NaHCO ₃ until no bubbles are generated, extract 4 times with ethyl acetate, discard the aqueous layer, wash the organic layer 3 times with water and 2 times with saturated brine, and dry over anhydrous sodium sulfate. Silica gel column chromatography (PE:EA=1:5) gave 3.11 g of a yellow oily substance, with a yield of 60%.

Example 22 Synthesis of N-Fmoc-4-(cis-N ₃ )-Pro-OBn(22)

100 mL of 6wt.% Na ₂ CO ₃ was added to compound 21 (3.11 g, 12.6 mmol), Fmoc-OSu (4.665 g, 13.83 mmol) was dissolved in 100 mL of dioxane and added to the above reaction solution for 24 h of reaction. Add double-distilled water to the reaction solution until no substance is precipitated, extract 4 times with n-hexane, collect the water layer, adjust the pH to 2 with 2mol/L hydrochloric acid, extract 4 times with ethyl acetate, discard the water layer, and wash the organic layer with water for 3 times, washed twice with saturated saline, and dried over anhydrous sodium sulfate. Silica gel column chromatography (PE:EA=5:1) gave 4.55 g of a white solid with a yield of 78%. MS(ESI) calcd for C ₂₇ H ₂₄ N ₄ O ₄ [M+Na] ⁺ : 491.18; found: m/z 491.2.

Example 23 Synthesis of N-Fmoc-4-(cis-NH ₂ )-Pro-OH (23)

Compound 22 (4.55 g, 9.7 mmol) was added to 200 mL of methanol to dissolve, 0.3 g of 10% Pd/C was added, and the reaction was carried out in an autoclave for 72 h at 8 atmospheres. After the reaction stopped, it was concentrated, cooled, and about 70 mL of ether was added, and 0.9 g of a white solid was precipitated, with a yield of 30%. MS(ESI) calcd for C ₂₀ H ₂₀ N ₂ O ₄ [M+H] ⁺ : 353.14; found: m/z 353.2.

Example 24 Synthesis of N-Fmoc-4-(cis-Boc-NH)-Pro-OH (24)

Under ice-bath conditions, add 20mL of water, triethylamine (0.7mL, 5.1mmol) to the unpurified compound 23 (0.9g, 2.5mmol) in the previous step, and add Boc anhydride (0.52g, 24mmol) to 20mL After dissolving in dioxane, it was poured into the above reaction solution and reacted overnight. After the reaction stopped, spin the reaction liquid to a small volume, adjust the pH of the solution to 3-4 with 10wt.% citric acid, extract 4 times with ethyl acetate, discard the water layer, wash the organic layer with water twice, and wash with saturated saline 2 times, dried with anhydrous sodium sulfate. Silica gel column chromatography (DCM:MeOH=40:1-20:1) gave 0.5 g of a light yellow solid with a yield of 50%. ¹ H NMR (300MHz,DMSO-d ₆ )δ:10.38(s,1H),7.94-6.82(m,8H),4.33(s,1H),3.99-3.94(m,1H),3.80-3.78(m ,2H),3.58-3.50(m,1H),3.41-3.39(m,1H),2.76-2.72(t,J＝6.10Hz,2H),2.45-2.49(m,2H),1.46(s,3H ), 1.42(s,6H).MS(ESI) calcd for C ₂₅ H ₂₈ N ₂ O ₆ [M+H] ⁺ :451.14; found: m/z 451.1.

Example 25 Synthesis of N-Cbz-4-(cis-OH)-Pro-OH (25)

Cis-hydroxyproline (5g, 38.2mol) was dissolved in a saturated solution of saturated sodium bicarbonate (150mL) and tetrahydrofuran (150mL), and benzyl chloroformate (6.2mL, 6.2mmoL) was added dropwise in an ice bath , stirred overnight. After the reaction stopped, the pH was adjusted to 3-4 with 3 mol/L hydrochloric acid, extracted 4 times with ethyl acetate, and the organic layer was collected and dried over anhydrous sodium sulfate. It was directly used for the next reaction without purification.

Example 26 Synthesis of N-Cbz-4-(cis-OH)-Pro-OBn(26)

Compound 25 (8.2g, 32.1mmoL) was dissolved in tetrahydrofuran (50mL), and triethylamine (4.9mL, 35.2mmoL) was added under ice-cooling conditions, and benzyl bromide (4.3mL, 35.2mmoL) was added for reaction for 22h. After the reaction stopped, it was concentrated and dissolved in dichloromethane (100mL), washed with 1mol/L hydrochloric acid, water, 5wt.% sodium carbonate and water successively, and dried. Silica gel column chromatography (PE:EA=2:1) gave a pale yellow oil (9.3 g), with a yield of 82.4%. MS(ESI) calcd for C ₂₀ H ₂₁ NO ₅ [M+Na] ⁺ : 378.1; found: m/z 378.

Example 27 Synthesis of N-Cbz-4-(cis-OTs)-Pro-OBn(27)

Compound 26 was dissolved in dichloromethane solution (9.3g, 26.2mmoL), and p-methylsulfonyl chloride (5.5g, 28.82mmoL) was added in batches under ice-cooling conditions, and reacted for 12h. After the reaction stopped, it was cooled to room temperature, washed with water and saturated sodium chloride successively, and dried. Silica gel column chromatography (PE:EA=5:1-PE:EA=2:1) gave a pale yellow oil (7.4 g), yield 58%. MS(ESI) calcd for C ₂₇ H ₂₇ NO ₇ S[M+Na] ⁺ : 532.1; found: m/z 532.1.

Example 28 Synthesis of N-Cbz-4-(trans-N ₃ )-Pro-OBn(28)

Compound 27 (7.4g, 14.7mmoL) was dissolved in anhydrous DMF (50mL), sodium azide (5.1g, 78.5mmoL) was added, heated to 70°C, and stirred overnight. After the reaction was completed, after cooling to room temperature, an appropriate amount of ice water was added, extracted 4 times with ethyl acetate, the organic layer was collected, washed twice with water and saturated sodium chloride, and dried. Without purification, it was directly used for the next reaction.

Example 29 Synthesis of N-Cbz-4-(trans-NH ₂ )-Pro-OBn(29)

Compound 28 (5.1g, 13.4mmoL) was dissolved in tetrahydrofuran (50mL), added triphenylphosphine (7.0g, 26.8mmoL), water (0.5mL, 26.8mmoL), and refluxed overnight. After the reaction stopped, cool to room temperature, concentrate, dissolve in ether (70mL) and 0.1mol/L hydrochloric acid (90mL) mixed solution, collect the water layer, wash with ether three times, 10wt.% sodium bicarbonate once, twice Chloromethane extracted 3 times, and the organic layer was collected and dried. Without purification, it was directly used for the next reaction. MS(ESI) calcd for C ₂₀ H ₂₂ N ₂ O ₄ [M+H] ⁺ : 355.2; found: m/z 355.2.

Example 30 Synthesis of N-Cbz-4-(trans-Boc-NH)-Pro-OBn(30)

Compound 29 (4.2g, 10.9mmoL) was dissolved in a mixed solution of water (50mL) and TEA (3.1mL, 21.8moL); dioxane (50mL) dissolved in (Boc) ₂ O (2.2g, 12.9mmoL) , and at 0°C, added to the above solution, stirred for ten minutes and removed the ice bath, and reacted for 26h. After the reaction stopped, the reaction liquid was concentrated to a small volume, adjusted to pH 3-4 with 10wt.% citric acid, extracted 3 times with ethyl acetate, and after collecting the organic layer, washed 2 times with water, 2 times with saturated saline, and washed with anhydrous sodium sulfate dry. Silica gel column chromatography (PE:EA=5:1) gave a colorless oil (3.0 g). MS(ESI) calcd for C ₂₅ H ₃₀ N ₂ O ₆ [M+Na] ⁺ : 477.2; found: m/z 477.2.

Example 31 Synthesis of H-4-(trans-BocNH)-Pro-OH (31)

Compound 30 (3 g, 6.54 mmoL) was dissolved in methanol (300 mL), and reacted for 10 h under the condition of 10% Pd/C (0.2 g) and H ₂ . After the reaction stopped, it was suction filtered and spin-dried, and it was directly put into the next reaction without purification.

Example 32 Synthesis of N-Fmoc-4-(trans-Boc-NH)-Pro-OH (32)

Compound 31 (1.2g, 5.2mmoL) was dissolved in 6wt.% sodium carbonate solution (35mL); FmocOSu (1.9g, 5.72mmoL) was dissolved in dioxane (35mL), and at 0°C, the above In the reaction solution, react for 20h. After the reaction stopped, add double-distilled water until no flocs were precipitated, washed 3 times with n-hexane, adjusted the pH to 2 with hydrochloric acid (2moL/L), extracted 3 times with ethyl acetate, collected the organic layer, and separated them with water and saturated saline successively. Wash twice and dry over anhydrous sodium sulfate. Silica gel column chromatography (DCM-DCM:MeOH=50:1-DCM:MeOH=15:1) gave a pale yellow solid (0.7g). MS(ESI) calcd for C ₂₇ H ₂₇ NO ₇ S[MH] ⁺ :451.2; found: m/z 451.2.1H NMR(300MHz,DMSO)δ7.89(t,J＝6.4Hz,1H),7.77–7.59 (m,1H),7.38(d,J=26.7Hz,2H),7.14(s,0H),6.28(s,0H),4.20(d,J=23.2Hz,2H),3.60(s,1H) ,2.73(s,1H),2.15(d,J=54.5Hz,1H),1.40(s,3H),1.3–1.20(m,2H),0.86(s,1H).

Example 33 Synthesis of N-Cbz-4-(trans-diBoc-guanidyl)-Pro-OBn(33)

Compound 29 (1.1g, 2.8mmoL) and triethylamine (0.4mL, 2.8mmoL) were dissolved in dichloromethane (15mL), cis-VZ-4 (0.43g, 0.28mmoL) was dissolved in dichloromethane (5mL) , and at 0 ° C, added to the above solution, stirred at room temperature, and reacted for 24 hours. After the reaction stopped, spin dry. Silica gel column chromatography (PE:EA=5:1) gave a pale yellow oil (0.92 g), yield 55.3%. MS(ESI) calcd for C ₃₁ H ₄₀ N ₄ O ₈ [M+Na] ⁺ : 619; found: m/z 619.

Example 34 Synthesis of NH ₂ -4-(trans-diBoc-guanidyl)-Pro-OH (34)

Compound 33 (0.92g, 1.54mmoL) was dissolved in methanol (30mL), 10%Pd/C (0.5g) was added, and the balloon was filled with H ₂ for 15h. After the reaction stopped, it was suction filtered and spin-dried. It was directly used for the next reaction without purification.

Example 35 Synthesis of N-Fmoc-4-(trans-diBoc-guanidyl)-Pro-OH (35)

Compound 34 (1g, 2.7mmoL) was dissolved in 6% sodium carbonate solution (20mL), and a solution of Fmoc-OSu (1g, 2.96mmoL) in dioxane (20mL) was added to the reaction solution at 0°C, React overnight. After the reaction stopped, add an appropriate amount of double-distilled water, wash twice with n-hexane, adjust the pH to 3-4, extract four times with ethyl acetate, wash once with water and saturated sodium chloride, and dry. Silica gel column chromatography (DCM:MeOH=30:1) gave 0.47 g of a pale yellow solid. MS (ESI) calcd for C ₃₁ H ₃₈ N ₄ O ₈ [MH] ^- : 593; found: m/z 593. ¹ H NMR (300MHz, DMSO) δ 11.56 (s, 1H), 8.07 (s, 2H) ,7.84(s,2H),7.56(m,4H),4.83(m,3H),4.39(d,J=26.9Hz,4H),3.76(s,2H),1.52(m,18H).

Synthesis of Example 36 Peptide

Rink amine resin (substitution degree 0.684mmol/g, particle size 100-200 mesh) was used for peptide synthesis, PyBop was used as coupling agent, and the manual peptide synthesis reactor was made of glass with a reaction volume of 20mL. The specific method is as follows: Accurately weigh 1 g of dry Rink Amide resin (0.684 mmoL), add 9 mL of DMF and shake for 30 min to fully swell the resin, and drain it after swelling. Wash 6 times with DMF, shake for 5 min each time, and drain. Add 9 mL of 20% piperidine in DMF to the reactor, shake for 5 min, and drain; add 9 mL of 20% piperidine in DMF again, shake for 40 min, and drain; then wash with DMF 6 times, each time Shake for 5 minutes and drain. Add Fmoc-Xaa-OH (1.7mmol, 2.5eq), PyBop (1.7mmol, 2.5eq), HOBt (1.7mmol, 2.5eq), DIPEA (2.74mmol, 4eq) DMF (9mL) solution to the above system, 26 Shake at constant temperature for 4 h under the condition of ℃, and use the Kaise chromogenic method to detect the progress of the reaction. After the reaction was completed, it was drained, washed with DMF for 6 times, shaken for 5 min each time, and drained. Repeat the above two steps in sequence until the connection of the peptide chains is completed. After the coupling of the last amino acid is completed, add 9 mL of 20% piperidine in DMF to the reactor, shake for 5 min, and drain; add 9 mL of 20% piperidine in DMF again, shake for 40 min, and drain. Afterwards, the resin was washed 3 times with DMF, 3 times with DCM, 3 times with MeOH, 3 times with DCM, and 3 times with MeOH, each time for 5 min. After draining, put the reactor into a vacuum dryer to fully dry. Add cleavage reagent 9.5 mL of trifluoroacetic acid and 0.5 mL of anisole to the dried resin, shake and react at 26° C. for 2 h, filter with suction, and save the filtrate for future use. This step is repeated twice. The filtrates obtained from the three cuts were combined and concentrated under reduced pressure. Glacial ether and n-hexane were added to precipitate a precipitate, which was filtered and dried to obtain a crude product. The crude product was purified by liquid chromatography and freeze-dried to obtain the final product.

H-Dmt-cis-4-amino-Pro-Trp-Lys-NH ₂ (37)

MS[M+H] ⁺ 635.4. ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 10.93(s, 1H), 9.35–9.24(m, 1H), 8.71(d, J=7.2Hz, 1H), 8.48(s,3H),8.11(s,2H),7.95(s,3H),7.62(d,J＝8.0Hz,1H),7.37–7.21(m,3H),7.08–7.00(m,3H) ,6.41(s,2H),4.56(s,1H),4.16–4.12(m,1H),3.82(s,1H),3.15–2.90(m,5H),2.72(s,2H),2.46–2.25 (m,2H),2.09–1.91(m,6H),1.82–1.17(m,8H).

H-Dmt-cis-4-amino-Pro-Tmp-Lys- _NH2 (38)

MS(M+H) ⁺ 638.4.1H NMR(400MHz, DMSO-d ₆ )δ: 9.43–9.28(s, 1H), 8.92(d ^, J=6.1Hz, 1H), 8.51(s, 3H), 7.99–7.83(m,6H),6.99(s,1H),6.75(s,2H),6.59(s,1H),6.50–6.41(m,2H),4.59–4.56(m,1H),4.37( d,J＝5.0Hz,1H),4.17(s,1H),4.08–4.05(m,1H),3.91–3.80(m,1H),3.28–2.83(m,4H),2.72(s,2H) ,2.42–2.33(m,1H),2.27–2.10(m,15H),1.80(s,1H),1.63(s,1H),1.50(d,J＝6.3Hz,3H),1.20(d,J =5.5Hz,2H).

H-Dmt-cis-4-amino-Pro-Phe-Arg-NH ₂ (39)

MS (M+H) ⁺ 624.4. ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 8.72 (d, J = 7.5Hz, 1H), 8.45–8.34 (m, 4H), 8.13–7.99 (m, 3H ),7.40–7.19(m,8H),7.10(s,1H),6.41(s,2H),4.55(s,1H),4.18–4.15(m,2H),3.83–3.79(m,2H), 3.64(s,1H),3.11–2.80(m,7H),2.45–2.22(m,1H),2.10–1.93(m,6H),1.81–1.67(m,2H),1.53–1.44(m,3H ).

H-Dmt-cis-4-amino-Pro-Tmp-Arg- _NH2 (40)

MS (M+H) ⁺ 666.4. ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 8.88 (d, J = 6.8Hz, 1H), 8.62–8.34 (m, 4H), 8.03–7.90 (m, 5H ),7.02(s,1H),6.76(s,2H),6.63(s,1H),6.50–6.41(m,2H),4.59(dd,J＝8.1,5.0Hz,1H),4.40(dd, J＝14.1,7.9Hz,1H),4.17–4.09(m,2H),3.90–3.66(m,2H),3.26–2.83(m,6H),2.46–2.33(m,2H),2.28–2.11( m,15H),1.82–1.79(m,1H),1.66(s,1H),1.46–1.38(m,3H).

H-Dmt-trans-4-amino-Pro-Phe-Arg-NH ₂ (41)

MS(M+H) ⁺ 624.4. ¹ H NMR(400MHz,DMSO-d ₆ )δ:9.42–9.27(m,1H),8.47–8.23(m,6H),8.02–7.96(m,1H),7.57 –6.83(m,10H),6.42(m,2H),4.59–4.47(m,1H),4.22–4.14(m,1H),3.83–3.74(m,1H),3.54–3.41(m,5H) ,3.10–2.90(m,6H),2.39–2.25(m,1H),2.09–2.00(m,6H),1.69(s,1H),1.51–1.45(m,3H).

H-Dmt-trans-4-guanidino-Pro-Phe-Lys-NH ₂ (42)

MS(M+H) ⁺ 638.4. ¹ H NMR(400MHz,DMSO-d ₆ )δ:9.33–9.20(m,1H),8.45–8.16(m,4H),8.05–7.93(m,1H),7.83 (s,3H),7.55–7.48(m,3H),7.26–7.15(m,6H),7.04(s,1H),6.39(d,J＝10.6Hz,2H),4.46–4.38(m,1H ),4.25–4.06(m,1H),3.96–3.74(m,1H),3.40(s,4H),3.07–2.78(m,4H),2.70–2.59(m,2H)2.20–1.95(m, 6H), 1.83–1.60(m,2H), 1.47(s,3H), 1.22(s,2H).

H-Dmt-cis-4-aminoethyl-Pro-Trp-Arg-NH ₂ (43)

MS (M+H) ⁺ 691.7. ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 10.88–10.83 (m, 1H), 9.30–9.19 (m, 1H), 8.34 (s, 1H), 8.25–8.12 (m,2H),8.05–7.91(m,1H),7.79–7.76(m,3H),7.67–7.60(m,2H),7.44(s,1H),7.37–7.31(m,2H),7.23 (d,J=2.3Hz,1H),7.16(d,J=2.3Hz,1H),7.12–6.96(m,4H),6.41(d,J=10.6Hz,2H),4.55–4.48(m, 1H),4.34–4.14(m,2H),3.19–2.92(m,6H),2.84–2.66(m,4H),2.18(s,3H),1.92(s,3H),1.67–1.10(m, 9H).

H-Dmt-cis-4-aminoethyl-Pro-Tmp-Arg-NH ₂ (44)

MS(M+H) ⁺ 694.7. ¹ H NMR(400MHz,DMSO-d ₆ )δ:9.39–9.19(m,1H),8.37–8.10(m,3H),7.84–7.68(m,4H),7.16 –7.03(m,2H),6.78(d,J＝10.4Hz,2H),6.61(s,1H),6.45(m,2H),4.42–4.08(m,4H),3.83–3.79(m,2H ),3.66–3.52(m,1H),3.47–3.43(m,1H),3.08–2.86(m,8H),2.26(d,J＝6.1Hz,6H),2.18(d,J＝8.2Hz, 6H),2.08(s,3H),2.01–1.03(m,9H).

H-Dmt-cis-4-aminoethyl-Pro-1-Nal-Arg-NH ₂ (45)

MS(M+H) ⁺ 702.7. ¹ H NMR(400MHz,DMSO-d ₆ )δ:9.29–9.18(m,1H),8.38–8.29(m,2H),8.24–8.17(m,2H),7.93 (t,J=6.8Hz,1H),7.82–7.77(m,4H),7.69–7.36(m,5H),7.18–7.11(m,2H),6.41(m,2H),4.72–4.59(m ,1H),4.33–4.15(m,2H),3.76–3.73(m,2H),3.67–3.63(m,3H),3.60–3.56(m,2H),3.43–3.30(m,2H),3.12 –2.66(m,6H),2.21–1.96(m,6H),1.86–1.21(m,8H).

H-Dmt-cis-4-guanidylethyl-Pro-Trp-Lys-NH ₂ (46)

MS (M+H) ⁺ 705.5. ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 10.85 (dd, J = 13.3, 2.0 Hz, 1H), 9.31–9.17 (m, 1H), 8.34 (s, 1H ),8.20(s,1H),8.12–8.00(m,1H),7.85–7.72(m,4H),7.63(t,J＝8.7Hz,1H),7.41(s,1H),7.36–7.31( m,1H),7.25–7.23(m,1H),7.17–6.98(m,5H),6.41(d,J＝12.1Hz,2H),4.54–4.49(m,1H),4.32(t,J＝ 8.3Hz,1H),4.18–4.11(m,3H),3.76–3.70(m,1H),3.60–3.46(m,1H),3.20–2.71(m,9H),2.19(s,3H),1.94 (s,3H),1.66–1.05(m,10H).

H-Dmt-cis-4-guanidylethyl-Pro-Tmp-Lys-NH ₂ (47)

MS(M+H) ⁺ 708.5. ¹ H NMR(400MHz,DMSO-d ₆ )δ:9.34–9.16(m,1H),8.37(s,1H),8.28–8.08(m,2H),7.87–7.77 (m,5H),7.12(s,1H),7.03–6.91(m,1H),6.77(d,J＝6.2Hz,2H),6.52–6.42(m,2H),4.42–4.30(m,2H ),4.25–4.21(m,1H),4.18–4.03(m,2H),3.82–3.76(m,1H),3.62–3.47(m,2H),3.08–2.98(m,4H),2.91–2.70 (m,5H),2.25(d,J=8.9Hz,6H),2.18(d,J=12.0Hz,6H),2.08(s,3H),1.99–1.86(m,1H),1.65–1.15( m, 9H).

H-Dmt-cis-4-guanidylethyl-Pro-1-Nal-Lys-NH ₂ (48)

MS (M+H) ⁺ 716.5. ¹ H NMR (400MHz, DMSO-d ₆ ) δ: 9.27–9.18 (m, 1H), 8.35 (d, J=7.2Hz, 2H), 8.23–8.14 (m, 3H ),7.95–7.90(m,1H),7.83–7.72(m,5H),7.61–7.36(m,5H),7.16–7.08(m,2H),6.41(m,2H),4.72–4.58(m ,1H),4.32–4.12(m,3H),3.75(dd,J＝11.5,6.8Hz,1H),3.60–3.55(m,1H),3.47–3.31(m,2H),3.04–2.69(m ,7H),2.21(s,3H),1.97(s,3H),1.80–0.94(m,11H).

Example 37 Receptor Binding Activity Analysis

Sprague-Dawley rat brain P2 membranes and guinea pig brain membranes were prepared to test the affinity activity of ligands for opioid receptors. Synaptic membranes are pretreated at low temperature in a buffer containing excess protease inhibitors to remove endogenous opioid ligands. Synaptic membranes were placed in buffered glycerol and protease inhibitors and kept at -80°C. The μ, δ, and κ receptors in the synaptic membrane were labeled with [ ³ H]DAMGO and [ ³ H]DSLET, [ ³ H]U69,593, respectively, and non-radioactive peptides were used to determine the non-specific binding of the synaptic membrane level. After the synaptic membrane was treated with the compound to be tested, it was washed with cold BSA buffer and dried at 75°C. The binding ability of the compound to the receptor is determined according to the difference of the radioactive intensity on the synaptic membrane before and after the compound treatment, and then deducting the non-specific binding part.

The functional activity analysis of embodiment 38 peptides

Guinea pig ileal longitudinal muscle (GPI) and rat vas deferens (MVD) are classic models for evaluating the in vitro activity of opioids. All isolated tissues were maintained in physiological buffer containing balanced salt solution. Place the guinea pig ileal longitudinal muscle (GPI) or rat vas deferens (MVD) specimen in a 37°C bath tube, and apply an electric field to the platinum electrodes at both ends of the bath tube to stimulate the specimen to cause longitudinal contraction. Contractions were recorded with a balance recorder. Ability to inhibit electrical stimulation-induced contraction indicates agonistic activity. The agonistic activity of the ligand was calculated from the concentration-response curve. The antagonistic activity of the ligand is represented by the K _e value, where K _e is the IC of the mu opioid receptor agonist TAPP or the delta opioid receptor agonist DPDPE or the kappa opioid receptor agonist U69,593 with or without the treatment of the test compound A ratio of ₅₀ .

Table 1. Affinity activity analysis of endomorphin analogs

Table 1: Receptor affinity activity of endomorphin analogs. Except for 40-45 which showed weak mu-receptor affinity activity (84-300nM), other compounds showed strong mu-receptor binding activity, among them, the binding of 37 and 41 to mu-receptor may be the same as positive The drug CTOP is comparable, and CTOP is a commonly used μ-receptor antagonist. The compound of the invention has very weak binding activity to δ- and κ-receptors, and the system has strong mu-receptor selectivity.

Table 2. Functional Activity ^Analysis of Endomorphin Analoguesa

^a Mean ± SEM of 3-4 assays. ^b Antagonized TAPP. ^c Antagonized U50,488. ^d Antagonized DPDPE. ^e PA = partial agonist.

Table 2: In vitro functional activity of endomorphin analogs. The ileum muscle (GPI) of guinea pig mainly contains μ-receptors and was used to test the agonistic or antagonistic activity of μ-receptors. The vas deferens of mice mainly contain δ-receptors, which were used to test the activity of δ-receptors, and the antagonistic activity of κ-receptors was also measured with GPI. TAPP is a mu-receptor selective agonist, U50488κ-receptor selective agonist, DPDPE is a delta-receptor selective agonist. IC ₅₀ indicates agonistic activity, K _e indicates antagonistic activity. As shown in Table 2, the compounds of the present invention have weak agonistic activity on μ- or δ-receptors, and compounds 37, 38, and 40 exhibit strong μ-receptor antagonistic activity (K _e ^μ : 16.8-33.5 nM), which is stronger than the commonly used μ-receptor antagonist CTOP antagonistic activity (K _e ^μ : 40.9nM), wherein compound 37 (H-Dmt-cis-4-amino-Pro-Trp-Lys-NH ₂ ) and The antagonistic activity of 40 (H-Dmt-cis-4-amino-Pro-Tmp-Arg-NH ₂ ) was about twice that of CTOP. All compounds have weak agonistic and antagonistic activities on κ-receptors.

Claims (4)

1. Endomorphin analog characterized by the structure:

2. use of an endomorphin analogue according to claim 1 for the preparation of a medicament for the prevention or treatment of withdrawal symptoms of opiate addiction.

3. Use of an endomorphin analogue according to claim 1 for the preparation of a medicament for alleviating respiratory depression due to opioid.

4. The use of the endomorphin analogue of claim 1 for preparing medicaments for neurodegenerative diseases, diabetic complications, cerebral apoplexy, myocardial infarction, intervention therapy of coronary heart disease, inflammation, glaucoma or cataract and the like.

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