Synthesis and application of delta opioid receptor biased agonistic compounds and analgesic drugs

Abstract

The application discloses synthesis of a diphenyl ether substituted tetrahydroisoquinoline compound and application of the compound in preparation of analgesic drugs. The compound has a structural formula as shown in formula I, and is obtained through total synthesis. Cell experiments show that the compound is a biased agonist of an opioid receptor Delta subtype; animal experiments show that the compound has a significant analgesic effect on acute and chronic pain models, has no side effects such as drug resistance, constipation, convulsion and excitation movement, and acute toxicity experiments show that the compound has good safety; pharmacokinetics related experiments show that the compound can penetrate through a blood-brain barrier. The compound can be used for treatment of diseases related to acute pain, chronic pain, inflammatory pain and neuropathic pain.

Description

Technical Field

[0001] The present invention relates to the field of new drug discovery, and more specifically, to a method for synthesizing diphenyl ether-substituted tetrahydroisoquinoline compounds and the use thereof in the preparation of analgesic drugs. Background Art

[0002] Pain refers to the unpleasant sensory and emotional experience caused by actual or potential tissue damage, and it is one of the most common symptoms among all diseases. Meanwhile, chronic pain itself is also a disease. Currently, the main clinical analgesics are opioids and non-steroidal anti-inflammatory drugs. These drugs play a great role in relieving patients' pain, but repeated use can cause serious adverse reactions such as tolerance, constipation, respiratory depression, addiction and gastrointestinal ulcer. Therefore, the research and development of analgesics with low side effects is a consensus goal in the field of new drugs, and also an urgent clinical demand.

[0003] Opioid receptors are the most important proteins in pain signal transmission and analgesia, and are also the therapeutic targets of opioid drugs. Opioid receptors are a class of G protein-coupled receptors, mainly including three subtypes: Mu, Delta and Kappa. Their regulatory mechanism is as follows: exogenous opioids or endogenous opioid peptides activate opioid receptors on the pre- and postsynaptic membranes of sensory neurons. Through coupling with Gi protein, they inhibit adenylate cyclase (AC) from degrading adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP), thereby inhibiting the release of neurotransmitters from the presynaptic membrane, and simultaneously promoting K + efflux and reducing Ca 2+ influx, ultimately weakening or blocking the transmission of pain signals and producing an analgesic effect. Current studies have shown that poor agonist subtype selectivity of receptors is the main cause of drug side effects. Therefore, discovery of agonists with good subtype selectivity is the research direction for new analgesic drugs with low side effects.

[0004] Traditional Chinese medicine has good clinical analgesic efficacy with low side effects, and the compounds contained therein have diverse structures, making it an important source for the discovery of analgesic drugs. Menispermum dauricum DC. is a plant of the genus Menispermum in the family Menispermaceae, and its rhizome is a traditional Chinese medicine also known as Beidougen. It is clinically commonly used to treat diseases such as tonsillitis, rheumatoid arthritis, and gastroenteritis. The main components of Menispermi Rhizoma are isoquinoline alkaloids, which exhibit extensive biological activities. Isoquinoline alkaloids mainly include multiple subclasses such as dibenzylisoquinolines, benzylisoquinolines, aporphines and protoberberines. In our previous study, we found that the compound of structural formula I has agonist activity on Delta opioid receptor subtype, and the preparation method via separation and purification from Beidougen has been applied for a patent (Patent No.: 202210357329.8). No report has been published on the chemical synthesis method of this compound, nor on the research related to its biased agonist activity or its application in acute and chronic pain. Summary of the Invention

[0005] The purpose of this invention is to provide a method for synthesizing a diphenyl ether-substituted tetrahydroisoquinoline compound and its application in the preparation of analgesic drugs. Specifically, one objective of this invention is to provide a method for synthesizing a compound with the structural formula shown in Formula I; another objective is to provide applications of the compound, namely, demonstrating through cell experiments that the compound is a delta-opioid receptor biased agonist, and demonstrating through animal experiments that the compound has significant analgesic effects on acute and chronic pain without side effects such as drug resistance, constipation, convulsions, or excitatory movement, thus providing a foundation for the development of analgesic drugs.

[0006]

[0007] Compound I, white powder, molecular formula C 26 H 29 NO5, molecular weight 435.2.

[0008] This invention provides a method for synthesizing compound I, the synthetic route of which is as follows:

[0009] Starting with 3,4-dihydroxybenzaldehyde, a benzyl protection reaction was carried out via method A, followed by column chromatography purification to obtain intermediate 1. Intermediate 1 was then reacted with ethyl 4-bromophenylacetate via method B, and after rotary evaporation to remove the solvent, column chromatography purification was performed to obtain intermediate 2. Intermediate 2 was hydrolyzed via method C, the solvent was removed by vacuum concentration, and after neutralization to acidity, column chromatography purification was performed to obtain intermediate 3. Intermediate 3 and 3, 4-Dimethoxyphenethylamine was condensed using method D, quenched with water, and then purified by dichloromethane extraction and column chromatography to obtain intermediate 4. Intermediate 4 was cyclized using method E to obtain intermediate 5, which was then concentrated under reduced pressure and directly proceeded to the next reaction. Intermediate 5 was further reduced using method F, and then purified by dichloromethane extraction and column chromatography to obtain intermediate 6. Intermediate 6 was methylated using method G, and then neutralized to alkaline conditions and purified by dichloromethane extraction and column chromatography to obtain intermediate 7. Intermediate 7 was reduced and debenzylated using method H, and then purified by filtration to remove the catalyst and concentration under reduced pressure to obtain intermediate 8. Racemic intermediate 8 was separated by high-performance liquid chromatography (HPLC) to obtain compound I of claim 1 with configuration R. The preparation methods of each intermediate are as follows: (1) Intermediate 1 and ethyl 4-bromophenylacetate are reacted by method B to obtain intermediate 2. The preferred reaction solvent in method B is pyridine, the preferred reaction reagents are potassium carbonate (1-4 equivalents) and cuprous oxide (0.01-1 equivalents), and the preferred reaction conditions are 80-140 degrees Celsius for 3-40 hours.

[0010] (2) Intermediate 2 is hydrolyzed by method C to obtain intermediate 3. The preferred reaction solvent for method C is a solution of tetrahydrofuran / methanol / water in any proportion. The preferred reaction reagents are potassium carbonate, sodium carbonate, cesium carbonate, lithium hydroxide, sodium hydroxide, and potassium hydroxide (1-10 equivalents). The preferred reaction conditions are 40-90 degrees Celsius and 5-30 hours.

[0011] (3) Intermediate 3 is condensed with 3,4-dimethoxyphenylethylamine by method D to obtain intermediate 4. The preferred reaction solvent for method D is dichloromethane, tetrahydrofuran or DMF. The preferred conditions are the condensation of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1,3-dicyclohexylcarbodiimide (DCC) and N,N-diisopropylcarbodiimide (DIC) (1-4 equivalents) in the presence of triethylamine or N,N-diisopropylethylamine (DIPEA) (1-4 equivalents). The preferred reaction conditions are 10-60 degrees Celsius for 5-30 hours.

[0012] (4) Intermediate 4 is cyclized by method E to obtain intermediate 5. The preferred reaction solvents for method E are acetonitrile, tetrahydrofuran, methanol, ethanol, dichloromethane, chloroform, and dioxane. The preferred reagents are phosphorus oxychloride, aluminum trichloride, and boron trifluoride (1-5 equivalents). The preferred reaction conditions are 30-100 degrees Celsius for 2-24 hours.

[0013] (5) Intermediate 5 is further reduced by method F to obtain intermediate 6. The preferred reaction solvents for method F are acetonitrile, tetrahydrofuran, and methanol, and the preferred reagents are sodium borohydride or sodium cyanoborohydride (2-10 equivalents). The preferred reaction conditions are -10 to 40 degrees Celsius and 0.1-3 hours.

[0014] (6) Intermediate 6 is methylated by method G to obtain intermediate 7. The preferred reaction solvents for method G are acetonitrile, tetrahydrofuran, and methanol. The preferred methylating agent is paraformaldehyde or formaldehyde solution (2-50 equivalents). The preferred catalysts are formic acid, acetic acid, trifluoroacetic acid, and hydrochloric acid (2-50 equivalents). The preferred reaction conditions are 30-80 degrees Celsius for 5-30 hours.

[0015] (7) Intermediate 7 is reduced and debenzylated by method H to obtain intermediate 8. The preferred reaction solvents for method H are acetonitrile, tetrahydrofuran, and methanol. The preferred debenzylating agent is palladium on carbon or sponge palladium black (0.05-1 equivalent) reacting with hydrogen. The preferred reaction conditions are 30-100 degrees Celsius for 5-40 hours.

[0016] (8) Intermediate 8 is separated and purified by high performance liquid chiral chromatography to obtain compound I as described in claim 1. The chiral separation method is preferably solid phase chiral packing method, and the eluent is preferably a mixed solution of n-hexane / ethanol / diethylamine, n-hexane / ethanol / triethylamine, n-hexane / methanol / diethylamine, n-hexane / methanol / triethylamine, n-hexane / isopropanol / diethylamine, or n-hexane / isopropanol / triethylamine in any proportion. The chiral compound is prepared with an enantiomeric excess percentage greater than 95%.

[0017] The described biased activation refers to biased activation of the delta-opioid receptor Gi protein, and the pain includes acute, chronic, neuropathic, inflammatory, and cancer pain. The neuropathic pain is primarily shingles pain, and the inflammatory pain includes rheumatoid arthritis and knee osteoarthritis. The application of the compound in the preparation of analgesic drugs is characterized by the drug being a pharmaceutical composition made from structural formula I as the active ingredient and a conventional pharmaceutical carrier. The pharmaceutical composition is in the form of tablets, capsules, granules, suspensions, drops, injections, or aerosols.

[0018] The application of compound I in this invention is specifically demonstrated by its good analgesic effect in mouse hot plate tests, formalin-induced inflammation models, and complete Freund's adjuvant-induced chronic inflammation models, without significant drug resistance or side effects such as constipation, convulsions, or excitatory movement. Furthermore, acute toxicity tests show its good safety profile and a certain degree of blood-brain barrier permeability. The discovery of this molecule provides a candidate molecule for the development of low-side-effect analgesics.

[0019] Cellular experiments showed that this compound is a biased agonist of the Delta subtype of opioid receptors; animal experiments showed that this compound has significant analgesic effects in acute and chronic pain models, with no side effects such as drug resistance, constipation, convulsions, or excitatory movement, and acute toxicity experiments showed that it has good safety; pharmacokinetic experiments showed that this compound can cross the blood-brain barrier. This compound can be used to treat acute pain, chronic pain, inflammatory pain, and neuropathic pain.

[0020] The present invention discloses a method for synthesizing and applying a diphenyl ether-substituted tetrahydroisoquinoline compound. This compound exhibits significant analgesic effects on both acute and chronic pain, without side effects such as drug resistance, constipation, convulsions, or excitatory movement. This compound shows promising potential for the treatment of acute pain, chronic pain, inflammatory pain, and neuropathic pain.

[0021] Explanation of the attached diagram Figure 1 This is the structural formula of compound I.

[0022] Figure 2Assay for Gi bias activity of compound I Figure 3 Assay for the β-arrestin2 activity of compound I Figure 4 Analgesic effect of compound I in a mouse hot plate model Figure 5 The analgesic effect of compound I in a formalin-induced pain experiment in mice.

[0023] Figure 6 The analgesic effect of compound I in a fully Freund's adjuvant-induced inflammation model.

[0024] Figure 7 This is for the evaluation of drug resistance to compound I.

[0025] Figure 8 Evaluation of the constipation side effect of compound I.

[0026] Figure 9 Evaluation of the convulsive side effects of compound I.

[0027] Figure 10 Evaluation of the excitatory motor side effects of compound I.

[0028] Figure 11 The results are from an acute toxicity test of compound I in mice.

[0029] Figure 12 This study evaluates the blood-brain barrier permeability of compound I. Detailed Implementation

[0030] The present invention will be described below through specific embodiments, but the present invention is not limited thereto.

[0031] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and biological materials described are commercially available unless otherwise specified.

[0032] Example 1: Synthesis and structural characterization of compound I

[0033] (1) Synthesis of intermediate 1: 3,4-Dihydroxybenzaldehyde (5 g, 36.2 mmol) and potassium carbonate (7.5 g, 54.3 mmol) were weighed and placed in a 50 mL single-necked round-bottom flask. 25 mL of acetonitrile was added, and under nitrogen protection, benzyl bromide (4.3 mL, 36.2 mmol) was added dropwise to the reaction solution. The reaction was carried out at room temperature for 12 hours, and the reaction was checked to confirm the completion of the reaction. After quenching with water, the product was extracted with dichloromethane and purified by column chromatography to obtain 3.429 g of white solid intermediate 1, with a yield of 59.3%. MS (m / z): 229.08 [M+H] + . 1HNMR (400 MHz, CDCl3) δ 9.86 (s, 1H), 7.49 – 7.48 (m, 1H), 7.47 – 7.39 (m,6H), 7.06 (d, J = 8.3 Hz, 1H), 5.85 (s, 1H), 5.23 (s, 2H).

[0034] (2) Synthesis of intermediate 2: Intermediate 1 (5 g, 21.9 mmol), ethyl p-bromophenylacetate (0.65 g, 43.8 mmol), and anhydrous potassium carbonate (6.05 g, 43.8 mmol) were weighed and placed in a double-necked flask. After vacuum deoxygenation and nitrogen protection, 50 mL of anhydrous pyridine was added to dissolve the product, followed by the addition of cuprous oxide (0.627 g, 4.38 mmol). The mixture was heated to 120 °C and reacted for 17 hours. The reaction proceeds were found to be almost complete. After concentration under reduced pressure, the product was purified by column chromatography to obtain 3.18 g of intermediate 2, with a yield of 36.8%. MS (m / z): 391.20 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ 9.85 (s, 1H), 7.65 (dd, J = 8.4, 2.0Hz, 1H), 7.56 (d, J = 2.0 Hz, 1H), 7.34 – 7.31 (m, 3H), 7.28 – 7.23 (m, 4H), 7.15 (d, J = 8.4 Hz, 1H), 6.95 (d, J = 8.4 Hz, 2H), 5.22 (s, 2H), 4.18 (q, J =7.2 Hz, 2H), 3.62 (s, 2H), 1.28 (t, J = 7.2 Hz, 3H).

[0035] (3) Synthesis of intermediate 3: Intermediate 2 (1.12 g, 2.87 mmol) was weighed and dissolved in 46 mL of tetrahydrofuran and 12 mL of ethanol. Potassium carbonate (2 g, 5 eq, 14.35 mmol) was dissolved in 17 mL of water and added to the reaction solution. The reaction was carried out at 70 °C for 22 hours, and the reaction was checked to ensure that the starting material was completely reacted. The solvent was removed by concentration under reduced pressure, and the pH was adjusted to acidity to 5 with 1 M dilute hydrochloric acid. The product was then extracted with dichloromethane and purified by column chromatography to obtain 0.8 g of intermediate 3 as a white solid, with a yield of 77%. MS (m / z): 361.37 [M+H] + . 1H NMR (400 MHz, DMSO) δ 12.31 (s, 1H), 9.85 (s, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 1.6 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.36 – 7.28 (m, 3H), 7.27 – 7.21 (m, 4H), 6.90 (d, J = 8.4 Hz, 2H), 5.25 (s, 2H), 3.57 (s, 2H).

[0036] (4) Synthesis of intermediate 4: Intermediate 3 (0.25 g, 0.436 mmol) was dissolved in 10 mL of dichloromethane. Triethylamine (0.24 mL, 2.5 eq) was added dropwise to the reaction solution. EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (0.21 g, 2.5 eq) was added to the reaction solution. 3,4-Dimethoxyphenylethylamine (0.25 g, 2 eq) was slowly added to the reaction solution. The reaction was initiated at room temperature and carried out for 17 hours. The reaction was checked to confirm the completion of the reaction. The reaction was quenched with water, extracted with dichloromethane, and purified by column chromatography to obtain 0.33 g of intermediate 4, with a yield of 90%. MS (m / z): 526.25 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ 9.86 (s, 1H), 7.66 (dd, J = 8.4, 1.8 Hz, 1H), 7.57 (d, J = 1.7 Hz, 1H), 7.33 (d, J = 6.3 Hz, 3H), 7.25 (d, J = 7.5 Hz, 2H), 7.19 – 7.10 (m, 3H), 6.94 (d, J = 8.5 Hz, 2H), 6.75 (d, J = 8.1 Hz, 1H), 6.65 (s, 1H), 6.59 (d, J = 8.1 Hz, 1H), 5.40 (s, 1H), 5.21 (s, 2H), 3.84 (s, 6H), 3.53 (s, 2H), 3.47 (dd, J = 13.0, 6.7Hz, 2H), 2.70 (t,J = 6.9 Hz, 2H).

[0037] (5) Synthesis of intermediate 5: Intermediate 4 (0.09 g, 0.171 mmol) was dissolved in 10 mL of acetonitrile, and phosphorus oxychloride (0.05 mL, 3 eq) was added. The mixture was refluxed at 70 °C for 2.5 h to determine the completeness of the reaction. After quenching with saturated sodium carbonate solution, the mixture was extracted directly with dichloromethane and purified by rapid column chromatography to obtain a total of 55 mg of intermediate 5, with a yield of 63.3%. MS (m / z): 508.33 [M+H] + .

[0038] (6) Synthesis of intermediate 6: Intermediate 5 (0.055 g, 0.108 mmol) was dissolved in 10 mL of methanol and 2 mL of tetrahydrofuran, cooled to 0°C in an ice bath, and sodium borohydride (0.035 g, 8 eq) was added. The reaction was carried out at 0°C for 20 minutes to check the completion of the reaction. The reaction was quenched with formic acid, the solvent was removed by rotary evaporation, and the product was neutralized to neutral with sodium carbonate. After extraction with dichloromethane, the product was purified by column chromatography to obtain 35 mg of intermediate 6, with a yield of 63%. MS (m / z): 512.28 [M+H] + . 1 H NMR (400MHz, CDCl3) δ 7.34 – 7.29 (m, 3H), 7.27 – 7.25 (m, 2H), 7.19 (d, J = 8.4 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 10.0 Hz, 2H), 5.13 (s, 2H), 4.60 (s, 2H), 4.20 (dd, J = 8.8, 4.4 Hz, 1H), 3.88 (s, 3H), 3.84 (s, 3H), 3.27 – 3.17 (m, 2H), 2.98 – 2.93 (m, 2H), 2.79 – 2.74 (m, 2H).

[0039] (7) Synthesis of intermediate 7: Intermediate 6 (0.08 g, 0.156 mmol) was dissolved in 10 mL of methanol, and paraformaldehyde (0.15 g, 30 eq) and formic acid (0.2 mL, 30 eq) were added. The mixture was heated to 75 °C and refluxed for 15 h. The reaction was monitored by LCMS to ensure complete reaction of the starting material. The solvent was removed by concentration under reduced pressure, and the pH was adjusted to alkaline level with 1 M sodium carbonate solution. After extraction with dichloromethane, the product was purified by column chromatography to obtain 82 mg of intermediate 7 as a white solid, with a yield of 97%. MS (m / z): 526.37 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ 7.33 – 7.26 (m, 5H), 7.08 – 7.00 (m, 5H), 6.89 (d, J = 8.4 Hz, 2H),6.57 (s, 1H), 6.07 (s, 1H), 5.13 (s, 2H), 4.59 (s, 2H), 3.85 (s, 3H), 3.76 –3.69 (m, 1H), 3.57 (s, 3H), 3.25 – 3.14 (m, 2H), 2.92 – 2.75 (m, 3H), 2.64 –2.59 (m, 1H), 2.55 (s, 3H).

[0040] (8) Synthesis of intermediate 8: Intermediate 7 (80 mg, 0.15 mmol) was dissolved in 10 ml of methanol and placed in a single-necked round-bottom flask. 20 mg of wet palladium on carbon (palladium content was 10% Pd by mass, and water content was 55% by mass) was added, and the mixture was replaced with hydrogen gas and reacted at 60 degrees Celsius for 17 h. The reaction was monitored by LCMS until it was complete. The palladium on carbon was removed by filtration to obtain intermediate 8. (9) Intermediate 8 was prepared by high performance liquid chiral chromatography (column: S-Chiral C, 7 mm, 20*250 mm), with a mobile phase of 71% n-hexane + 29% ethanol + 0.01% diethylamine (volume ratio) for isocratic elution. Compound I, with a chiral enantiomer excess percentage greater than 99%, was prepared at a concentration of 31 mg, and its structure is as follows: Figure 1 MS (m / z): 436.25 [M+H] + . 1 H NMR (600MHz, MeOD) δ 8.51 (s, 1H), 7.06 (d, J = 8.4 Hz, 2H), 7.03 (dd, J = 8.4, 1.8 Hz, 1H), 6.93 (d, J= 2.4 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.87 (d, J = 8.4 Hz, 2H), 6.78 (s, 1H), 5.90 (s, 1H), 4.47 (s, 2H), 4.36 (dd, J = 9.6, 4.8 Hz, 1H), 3.79(s, 3H), 3.66 – 3.63 (m, 1H), 3.47 (s, 3H), 3.36 (dd, J = 12.6, 4.8 Hz, 1H), 3.30 – 3.26 (m, 1H), 3.13 – 3.08 (m, 1H), 3.02 – 2.92 (m, 2H), 2.87 (s, 3H). 13 C NMR (151 MHz, MeOD) δ 169.89, 159.04, 150.42, 149.80, 148.50, 144.33,134.89, 132.34, 131.28, 125.42, 124.39, 123.86, 121.65, 118.04, 117.96,112.83, 112.75, 65.99, 64.70, 56.43, 56.32, 46.57, 40.99, 40.11, 23.82.

[0041] Example 2: Determination of Gi bias activity of compound I The delta opioid receptor couples to the Gi protein-cyclic adenosine monophosphate (cAMP) pathway. The Gi-biased agonist activity of compound I can be evaluated by detecting changes in cAMP concentration using the GloSensor™ cAMPAssay method. The specific steps are as follows: HEK293T cells were seeded at 20,000 cells / well in 96-well plates and cultured for 24 h. Afterward, pGloSensor™-22F cAMP plasmid and Delta receptor plasmid were co-transfected using lipofectamine 3000 at a transfection volume / mass ratio of 1:1. 24 h after transfection, the medium was removed and replaced with balanced medium containing 2% (v / v) GloSensor™ cAMP. After incubation for 2 h, the initial luminescence intensity was detected using a microplate reader. Then, 25 μL of the test compound was added and incubated at room temperature for 5 min. The concentrations of compound I were 10000 nM, 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, and 0.64 nM. SNC80 compound was used as a positive control at concentrations of 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, and 0.064 nM. nM and 0.0128 nM; then add 25 μL of saliva extract (10 µM), incubate at room temperature for 30 min, and detect the luminescence intensity using a microplate reader. Divide the result by the pre-read value to obtain the response fold. The Gi pathway activities of compound I and SNC80 are as follows: Figure 2 As shown, they can inhibit cAMP production in a concentration-dependent manner, achieving IC50. 50 The values ​​were 5.42 nM and 50.49 nM, respectively. These results indicate that compound I can activate the Gi pathway.

[0042] Example 3: Determination of β-arrestin2 activity of compound I In addition to the Gi pathway, the downstream pathway of the Delta opioid receptor also includes the β-arrestin2 pathway. The β-arrestin2-biased agonist activity of compound I can be evaluated using bioluminescent resonance energy transfer (BRET). The specific steps are as follows: CHO-K1 cells were seeded in 10 cm culture dishes and cultured for 24 h. Then, β-arrestin2-Rluc plasmid and DOR-EYFP plasmid were co-transfected using lipofectamine 3000. After 24 h of transfection, 50,000 cells / well were seeded in 96-well plates and cultured for 24 h. The plates were then replaced with HBSS buffer, and 6 µM of coelenterazine (a substrate of Renilla luciferase) was added. oIncubate at C for 15 min, then add the test compound and incubate at room temperature for 5 min. The concentrations of compound I were 100 μM, 20 μM, 4 μM, 0.8 μM, 0.16 μM, 0.032 μM, 0.0064 μM, and 0.0013 μM. SNC80 was used as a positive control at concentrations of 10 μM, 2 μM, 0.4 μM, 0.08 μM, 0.016 μM, 0.0032 μM, 0.00064 μM, and 0.00013 μM. Fluorescence intensity was detected using a microplate reader; the excitation wavelength was 460 ± 40 nm, and the emission wavelength was 540 ± 25 nm. The β-arrestin2 pathway activities of compound I and SNC80 were as follows: Figure 3 As shown, SNC80 can recruit β-arrestin2 in a concentration-dependent manner, and its EC50... 50 The concentration was 1.35 μM, and compound I showed no recruitment activity for β-arrestin2 even at the highest concentration. These results indicate that compound I cannot activate the β-arrestin2 pathway and is a Gi protein pathway-biased agonist.

[0043] Example 4: Analgesic effect of compound I in a mouse hot plate model The effect of compound I on acute pain was studied using a hot plate model in mice. Six- to eight-week-old male C57BL / 6 mice were selected and divided into five groups of seven mice each. Morphine (5 mg / kg) served as the positive control. Compound I experimental groups were administered the compound I compound intraperitoneally at doses of 10 mg / kg, 20 mg / kg, and 40 mg / kg, respectively. The blank control group served as the solvent control, using 0.5% sodium carboxymethyl cellulose (mass concentration). The same method was used for the blank control group below. Pain thresholds were measured at 30, 60, 90, and 120 minutes after administration under a hot plate heated to 55°C. The criteria for pain threshold assessment included jumping, licking the hind paw, repeated hopping of the hind paw, and continuous rapid backward or forward lunging. To prevent burns to the mice's paws, mice that did not exhibit pain threshold indicators after 20 seconds had their pain threshold calculated based on 20 seconds. Results are as follows: Figure 4 As shown, different concentrations of compound I significantly increased the duration of heat pain tolerance in mice, significantly raising the pain threshold in a dose-dependent manner. At a dose of 40 mg / kg, the analgesic effect of compound I lasted for at least 3 hours. Therefore, compound I exhibits good analgesic effects for acute pain.

[0044] Example 5: Analgesic effect of compound I in formalin-induced pain experiment in mice A formalin-induced pain model was used in mice to investigate the analgesic effect of compound I on inflammatory pain. Six- to eight-week-old male C57BL / 6 mice were divided into five groups (blank group, morphine group (10 mg / kg), and different dosages of the compound: 10, 20, and 40 mg / kg), with seven mice in each group. The hind paws of the mice to be injected with formalin were marked, and the mice were placed in cages for 30 minutes to acclimatize. Each mouse was injected with the appropriate dose of the drug, and 15 minutes later, a 5% formalin solution was subcutaneously injected into the sole of the marked paw. The mice quickly exhibited paw-licking behavior. The mice were observed continuously for 45 minutes, and the cumulative paw-licking time was recorded in 5-minute intervals during phase I (0-10 min) and phase II (25-45 min). Figure 5 As shown, the experimental results indicate that the compound has significant analgesic effects in both phase I and phase II of the analgesic model at doses of 20 and 40 mg / kg, suggesting that compound I is effective for both acute and chronic inflammatory pain.

[0045] Example 6: Analgesic effect of compound I in a fully Freund's adjuvant-induced inflammation model The analgesic effect of compound I was investigated in mice with acute and chronic inflammatory pain induced by complete Freund's adjuvant (CFA) injection into the plantar fossa. Six- to eight-week-old male C57BL / 6 mice were randomly divided into five groups (blank group, CFA group, morphine group (5 mg / kg), and different doses of the compound: 10, 20, and 40 mg / kg), with seven mice in each group. The acute inflammatory pain model was as follows: Figure 6 As shown on the left, 24 hours after CFA modeling, 1 hour after intraperitoneal injection, the mice's tolerance to mechanical pain was measured using Von Frey fibers. Measurements were then taken every hour until the drug effect completely disappeared. The mechanical pain threshold in the control group showed no significant difference from baseline, while the mechanical pain threshold in the CFA group was significantly reduced. Compared to the CFA group, the compound at 40 mg / kg significantly increased the mice's tolerance to mechanical pain, and this analgesic effect lasted for 3 hours. Chronic inflammatory pain model as follows: Figure 6 As shown on the right, mice were administered compound I via intraperitoneal injection on days 1, 3, 7, and 14 after CFA modeling. The tolerance to mechanical pain was measured using Von Frey fibers. Compared to the CFA model group, continuous administration of compound I significantly improved the mice's tolerance to mechanical stimulation, with the analgesic effect at 40 mg / kg lasting for 7 days. Therefore, compound I has a good analgesic effect on both acute and chronic inflammatory pain.

[0046] Example 7: Evaluation of drug resistance to compound I Having confirmed the good analgesic activity of compound I, the study further investigated its potential for drug resistance. Mice were divided into three groups: a blank control group (solvent group), an administration group (compound 20 mg / kg), and a positive control group (morphine 10 mg / kg). Administration was performed intraperitoneally for 7 consecutive days, and changes in the mice's acute pain threshold were observed daily using a hot plate test. Figure 7 As shown, the pain tolerance of mice in the positive control morphine administration group decreased day by day; compared with the solvent control group, the pain threshold of mice in the compound administration group did not change significantly for 7 consecutive days. Therefore, the compound did not produce drug resistance within 7 days of administration, and the drug resistance side effect was lower than that of morphine.

[0047] Example 8: Evaluation of the constipation side effect of compound I Constipation is a serious adverse reaction caused by the analgesic morphine, therefore this study investigated whether compound I could cause constipation. Mice were divided into three groups: a blank control group (solvent group), an administration group (compound 20 mg / kg), and a positive control group (morphine 10 mg / kg). After intraperitoneal injection, mice were placed in cages with a mesh grid, and an A4 sheet of paper was placed under the grid to collect feces. Fecal weight was measured every hour for 6 consecutive hours. Figure 8 As shown, mice in the positive control morphine administration group exhibited significant constipation, while compound I produced some constipation in the first 3 hours, but this effect gradually subsided thereafter. The amount of stool produced within 6 hours was not significantly different from that in the control group, indicating that the constipation side effect caused by compound I was lower than that caused by morphine.

[0048] Example 9: Evaluation of the convulsive side effects of compound I Agonists of the opioid receptor subtype Delta often cause convulsions as a side effect. This study investigated whether compound I would produce a convulsive effect. Mice were divided into three groups: a blank control group (solvent group), an administration group (compound 20 mg / kg), and a positive control group (SNC80 10 mg / kg). After intraperitoneal injection, mice were placed in a 20 cm × 20 cm × 20 cm test box with bedding at the bottom, and timing was started. The presence of head and forepaw convulsions was observed for 40 minutes. The severity of convulsions was scored using the Racine scale, and the duration of generalized rigidity was recorded. Figure 9 As shown, mice in the positive control group treated with SNC80 exhibited significant twitching behavior that lasted for approximately 400 seconds, while the Racine score in the compound group was far below 3, thus there was no twitching as a side effect.

[0049] Example 10: Evaluation of the excitatory motor side effects of compound I Agonists of the opioid receptor subtype Delta often produce excitatory kinesia as a side effect; therefore, excitatory kinesia is an important indicator for evaluating the side effects of analgesics. We further explored whether compound I induces excitatory kinesia through a mine field experiment. Mice were divided into three groups: a blank control group (solvent group), an administration group (compound 20 mg / kg), and a positive control group (SNC80 10 mg / kg). Mice were first placed in a 26 cm long, 26 cm wide, and 33 cm high testing chamber. After 30 minutes of video recording, the mice were randomly administered the drug and immediately returned to the testing chamber, and recording was repeated for another 150 minutes. After the test, the built-in analysis tool of the software was used to analyze the video, using movement distance as the key indicator to assess the mice's activity level. Figure 10 As shown, there was no significant difference in movement distance among the three groups of mice 30 minutes before administration. Within 150 minutes after administration, the movement distance of the SNC80 group was significantly higher than that of the blank group, while the compound administration group did not cause an increase in movement distance, indicating that compound I did not produce the side effect of excitatory movement.

[0050] Example 11: Acute toxicity evaluation of compound I in mice Acute toxicity testing was performed using 12 SPF-grade ICR mice (25±1 g, half male and half female), randomly divided into two groups: a blank group (solvent group) and an administration group (compound I). Mice were allowed a one-week acclimatization period, with fasting for 16 hours prior to administration. The drug was administered intraperitoneally at a dose of 100 mg / kg. For 14 days after administration, mouse weight and condition were recorded at fixed times daily. At the end of the experiment, mice were sacrificed, and the heart, liver, spleen, lungs, and kidneys were harvested, weighed, and their proportions to body weight were calculated. The color and texture of the tissues were also observed. Results showed (…). Figure 11 The mice were in normal condition during the experiment, and there was no statistically significant difference in daily body weight and the weight of each organ compared with the control group, indicating that compound I has good safety at 100 mg / kg.

[0051] Example 12: Evaluation of blood-brain barrier permeability of compound I Blood-brain barrier permeability is a key factor in evaluating drug efficacy. We used UPLC-MS quantitative analysis to evaluate whether compound I could cross the blood-brain barrier. Eighteen healthy male SPF-grade mice (~25 g) were housed in a barrier environment. After one week of acclimatization, the mice were observed to be in good condition and were administered the drug intraperitoneally at 20 mg / kg. Blood samples were collected at 10 min, 30 min, 1 h, 2 h, 3 h, and 4 h after drug administration, and intact brain tissue was extracted. Approximately 300 μL of whole blood was collected in anticoagulant tubes. After standing at room temperature for 1 h, the samples were centrifuged at 4°C, 3000 r / min for 10 min, and the supernatant was collected as plasma and stored at -80°C. Brain tissue was rapidly frozen in liquid nitrogen and then stored at -80°C. Post-processing of plasma samples: Proteins in serum samples were removed by organic solvent precipitation (serum:acetonitrile = 1:3, volume ratio), followed by vortexing and centrifugation (15,000 r / min, 15 min, 20°C). The supernatant was collected for analysis. Post-processing of brain tissue samples: Distilled water of the same volume as the brain tissue mass was added (e.g., 310.31 mg brain tissue to 620.62 L distilled water), followed by grinding (70 Hz, 90 s), sonication (30 s), and centrifugation (15,000 r / min, 10 min, 4°C). The supernatant was collected. After protein precipitation, the supernatant was injected for analysis, following the same procedure as for plasma. The liquid chromatograph used was a Shimadzu LC-30AD, the mass spectrometer was an AB SCIEX QTRAP-6500, the column was a Waters ACQUITY UPLC BEH C18 (2.1*100 mm, 1.7 m), and the mobile phase was 0.1% formic acid / acetonitrile. The mobile phase elution conditions were: 0-2 min, 30-85% acetonitrile; 2-4 min, 85-90% acetonitrile; 4.1-6 min, 95% acetonitrile (volume ratio). The scanning mode was positive ion mode. The drug-time curves under different test conditions are shown below. Figure 12 As shown, the highest plasma concentration of the compound is 880 ng / mL, and the brain tissue concentration is 44 ng / g. Therefore, the plasma-to-brain tissue ratio is 20, which is much lower than the blood-brain barrier permeability of morphine. This also indicates that the compound has a moderate blood-brain permeability and exhibits fewer side effects than morphine.

Claims

1. A method for synthesizing a Delta opioid receptor-biased agonist compound, characterized in that, Its structural formula is as follows: (Equation I); Includes the following steps: Intermediate 1 was obtained by benzyl protection of 3,4-dihydroxybenzaldehyde as the starting material via method A; Intermediate 1 and ethyl 4-bromophenylacetate were reacted by method B to obtain intermediate 2; Intermediate 2 is hydrolyzed using method C to obtain intermediate 3; Intermediate 3 and 3,4-dimethoxyphenylethylamine were condensed by method D to obtain intermediate 4; Intermediate 4 is loop-closed using method E to obtain intermediate 5; Intermediate 5 is further reduced by method F to obtain intermediate 6; Intermediate 6 was methylated by method G to obtain intermediate 7; Intermediate 7 was reduced and debenzylated by method H to obtain intermediate 8; Intermediate 8 was chirally isolated to obtain compound I with the R configuration. The specific synthetic route is as follows: 。 2. The synthesis method according to claim 1, characterized in that: Intermediate 1 and ethyl 4-bromophenylacetate were reacted with method B to obtain intermediate 2. In method B, the reaction solvent was pyridine, the reaction reagents were potassium carbonate and cuprous oxide, and the reaction conditions were 80-140 degrees Celsius for 3-40 hours. Intermediate 2 is hydrolyzed by method C to obtain intermediate 3. The reaction solvent of method C is one or more solutions of tetrahydrofuran, methanol, and water in any proportion. The reaction reagent is one or more of potassium carbonate, sodium carbonate, cesium carbonate, lithium hydroxide, sodium hydroxide, and potassium hydroxide. The reaction conditions are 40-90 degrees Celsius for 5-30 hours. Intermediate 3 is condensed with 3,4-dimethoxyphenethylamine via method D to obtain intermediate 4. The reaction solvent in method D is one or more of dichloromethane, tetrahydrofuran, or DMF, and the reaction conditions are condensation under the action of one or more of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1,3-dicyclohexylcarbodiimide (DCC), and N,N-diisopropylcarbodiimide (DIC); the reaction conditions are 10-60 degrees Celsius for 5-30 hours. Intermediate 4 is cyclized to obtain intermediate 5 via method E. The reaction solvent in method E is one or more of acetonitrile, tetrahydrofuran, methanol, ethanol, dichloromethane, trichloromethane, and dioxane, and the reagent is one or more of phosphorus oxychloride, aluminum trichloride, and boron trifluoride. The reaction conditions are 30-100 degrees Celsius for 2-24 hours. Intermediate 5 is further reduced by method F to obtain intermediate 6. The reaction solvent of method F is one or more of acetonitrile, tetrahydrofuran, and methanol, and the reagent is sodium borohydride and / or sodium cyanoborohydride; the reaction conditions are -10 to 40 degrees Celsius and 0.1 to 3 hours. Intermediate 6 is methylated by method G to obtain intermediate 7. The reaction solvent of method G is one or more of acetonitrile, tetrahydrofuran, and methanol, the methylating agent is paraformaldehyde and / or formaldehyde solution, and the catalyst is one or more of formic acid, acetic acid, trifluoroacetic acid, and hydrochloric acid. The reaction conditions are 30-80 degrees Celsius for 5-30 hours. Intermediate 7 is reduced and debenzylated by method H to obtain intermediate 8. The reaction solvent of method H is one or more of acetonitrile, tetrahydrofuran, and methanol. The debenzylating reagent is palladium on carbon and / or sponge palladium black reacting with hydrogen. The reaction conditions are 30-100 degrees Celsius and 5-40 hours.

3. The synthesis method according to claim 1, characterized in that: Intermediate 8 was purified by high-performance liquid chromatography (HPLC) for the separation of R and S configuration chiral products to obtain compound I. The chiral separation method employed a solid-phase chiral packing method, using any one of the following eluents: hexane / ethanol / diethylamine, hexane / ethanol / triethylamine, hexane / methanol / diethylamine, hexane / methanol / triethylamine, hexane / isopropanol / diethylamine, or a mixed solution of hexane / isopropanol / triethylamine, to prepare a chiral compound with an enantiomeric excess percentage greater than 95%.

4. The use of the compound of claim 1 in the preparation of a medicament for treating chronic analgesia, characterized in that: The chronic pain is one or more of the following: rheumatoid arthritis, osteoarthritis, and postherpetic neuralgia.

5. The application according to claim 4, characterized in that: The drug is a pharmaceutical composition made by using the compound of claim 1 as the active ingredient and a pharmaceutical carrier, and the pharmaceutical composition is in the form of tablets, capsules, granules, suspensions, drops, injections, or aerosols.

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