Water-soluble phenyl spirocyclohexane compound and pharmaceutical composition thereof

Abstract

The invention relates to the field of medicinal chemistry, provides a novel phenyl spirocyclohexane compound and a pharmaceutical composition thereof, and particularly relates to a compound with excellent water solubility and stability and a pharmaceutically acceptable salt thereof, which are obtained by modification on the basis of Cebranopadol and can be used for preparing a medicine for treating pain.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to a novel phenylspirocyclohexane compound and its pharmaceutical composition, specifically to a Cebranopadol analog with excellent water solubility and stability. Background Technology

[0002] The discovery of novel prodrugs has become an indispensable part of the current pharmaceutical industry. Prodrugs are alternative forms of drugs used to improve the absorption, distribution, metabolism, and excretion (ADME) properties of drugs. Prodrugs are administered in an inactive or less active form and are subsequently converted into their active form through the subject's normal metabolic processes, such as hydrolysis or other chemical reactions.

[0003] Pain is an unpleasant sensory and emotional experience, or a similar experience, associated with actual or potential tissue damage. Opioid analgesics are currently recognized as the most effective analgesics for moderate to severe pain, and are recognized by the World Health Organization as standard medications in the three-step analgesic ladder for cancer pain management, as well as first-line drugs for controlling acute pain. Traditional opioids are the most effective analgesics, but their abuse is the most challenging issue. Ideally, opioid analgesics should target pain tissue, act only on pain pathways without activating addiction pathways, and simultaneously alleviate the negative emotions induced by pain. Therefore, scientists have dedicated themselves to the research of low-addiction opioid analgesics from all fields of pharmaceutics, medicinal chemistry, pharmacokinetics, and pharmacology.

[0004] Cebranopadol (GRT-6500) (trans-6′-fluoro-4′,9′-dihydro-N,N-dimethyl-4-phenylspiro[cyclohexane-1,1′-(3′H)-pyran[3,4-b]indole]-4-amine) is an analgesic nociceptin / orphanone FQ peptide (NOP) and opioid receptor agonist (WO 2004 / 043967, WO 2008 / 040481, WO 2012 / 016703, WO 2012 / 016699, WO 2012 / 016695, WO 2012 / 016698, WO 2012 / 016697, WO 2013 / 007361) developed by Grundenthal. Its structural formula is as follows: , Cebranopadol demonstrated potent anti-nociceptive and anti-hypersensitivity effects in several rat models of acute and chronic pain, with ED50 values ​​of 0.5-5.6 μg / kg after intravenous administration and 25.1 μg / kg after oral administration. Compared with selective MOP receptor agonists, cebranopadol was more effective in chronic neuropathic pain models than in acute nociceptive pain models.

[0005] Physicochemical properties, therapeutic effective dose, and route of administration all affect the pharmacokinetic characteristics of drug molecules. The therapeutic effective dose of a particular drug is fixed. Nevertheless, changing the route of administration may reduce the drug dose if a new route provides higher bioavailability. For example, given suitable physicochemical properties, a drug with poor oral bioavailability and requiring a high dose can be formulated for lower parenteral administration due to its improved bioavailability. However, different routes of administration are generally only possible when the physicochemical properties of a given drug molecule are suitable for a new dosage form. The physicochemical composition of many existing drugs limits their oral administration, resulting in high doses and unfavorable pharmacokinetic characteristics. Therefore, efforts have been made to modify the physicochemical properties of existing drugs and / or their formulations.

[0006] Drugs with poor solubility often exhibit poor bioavailability, which can hinder drug development and necessitate high doses to achieve therapeutically effective blood levels. For example, Tricor® (fenofibrate) is marketed as 300 mg capsules. Reducing the particle size to a fine powder increases drug solubility and allows for dose reductions down to 200 mg. With the addition of surfactants to the fine powder, formulations similar to the 300 mg and 200 mg doses can be obtained using only 160 mg tablets. Another bioequivalent formulation containing drug nanoparticles allows for an effective dose of 145 mg. Thus, by increasing its solubility, Tricor® significantly reduces the dose (by more than 100%), thereby improving bioavailability. However, while there are examples demonstrating that particle size reduction improves solubility, the inherent conditions of oral administration (e.g., the limited aqueous medium in the gastrointestinal tract) may limit improvements in the solubility and bioavailability of certain drugs.

[0007] Another technique for increasing solubility is to form molecular complexes with insoluble / poorly soluble drugs and more soluble molecules, such as cyclodextrin. Itraconazole (Sporanox®), voriconazole (Vfend®), and ziprasidone (Geodon®) are examples of successful applications of this technique. However, this application typically requires a large excess of cyclodextrin relative to the amount of drug being dissolved and may not provide the desired increase in solubility for the entire drug sample (e.g., 10 mg of itraconazole, 200 mg of voriconazole, or 20 mg of ziprasidone require 400 mg, 3200 mg, or 294 mg of cyclodextrin, respectively).

[0008] While the importance of discovering new drugs cannot be overstated, the ability to improve the physicochemical properties of existing drugs is also beneficial. Therefore, there remains a significant and unmet need for improved drugs, such as prodrugs of existing drugs.

[0009] WO2013 / 007361 discloses the crystalline polymorphs of cebranopadol and cebranopadol solvates, which can be distinguished by X-ray diffraction. It further reports that the crystalline polymorphs can interconvert, for example, in the presence of a solvent or upon application of heat or mechanical energy. However, this behavior (the occurrence of morphological changes) can be detrimental, for example, for dosage forms such as tablets, as it can lead to solid-state changes in the dosage form, typically resulting in different solubility and pharmacokinetic properties. Therefore, such changes may require strict temperature control of the dosage form, especially in summer. Furthermore, this solid-state change can lead to regulatory and commercial disadvantages.

[0010] Furthermore, cebranopadol has been reported to have poor solubility, requiring very small doses. The oral bioavailability of cebranopadol is only 13–23% (J Pharmacol Exp Ther. 2014, 349, 535–548). However, due to its low water solubility, cebranopadol suffers from inadequate intestinal absorption, limiting its dosage forms and routes of administration. Although efforts have been made to address these issues, such as the fat emulsion administration of cebranopadol (CN115813856) and compositions containing soluble cebranopadol (EP3253374), the dosage forms and routes of administration for cebranopadol remain limited in this application.

[0011] The prior art compounds in these references do not contain methyl dihydrogen phosphate (or phosphate, monoester, or diester group) or methyl diacid (diacid monoester or monosalt) groups linked to N-1 nitrogen; therefore, the compounds of this invention represent novel material components. The applicant's application 202511687899.3 is incorporated herein by reference. This part dramatically increases the utility of the parent compound through prodrug modification, which dramatically increases the maximum systemic exposure of the parent molecule in preclinical human exposure models. The inventors believe that no substance in the prior art references can be considered as a disclosure or indication of the invention of a novel compound and its use in the treatment of pain.

[0012] This invention describes specific azaindole prodrugs that are highly effective in improving the oral efficacy of parent molecules used as pain medications, particularly centrally active analgesics. The parent molecules are relatively insoluble and subject to dissolution-limited or solubility-absorption-limited conditions, meaning that as the dose increases beyond a maximum level, less and less drug dissolves and is absorbed into the circulation; instead, it is eliminated from the body as waste. Improvements must be provided by prodrugs because they significantly increase drug levels in the body, thus providing greater efficacy against dual nociceptor / orphanin FQ peptide (NOP) receptors and μ-opioid peptide (MOP) receptors (dual NMR) agonists. Therefore, the highest exposure fold is required. Because using prodrugs allows for greater drug absorption to the target, drug burden, patient costs, and dosing intervals can be reduced. Prodrugs with these properties are difficult to identify; successful prodrug designs disclosed in the literature are neither straightforward nor have feasible clarification pathways. There is no clear explanation of existing techniques regarding which prodrug chemistry is most effective. The following discussion and data will demonstrate that the prodrugs described in this invention have unexpectedly good effects. They release the parent drug very rapidly and efficiently, increasing exposure levels higher than many reported prodrugs.

[0013] In certain circumstances, prodrug strategies or methods can be used to significantly enhance drug efficacy by improving drug properties or overcoming inherent pharmacological or pharmacokinetic defects. Prodrugs differ from formulations because chemical modifications result in entirely new chemical entities that, when administered to a patient, regenerate the parent molecule in vivo. Numerous prodrug strategies exist that provide conditions for modulating the regeneration of the parent drug, the physical, pharmacological, or pharmacokinetic properties of the prodrug, and functional groups that can be linked to prodrug modifications. However, none of these publications teaches the methods used to produce the specific prodrugs invented herein. Numerous reviews and discussions on prodrug strategies have been published; the following is an incomplete list: 1) Phosphate moiety in FDA-approved pharmaceutical salts and Prodrugs: Drug DevRes. 2022;83:1059–1074; 2) FimH Antagonists - Phosphate Prodrugs Improve Oral Bioavailability. J. Med. Chem, 2016:1-86; While some techniques are known to have specific applications, such as improving solubility or absorption, prodrug development remains largely empirical. Therefore, it is often necessary to study numerous strategies or chemical modifications and evaluate the resulting compounds in biological models to determine and measure the success of prodrug strategies. Summary of the Invention

[0014] This invention addresses the limitation of cebranopadol's therapeutic potential by exploiting its extremely poor solubility and bioavailability in water. By modifying its structure, we prepared bioavailable analogs of this active compound, providing novel phenylspirocyclohexane compounds to improve the solubility and bioavailability of cebranopadol in water.

[0015] The inventors of this invention conducted extensive research to solve the aforementioned problems and discovered that when cebranopadol is derived to a hydroxymethyl form and simultaneously modified into a phosphate ester or succinic acid monoester form, its water solubility can be significantly improved. This invention is based on this discovery.

[0016] The present invention provides compounds of Formula I or pharmaceutically acceptable salts thereof.

[0017] , Where R is selected from C3-C 10 Straight-chain or branched fatty acid ester groups or phosphate ester groups.

[0018] Preferably, R is a C3-C5 straight-chain or branched fatty acid ester group.

[0019] In some embodiments, the present invention provides compounds represented by Formula II-a or Formula II-b: , Wherein, n1 is selected from an integer from 1 to 8, preferably from an integer from 1 to 5, and even more preferably from an integer from 1 to 3.

[0020] Thirdly, the present invention provides compounds represented by Formula II-c: , Where n is an integer from 1 to 3, preferably an integer from 1 to 2; M is a monovalent metal ion, a divalent metal ion, an organic base, a basic amino acid, or an active alkaloid.

[0021] Furthermore, the present invention provides compounds represented by formula II-c that satisfy one or more of the following conditions: 1) The monovalent metal ion is selected from one or both of potassium ions and sodium ions; 2) The divalent metal ions are selected from one or both of calcium ions and magnesium ions; 3) The organic base is selected from one, two, or three of ammonia, meglumine, and tromethamine; 4) The basic amino acid is selected from one, two or three of arginine, lysine and histidine.

[0022] Furthermore, the present invention provides that the following compounds or their pharmaceutically acceptable salts are any of the following compounds: Compounds of Formula I containing one or more chiral centers may exist as racemic mixtures, enantiomer-enriched mixtures, or enantiomerically pure individual stereoisomers. Various stereoisomers containing one or more asymmetric centers can be resolved by methods known to those skilled in the art.

[0023] , , Suitable addition salts of the present invention, acid addition salts, are selected from acetates, p-aminobenzoates, ascorbic acid salts, aspartate salts, benzenesulfonates, benzoates, bicarbonates, bisulfates, tartrates, calcium edetate, camphorsulfonates, carbonates, citrates, ethanedisulfonates, ethanesulfonates, formates, fumarates, gluconates, glutamates, hydrobromide, hydrochlorides, hydrogen phosphates, hydroiodates, hydrogen maleate, hydrogen succinate, hydroxynaphthylcarboxylate, hydroxyethylsulfonate, lactates, lacturonates, malates, and mandelates. Base addition salts include sodium, potassium, calcium, magnesium, aluminum, zinc, ammonium, morpholine, pyridine, piperidine, methylpyridine, dicyclohexylamine, glucosamine, N-methylglucosamine, trimethylpyridine, lysine, and arginine. In one embodiment, pharmaceutically acceptable base addition salts are sodium or potassium salts. In another embodiment, the pharmaceutically acceptable base addition salt is meglumine salt, lysine, and arginine.

[0024] Furthermore, the present invention provides a pharmaceutical composition comprising the above-described compound and a pharmaceutically acceptable excipient carrier. Advantageously, the compounds disclosed herein have increased bioavailability relative to the cebranopadol parent compound, and therefore certain embodiments relate to the formulation of the aforementioned pharmaceutical compositions for oral delivery. Any carriers and / or excipients known in the art for use in oral formulations, other than those available to those skilled in the art, may be used in these embodiments.

[0025] The compounds of the present invention or their pharmaceutically acceptable salts can be administered via any acceptable method of reagent administration used for similar applications. Pharmaceutical compositions of embodiments of the present invention can be prepared by combining the compounds of the present invention with suitable pharmaceutically acceptable carriers, diluents, or excipients, and said pharmaceutical compositions can be formulated into solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, solutions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, and intranasal administration. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques.

[0026] Fourthly, the composition provided by this invention is used in the preparation of pain medications. The pain refers to one or more of the following: chronic pain, nociceptive pain, neuropathic pain, malignant pain, or inflammatory pain.

[0027] In another aspect, the present invention provides a method for preparing a compound of formula I, comprising the following steps: , The definition of R is as defined for compound I.

[0028] The present invention also provides an intermediate compound, namely compound of formula I-1.

[0029] , The key points of this invention are: 1) The compounds or salts thereof of the present invention have excellent water solubility and excellent absorbability, etc. 2) The compounds of the present invention or their salts facilitate the production of the active ingredient cebranopadol; 3) The compounds or their salts of the present invention provide multiple routes of drug administration, wherein oral bioavailability is significantly improved, and the compounds or their salts can be prepared in the form of aqueous solutions for injection. Attached Figure Description

[0031] Figure 1 HPLC analysis of compound I-7 (hydroxymethylcebranopadol succinic acid); Figure 2 The 1H NMR spectrum of compound I-7 (hydroxymethylcebranopadol succinic acid); Figure 3 Carbon 160-carbon spectrum of compound I-7 (hydroxymethylcebranopadol succinic acid); Figure 4IR for compound I-7 (hydroxymethylcebranopadol succinic acid); Figure 5 HPLC analysis of compound I-1 (hydroxymethyl cebranopadol phosphate); Figure 6 The 1H NMR spectrum of compound I-1 (hydroxymethyl cebranopadol phosphate); Figure 7 The carbon spectrum of compound I-1 (hydroxymethyl cebranopadol phosphate); Figure 8 The results are from the pKa determination experiment for compound I-1; Figure 9 The change in serum concentration of cebranopadol generated by tail vein injection of compounds I-1 and I-7 in rats over time; Figure 10 The graph shows the changes in blood concentrations of Cebranopadol, compound I-1, and compound I-7 over time. Figure 11 The results of cytotoxicity assays for Cebranopadol, compound I-1, and compound I-7 in Caco-2 cells. Detailed Implementation

[0032] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] The following embodiments can further describe the present invention; however, these embodiments should not be construed as limiting the scope of the present invention.

[0034] Preparation Example: Preparation of Cebranopadol.

[0035] Preparation steps 1+2: , 220.00g of 1,4-cyclohexanedione monoethylene glycol ketal and 583.00g of methylamine solution (30% ethanol solution) were added to a 1000ml reaction flask and stirred until dissolved. Then 440.00g of anhydrous sodium sulfate was added. The reaction was carried out at 25–35°C for 24 hours. After the reaction was completed, the mixture was filtered, the filtrate was concentrated to dryness, and 220 ml of isopropyl ether was added twice to obtain 230.00 g of black oily substance (intermediate one).

[0036] Add 1000 ml of lithium phenylene solution (1.5 M n-butyl ether solution) to a 3000 ml reaction flask, cool to 0–5 °C, and protect under nitrogen. Then, dissolve 230.00 g of intermediate one in 440 ml of dibutyl ether and slowly add the solution dropwise to the reaction flask, ensuring the addition temperature does not exceed 20 °C, under nitrogen protection. After the addition is complete, heat to room temperature (25–30 °C) and stir for 1 hour. After the reaction is complete, wash once with 440 ml of saturated ammonium chloride solution, wash three times with 440 ml x 3 ml of water, concentrate to dryness at 90 °C, and wash once with 220 ml of isopropanol. Cool to room temperature and dissolve completely in 220 ml of isopropanol. Adjust the pH to 3–4 with 25% isopropanol hydrochloride solution. The mixture was cooled to 0–5 °C and allowed to crystallize for 1 hour. It was then filtered, washed with cold isopropanol (0.5 V), washed with methyl tert-ethyl ether (1.0 V), and dried at 60 °C to give 131.00 g of white solid (intermediate II). The total yield of the two steps was 32.77%.

[0037] Preparation step 3: , 130.00 g of intermediate II was dissolved in 260 ml of water and 1300 ml of dichloromethane, and the pH was adjusted to 12-13 with 25% sodium hydroxide solution. The mixture separated into layers, and the organic phase was washed once with 260 ml of water. The organic phase was concentrated to dryness, and 260 ml of isopropanol was added. The temperature was raised to 65°C. 63.00 g of anhydrous formic acid was added dropwise, and after the addition was complete, 70.00 g of formaldehyde solution was quickly added. The mixture was stirred at 65°C for 16 hours. After the reaction was complete, the mixture was concentrated to dryness under vacuum at 85°C. 156 ml of hydrochloric acid and 39 ml of water were added, and the mixture was stirred at 65°C for 3 hours. After the reaction was complete, the mixture was washed once with 260 ml of isopropyl ether. The temperature was lowered to 0-5°C, and the pH was adjusted to 12-13 with 25% sodium hydroxide solution. The mixture was extracted with 1300 ml of dimethyltetrahydrofuran, washed once with 260 ml of water, and concentrated to dryness. 117 ml of isopropanol and 78 ml of water were added, and the mixture was stirred until dissolved. The solution was cooled to 0–5 °C and allowed to crystallize for 1 hour. After filtration, the solution was washed with 65 ml of cold isopropanol-water mixture and dried at 60 °C to obtain 45.41 g of off-white solid (intermediate tri), with a yield of 45.62%.

[0038] Preparation step 4:

[0039] 70.00 g ammonium chloride and 930 ml water were added to a 3000 ml reaction flask and stirred until dissolved. Then, 500 ml of 2-methyltetrahydrofuran and 100 g of 4-fluorophenylhydrazine hydrochloride were added, and the mixture was heated to 70 °C. 43.11 g of 2,3-dihydrofuran was dissolved in 480 ml of 2-methyltetrahydrofuran and then slowly added dropwise to the reaction flask. The mixture was stirred at 70 °C for 16 hours. The mixture was cooled to 50 °C, and the layers separated; the aqueous phase was discarded. The organic phase was washed once with 700 ml of 5% sodium chloride solution. The organic phase was concentrated to dryness, and 600 ml of toluene and 250 ml of water were added and stirred until the layers separated; the aqueous phase was discarded. The organic phase was concentrated to remove half of the solid, cooled to 0–5 °C, and crystallized for 5 hours. The crystals were filtered, washed with 50 ml of cold toluene solution, and dried at 40 °C to give 59.70 g of a reddish-brown solid (intermediate tetrahydrofuran), with a yield of 54.17%.

[0040] Preparation step 5: , 45.00 g of intermediate III, 37.11 g of intermediate IV, and 2250 ml of dichloromethane were added to a 3000 ml reaction flask, and the mixture was cooled to 0–5 °C. 55.23 g of trimethylsilyl trifluoromethanesulfonate was dissolved in 45 ml of dichloromethane and quickly added to the reaction flask. After addition, the mixture was stirred at 0–5 °C for 20 minutes. The temperature was then raised to room temperature (25–30 °C), and the reaction was stirred for 3 hours. After the reaction was complete, the mixture was concentrated to remove approximately 3 / 4 of the dichloromethane, and then 765 ml of 1 N sodium hydroxide solution and 450 ml of water were added. The mixture was stirred and cooled to 0–5 °C, and crystallization was allowed to occur for 1 hour. The crystals were filtered, and the filter cake was slurried with 450 ml of isopropanol, filtered again, and dried at 60 °C to obtain 75.00 g of a white solid (intermediate V), with a yield of 95.70%.

[0041] Example 1: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0042] 2.0 g of Cebranopadol, 3.55 g of paraformaldehyde, 0.13 g of DMAP, and 10 ml of DMF were added to a 50 ml reaction flask and heated to 50–60 °C for 6 hours. After the reaction was complete, the mixture was filtered, and the filtrate was washed three times with 200 ml of dichloromethane and 200 ml of water (3 x 3). The filtrate was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (eluent: PE:EA = 2:1) to give 0.63 g of a white solid (hydroxymethyl Cebranopadol), with a yield of 29.18%.

[0043] 0.30 g of hydroxymethyl cebranopadol, 0.07 g of succinic anhydride, and 30 mL of dichloromethane were added to a 50 mL reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed, 0.22 g of cebranopadol succinate was obtained, with a yield of 59%.

[0044] Example 2: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0045] 2.0 g of Cebranopadol, 3.55 g of paraformaldehyde, 5.25 g of cesium carbonate, and 10 ml of DMF were added to a 50 ml reaction flask and heated to 50–60 °C for 10 hours. After the reaction was complete, the mixture was filtered, and the filtrate was washed three times with 200 ml of dichloromethane and 200 ml of water (3 x 3). The filtrate was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (eluent: PE:EA = 2:1) to give 0.11 g of a white solid (hydroxymethyl Cebranopadol), with a yield of 5.09%.

[0046] 0.10 g of hydroxymethyl cebranopadol, 0.02 g of succinic anhydride, and 10 mL of dichloromethane were added to a 50 mL reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed, 0.07 g of cebranopadol succinate was obtained, with a yield of 58.7%.

[0047] Example 3: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0048] 2.0 g of Cebranopadol, 3.55 g of paraformaldehyde, 1.13 g of pyridine, and 10 ml of DMF were added to a 50 ml reaction flask and heated to 50–60 °C for 5 hours. After the reaction was complete, the mixture was filtered, and the filtrate was washed three times with 200 ml of dichloromethane and 200 ml of water (3 x 3). The filtrate was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (eluent: PE:EA = 2:1) to give 0.35 g of hydroxymethyl Cebranopadol, with a yield of 16.21%.

[0049] 0.10 g of hydroxymethyl cebranopadol, 0.02 g of succinic anhydride, and 10 mL of dichloromethane were added to a 50 mL reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 0.06 g of hydroxymethyl cebranopadol succinic acid was obtained, with a yield of 48.78%.

[0050] Example 4: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0051] 2.0 g of Cebranopadol, 2.55 g of paraformaldehyde, 0.54 g of DBU, and 10 ml of DMF were added to a 50 ml reaction flask and heated to 50–60 °C for 5 hours. After the reaction was complete, the mixture was filtered, and the filtrate was washed three times with 200 ml of dichloromethane and 200 ml of water (3 x 3). The filtrate was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (eluent: PE:EA = 2:1) to give 0.45 g of hydroxymethyl Cebranopadol, with a yield of 20.85%.

[0052] 0.30 g of hydroxymethyl cebranopadol, 0.07 g of succinic anhydride, and 30 mL of dichloromethane were added to a 50 mL reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed, post-treatment yielded hydroxymethyl cebranopadol succinic acid, with a yield of 58.6%.

[0053] Example 5: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0054] 20.0 g of Cebranopadol, 35.47 g of paraformaldehyde, 1.31 g of DMAP, and 100 ml of DMF were added to a 500 ml reaction flask and heated to 50–60 °C for 16 hours. After the reaction was complete, the mixture was filtered, and the filtrate was washed three times with 300 ml of dichloromethane and 200 ml x 3 ml of water. The solution was dried over anhydrous sodium sulfate, concentrated, and recrystallized from 120 ml of methanol to give 6.81 g of a white solid (hydroxymethyl Cebranopadol), with a yield of 31.54%.

[0055] 0.30 g of hydroxymethyl cebranopadol, 0.07 g of succinic anhydride, and 30 ml of tetrahydrofuran were added to a 50 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 0.11 g of cebranopadol succinate was obtained, with a yield of 29.73%.

[0056] Example 6: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0057] 20.0 g of Cebranopadol, 35.47 g of paraformaldehyde, 1.31 g of DMAP, and 100 ml of DMF were added to a 500 ml reaction flask and heated to 50–60 °C for 16 hours. After the reaction was complete, the mixture was filtered, and the filtrate was washed three times with 300 ml of dichloromethane and 200 ml x 3 ml of water. The solution was dried over anhydrous sodium sulfate, concentrated, and recrystallized from 100 ml of methanol to give 6.59 g of a white solid (hydroxymethyl Cebranopadol), with a yield of 30.53%.

[0058] 0.30 g of hydroxymethyl cebranopadol, 0.07 g of succinic anhydride, and 30 ml of acetone were added to a 50 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 0.08 g of cebranopadol succinate was obtained, with a yield of 21.62%.

[0059] Example 7: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0060] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to allow crystals to crystallize. After filtration, 69.26g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0061] 3.0 g of hydroxymethyl cebranopadol, 0.7 g of succinic anhydride, and 300 ml of dichloromethane were added to a 500 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 2.26 g of cebranopadol succinate was obtained, with a yield of 61.08%.

[0062] Example 8: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0063] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to allow crystals to crystallize. After filtration, 69.26g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0064] 3.0 g of hydroxymethyl cebranopadol, 0.7 g of succinic anhydride, and 300 ml of tetrahydrofuran were added to a 500 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 2.37 g of cebranopadol succinate was obtained, with a yield of 64.05%.

[0065] Example 9: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0066] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to allow crystals to crystallize. After filtration, 69.26g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0067] 3.0 g of hydroxymethyl cebranopadol, 0.7 g of succinic anhydride, and 300 ml of acetone were added to a 500 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 1.36 g of cebranopadol succinate was obtained, with a yield of 36.75%.

[0068] Example 10: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0069] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to allow crystals to crystallize. After filtration, 69.26g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0070] 3.0 g of hydroxymethyl cebranopadol, 0.7 g of succinic anhydride, and 300 ml of dioxane were added to a 500 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 3.06 g of cebranopadol succinate was obtained, with a yield of 82.70%.

[0071] Example 11: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0072] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to allow crystals to crystallize. After filtration, 69.26g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0073] 3.0 g of hydroxymethyl cebranopadol, 0.7 g of succinic anhydride, and 300 ml of dioxane were added to a 500 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 3.17 g of cebranopadol succinate was obtained, with a yield of 85.40%.

[0074] Example 12: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0075] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to allow crystals to crystallize. After filtration, 69.26g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0076] 3.0 g of hydroxymethyl cebranopadol, 0.7 g of succinic anhydride, and 300 ml of acetone were added to a 500 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 1.15 g of cebranopadol succinate was obtained, with a yield of 31.08%.

[0077] Example 13: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0078] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to allow crystals to crystallize. After filtration, 69.26g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0079] 3.0 g of hydroxymethyl cebranopadol, 0.7 g of succinic anhydride, and 300 ml of dioxane were added to a 500 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 2.54 g of cebranopadol succinate was obtained, with a yield of 68.65%.

[0080] Example 14: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0081] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to allow crystals to crystallize. After filtration, 69.26g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0082] 3.0 g of hydroxymethyl cebranopadol, 0.7 g of succinic anhydride, and 300 ml of dioxane were added to a 500 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 3.12 g of cebranopadol succinate was obtained, with a yield of 84.32%.

[0083] Example 15: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0084] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to allow crystals to crystallize. After filtration, 69.26g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0085] 3.0 g of hydroxymethyl cebranopadol, 0.7 g of succinic anhydride, and 300 ml of dioxane were added to a 500 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 3.0 g of cebranopadol succinate was obtained, with a yield of 81.08%.

[0086] Example 16: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0087] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to crystallize, and then filtered to give 69.26g of a white solid (hydroxymethyl Cebranopadol), with a yield of 64.13%.

[0088] 3.0 g of hydroxymethyl cebranopadol, 0.7 g of succinic anhydride, and 300 ml of dioxane were added to a 500 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 2.63 g of cebranopadol succinate was obtained, with a yield of 71.08%.

[0089] Example 17: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0090] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to crystallize, and then filtered to give 69.26g of a white solid (hydroxymethyl Cebranopadol), with a yield of 64.13%.

[0091] 3.0 g of hydroxymethyl cebranopadol, 0.7 g of succinic anhydride, and 300 ml of dioxane were added to a 500 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 2.98 g of cebranopadol succinate was obtained, with a yield of 80.54%.

[0092] Example 18: Preparation of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0093] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to allow crystals to crystallize. After filtration, 69.26g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0094] 3.0 g of hydroxymethyl cebranopadol, 0.7 g of succinic anhydride, and 300 ml of acetonitrile were added to a 500 ml reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 2.59 g of cebranopadol succinate was obtained, with a yield of 70%.

[0095] Example 19: Structural testing of compound I-7 (hydroxymethylcebranopadol succinic acid).

[0096] HPLC purity: 99.04%, method as follows: HPLC conditions.

[0097]

[0098] Hydrogen spectrum analysis: 1 H-NMR (400 MHz, DMSO-d6) δ 12.33 (s, 1H), 7.50 (dd, J =8.9, 4.3 Hz, 1H), 7.43~7.36 (m, 4H), 7.33~7.25 (m, 2H), 7.04 (td, J =9.2, 2.2 Hz, 1H), 6.36 (s, 2H), 3.89 (t, J = 4.8 Hz, 2H), 2.69 (t, J = 4.7Hz, 2H), 2.62~2.54 (m, 3H), 2.51~2.43 (m, 3H), 2.38~2.22 (m, 2H), 2.00 (s, 6H), 1.88~1.79 (m, 2H), 1.77~1.65 (m, 2H); Carbon spectrum analysis: 13 C-NMR (151 MHz, DMSO) δ 173.67, 172.09, 159.11, 157.56,141.46, 139.13, 133.90, 127.83, 126.99, 111.69, 111.62, 110.46, 110.44,110.27, 103.88, 103.73, 72.44, 69.02, 58.66, 58.19, 37.90, 30.27, 29.48,29.14, 29.06, 28.68, 22.73; Infrared Spectrum (IR) Analysis: .

[0099] Example 20: Preparation of compound I-8 (hydroxymethylcebranopadol sodium succinate).

[0100] 2.0 g of Cebranopadol, 3.55 g of paraformaldehyde, 5.25 g of cesium carbonate, and 10 ml of DMF were added to a 50 ml reaction flask and heated to 50–60 °C for 10 hours. After the reaction was complete, the mixture was filtered, and the filtrate was washed three times with 200 ml of dichloromethane and 200 ml of water (3 x 3). The filtrate was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (eluent: PE:EA = 2:1) to give 0.11 g of a white solid (hydroxymethyl Cebranopadol), with a yield of 5.09%.

[0101] 0.10 g of hydroxymethyl cebranopadol, 0.02 g of succinic anhydride, and 10 mL of dichloromethane were added to a 50 mL reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 0.07 g of cebranopadol succinate was obtained, with a yield of 58.7%.

[0102] 0.05 g of cebranopadol succinate was added to 10 ml of acetone and 1 ml of water. The pH was adjusted to 7-8 with 2N NaHCO3 solution. The mixture was stirred at 0-5℃. After the reaction was completed, 0.04 g of sodium hydroxymethyl cebranopadol succinate was obtained after post-treatment.

[0103] Example 21: Preparation of compound I-9 (hydroxymethylcebranopadol potassium succinate) 2.0 g of Cebranopadol, 3.55 g of paraformaldehyde, 5.25 g of cesium carbonate, and 10 ml of DMF were added to a 50 ml reaction flask and heated to 50–60 °C for 10 hours. After the reaction was complete, the mixture was filtered, and the filtrate was washed three times with 200 ml of dichloromethane and 200 ml of water (3 x 3). The filtrate was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (eluent: PE:EA = 2:1) to give 0.11 g of a white solid (hydroxymethyl Cebranopadol), with a yield of 5.09%.

[0104] 0.10 g of hydroxymethyl cebranopadol, 0.02 g of succinic anhydride, and 10 mL of dichloromethane were added to a 50 mL reaction flask, and the mixture was heated to reflux and reacted overnight. After the reaction was completed and post-processed, 0.07 g of cebranopadol succinate was obtained, with a yield of 58.7%.

[0105] 0.05 g of cebranopadol succinate was added to 10 ml of acetone and 1 ml of water. The pH was adjusted to 7-8 with 2N K2CO3 solution. The mixture was stirred at 0-5℃. After the reaction was completed, 0.04 g of potassium hydroxymethyl cebranopadol succinate was obtained after post-treatment, with a yield of 74.07%.

[0106] Example 22: Preparation of compound I-1 (hydroxymethylcebranopadol phosphate).

[0107] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to allow crystals to crystallize. After filtration, 69.26g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0108] 0.50 g of hydroxymethyl cebranopadol, 10 ml of pyridine, and 20 ml of dichloromethane were added to a 100 ml reaction flask. The mixture was cooled to 0–5 °C, and 0.23 g of phosphorus oxychloride was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 1 hour. After post-treatment, 0.42 g of hydroxymethyl cebranopadol monophosphate was obtained, with a yield of 70%. HPLC: 97.9%.

[0109] MS (ESI): M-H2O3P+H + 409.1.

[0110] 1 H-NMR (400 MHz, Chloroform-d) δ 7.41~7.35 (m, 4H), 7.33~7.28 (m,2H), 7.19~7.12 (m, 2H), 6.94 (td, J = 9.0, 2.5 Hz, 1H), 5.84 (s,2H), 3.98(t, J = 5.4 Hz, 2H), 2.79 (t, J = 5.3 Hz, 2H), 2.57~2.43 (m, 4H), 2.05 (s,6H), 2.02~1.89 (m, 4H), 1.62 (s, 2H). 13 C-NMR (151 MHz, DMSO-D6) δ 158.69, 157.12, 141.60, 139.61, 133.59,127.90, 127.08, 112.03, 109.45, 109.27, 108.45, 103.31, 103.16, 72.87, 66.73,58.75, 58.38, 38.36, 30.48, 28.83, 22.89.

[0111] Example 23: Preparation of compound I-1 (hydroxymethylcebranopadol phosphate).

[0112] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to allow crystals to crystallize. After filtration, 69.26g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0113] 0.50 g of hydroxymethyl cebranopadol, 10 ml of triethylamine, and 20 ml of dichloromethane were added to a 100 ml reaction flask. The mixture was cooled to 0–5 °C, and 0.23 g of phosphorus oxychloride was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 1 hour. After post-treatment, 0.12 g of hydroxymethyl cebranopadol monophosphate was obtained, with a yield of 20%.

[0114] Example 24: Preparation of compound I-1 (hydroxymethylcebranopadol phosphate).

[0115] 100g of Cebranopadol, 177.33g of paraformaldehyde, 6.56g of DMAP, and 6000ml of DMF were added to a 1000ml reaction flask and heated to 50-60℃ for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000ml of dichloromethane and 500ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000ml of methanol, and concentrated to approximately 500ml. The solution was cooled to 0-5℃ for 2 hours to allow crystals to crystallize. After filtration, 69.26g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0116] 0.50 g of hydroxymethyl cebranopadol, 10 ml of DIPEA, and 20 ml of dichloromethane were added to a 100 ml reaction flask. The mixture was cooled to 0–5 °C, and 0.23 g of phosphorus oxychloride was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 1 hour. After post-treatment, 0.33 g of hydroxymethyl cebranopadol monophosphate was obtained, with a yield of 55%.

[0117] Example 25: Preparation of compound I-1 (hydroxymethylcebranopadol phosphate).

[0118] 100 g of Cebranopadol, 177.33 g of paraformaldehyde, 6.56 g of DMAP, and 6000 ml of DMF were added to a 1000 ml reaction flask and heated to 50–60 °C for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000 ml of dichloromethane and 500 ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000 ml of methanol, and concentrated to approximately 500 ml. The mixture was then cooled to 0–5 °C for 2 hours to allow crystals to crystallize. After filtration, 69.26 g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0119] 0.50 g of hydroxymethyl cebranopadol, 10 ml of DBU, and 20 ml of dichloromethane were added to a 100 ml reaction flask. The mixture was cooled to 0–5 °C, and 0.23 g of phosphorus oxychloride was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 1 hour. After post-treatment, 0.39 g of hydroxymethyl cebranopadol monophosphate was obtained, with a yield of 65%.

[0120] Example 26: Preparation of compound I-1 (hydroxymethylcebranopadol phosphate).

[0121] 100 g of Cebranopadol, 177.33 g of paraformaldehyde, 6.56 g of DMAP, and 6000 ml of DMF were added to a 1000 ml reaction flask and heated to 50–60 °C for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000 ml of dichloromethane and 500 ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000 ml of methanol, and concentrated to approximately 500 ml. The mixture was then cooled to 0–5 °C for 2 hours to allow crystals to crystallize. After filtration, 69.26 g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0122] 0.50 g of hydroxymethyl cebranopadol, 0.3 g of DMAP, 10 ml of triethylamine, and 20 ml of dichloromethane were added to a 100 ml reaction flask. The mixture was cooled to 0–5 °C, and 0.23 g of phosphorus oxychloride was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 1 hour. After post-treatment, 0.55 g of hydroxymethyl cebranopadol monophosphate was obtained, with a yield of 91.6%.

[0123] Example 27: Preparation of compound I-1 (hydroxymethylcebranopadol phosphate).

[0124] 100 g of Cebranopadol, 177.33 g of paraformaldehyde, 6.56 g of DMAP, and 6000 ml of DMF were added to a 1000 ml reaction flask and heated to 50–60 °C for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000 ml of dichloromethane and 500 ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000 ml of methanol, and concentrated to approximately 500 ml. The mixture was then cooled to 0–5 °C for 2 hours to allow crystals to crystallize. After filtration, 69.26 g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0125] 0.50 g of hydroxymethyl cebranopadol, 0.3 g of DMAP, 10 ml of pyridine, and 20 ml of dichloromethane were added to a 100 ml reaction flask. The mixture was cooled to 0–5 °C, and 0.23 g of phosphorus oxychloride was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 1 hour. After the reaction was completed and post-processed, 0.53 g of hydroxymethyl cebranopadol monophosphate was obtained, with a yield of 88.33%.

[0126] Example 28: Preparation of compound I-2 (hydroxymethylcebranopadol disodium phosphate).

[0127] 100 g of Cebranopadol, 177.33 g of paraformaldehyde, 6.56 g of DMAP, and 6000 ml of DMF were added to a 1000 ml reaction flask and heated to 50–60 °C for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000 ml of dichloromethane and 500 ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000 ml of methanol, and concentrated to approximately 500 ml. The mixture was then cooled to 0–5 °C for 2 hours to allow crystals to crystallize. After filtration, 69.26 g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0128] 0.50 g of hydroxymethyl cebranopadol, 0.3 g of DMAP, 10 ml of pyridine, and 20 ml of dichloromethane were added to a 100 ml reaction flask. The mixture was cooled to 0–5 °C, and 0.23 g of phosphorus oxychloride was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 1 hour. After the reaction was completed and post-processed, 0.53 g of hydroxymethyl cebranopadol monophosphate was obtained, with a yield of 88.33%.

[0129] 0.5 g of cebranopadol hydroxymethyl monophosphate was added to 10 ml of acetone and 2 ml of water. The pH was adjusted to 7-8 by adding 2N NaHCO3 solution. The mixture was stirred at 0-5℃. After the reaction was completed, 0.41 g of cebranopadol disodium phosphate was obtained after post-treatment, with a yield of 75.93%.

[0130] Example 29: Preparation of compound I-3 (hydroxymethylcebranopadol dimethylglucamine phosphate).

[0131] 100 g of Cebranopadol, 177.33 g of paraformaldehyde, 6.56 g of DMAP, and 6000 ml of DMF were added to a 1000 ml reaction flask and heated to 50–60 °C for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000 ml of dichloromethane and 500 ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000 ml of methanol, and concentrated to approximately 500 ml. The mixture was then cooled to 0–5 °C for 2 hours to allow crystals to crystallize. After filtration, 69.26 g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0132] 0.50 g of hydroxymethyl cebranopadol, 0.3 g of DMAP, 10 ml of pyridine, and 20 ml of dichloromethane were added to a 100 ml reaction flask. The mixture was cooled to 0–5 °C, and 0.23 g of phosphorus oxychloride was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 1 hour. After the reaction was completed and post-processed, 0.53 g of hydroxymethyl cebranopadol monophosphate was obtained, with a yield of 88.33%.

[0133] 0.5 g of cebranopadol hydroxymethyl monophosphate and 0.36 g of meglumine were added to 10 ml of acetone, heated to reflux for 0.5 hours, cooled to 0-5 °C and stirred. After the reaction was completed and post-processed, 0.21 g of cebranopadol dimethyl meglumine phosphate was obtained, with a yield of 23.3%.

[0134] Example 30: Preparation of compound I-4 (hydroxymethylcebranopadol diaminobutyridine phosphate).

[0135] 100 g of Cebranopadol, 177.33 g of paraformaldehyde, 6.56 g of DMAP, and 6000 ml of DMF were added to a 1000 ml reaction flask and heated to 50–60 °C for 24 hours. After post-treatment, 69.26 g of hydroxymethyl Cebranopadol was obtained, with a yield of 64.16%.

[0136] 0.50 g of hydroxymethyl cebranopadol, 0.3 g of DMAP, 10 ml of pyridine, and 20 ml of dichloromethane were added to a 100 ml reaction flask. The mixture was cooled to 0–5 °C, and 0.23 g of phosphorus oxychloride was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 1 hour. After the reaction was completed and post-processed, 0.53 g of hydroxymethyl cebranopadol monophosphate was obtained, with a yield of 88.33%.

[0137] 0.5 g of cebranopadol hydroxymethyl monophosphate and 0.25 g of tromethamine were added to 10 ml of acetone, heated to reflux for 0.5 hours, cooled to 0-5 °C and stirred. After the reaction was completed and post-processed, 0.30 g of cebranopadol hydroxymethyl phosphate diaminobutyrol was obtained, with a yield of 32.6%.

[0138] Example 31: Preparation of compound I-5 (hydroxymethylcebranopadol di-L-arginine phosphate).

[0139] 100 g of Cebranopadol, 177.33 g of paraformaldehyde, 6.56 g of DMAP, and 6000 ml of DMF were added to a 1000 ml reaction flask and heated to 50–60 °C for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000 ml of dichloromethane and 500 ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000 ml of methanol, and concentrated to approximately 500 ml. The mixture was then cooled to 0–5 °C for 2 hours to allow crystals to crystallize. After filtration, 69.26 g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0140] 0.50 g of hydroxymethyl cebranopadol, 0.3 g of DMAP, 10 ml of pyridine, and 20 ml of dichloromethane were added to a 100 ml reaction flask. The mixture was cooled to 0–5 °C, and 0.23 g of phosphorus oxychloride was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 1 hour. After the reaction was completed and post-processed, 0.53 g of hydroxymethyl cebranopadol monophosphate was obtained, with a yield of 88.33%.

[0141] 0.5 g of cebranopadol hydroxymethyl monophosphate and 0.4 g of L-arginine were added to 10 ml of acetone, heated to reflux for 0.5 hours, cooled to 0-5 °C and stirred. After the reaction was completed and post-processed, 0.46 g of cebranopadol phospho-L-arginine was obtained, with a yield of 54.11%.

[0142] Example 32: Preparation of compound I-6 (hydroxymethylcebranopadol di-L-lysine phosphate).

[0143] 100 g of Cebranopadol, 177.33 g of paraformaldehyde, 6.56 g of DMAP, and 6000 ml of DMF were added to a 1000 ml reaction flask and heated to 50–60 °C for 24 hours. After the reaction was completed, the mixture was filtered, and the filtrate was washed three times with 1000 ml of dichloromethane and 500 ml x 3 ml of water. The filtrate was dried over anhydrous sodium sulfate, concentrated, dissolved in 1000 ml of methanol, and concentrated to approximately 500 ml. The mixture was then cooled to 0–5 °C for 2 hours to allow crystals to crystallize. After filtration, 69.26 g of a white solid (hydroxymethyl Cebranopadol) was obtained, with a yield of 64.16%.

[0144] 0.50 g of hydroxymethyl cebranopadol, 0.3 g of DMAP, 10 ml of pyridine, and 20 ml of dichloromethane were added to a 100 ml reaction flask. The mixture was cooled to 0–5 °C, and 0.23 g of phosphorus oxychloride was slowly added dropwise. After the addition was complete, the mixture was heated to room temperature and stirred for 1 hour. After the reaction was completed and post-processed, 0.53 g of hydroxymethyl cebranopadol monophosphate was obtained, with a yield of 88.33%.

[0145] 0.5 g of cebranopadol hydroxymethyl monophosphate and 0.36 g of L-lysine were added to 10 ml of acetone, heated to reflux for 0.5 hours, cooled to 0-5 °C and stirred. After the reaction was completed and post-processed, 0.35 g of cebranopadol di-L-lysine phosphate was obtained, with a yield of 43.75%.

[0146] Test Example 1: Solubility Experiment.

[0147] The present invention and the Cebranopadol compound were added in excess to a sodium phosphate buffer solution at pH 7.4 and shaken continuously at room temperature for 24 h. Each solution was filtered through a 0.45 μm filter, and the solubility of the compound was determined by absolute calibration under the following HPLC conditions.

[0148]

[0149] The results are as follows: , The literature reports the solubility of Cebranopadol compounds as 0.00106 mg / mL, while the actual measured solubility is 0.00101 mg / mL. The solubility of compounds 1 and 7 is significantly increased. Specifically, the solubility of compound I-1 is about 630 times higher than that of Cebranopadol compounds, and the solubility of compound I-7 is about 529 times higher than that of Cebranopadol compounds. The solubility of compounds I-2 to I-6 and I-8 to I-9 are all significantly increased by more than 4,000 times or 43,000 times, showing good drug-like advantages.

[0150] Test Example 2: pKa2 determination of compound I-1.

[0151] In solution, depending on pH, compound I-1 will exist in a diacidic, monobasic, or dibasic state, as represented by the following formula. The pharmaceutical relevance of pKa1 is limited because most physiologically acceptable solutions at these pH values ​​will ensure the ionization of diacidic substances. However, pKa2 is significant given its influence on water solubility, chemical properties, stability, and effectiveness as an enzyme substrate to ensure biotransformation. Therefore, within a pharmaceutically relevant pH range, compound I-1 will exist in a balance between monobasic and dibasic states.

[0152]

[0153] 1. Determination method.

[0154] An isotonic solution of 4 mM compound I-1 was prepared using 10% D2O, and a stock solution of 15 mL was prepared. The pH values ​​were recorded by adding small volumes (μL) of 1N HCl or 1N NaOH solution (using an NMR spectrometer). Twelve to fifteen samples were prepared within the pH range of 3–10. After each pH change, 0.5 mL of the stock solution was transferred to an NMR tube for analysis. The analytical solutions were tested using a 400 MHz NMR spectrometer. 31 p-NMR spectroscopy. Chemical shift changes are recorded as a function of pH. Calibration was performed using 85% H3PO4 as a standard for chemical shifts. 31 P-spectrum.

[0155] The pKa2 of compound I-1 is approximately 6.54, indicating that at a physiological pH of 7.4, compound I-1 will exist primarily as a diion, thus ensuring high solubility.

[0156] Test Example 3: Chemical Stability.

[0157] Following oral administration, compounds I-1 and I-7 are exposed to a wide pH range in the gastrointestinal tract, being acidic in the stomach and neutral in the colon. Furthermore, hydrolytic enzymes are present in the body, such as pepsin and pancreatic enzymes in the gastrointestinal tract. These enzymes have natural digestive functions, serving as digestive agents for large molecules used as nutrients, but they can also bind to and hydrolyze drug compounds. Therefore, compounds I-1 and I-7 must possess a certain degree of stability.

[0158] 1. Stability at different pH levels.

[0159] Following the steps in the 2025 edition of the Chinese Pharmacopoeia, hydrochloric acid solutions with pH 1.2 and phosphate buffer solutions with pH 4.5, 6.8, and 7.4 were prepared and refrigerated for later use. Compounds I-1 and I-7 were accurately weighed and dissolved separately in ultrapure water using ultrasonication to obtain 1.0 mg / mL solutions of compounds I-1 and I-7. 0.1 mL of each (1.0 mg / mL) solution was added to a 10 mL volumetric flask using a pipette, and then diluted with hydrochloric acid and phosphate buffer solutions of different pH values. The volumetric flasks were incubated at 37°C in a water bath, and samples were taken periodically, filtered through a filter membrane, and then injected for analysis. The peak areas of compounds I-1 and I-7 were measured by HPLC, and the degradation rate constants were calculated using a first-order kinetic model.

[0160] 2. Stability in artificial small intestinal fluid and artificial gastric fluid.

[0161] Preparation of artificial gastric juice (containing pepsin): According to the steps in the 2025 edition of the Chinese Pharmacopoeia, accurately measure 1.64 mL of dilute hydrochloric acid and 10 g of pepsin, add water to 1000 mL to obtain artificial gastric juice containing pepsin.

[0162] Preparation of artificial intestinal fluid (containing pancreatic enzyme): Weigh 1.36 g of potassium dihydrogen phosphate according to the 2025 edition of the Chinese Pharmacopoeia, add 100 mL of water to dissolve it, and adjust the pH to 6.8 with 0.1 mol / L NaOH; separately weigh 2 g of pancreatic enzyme, dissolve it in water, mix the two solutions, and add water to 200 mL to obtain the final solution.

[0163] Accurately weigh compounds I-1 and I-7, dissolve them in ultrapure water, and dilute to obtain a 0.2 mg / mL solution of compounds I-1 and I-7 for later use. Dilute the prodrug solution with prepared artificial gastric and small intestinal fluids, following the same steps as above. Calculate the stability of compounds I-1 and I-7 in artificial small intestinal and gastric fluids using HPLC analysis.

[0164] 3. Results.

[0165]

[0166] The results showed that compounds I-1 and I-7 exhibited good preliminary drug-like properties.

[0167] Test Example 4: Determination of Cebranopadol Concentration in Rats by Tail Vein Injection.

[0168] Sodium bicarbonate solutions of compounds I-1 and I-7 at pH 8.5 were prepared to obtain samples containing 0.5 mg of cerebranopadol per ml (0.65 mg / ml for compound I-1 and 0.67 mg / kg for compound I-7). Both solutions were rapidly administered to female rats via the tail vein at a dose of 0.5 mg cerebranopadol per kilogram of body weight. Jugular venous blood was collected periodically under mild ether anesthesia, and serum cerebranopadol concentrations were measured by high-performance liquid chromatography (HPLC). Results are shown below. Figure 9 When compounds I-1 and I-7 were injected, Cebranopadol was detected in vivo in 3 minutes after the injection of compound I-1, and in 7 minutes after the injection of compound I-7, indicating that compounds I-1 and I-7 can be rapidly converted into Cebranopadol.

[0169] Test Example 5: In vivo pharmacokinetic study.

[0170] 1. In vivo high performance liquid chromatography analysis method.

[0171] The HPLC method is the same as that used in Example 19.

[0172] 2. Preparation of plasma samples.

[0173] Blank plasma sample: Take blank plasma from rats, thaw it, and accurately measure 100 µL into a 1.5 mL centrifuge tube. Add 700 µL of 4% glacial acetic acid methanol, vortex for 10 min, and centrifuge at 9000 rpm for 10 min to precipitate proteins. Transfer the supernatant to a 1.5 mL centrifuge tube and evaporate to dryness at 40 °C. Add 50 µL of 50% acetonitrile to the residue to reconstitute it, vortex for 6 min, sonicate for 15 min, and centrifuge at 13000 rpm for 20 min. The supernatant is the blank plasma sample solution.

[0174] Plasma samples after drug administration: After thawing, accurately measure 100 µL of rat plasma into a 1.5 mL centrifuge tube. Add 10 µL of internal standard (pentazocine 1 μg / mL, dissolved in 50% acetonitrile), and vortex for 1 min to mix thoroughly. Add 700 µL of 4% glacial acetic acid methanol, vortex for 5 min, and centrifuge at 9000 rpm for 1 min to precipitate proteins. Transfer the supernatant to a 1.5 mL centrifuge tube and evaporate to dryness at 40°C. Redissolve the residue in 50 µL of 50% acetonitrile, vortex for 6 min, and sonicate for 15 min to ensure complete drug dissolution. Centrifuge at 13000 rpm for 20 min. The supernatant is the plasma sample solution after drug administration.

[0175] 3. Dosing regimen and sample collection.

[0176] Twenty-four rats were randomly divided into four groups of six each: an intravenous injection group of Cebranopadol (10% Tween 80 aqueous solution), an oral administration group of Cebranopadol (0.5% CMC-Na aqueous suspension), an oral administration group of compound I-1 (0.5% CMC-Na aqueous suspension), and an oral administration group of compound I-7 (0.5% CMC-Na aqueous suspension). The dosages were 46 mg / kg for Cebranopadol, 59 mg / kg for compound I-1, and 61.8 mg / kg for compound I-7, with equimolar doses of Cebranopadol, compound I-1, and compound I-7. Blood samples were collected from the eyes at 0.09, 0.25, 0.5, 0.75, 2, 4, 6, 8, and 12 hours after administration. Whole blood was centrifuged at 4000 rpm for 15 min, and the supernatant plasma was used for HPLC analysis to determine the drug content.

[0177] 4. Plasma sample testing and data processing.

[0178] Plasma samples were collected after drug administration and processed according to the method described in Section 2, "Preparation of Plasma Samples." The samples were injected under the chromatographic conditions described in Section 1, "In Vivo High Performance Liquid Chromatography Analysis Method." The peak area ratio of the active pharmaceutical ingredient to the internal standard peak was substituted into the standard curve to calculate the drug concentration. The results were processed using Phoenix software to calculate the relevant pharmacokinetic parameters.

[0179] 5. Results.

[0180]

[0181] Conclusion: The main pharmacokinetic parameters and plasma concentration-time curves of Cebranopadol after gavage administration to Kunming mice were compared with those of oral Cebranopadol. The areas under the curve (AUC) of compounds I-1 and I-7 after gavage administration of Cebranopadol to Kunming mice were significantly different. 0-∞ The bioavailability of compounds I-7 and I-1 was significantly improved compared to oral Cebranopadol. The data indicate that the oral bioavailability of this invention is significantly improved compared to oral Cebranopadol. In this iso-dose pharmacokinetic experiment, the pharmacokinetic parameters of compounds I-1 and I-7 were significantly improved compared to Cebranopadol, demonstrating promising prospects for further research.

[0182] Test Example 6: Toxicity detection of Caco-2 cells.

[0183] The Caco-2 cell line, derived from human colon cancer, can spontaneously differentiate into epithelial cells under appropriate culture conditions, forming absorptive intestinal cells with typical morphology. Their morphology and permeability are similar to intestinal epithelial cells, and they express various active transport proteins, including amino acid transporters and P-gp efflux pump proteins. This model effectively simulates in vivo intestinal tissue absorption and is now widely used in studies of oral drug absorption. The MTT assay is a commonly used method to detect the influence of samples on cell survival and growth. MTT is a yellow dye, its full name being 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide. Its detection principle is that succinate dehydrogenase present in the mitochondria of living cells can reduce MTT to formazan, a blue-purple crystal that is insoluble in water and deposits in the cell; dead cells do not have this function. Formazan is dissolved in DMSO solution, and then its absorbance is measured using an enzyme-linked immunosorbent assay (ELISA) reader at 490 nm or 570 nm. Within a certain cellular range, the number of living cells is directly proportional to the amount of formazan formed. The experimental group's viable cell count can be indirectly reflected by comparing it with the control group's values. The specific experimental steps are as follows: 1) Take Caco-2 cells that are in normal growth stage and in the logarithmic growth phase, digest them with trypsin to form a single cell suspension, and adjust the concentration of the Caco-2 cell suspension to 1×10⁻⁶. 4 cell / mL; 2) Spread the cell suspension evenly into a 96 cell culture plate, add 100 μL to each well, and fill the edge wells with 100 μL of PBS buffer. Incubate the plate at 37°C in a 5% CO2 cell culture incubator. 3) After 24 hours of culture, observe the cell state under a microscope; the cells show uniform monolayer growth. Carefully aspirate the old culture medium from each well and add different concentrations of Cebranopadol and its compounds I-1 and I-7 to each well. The final concentrations of Cebranopadol and its compounds I-1 and I-7 in each experimental group were 1.92, 15.47, 61.75, 123.5, 247, and 494 μmol / L, respectively. Each concentration group had three replicates. Control wells (containing an equal volume of Caco-2 cell suspension, without drug administration) and zeroing wells (containing no cells, no drug administration, and 100 μL of an equal volume of culture medium) were also included. The cells were then placed in a cell culture incubator for further culture. 4) After culturing for 4 hours, discard the old culture medium, add fresh culture medium, and add 20 μL of MTT solution (5 mg / mL) to each well of the 96-well plate under dark conditions, and incubate in an incubator. 5) After incubating for 4 h, gently remove the 96-well plate from the incubator, carefully aspirate the liquid from each well, add 150 μL of DMSO solution, and shake on a shaker at 37 °C for 10–15 min to completely dissolve the blue-purple crystals. 6) Use an ELISA reader to detect the absorbance (OD) value of each well at a wavelength of 490 nm, and calculate the cell inhibition rate of each experimental group.

[0184] The results showed that after 4 hours of treatment with Cebranopadol and its compounds I-1 and I-7 at concentrations of 1.92, 15.47, 61.75, 123.5, 247, and 494 μmol / L, the survival rate of Caco-2 cells remained above 90%, indicating that the drugs had almost no toxic effects on the cells within this uptake time and concentration range.

[0185] Test Example 7: Experimental Study on In Vitro Cytotoxicity of Normal Human Hepatocytes 1. Experimental materials.

[0186] 1.1 Cells: LO2 cells, a human hepatitis cell line 1.2 Drug: Compound of Example 11 of this invention, HPLC purity 99.54% 1.3 Reagents and Instruments: Modified RPMI-1640 culture medium, penicillin-streptomycin solution, 0.25% trypsin-EDTA, fetal bovine serum, MTT, dimethyl sulfoxide, CO-150 carbon monoxide incubator, SW-CJ-2F medical clean bench, CKX-41-32 inverted microscope, CU600 electric thermostatic water bath, RT-2100C enzyme-linked immunosorbent assay (ELISA) analyzer. 2. Experimental methods.

[0187] 2.1 Reagent preparation.

[0188] 2.1.1 Preparation of MTT: Weigh 0.25g of MTT using a precision balance and place it in a 50mL volumetric flask. Add an appropriate amount of PBS, incubate in a 50-60℃ water bath, and shake well to dissolve completely. Add PBS to the mark to prepare a 5mg / kg solution. Filter the solution through a 0.22μm microporous membrane for sterilization, aliquot, and store in a refrigerator at 4℃ protected from light.

[0189] 2.1.2 Preparation of cell cryopreservation solution: Mix 20% serum, 10% DMSO and 70% 1640 medium evenly and store at -20℃.

[0190] 2.1.3 The preparation of the representative compounds I-1 and I-7 of this invention uses DMSO to prepare the stock solution of the drug, and then dilutes it with culture medium to the concentration of the drug to be used. The final concentration of DMSO is controlled at ≤0.1%.

[0191] 2.2 LO2 cell culture: Normal human LO2 cells were placed in a 25cm² culture medium. 2Add approximately 4–5 mL of RPMI-1640 culture medium containing 10% FBS to cell culture flasks and incubate at 37°C in a 5% CO2 saturated humidity cell culture incubator. Change the culture medium every 2 days and observe cell growth daily. Once cells reach 80% confluence, passage or cryopreserve them. Use cells from passages 5–7 for formal experiments.

[0192] 2.3 Grouping and Dosing Experiments: The experiment was divided into a normal cell control group and different concentrations of the drug group of the present invention, based on the preliminary experimental results. The concentrations were 10.0, 100, 200, 1000, 2000, and 10000 μmol / L.

[0193] 2.4 Hepatocyte MTT assay: Logarithmic growth phase LO2 cells were prepared into a cell suspension of 5.0 × 10³ cells / mL and seeded into 96-well plates. A normal control group and groups treated with different concentrations of the compound of the present invention (0.65, 6.5, 13.0, 65, 130, and 650 μmol / L) were included. After 24 h of culture, the culture medium was aspirated, and the cells were washed 2–3 times with PBS. Different concentrations of the compound of the present invention were added, with 8 replicates per concentration. The plates were incubated at 37°C in a 5% CO₂ incubator. After 24, 48, and 72 h of culture, MTT solution was added at each time point, and the plates were incubated in the dark for 4 h. After 4 h, the supernatant was aspirated, and 150 L DMSO was added to each well to dissolve the thiazolyl blue crystals. The plates were gently shaken to ensure uniform dissolution. The absorbance of each well was measured at 490 nm using a microplate reader. The absorbance value of each well directly reflects the number of cells. The experiment was repeated three times. The cell viability rate was calculated as follows: Cell viability rate (%) = Absorbance of each group (OD490) × 100 / Absorbance of the control group (OD490).

[0194] 2.5 Statistical Analysis All data are expressed as mean ± standard deviation and were processed using SPSS 17.0 statistical software. t-tests were performed for statistical analysis; paired t-tests were used for self-comparisons, and unpaired t-tests were used for inter-group comparisons. The significance level was P < 0.05.

[0195] 3. Experimental results.

[0196]

[0197] Conclusion: After 72 h of administration, the OD values ​​of cells in each group showed an increasing trend at different concentrations of the present invention, but the growth rate decreased, indicating that the cells were still in the growth stage and the growth rate was somewhat inhibited. The typical representative compounds I-1 and I-4, at different concentrations, showed no inhibitory effect on cells. Compared with the normal group, the cell survival rate was greater than 90%. Within the range of 0.65, 6.5, 13.0, 65, 130, and 650 μmol / L, there was almost no inhibition of cell growth and no cytotoxicity.

[0198] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof, characterized in that, The compound has a structure as described in Formula I: 。 2. The compound of claim 1 of formula I or a pharmaceutically acceptable salt thereof, wherein The compound is a compound as described in Formula II-a or a compound as described in Formula II-b: ， wherein n1 is selected from an integer of 1 to 8, preferably n1 is selected from an integer of 1 to 5, preferably n1 is selected from an integer of 1 to 3.

3. A compound of formula I according to one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, wherein The compound as described in Formula I or a pharmaceutically acceptable salt thereof is any one of the following compounds: 。 4. A pharmaceutical composition comprising the compound as described in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

5. Use of the compound as described in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or the composition as described in claim 4 for the manufacture of a medicament for the treatment of pain.

6. The use as described in claim 5, wherein the pain refers to one or more of chronic pain, nociceptive pain, neuropathic pain, malignant pain or inflammatory pain.

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