A scopolamine derivative and a method for preparing the same

Abstract

This invention discloses a hyoscyamine derivative and its preparation method, relating to the field of drug synthesis, and aims to provide an effective and safe drug molecule for inhalation / local pulmonary administration. The invention prepares the hyoscyamine derivative by introducing a linear polyethylene glycol chain containing seven ethylene glycol repeating units at the hydroxyl position of hyoscyamine and quaternizing its nitrogen atom. This derivative is specifically designed for inhalation / local pulmonary administration. Through the above structural modification, it achieves reduced systemic absorption and central penetration of the compound, increased local lung exposure, and prolonged pulmonary retention time, while also maintaining good nebulization performance.

Description

Technical Field

[0001] This invention relates to the field of drug synthesis, specifically to a hyoscyamine derivative and its preparation method. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD), asthma, and allergic respiratory diseases are among the most prevalent chronic diseases globally. Clinical treatment typically relies on bronchodilators to quickly or continuously relieve dyspnea. Anticholinergic bronchodilators are an important class of drugs currently used clinically. Their mechanism of action involves blocking M3 cholinergic receptors, thereby inhibiting airway smooth muscle contraction and reducing mucus secretion. Representative drugs in this class include atropine, hyoscine, and their structurally modified derivatives. To improve the safety of anticholinergic drugs and reduce their crossing of the blood-brain barrier (BBB), the current mainstream strategy is quaternization, which permanently positively charges the tertiary amine nitrogen, making it difficult for it to enter the central nervous system. However, existing quaternized anticholinergic drugs still have shortcomings: limited lung retention time; limited solubility of some drugs in inhaled solutions; and limited means of improving mucosal adhesion.

[0003] In recent years, studies have proposed prolonging pulmonary retention by altering the hydrophilicity, hydratability, and adhesiveness of molecules. However, these studies mainly focus on polymeric carriers, micelle systems, or nanoparticle systems, and lack direct modification strategies targeting the bulk structure of small-molecule anticholinergic drugs. Regarding hyoscyamine and its derivatives, various structural modification strategies have been reported in patents, such as quaternization, esterification, and chiral resolution. However, no technical solution has yet been found that directly introduces short-chain PEG onto the hyoscyamine molecule and quaternizes it to simultaneously enhance water solubility and pulmonary adhesion while avoiding a significant increase in molecular weight.

[0004] Based on research on anticholinergic drugs, existing technology WO2018 / 154597A1 discloses a synthetic method for a class of anticholinergic drugs, with the representative product being the quaternary ammonium anticholinergic agent glycopyrronium bromide. This technology constructs a hyoscyamine core and quaternizes it on the nitrogen atom, introducing substituted alkyl or cycloalkyl side chains to improve receptor selectivity, reduce central penetration, and enhance the salt-forming stability of the compound. While these quaternized derivatives offer significant advantages in terms of safety, they lack structural means to enhance lung adhesion, have limited local airway retention time, insufficient hydration capacity and hydrophilicity, and the performance of inhaled solution formulations has not been specifically optimized.

[0005] In summary, designing a hyoscyamine derivative anticholinergic drug that improves water solubility, moderately enhances adhesion, and prolongs local retention time, while also possessing low BBB permeability and good nebulization performance, has become a pressing technical problem to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a hyoscyamine derivative and its preparation method. By performing the above-mentioned dual structural modification on the hyoscyamine molecule, a long-acting local bronchodilator with superior physicochemical properties and suitable for inhalation administration is obtained, providing a safer and more effective treatment option for respiratory diseases such as asthma and COPD, and overcoming the potential risks of limited lung retention time and central nervous system side effects of existing inhaled anticholinergic drugs.

[0007] The specific technical solution is as follows:

[0008] A hyoscyamine derivative, based on the hyoscyamine skeleton structure, is covalently modified, characterized in that the covalent modification involves covalently binding a polyether polymer segment to the hydroxyl position of the hyoscyamine skeleton, and simultaneously quaternizing the nitrogen atoms of the hyoscyamine skeleton.

[0009] Furthermore, the hyoscyamine derivative is m-(PEG7)-hyoscyamine. It is a quaternary ammonium salt bromide containing a linear PEG7 chain; the linear PEG7 chain is a linear polymer chain containing 7 ethylene glycol repeating units; the quaternary ammonium salt is formed by methylation of the nitrogen atom of scopolamine to form an N,N-dimethyl quaternary ammonium cation.

[0010] Furthermore, the structural formula of the hyoscyamine derivative is shown below:

[0011] .

[0012] Furthermore, the method for preparing the hyoscyamine derivative includes the following steps:

[0013] S1: Hyoscyamine was etherified with PEG reagent. After the etherification reaction was completed, the intermediate m-(PEG7)-hyoscyamine was obtained through post-processing, purification and drying.

[0014] S2: The nitrogen atom of the m-(PEG7)-hyoscyamine intermediate prepared in S1 is subjected to a methylation reaction, introducing a methyl group to form an N,N-dimethyl quaternary ammonium cation and then a bromide. After the methylation reaction is completed, the crude product is obtained through post-processing, followed by pulping purification and drying to finally obtain m-(PEG7)-hyoscyamine. .

[0015] Further, the etherification reaction described in step S1 is as follows: First, 1.0 molar equivalent of hyoscyamine is dissolved in DMF to prepare a solution with a concentration of 0.1 g / mL. The solution is stirred and the temperature is lowered to 0°C under nitrogen protection. 1.2 molar equivalent of a strong base is added to the reaction system, and the reaction temperature is kept below 20°C. After the addition is complete, the reaction mixture is stirred at room temperature for 30 minutes. The reaction system is then cooled to 0°C, and a PEG reagent / DMF solution with a concentration of 0.2 g / mL is added dropwise, wherein the PEG reagent is 1.1 molar equivalent. The temperature is kept below 5°C. After the addition is complete, the temperature is raised to 35°C, and the reaction is stirred at this temperature for 12 hours until the reaction is complete.

[0016] Furthermore, the PEG reagent is methyl-heptaethylene glycol-brominated; the strong base is sodium hydride, used to activate the hydroxyl groups on hyoscyamine.

[0017] Furthermore, the specific steps of the methylation reaction in step S2 include: dissolving 1.0 molar equivalent of the m-(PEG7)-hyoscyamine intermediate prepared in step S1 into DMF to prepare a solution with a concentration of 0.1 g / mL, stirring, and lowering the temperature of the reaction solution to -10°C under nitrogen protection, adding the methylation reagent, raising the temperature of the reaction system to 50°C, and continuing the reaction at this temperature for 10 hours until the reaction is completed.

[0018] Furthermore, the methylating agent is gaseous bromomethane, which needs to be introduced into the reaction system. The gas introduction time is controlled at 2 hours, during which the temperature of the reaction system is kept below -5°C.

[0019] Furthermore, the specific steps of the post-processing described in step S2 include: cooling the reaction system to 10°C, stirring for 30 minutes, filtering, washing the filter cake three times with acetonitrile to remove unreacted quaternizing reagents and impurities, collecting the filter cake, and drying it under reduced pressure to constant weight.

[0020] Furthermore, the specific method for pulping and purification in step S2 is as follows: the crude product is dispersed in ethyl acetate, stirred at room temperature for 2 hours, then stirred at 10°C for 30 minutes, and filtered to obtain the pure product.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) This invention significantly improves the hydrophilicity of the molecule by grafting a linear PEG7 chain onto it, endowing it with moderate hydration capacity and interaction with the biological mucus layer or epithelial cell surface. After inhalation, the PEG7 chain helps the drug molecule adhere better to the airway mucus layer and epithelial cell surface, making it less likely to be rapidly removed by the mucociliary clearance mechanism. Therefore, the effective concentration of the drug in the lungs is maintained for a significantly longer period, which helps to achieve a more sustained bronchodilatory effect, thereby reducing the number of daily doses, improving patient compliance, and reducing the total amount of drug entering the systemic blood circulation, further reducing systemic adverse reactions.

[0023] (2) This invention introduces a permanently positively charged N,N-dimethyl quaternary ammonium group onto the nitrogen atom of the tertiary amine of hyoscyamine, thereby increasing the molecular polarity and almost eliminating its ability to penetrate the BBB via passive diffusion. This design is specifically intended to reduce the amount of drug entering the central nervous system after inhalation, thereby significantly reducing the risk of centrally acting anticholinergic side effects.

[0024] (3) The compound of this invention maintains good affinity for M3 muscarinic receptors, ensuring the pharmacological effect of bronchodilation. The compound has excellent water solubility and can be stably dissolved in conventional inhalation solvents. It is not prone to crystallization or turbidity, making it suitable for development into nebulized inhalation solutions and related inhalation formulations. Furthermore, the quaternary ammonium salt form is selected as bromide as the para-anion, giving the resulting salt form clear crystal characteristics, good thermal stability, and controllable process reproducibility. This facilitates the preparation of high-purity active pharmaceutical ingredients and helps improve the quality consistency and long-term storage stability of the formulation. Attached Figure Description

[0025] Figure 1 This is a synthetic route diagram of a hyoscyamine derivative of the present invention;

[0026] Figure 2 The mass spectrum of the m-(PEG7)-hyoscyamine intermediate prepared in Example 1 of this invention;

[0027] Figure 3 m-(PEG7)-hyoscyamine prepared in Example 1 of this invention The mass spectrum;

[0028] Figure 4 The 1H NMR spectrum of the m-(PEG7)-hyoscyamine intermediate prepared in Example 1 of this invention;

[0029] Figure 5 m-(PEG7)-hyoscyamine prepared in Example 1 of this invention The proton NMR spectrum;

[0030] Figure 6 m-(PEG7)-hyoscyamine prepared in Example 1 of this invention The high performance liquid chromatogram. Detailed Implementation

[0031] The following embodiments further explain and illustrate the technical solutions of the present invention. It should be specifically noted that each specific embodiment is a concretization and explanation of the technical solution and should not be considered as a limitation on the scope of protection of the present invention. Those skilled in the art still have the right to modify the technical solutions of these embodiments and make equivalent substitutions for some or all of the technical features, and these modifications or substitutions do not change the essence of the corresponding technical solutions, nor do they cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions described in the present invention.

[0032] The core hyoscyamine skeleton in the compound of this invention can be natural hyoscyamine, and the skeleton completely retains the pharmacologically active groups that antagonize muscarinic receptors, especially the M3 receptor, thereby ensuring the compound's effective affinity for the target receptor. By covalently linking a short-chain PEG, the surface of the compound of this invention is enhanced with hydrophilicity and hydration capacity, and its interaction with airway mucus / biological proteins is strengthened, thereby prolonging its residence time on the pulmonary mucosa. Simultaneously, quaternization gives the compound a stable positive charge, increasing its polarity and making it virtually impermeable to the blood-brain barrier. Therefore, after systemic absorption, it is less likely to enter the central nervous system, significantly reducing potential central nervous system side effects. (See attached...) Figure 1 The diagram shows a synthetic route for a hyoscyamine derivative according to the present invention, which involves the following main steps to synthesize the target product:

[0033] S1: Etherification reaction

[0034] The hydroxyl group of hyoscyamine is a reactive nucleophilic group that can undergo a Williamson ether synthesis reaction with halogenated PEG reagents under basic catalysis to form a stable ether bond. The ether bond is chosen because it has high chemical stability and is not easily hydrolyzed in vivo, thus ensuring the persistence of the PEG7 chain in the lungs.

[0035] Starting material: hyoscyamine.

[0036] PEG reagent: Methyl-heptaethylene glycol-brominated (CH3(OCH2CH2)7Br).

[0037] Base: The hydroxyl group on hyoscyamine requires a strong base to activate it, converting it into a stronger nucleophile. Sodium hydride (NaH) is preferred as a strong base because it is anhydrous and the reaction byproduct is hydrogen gas, which is easy to remove.

[0038] Solvent: A nonprotic polar solvent is required, preferably N,N-dimethylformamide (DMF). DMF has a high dielectric constant and good solubility for ionic reactants, which is beneficial for the etherification reaction. All solvents must be strictly anhydrous to prevent water in the solvent from reacting with NaH.

[0039] Reaction apparatus: three-necked reaction flask; magnetic stirrer; oil bath; thermometer.

[0040] Reaction Procedure: First, dissolve 1.0 mol equivalent of hyoscyamine in DMF to a concentration of 0.1 g / mL. Place the solution in a three-necked reaction flask equipped with a thermometer. Start stirring, purge with nitrogen three times, and then apply nitrogen protection to lower the reaction solution temperature to 0°C. Add 1.2 mol equivalents of NaH to the reaction system in portions, maintaining the reaction temperature below 20°C. Too low a temperature results in a slow reaction rate and incomplete substrate activation; too high a temperature leads to a violent reaction, generating a large amount of gas and potentially causing an explosion. After the NaH addition is complete, stir the reaction mixture at room temperature for 30 minutes. Cool the reaction system to 0°C. Prepare a 0.2 g / mL PEG reagent / DMF solution, containing 1.1 mol equivalents of PEG reagent. Add the prepared PEG reagent / DMF solution dropwise to the reaction system, maintaining the temperature below 5°C to prevent side reactions. After the addition is complete, heat the reaction system to 35°C using an oil bath and stir at this temperature for 12 hours until the reaction is complete.

[0041] Post-processing: After the reaction was completed, the system temperature was lowered to 0°C, and a saturated ammonium chloride aqueous solution was slowly added to quench unreacted NaH. The target product was then extracted with ethyl acetate (EtOAc). The organic phase was washed with saturated sodium bicarbonate solution, water, and brine, dried with anhydrous sodium sulfate (Na2SO4), and the solvent was concentrated under reduced pressure using a rotary evaporator to obtain crude m-(PEG7)-hyoscyamine intermediate.

[0042] Purification: The intermediate was purified by silica gel column chromatography with dichloromethane / methanol as the eluent and the elution gradient was 20:1-10:1 by volume to finally obtain m-(PEG7)-hyoscyamine intermediate.

[0043] S2: Methylation reaction

[0044] The lone pair electrons on the nitrogen atom of the m-(PEG7)-hyoscyamine intermediate prepared by S1 can act as a nucleophilic attack methylating agent to form an N,N-dimethyl quaternary ammonium salt structure with a permanent positive charge. Since hyoscyamine itself already has an N-CH3 group, an additional methyl group needs to be introduced to convert it into an N,N-dimethyl quaternary ammonium salt.

[0045] Quaternizing reagent: Bromomethane (BrCH3) is preferred as the methylating reagent. Bromomethane has high reactivity and can directly introduce methyl groups and serve as a bromide ion precursor.

[0046] Reaction solvent: DMF is preferred. The reaction needs to be carried out in an aprotic polar solvent to dissolve the quaternary ammonium salt and promote nucleophilic attack. The solvent needs to be dry to avoid hydrolysis of the quaternizing agent.

[0047] Reaction apparatus: three-necked reaction flask; reflux condenser; magnetic stirrer; oil bath; thermometer.

[0048] Reaction procedure: Dissolve 1.0 molar equivalent of m-(PEG7)-hyoscyamine intermediate in DMF to prepare a solution with a concentration of 0.1 g / mL. Place the solution in a three-necked reaction flask equipped with a reflux condenser and a thermometer. Turn on the stirrer, purge the solution with nitrogen three times, and then apply nitrogen protection. Lower the temperature of the reaction solution to -10°C. Introduce 2.5 molar equivalents of BrCH3 into the reaction system for 2 hours, keeping the temperature below -5°C to prevent loss of BrCH3 gas. After the aeration is complete, heat the reaction system to 50°C in an oil bath and continue the reaction at this temperature for 10 hours until the reaction is complete.

[0049] Post-processing: Due to the low solubility of the quaternary ammonium salt product in organic solvents, the reaction system was cooled to 10°C after the reaction was completed. This temperature ensured that most of the product precipitated while impurities did not. The mixture was stirred for another 30 minutes, filtered, and the filter cake was washed three times with acetonitrile to remove unreacted quaternizing reagents and impurities. The filter cake was collected and dried under reduced pressure to constant weight to obtain m-(PEG7)-hyoscyamine. Crude product. The reduced pressure rotary evaporation drying process removes any remaining BrCH3 gas.

[0050] Purification: The obtained m-(PEG7)-hyoscyamine The crude product was placed in a reaction flask, and ethyl acetate, which was 10 times the mass of the crude product, was added. The mixture was stirred at room temperature for 2 hours, then cooled to 10°C and stirred for 30 minutes. The mixture was then filtered, and the filter cake was washed three times with ethyl acetate. The filter cake was collected and dried in a vacuum oven at 60°C to constant weight to finally obtain the target product.

[0051] Table 1. Reagents used in the following examples and comparative examples.

[0052]

[0053] Product testing:

[0054] 1. Nuclear magnetic resonance hydrogen spectrum (NMR) 1 H-NMR)

[0055] (1) Sample preparation: Dissolve the sample in a deuterated reagent at a concentration of 10 mg / mL and place it in an NMR tube.

[0056] (2) Instrument parameter settings: Bruker 500MHz nuclear magnetic resonance spectrometer; resonance frequency of 500.13MHz; spectral width of 20ppm; number of sampling points of 65536; relaxation delay of 5 seconds; 64 scans; temperature of 298K.

[0057] 2. Liquid chromatography-mass spectrometry (LC-MS)

[0058] (1) Sample preparation and injection: Take 1 mg of sample and dilute and dissolve it with 10 mL of methanol. Injection volume: 1 μL.

[0059] (2) Column and mobile phase system: reversed-phase column C18; mobile phase: pure water: acetonitrile: methanol = 50:25:25 (v / v). Flow rate 1.0 mL / min

[0060] (3) The mass spectrometry is in positive ion mode (ESI+).

[0061] 3. High-performance liquid chromatography (HPLC)

[0062] (1) Sample preparation and injection: Take 1 mg of sample and dilute and dissolve it with 10 mL of pure water. Injection volume: 1 μL.

[0063] (2) Chromatographic column and mobile phase system: reversed-phase column C18; mobile phase: phase A is pure water, phase B is acetonitrile, elution gradient from 95%A:5%B to 5%A:95%B by volume percentage. Flow rate 1.0 mL / min.

[0064] (3) The absorption peak was detected at 220 nm.

[0065] Example 1: m-(PEG7)-hyoscyamine-N + (CH3)2Br - Synthesis

[0066] S1: Introduction of short-chain polyethylene glycol

[0067] Hyoscyamine (2.9 g, 10 mmol) was dissolved in 29 mL of anhydrous DMF and placed in a three-necked reaction flask. Stirring was started, and the mixture was purged with nitrogen three times, followed by nitrogen protection. The reaction solution temperature was lowered to 0°C. NaH (60 wt%, 0.48 g, 12 mmol) was added in five portions, maintaining a temperature not exceeding 20°C. After the NaH addition was complete, the reaction mixture was stirred at room temperature for 30 minutes until the system became clear. The reaction system was then cooled to 0°C, and a DMF solution of CH3(OCH2CH2)7Br (4.4 g, 11 mmol) was added dropwise over 30 minutes, maintaining a temperature not exceeding 5°C. After the addition was complete, the reaction mixture was heated to 35°C and stirred for 12 hours. After the reaction was complete, the reaction solution was cooled to 0°C in an ice bath, and 20 mL of saturated ammonium chloride aqueous solution was slowly added to quench excess NaH. Extracted three times with EtOAc, 50 mL each time, and the organic phases were combined. The organic phase was washed twice each with saturated sodium bicarbonate solution, water, and saturated brine, 30 mL each time. The organic phase was collected, dried over anhydrous Na₂SO₄, filtered, and the solvent was concentrated under reduced pressure to obtain a yellow oily crude product. The crude product was purified by silica gel column chromatography with an eluent volume ratio of dichloromethane:methanol = 20:1-10:1, yielding 4.6 g of m-(PEG7)-hyoscyamine intermediate, 75% yield, LC-MS (ESI+): m / z = 612.5, see appendix. Figure 2 ; 1 H-NMR (CDCl3): See appendix Figure 4 .

[0068] S2: Preparation of Quaternary Ammonium Salts

[0069] The m-(PEG7)-hyoscyamine intermediate (3.06 g, 5 mmol) prepared in S1 was dissolved in 50 mL of dry DMF. Stirring was started, and after three purgings with nitrogen, the reaction flask was placed in an ice-salt bath at -10°C. BrCH3 (1.2 g, 12.5 mmol) was bubbled into the reaction system for 2 hours, with the temperature controlled to not exceed -5°C. After bubbling, the reaction system was heated to 50°C in an oil bath and the reaction continued for 10 hours. After the reaction was complete, the reaction system was cooled to 10°C, stirred for 30 minutes, filtered, and the filter cake was washed three times with 10 mL of acetonitrile each time. The filter cake was collected and dried under reduced pressure to constant weight to obtain m-(PEG7)-hyoscyamine-N + (CH3)2Br - 4.5 g of crude product. The crude product was thoroughly dispersed in 45 mL of EtOAc and homogenized at room temperature for 2 hours. The mixture was filtered, and the filter cake was washed three times with ethyl acetate. The filter cake was collected and dried to constant weight in a vacuum oven at 60°C to obtain 2.5 g of the target product m-(PEG7)-hyoscyamine-N.+ (CH3)2Br - Yield 71%, LC-MS (ESI+): m / z = 707.4, see appendix. Figure 3 ; 1 H-NMR (D2O): See appendix Figure 5 HPLC: RT 4.719 min, see appendix Figure 6 .

[0070] Comparative Example 1: Commercially available hyoscyamine was used directly. This is the parent compound of the present invention, which has a tertiary amine structure and is easily permeable to the BBB.

[0071] Comparative Example 2: Ipratropium Bromide, a commercially available drug, was used. This is a traditional quaternized anticholinergic drug.

[0072] Comparative Example 3: Synthesis of m-(PEG7)-hyoscyamine. This comparative example simulates the structure with only PEG7 modification but without quaternization, aiming to evaluate the effect of PEGylation itself on BBB permeability. m-(PEG7)-hyoscyamine was prepared in the same manner as in Example 1.

[0073] Comparative Example 4: Hyoscyamine The synthesis of this comparative example simulates the structure with only quaternization but without PEG7 modification, aiming to evaluate the effect of quaternization on pulmonary retention. Similar to Example 1, except that quaternization was performed directly using hyoscyamine as a substrate to prepare hyoscyamine. .

[0074] The following tests were conducted with reference to the Pharmacopoeia of the People's Republic of China: 2020 Edition.

[0075] Physicochemical property determination

[0076] 1. Water solubility determination:

[0077] Method: Weigh 10 mg of sample and add it to 10 mL of ultrapure water. Stir at a constant 25°C for 24 hours to reach equilibrium. After reaching equilibrium, centrifuge the sample at 10000 rpm for 10 minutes, collect the supernatant, filter and dilute it, and then inject it into the HPLC to determine the concentration of the sample in mg / mL.

[0078] 2. Determination of the distribution coefficient (LogP)

[0079] Method: Dissolve 5 mg of sample in 5 mL of phosphate buffer (pH 7.4), add 5 mL of n-octanol, and shake the mixture at 150 rpm for 24 hours at 25°C. After equilibration, shake thoroughly and allow to separate into layers. Take samples from the aqueous and organic phases separately, and determine the drug concentration by HPLC. The LogP value is obtained by taking the logarithm of the ratio of drug concentration in the organic phase to that in the aqueous phase.

[0080] Atomization performance test

[0081] Sample preparation: The compound of the present invention was prepared into an inhalation solution that simulates clinical use, with a concentration range of 2.5 mg / mL.

[0082] Nebulizer: Clinical nebulizer.

[0083] Particle size determination: The nebulizer is connected to the cascade impactor. After nebulization for a certain period of time, the drug at different levels of the cascade impactor is collected. The drug content at each level is quantified by HPLC. Based on the cumulative mass distribution of each level, the particle size corresponding to 50% of the cumulative mass is found on the logarithmic coordinates corresponding to the particle size. This is the mass median aerodynamic diameter (MMAD), in μm. Each sample is measured in parallel 3 times.

[0084] Pharmacokinetic test

[0085] 1. Blood-brain barrier (BBB) ​​permeability test

[0086] Methods: A hCMEC / D3 human brain microvascular endothelial cell monolayer model was constructed using a Transwell culture system. The compound was dissolved in culture medium on the simulated blood side at concentrations of 1-10 μmol / L and added to the upper chamber. The mixture was incubated at 37°C for 3 hours. Samples were taken from the lower chamber at different time points, and drug concentrations were determined by HPLC. The apparent permeability coefficient was calculated using the formula: Papp(A→B)=(dQ / dt) / (A×C0), where dQ / dt is the steady-state permeation rate in mol / s; and A is the effective membrane area of ​​the Transwell in cm². 2 C0 represents the initial concentration of the drug in the upper chamber, in mol / cm³. 3 Papp(A→B) unit 10 -6 cm / s. Simultaneously measure the reverse osmosis Papp(B→A) and calculate the efflux ratio = Papp(B→A) / Papp(A→B).

[0087] 2. Local retention time in the lungs

[0088] Methods: In a rabbit model, non-invasive inhalation administration was used. Animals were sacrificed at 0.5h, 1h, 2h, 4h, 8h, 12h, and 24h after administration, and lung tissue was collected and homogenized. The drug concentration in the lung tissue was determined by HPLC. Lung concentration-time curves were plotted, and the mean retention time (MRT) in the lungs was calculated in hours.

[0089] 3. In vitro affinity for M3 muscarinic receptors

[0090] Radioligand binding assay: CHO cells expressing human M3 muscarinic receptors were used. Different concentrations of the test compound were co-incubated with a radiolabeled M3 antagonist. The half-maximal inhibitory concentration (IC50) was calculated by measuring the amount of radiobinding on the composite membrane. 50 Then, calculate the suppression constant based on the equation: Ki = IC 50 / (1+([L] / Kd)), where [L] is the concentration of the radioactive ligand used in the experiment, in nmol / L; Kd is the dissociation constant of the radioactive ligand binding to the receptor, which was experimentally measured to be 0.2 nmol / L.

[0091] Table 2 Comparison results of physicochemical property determination and atomization performance test of the examples and comparative examples.

[0092]

[0093] Table 3 Comparison of pharmacokinetic test results between the examples and comparative examples

[0094]

[0095] Analyze the results in Tables 2 and 3:

[0096] (1) Example 1 exhibits good water solubility and extremely low LogP, indicating its highly hydrophilic characteristics. Compared with Comparative Examples 1 and 3, the apparent permeability coefficient Papp(A→B) of Example 1 is significantly reduced, and the BBB permeability is greatly decreased, indicating its extremely low central nervous system penetration tendency. The Papp level of Example 1 is comparable to that of Comparative Examples 2 and 4, both falling within the low permeability range typical of quaternary ammonium drugs. This result indicates that the combined modification of introducing the PEG7 chain and N-quaternization can synergistically lead to a significant reduction in the passive diffusion ability of the molecule, thereby reducing the risk of central nervous system side effects, and relying on high water solubility to reduce systemic absorption and improve overall safety.

[0097] (2) The lung retention time of Example 1 was significantly longer than that of all comparative examples, indicating a slower clearance rate and longer local exposure time in lung tissue. Although Comparative Example 3 was also modified with PEG7, its overall retention time was significantly shorter than that of Example 1 due to its lack of quaternization, suggesting that the hydration layer provided by the PEG7 chain and the local adhesion effect formed by the positive charge of quaternary ammonium jointly promoted a more prolonged lung retention. This performance supports the use of the compounds of the present invention as long-acting inhaled anticholinergic bronchodilators, which is expected to reduce the frequency of administration and improve compliance.

[0098] (3) Example 1 still maintains good affinity for the M3 muscarinic receptor, although slightly lower than Comparative Example 1, but better than Comparative Example 2, indicating that the introduction of the PEG7 chain and quaternization did not weaken its core pharmacological activity. In terms of nebulization performance, the MMAD of Example 1 falls within the ideal aerodynamic particle size range of the inhalation formulation, similar to and stable with the comparative example, indicating that the modification of the PEG7 chain will not cause deterioration of the nebulized particle size or spray abnormalities, ensuring that it can be successfully used in the development of nebulized inhalation formulations.

[0099] This invention, through the introduction of a PEG7 chain into the hyoscyamine structure and subsequent quaternization modification, yields a novel inhaled anticholinergic drug molecule exhibiting extremely low BBB permeability, high water solubility, good receptor activity, significantly prolonged pulmonary retention time, and excellent nebulization performance. These characteristics collectively demonstrate that the compound of this invention possesses significant advantages in terms of safety, long-lasting effect, and compatibility with inhaled formulations, exhibiting comprehensive performance superior to existing technologies.

Claims

1. A hyoscyamine derivative, characterized in that, The structural formula of the hyoscyamine derivative is: .

2. The method for preparing a hyoscyamine derivative according to claim 1, characterized in that, Includes the following steps: S1: Hyoscyamine was etherified with CH3(OCH2CH2)7Br. After the reaction was completed, the product was post-processed, purified and dried to obtain m-(PEG7)-hyoscyamine intermediate. S2: The nitrogen atom of the m-(PEG7)-hyoscyamine intermediate prepared in S1 is subjected to a methylation reaction to introduce a methyl group to form an N,N-dimethyl quaternary ammonium cation and a bromide. After the methylation reaction is completed, the crude product is obtained by post-processing, and then purified by pulping and drying to finally obtain the hyoscyamine derivative.

3. The method for preparing a hyoscyamine derivative as described in claim 2, characterized in that, The etherification reaction described in step S1 is as follows: First, 1.0 molar equivalent of hyoscyamine is dissolved in DMF to prepare a solution with a concentration of 0.1 g / mL. The solution is stirred and the temperature is lowered to 0°C under nitrogen protection. 1.2 molar equivalent of a strong base is added to the reaction system, and the reaction temperature is kept below 20°C. After the addition is complete, the reaction mixture is stirred at room temperature for 30 minutes. The reaction system is then cooled to 0°C, and a CH3(OCH2CH2)7Br / DMF solution with a concentration of 0.2 g / mL is added dropwise, wherein CH3(OCH2CH2)7Br is 1.1 molar equivalent. The temperature is kept below 5°C. After the addition is complete, the temperature is raised to 35°C, and the reaction is stirred at this temperature for 12 hours until the reaction is complete.

4. The method for preparing a hyoscyamine derivative as described in claim 3, characterized in that, The strong base is sodium hydride.

5. The method for preparing a hyoscyamine derivative as described in claim 2, characterized in that, The specific steps of the methylation reaction in step S2 include: dissolving 1.0 molar equivalent of the m-(PEG7)-hyoscyamine intermediate prepared in step S1 into DMF to prepare a solution with a concentration of 0.1 g / mL, stirring, and lowering the temperature of the reaction solution to -10°C under nitrogen protection, adding the methylation reagent, raising the temperature of the reaction system to 50°C, and continuing the reaction at this temperature for 10 hours until the reaction is completed.

6. The method for preparing a hyoscyamine derivative as described in claim 5, characterized in that, The methylating agent is gaseous bromomethane, which needs to be introduced into the reaction system. The gas introduction time is controlled at 2 hours, and the temperature of the reaction system is kept below -5°C during this period.

7. The method for preparing a hyoscyamine derivative as described in claim 2, characterized in that, The specific steps of the post-processing described in step S2 include: cooling the reaction system to 10°C, stirring for 30 minutes, filtering, washing the filter cake three times with acetonitrile to remove unreacted quaternizing reagents and impurities, collecting the filter cake, and drying it under reduced pressure to constant weight.

8. The method for preparing a hyoscyamine derivative as described in claim 2, characterized in that, The specific method for pulping and purification in step S2 is as follows: the crude product is dispersed in ethyl acetate, stirred at room temperature for 2 hours, then stirred at 10°C for 30 minutes, and filtered to obtain the pure product.

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