A technical method for chiral resolution of finerenone

Abstract

The application relates to a preparation method and application of a non-naliramide magnetic nano-resolving agent. The magnetic nano-resolving agent takes magnetic nano-Fe3O4 particles as a core, and the surface is a molecularly imprinted polymer obtained by removing a template molecule after polymerization of a functional monomer, a crosslinking agent and a non-naliramide R configuration isomer through an initiator. The nano-resolving agent is simple and convenient to operate, can efficiently resolve non-naliramide with high chiral purity, and has great application value and market prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing and applying a phenelzine magnetic nano-resolving agent. It mainly utilizes molecular imprinting technology to remove the R-configuration isomer in the racemic mixture of phenelzine to achieve the purpose of resolution. Background Technology

[0002] Finerenone, chemically known as (4S)4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthidine-3-carboxamide, is a nonsteroidal selective mineralocorticoid receptor antagonist developed by Bayer. In July 2021, the U.S. Food and Drug Administration (FDA) approved finerenone for marketing in the United States. It is currently approved for marketing in several other countries for the treatment of chronic kidney disease (CKD) in adults caused by type 2 diabetes (T2D). Finerenone is a chiral dihydropyridine compound with an S-configuration. The chemical structural formula of finerenone is shown below:

[0003]

[0004] Compound patent CN101641352B discloses a method for preparing a racemic phenelzine using 4-cyano-2-methoxybenzaldehyde as the key raw material, followed by separation by chiral HPLC to obtain phenelzine. This method uses chiral HPLC for resolution, resulting in low throughput and high cost. The synthetic route for phenelzine is as follows:

[0005]

[0006] Subsequently, Bayer disclosed an optimized process for this route in patents WO2016016287 and WO2017032678, and scaled it up to the kilogram level. The chiral HPLC was changed to simulated moving bed (SMB) chromatography to obtain fenelone by chiral separation, but the problem of low separation efficiency still exists.

[0007] Bayer's patent CN112041318A discloses a chemical resolution method for purifying phenelzine from APIs. Specifically, it involves using benzoyl-D-tartaric acid as a resolving agent to form a salt with a racemic compound, then using a sodium phosphate solution to release the resolving agent to obtain crude phenelzine, which is finally recrystallized to obtain phenelzine. This method requires multiple resolution and crystallization steps, is complex, involves large amounts of solvent, and carries the risk of resolving agent residue, making it difficult to control the quality of the active pharmaceutical ingredient.

[0008] Bayer published patent WO2021254896A1 in 2021, which uses 4-amino-5-methyl-2-hydroxypyridine as a raw material and dihydropyridine and chiral phosphine catalyst (S)-PA as induction to obtain a single enantiomer. The chiral phosphine catalyst (S)-PA needs to be custom synthesized, which is expensive.

[0009] With the development of chiral separation technology, molecularly imprinted polymer (MIP) technology has been applied to the resolution of chiral dihydropyridine compounds. The literature "Preparation and Thermodynamic Properties of Molecularly Imprinted Polymers for Nimodipine" reports the use of imprinted polymers to recognize the dihydropyridine drug nimodipine and points out its potential application in chiral resolution, but no further research was conducted. The literature "Preparation and Properties of Magnetic Imprinted Materials for Amlodipine" reports the preparation of imprinted polymer materials that recognize S-amlodipine and their application in chiral drug separation; however, the material prepared in this paper has a low adsorption capacity of only 243 mg / g, making it unsuitable as a chiral resolving agent. Summary of the Invention

[0010] To address the shortcomings of existing technologies, this invention aims to prepare a magnetic nano-resolving agent for the convenient, low-cost, and efficient separation of phenelzine racemates. This resolving agent is a magnetic nanomaterial prepared using molecular imprinting technology with the R-configuration isomer of phenelzine as a template molecule. It can recognize and capture the R-configuration isomer in the phenelzine racemate, and rapidly separate it through a magnetic field, retaining the S-configuration of phenelzine to achieve the separation effect. The captured R-configuration phenelzine nanomaterial is removed by acid treatment, allowing the nanomaterial to be reused. The phenelzine prepared using this resolving agent has a chiral purity greater than 99.9%, and the chiral purity of phenelzine prepared after separation and reuse can still reach 99.7%.

[0011] The phenelzine nanomagnetic resolving agent of this invention has a core of magnetic iron oxide nanoparticles encapsulated in mesoporous silica, and a surface of phenelzine enantiomer molecularly imprinted polymer. Vinyl groups are modified on the surface of the mesoporous silica surrounding the magnetic nanocore, and then polymerized and grafted with functional monomers of the molecularly imprinted polymer. Specifically, this is achieved through the following technical solution:

[0012] Step 1: Prepare magnetic nanoparticles (Fe3O4@mSiO2) encapsulated in mesoporous silica.

[0013] Ferric chloride hexahydrate was dissolved in alcohol, followed by the addition of sodium acetate and polyethylene glycol 10000. After stirring at room temperature, the reaction solution was transferred to a high-pressure reactor and heated for 8 hours. After the reaction was completed, the solution was allowed to cool naturally to room temperature, washed several times alternately with deionized water and alcohol, and then vacuum dried to obtain Fe3O4 magnetic nanoparticles.

[0014] The prepared Fe3O4 magnetic nanoparticles were ultrasonically dispersed in 0.1M dilute hydrochloric acid and then washed several times with water. The pretreated Fe3O4 particles were then dispersed in a mixture of alcohol and water, and ammonia was added. Tetraethyl orthosilicate was added dropwise under vigorous stirring. After reacting at a constant temperature for 6 hours, black nanospheres were separated and washed repeatedly with distilled water. The washed nanospheres were then dispersed in a mixture of alcohol and water containing the porogen cetyltrimethylammonium bromide (CTAB), and concentrated ammonia was added. Tetraethyl orthosilicate was added dropwise under vigorous stirring, and the reaction was carried out at a constant temperature for 6 hours. After the reaction, the magnetic nanoparticles were separated under an external magnetic field, and repeated washing with alcohol and water was performed. The mixture was then refluxed in alcohol for 12 hours to remove the porogen. The nanoparticles were collected, washed, and dried to obtain magnetic nanoparticles (Fe3O4@mSiO2) encapsulated in mesoporous silica.

[0015] Preferably, the alcohol solvent used in step 1 can be ethanol, ethylene glycol, glycerol, etc., with ethylene glycol being the preferred solvent.

[0016] Preferably, the Fe3O4 magnetic nanoparticles are prepared at a temperature of 100–250°C in step 1, more preferably 180–220°C.

[0017] Preferably, the reaction temperature for preparing Fe3O4@mSiO2 in step 1 is 0–30°C, more preferably 20–25°C.

[0018] Step 2: Prepare vinyl-modified magnetic nanoparticles (Fe3O4@mSiO2-Vi).

[0019] Fe3O4@mSiO2 was added to an organic solvent, and a vinyl silane coupling agent was added dropwise under vigorous stirring. After the addition was complete, the reaction was carried out at a constant temperature for 24 hours. After the reaction was completed, the product was separated by centrifugation to obtain a black solid product. The product was washed thoroughly with anhydrous ethanol to obtain vinyl-modified magnetic nanoparticles (Fe3O4@mSiO2-Vi).

[0020] Preferably, the organic solvent used in step 2 can be methanol, ethanol, toluene, dichloromethane, etc., with toluene being the preferred solvent.

[0021] Preferably, the silane coupling agent used in step 2 is selected from vinyltrimethoxysilane (VTMO, A-171), vinyltriethoxysilane (VTEO, A-151), vinyltri(2-methoxyethoxy)silane (VTMOEO), vinyltrichlorosilane (VTCS, A-150), and vinylmethyldimethoxysilane (VMDMS), with VTEO being the most preferred.

[0022] Preferably, the reaction temperature in step 2 is 0–100°C, more preferably 20–30°C.

[0023] Step 3: Preparation of phenelzine magnetic nano-resolving agent (Fe3O4@mSiO2-MIP).

[0024] Magnetic nanoparticles Fe3O4@mSiO2-Vi, the template molecule phenelzine R-configuration isomer, functional monomers, and an initiator were added to a reaction flask. Methanol was added, and nitrogen gas was introduced into the reaction system. After stirring and heating to 30°C, a crosslinking agent was added, and the reaction was carried out under constant temperature for 6 hours. After the reaction was completed, the resulting microparticles were separated from the solution under the action of an external magnetic field. The magnetic microparticles obtained from the reaction were added to a 100 mL four-necked flask, and acetic acid and methanol were added. The mixture was stirred at 60°C for 3 hours to wash away the template molecule. After separation, washing, and vacuum drying to constant weight, the phenelzine enantiomerically magnetic surface molecularly imprinted material Fe3O4@mSiO2-MIP was obtained.

[0025] Preferably, the functional monomer used in step 3 may be 4-vinylphenylboronic acid, methacrylic acid (MMA), or methacrylamide, with 4-vinylphenylboronic acid being the most preferred.

[0026] Preferably, the crosslinking agent used in step 3 can be ethylene glycol diglycidyl ether (EGDE), ethylene glycol dimethacrylate (EGDMA), or trimethylolpropane triacrylate (TMPTA), with ethylene glycol dimethacrylate (EGDMA) being the most preferred.

[0027] Preferably, the initiator used in step 3 can be selected from benzoyl peroxide (BPO), azobisisobutyronitrile (AIBN), or azobisisopentyl cyanide.

[0028] Preferably, in step 3, the weight ratio of template molecule: functional monomer: crosslinking agent: initiator is 1:1.8:1:0.1.

[0029] Step 4: Preparation of fenelazol

[0030] Deionized water, methanol, and phenelzine racemic compound were added to the reaction flask. Nitrogen gas was introduced into the reaction system, and the mixture was stirred and heated to 50°C. Fe3O4@mSiO2-MIP was then added, and the mixture was stirred at a constant temperature for 6 hours. After the reaction was completed, the particles obtained from the reaction were separated from the solution under the action of an external magnetic field. After removing the particles, the reaction solution was retained and concentrated under reduced pressure to obtain solid phenelzine. Attached Figure Description

[0031] Appendix Figure 1 Transmission electron microscope (TEM) image of Fe3O4 magnetic nanoparticles.

[0032] Appendix Figure 2 : Transmission electron microscope (TEM) image of Fe3O4@mSiO2 magnetic nanoparticles.

[0033] Appendix Figure 3Magnified image of Fe3O4@mSiO2 magnetic nanoparticles obtained by transmission electron microscopy (TEM).

[0034] Appendix Figure 4 Scanning electron microscope (SEM) image of Fe3O4@mSiO2-MIP magnetic nano-resolving agent.

[0035] Appendix Figure 5 Transmission electron microscope (TEM) image of Fe3O4@mSiO2-MIP magnetic nano-resolving agent.

[0036] Appendix Figure 6 Example 3: Chiral liquid chromatogram.

[0037] Appendix Figure 7 Example 4: Chiral liquid chromatogram.

[0038] Appendix Figure 8 Example 5: Chiral liquid chromatography phase spectrum. Detailed Implementation

[0039] The following typical embodiments are used to illustrate the present invention. Simple substitutions or improvements made to the present invention by those skilled in the art are all within the scope of the technical solutions protected by the present invention.

[0040] Preparation Example 1: Preparation of Fe3O4 Magnetic Nanoparticles

[0041] 17g of ferric chloride hexahydrate, 250mL of ethylene glycol, 45g of sodium acetate, and 6.3g of polyethylene glycol 10000 were added to a pressure vessel. The mixture was stirred until dissolved, the pressure vessel was sealed, and the mixture was heated to 200℃ and reacted for 8 hours. After cooling to room temperature, the solid was filtered out, washed several times with deionized water and ethanol, and then dried under vacuum to obtain Fe3O4 magnetic nanoparticles.

[0042] Preparation Example 2: Preparation of Fe3O4@mSiO2 magnetic nanoparticles

[0043] Add 3.0g of freshly prepared Fe3O4 magnetic nanoparticles to the reaction flask and mix with 100mL of 0.1M hydrochloric acid. Sonicate for 20 minutes, centrifuge, and wash the solid with water multiple times. Then, 100 mL of deionized water, 200 mL of ethanol, and 10 mL of concentrated ammonia were added. After sonication for 20 min, 5 mL of tetraethyl orthosilicate was slowly added dropwise. The mixture was stirred at 20 °C for 6 h and filtered to obtain silica-modified Fe3O4@SiO2 magnetic nanoparticles. The obtained product was then dispersed again in a solution containing 0.5 g of hexadecyltrimethylammonium bromide (CTAB), 200 mL of ethanol, 100 mL of deionized water, and 5 mL of concentrated ammonia. Under vigorous stirring, 5 mL of tetraethyl orthosilicate was slowly added dropwise to the solution. The mixture was stirred vigorously at 20 °C for 6 h. The product was collected by magnetic separation and washed repeatedly with ethanol and deionized water. Then, the mixture was refluxed in ethanol for 12 h. The solid product was separated by an external magnetic field. After washing with ethanol and water multiple times, the product was vacuum dried at 60 °C to obtain magnetic nanoparticles Fe3O4@mSiO2 encapsulated in mesoporous silica.

[0044] Preparation Example 3: Preparation of vinyl-modified magnetic nanoparticles Fe3O4@mSiO2-Vi

[0045] 3.0 g of Fe3O4@mSiO2 and 150 mL of toluene were added to a reaction flask, and 20 mL of vinyltriethoxysilane was added dropwise under vigorous stirring at 20 °C. The mixture was stirred vigorously at 20 °C for 24 h. The solid was obtained by centrifugation and washed several times with deionized water and ethanol to prepare Fe3O4@SiO2-Vi magnetic nanoparticles.

[0046] Example 1: Preparation of magnetic nano-resolving agent Fe3O4@mSiO2-MIP

[0047] 50 mL of methanol was added to the reaction flask, followed by 1.0 g of magnetic nanoparticles Fe3O4@mSiO2-Vi, 1.5 g of phenelzine enantiomer (R configuration), 2.7 g of 4-vinylphenylboronic acid, and 0.2 g of azobisisobutyronitrile. Nitrogen gas was introduced into the reaction system, and the mixture was stirred and heated to 30 °C. Then, 1.5 g of crosslinking agent ethylene glycol dimethacrylate was added, and the reaction was carried out at a constant temperature for 8 h. After the reaction was completed, the resulting microparticles were separated from the solution under an external magnetic field. The magnetic microparticles obtained from the reaction were added to a 100 mL four-necked flask, along with acetic acid and methanol. The mixture was stirred at 60 °C for 3 h to wash away the phenelzine R configuration isomer. The microparticles were dried under vacuum at 60 °C to constant weight to obtain 1.2 g of phenelzine enantiomerically magnetic surface molecularly imprinted material Fe3O4@mSiO2-MIP.

[0048] Example 2: Preparation of fenelazol

[0049] 5 mL of deionized water, 50 mL of methanol, and 0.5 g of phenelzine racemic compound were added to a reaction flask. Nitrogen gas was introduced into the reaction system, and the mixture was stirred and heated to 50 °C. Then, 0.6 g of Fe3O4@mSiO2-MIP was added, and the mixture was stirred at a constant temperature for 6 h. After the reaction was completed, the particles obtained from the reaction were separated from the solution under the action of an external magnetic field. After removing the particles, the reaction solution was retained and concentrated under reduced pressure to obtain 0.20 g of phenelzine solid, ee% = 99.80%.

[0050] Example 3: Preparation of fenelazol

[0051] 10 mL of deionized water, 80 mL of methanol, and 1.5 g of racemic phenelzine were added to a reaction flask. Nitrogen gas was introduced into the reaction system, and the mixture was stirred and heated to 50 °C. Then, 1.0 g of Fe3O4@mSiO2-MIP was added, and the mixture was stirred at a constant temperature for 6 h. After the reaction was completed, the particles obtained from the reaction were separated from the solution under an external magnetic field. After removing the particles, the reaction solution was concentrated under reduced pressure to obtain 0.5 g of solid phenelzine (ee%) = 99.85%. The chiral liquid chromatogram is attached. Figure 6 .

[0052] Example 4: Preparation of fenelazol

[0053] 15 mL of deionized water, 100 mL of methanol, and 3.0 g of racemic phenelzine were added to a reaction flask. Nitrogen gas was introduced into the reaction system, and the mixture was stirred and heated to 50 °C. Then, 1.6 g of Fe3O4@mSiO2-MIP was added, and the mixture was stirred at a constant temperature for 6 h. After the reaction was completed, the particles obtained from the reaction were separated from the solution under an external magnetic field. After removing the particles, the reaction solution was concentrated under reduced pressure to obtain 1.1 g of solid phenelzine (ee%) = 99.85%. The chiral liquid chromatogram is attached. Figure 7 .

[0054] Example 5: Reuse of Fe3O4@mSiO2-MIP magnetic nanoparticles

[0055] 2.0 g of the magnetic nanoparticles separated in a magnetic field, 50 mL of methanol, and 30 mL of acetic acid were added to a reaction flask. Nitrogen gas was introduced into the reaction system, and the mixture was stirred at 60 °C for 3 h to wash away the R-configuration isomer of the template molecule phenelzine. After magnetic separation, washing, and vacuum drying to constant weight, 1.8 g of the phenelzine enantiomer magnetic surface molecularly imprinted material Fe3O4@mSiO2-MIP was reconstituted. 5 mL of deionized water, 50 mL of methanol, and 2.5 g of the racemic phenelzine were added to a reaction flask. Nitrogen gas was introduced into the reaction system, and the mixture was stirred and heated to 50 °C. Then, 1.8 g of Fe3O4@mSiO2-MIP was added, and the mixture was stirred at a constant temperature for 6 h. After the reaction was completed, the microparticles obtained from the reaction were separated from the solution under an external magnetic field. After removing the microparticles, the reaction solution was concentrated under reduced pressure to obtain 0.15 g of phenelzine solid (ee% = 99.53%). The chiral liquid chromatogram is attached. Figure 8 .

Claims

1. A novel method for resolving fenelone: ​​A fenelone magnetic nano-resolving agent and a racemic fenelone are stirred in a mixed solvent of methanol and water. After separating the resolving agent, the solution is concentrated to obtain fenelone.

2. The non-nelitone magnetic nano-resolving agent according to claim 1, characterized in that, The core of the nano-resolving agent is magnetic nano-iron oxide encapsulated in mesoporous silica, and the surface is a non-nelitone enantiomer molecularly imprinted polymer.

3. The molecularly imprinted polymer according to claim 2 is prepared by polymerizing functional monomers, crosslinking agents and template molecules phenelzine enantiomers with an initiator and then removing the template molecules to obtain the molecularly imprinted polymer.

4. The method for preparing the molecularly imprinted polymer according to claim 3, characterized in that, The functional monomers can be selected from 4-vinylphenylboronic acid, methacrylic acid (MMA), and methacrylamide.

5. The method for preparing the molecularly imprinted polymer according to claim 3, characterized in that, Crosslinking agents can be selected from ethylene glycol diglycidyl ether (EGDE), ethylene glycol dimethacrylate (EGDMA), and trimethylolpropane triacrylate (TMPTA).

6. The non-nelitone magnetic nano-resolving agent according to claim 2, characterized in that, The surface of magnetic nano-iron oxide wrapped in mesoporous silica is modified with vinyl groups by silane coupling agent, and the vinyl groups are polymerized and grafted with functional monomers.

7. The silane coupling agent according to claim 6 is selected from vinyltrimethoxysilane (VTMO, A-171), vinyltriethoxysilane (VTEO, A-151), vinyltris(2-methoxyethoxy)silane (VTMOEO), vinyltrichlorosilane (VTCS, A-150), and vinylmethyldimethoxysilane (VMDMS).

8. After use, the non-nelitone magnetic nano-resolving agent according to claim 1 is separated under the action of a magnetic field or centrifugation.

9. The resolving agent according to claim 8, after separation, is heated in a mixed solvent of acetic acid and methanol to remove non-nelitone enantiomers, and the resolving agent can be reused for the separation of non-nelitone after separation.

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