Preparation method of compound as shown in formula III and application of compound in stress-induced cells

Abstract

The invention relates to a preparation method of a compound shown as a formula III, which comprises the following step of: demethylating a compound 6 in the presence of a demethylation reagent to prepare the compound shown as the formula III. The compound shown in the formula III is used for stress induction of stem cells to generate functional protein with a repairing effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a preparation method of a compound of formula III and an application thereof in stress-induced cells. Background Art

[0002] Cells are the basic units of life and the foundation of body health. When the body's redox balance is disrupted, redox signaling and control are interrupted, triggering oxidative stress damage and causing a variety of diseases. Brain ischemia and hypoxia (such as stroke, cerebral hemorrhage, long-term high-altitude work, internal carotid artery and vertebral artery occlusion and / or stenosis, etc.) and oxidative stress damage to nerve cells can cause a variety of neurodegenerative diseases caused by the loss of neurons or glial cells, including memory loss, Alzheimer's disease, Parkinson's disease, dementia or senile dementia, multiple sclerosis, ataxia, Huntington's disease, amyotrophic lateral sclerosis (ALS, also known as motor neuron disease, Lou Gehrig's disease), etc.

[0003] Stroke (stenosis or occlusion of the cerebral arteries, resulting in blood flow to the brain) and cerebral hemorrhage (sudden rupture of a cerebral blood vessel) are common central nervous system diseases. Insufficient blood supply to the brain causes ischemic and hypoxic damage to brain tissue, manifesting as corresponding neurological deficits, including balance problems, hemiplegia, loss of sensation and vibration, numbness, decreased reflexes, ptosis, visual field defects, aphasia, and apraxia. Oxidative damage caused by ischemia, hypoxia, reperfusion, and excessive free radicals is the primary cause of the exacerbation of ischemia-reperfusion injury in early acute ischemic stroke.

[0004] Ataxia often results from cerebellar, proprioceptive, and vestibular dysfunction, leading to clumsy and uncoordinated movements. Involvement of the trunk, limbs, and throat muscles can cause balance, posture, gait, and speech disorders. Ataxia includes cerebellar ataxia, cerebral ataxia, sensory ataxia, and vestibular ataxia. The causes of ataxia are complex and often represent a combination of multiple diseases, including acute cerebral ischemia, acute brain swelling, and acute cerebral hemorrhage caused by infarction, edema, or hemorrhage.

[0005] Traumatic brain injury, often caused by external objects, often results in varying degrees of permanent functional impairment, including focal abnormalities in motor, sensory, speech, vision, and hearing. Peripheral nerve injury, a common clinical condition caused primarily by trauma, tumors, and metabolic diseases (such as diabetes and its complications), often leads to partial or complete loss of motor, sensory, and autonomic function in the affected segments, and can even cause persistent neuralgia, severely impacting patients' quality of life.

[0006] Amyotrophic lateral sclerosis (ALS) is a disease caused by damage to upper and lower motor neurons, leading to gradual weakness and atrophy of muscles in the bulbus, limbs, trunk, chest, and abdomen. On May 11, 2018, the National Health Commission and five other government departments jointly developed the "First List of Rare Diseases," which included ALS.

[0007] When the blood supply and nerve innervation of articular cartilage fail, joint damage can occur, which in turn affects the joint's ability to repair itself. If joint and cartilage damage are not repaired promptly, it can lead to irreversible pathological changes and even joint degeneration, compromising the patient's quality of life.

[0008] Arthritis, often called the "immortal cancer," has a high disability rate and severely impacts patients' quality of life. Joint injuries (such as traumatic joint lesions and degenerative osteoarthritis) are common clinical conditions, including refractory wounds, osteoarthritis of the knee, and cartilage wear and tear.

[0009] Treatment or repair methods for joint injuries and articular cartilage injuries include anti-inflammation, analgesia (pain relief), physical therapy, acupuncture, joint replacement, stem cell therapy, radiofrequency ablation, chondrocyte transplantation, osteochondral transplantation, and engineered cartilage, such as physical therapy and nutrition represented by traditional Chinese medicine physiotherapy and glucosamine, lubrication relief represented by chitosan and hyaluronic acid, anti-inflammatory treatment represented by PRP, and joint replacement represented by arthroscopy and artificial joints. Except for joint replacement, all of them are mainly aimed at relieving symptoms and delaying the course of the disease.

[0010] Articular cartilage lacks blood vessels, nerves, or a lymphatic system, and contains a small number of chondrocytes with limited migration and proliferation capabilities, making it difficult for articular cartilage injuries to heal on their own. Failure of the articular cartilage's blood supply and innervation can easily lead to joint damage, which in turn affects the joint's ability to repair itself. Without prompt and effective treatment and repair, the damage can worsen and trigger osteoarticular lesions, leading to joint pain, swelling, or movement disorders, and even irreversible pathological changes and joint degeneration, affecting the patient's mobility and causing disability.

[0011] Mesenchymal stem cells (MSCs) have the potential for self-replication and multidirectional differentiation. They are widely present in tissues and parts such as bone marrow, fat, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amniotic membrane, peripheral blood, muscle, and urine. They have the characteristics of wide source, no need for matching, low infection rate, strong differentiation potential, strong proliferation ability, and easy collection. They can produce active factors such as stem cell growth factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), and interferon (IFN), and participate in regulating cell growth, cell apoptosis, cell differentiation, antiviral, and immune maturation. They can be used for immune regulation, tissue repair, and the treatment of diseases such as acute lung injury, severe pneumonia, and acute respiratory distress syndrome. However, MSCs products are living organisms and require stringent production and storage conditions (such as refrigeration) during their production, storage, transportation, and application, and their cell viability must be maintained for ≤12 hours, which limits their effective treatment radius and clinical application.

[0012] When cells and microorganisms are subjected to stress induction or stress stimulation from external and exogenous stressors (including cold, heat, acid, alkali, high pressure, ultraviolet radiation, chemicals, etc.), they produce stress proteins due to the stress response. Reference 1 (New limonophyllines AC from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2 cell lines, Arch. Pharm. Res. (2018) 41:431–437) discloses that compounds isolated from extracts of the Rutaceae plant Atalantia monophylla and their analogs have activity in inhibiting tumor cell growth.

[0013] Injury repair is a long, gradual, and slow process. Timely repair of damaged cells or organs can significantly improve, prevent, and treat related lesions and their complications. Cytokines secreted by stem cells are used to repair various injuries. To this end, it is necessary to screen the type and dosage of exogenous stressors, as well as the culture environment of cells and microorganisms, to induce the production of stress proteins with repair efficacy, safety, and efficacy, to meet urgent clinical needs such as injury repair. Summary of the Invention

[0014] The object of the present invention is to provide a method for preparing a compound of formula III, wherein compound 6 is demethylated in the presence of a demethylating agent to obtain a compound of formula III, wherein the demethylating agent is selected from any one of boron tribromide, hydrobromic acid, sulfuric acid, sodium ethanethiolate, sodium amide, and trimethylsilyl iodide, or a combination thereof.

[0015]

[0016] In a preferred technical solution of the present invention, compound 6 is prepared by reacting compound 5 with an alkylating agent in the presence of a base, wherein the base is selected from any one or a combination of NaH, KH, sodium methoxide, sodium ethoxide, NaOH, KOH, and cesium carbonate, and the alkylating agent is selected from any one or a combination of iodomethane, dimethyl sulfate, dimethyl carbonate, methyl trifluoromethanesulfonate, methyl fluorosulfonate, methyl methanesulfonate, 2,2-dimethoxypropane, dimethyl pyrocarbonate, N-methyl-N-nitrosourea, and dicumyl peroxide.

[0017]

[0018] In a preferred technical solution of the present invention, the compound 5 is prepared by reacting compound 4 and 2-methyl-3-butene-2-ol under Lewis acid catalysis conditions, wherein the Lewis acid is selected from any one or a combination of boron trifluoride etherate, boron trifluoride acetonitrile, boron trifluoride, and aluminum chloride.

[0019]

[0020] In a preferred technical solution of the present invention, the compound 4 is prepared by reacting the compound 3 with isopentenal in the presence of a titanium catalyst, wherein the titanium catalyst is selected from any one of tetraisobutoxytitanium and titanium tetrachloride or a combination thereof.

[0021]

[0022] In a preferred technical solution of the present invention, the compound 3 is prepared by reacting 2-amino-3-methoxybenzoic acid and phloroglucinol under catalytic conditions of an activator, wherein the activator is selected from any one of p-toluenesulfonic acid, methanesulfonic acid, and benzenesulfonic acid, or a combination thereof.

[0023]

[0024] Another object of the present invention is to provide a method for preparing the compound of formula III, comprising the following steps:

[0025]

[0026] 1) 2-amino-3-methoxybenzoic acid and phloroglucinol react under catalytic conditions of an activator to prepare compound 3;

[0027] 2) Compound 3 reacts with isopentenal in the presence of a titanium catalyst to produce compound 4;

[0028] 3) Compound 4 and 2-methyl-3-butene-2-ol react under Lewis acid catalysis to produce compound 5;

[0029] 4) Compound 5 reacts with an alkylating agent in the presence of a base to obtain compound 6;

[0030] 5) Compound 6 is demethylated in the presence of a demethylating agent to obtain a compound of formula III.

[0031] In a preferred technical solution of the present invention, in step 1), the activating agent is selected from any one of p-toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, or a combination thereof.

[0032] In a preferred technical solution of the present invention, in step 1), the reaction temperature is from 80°C to reflux temperature.

[0033] In a preferred technical solution of the present invention, in step 2), the titanium catalyst is selected from any one of tetraisobutoxytitanium and titanium tetrachloride, or a combination thereof.

[0034] In a preferred technical solution of the present invention, in step 2), the titanium catalyst is added dropwise to the reaction system, wherein the dropping temperature is -78°C to -50°C, preferably -78°C to -60°C.

[0035] In a preferred technical solution of the present invention, in step 2), after the dropwise addition is completed, the temperature is raised to 0° C. to room temperature for reaction.

[0036] In a preferred technical solution of the present invention, in step 2), the reaction is carried out under inert gas protection conditions, wherein the inert gas is selected from any one of argon and nitrogen or a combination thereof.

[0037] In a preferred technical solution of the present invention, in step 3), the Lewis acid is selected from any one of boron trifluoride etherate, boron trifluoride acetonitrile, boron trifluoride, aluminum trichloride, or a combination thereof.

[0038] In a preferred technical solution of the present invention, in step 3), the reaction is carried out under a microwave environment at a reaction temperature of 100-150°C, preferably 120-130°C.

[0039] In a preferred technical solution of the present invention, in step 4), the base is selected from any one of NaH, KH, sodium methoxide, sodium ethoxide, NaOH, KOH, and cesium carbonate, or a combination thereof.

[0040] In a preferred technical solution of the present invention, in step 4), the alkylating agent is selected from any one of iodomethane, dimethyl sulfate, dimethyl carbonate, methyl trifluoromethanesulfonate, methyl fluorosulfonate, methyl methanesulfonate, 2,2-dimethoxypropane, dimethyl pyrocarbonate, N-methyl-N-nitrosourea, and dicumyl peroxide, or a combination thereof.

[0041] In a preferred technical solution of the present invention, in step 4), the molar ratio of compound 5:base is 1:4-6, preferably 1:4.5-5.5.

[0042] In a preferred technical solution of the present invention, in step 4), the molar ratio of compound 5:alkylating agent is 1:2-5, preferably 1:3-4.

[0043] In a preferred technical solution of the present invention, in step 5), the demethylation agent is selected from any one of boron tribromide, hydrobromic acid, sulfuric acid, sodium ethanethiolate, sodium amide, and trimethylsilyl iodide, or a combination thereof.

[0044] In a preferred technical solution of the present invention, in step 5), the demethylation reagent is added dropwise to the reaction system, wherein the dropping temperature is -10°C to 5°C, preferably -5°C to 0°C.

[0045] In a preferred technical solution of the present invention, in step 5), after the dropwise addition is completed, the temperature is raised to 20°C to 35°C for reaction, preferably 25°C to 30°C.

[0046] In a preferred technical solution of the present invention, in step 5), the reaction is carried out under inert gas protection conditions, wherein the inert gas is selected from any one of argon and nitrogen or a combination thereof.

[0047] The purpose of the present invention is to provide an application of a compound of formula III for stress-induced cells to produce proteins with repair efficacy.

[0048] In the preferred technical solution of the present invention, the repair is selected from any one or a combination of cell repair, joint repair, hair follicle repair, cartilage repair, skin damage cell repair, nerve damage cell repair, organ damage cell repair, pulmonary fibrosis repair, liver damage repair, kidney damage repair, ovarian repair, Crohn's disease repair, sub-health repair, and aging repair.

[0049] Prior patent applications (CN202310042911X, CN2023100429105, CN2023100429139, CN2023100429143, PCTCN2023073582, PCTCN2023073566, PCTCN2023073590, PCTCN2023073596) serve as essential technical content and technical references for this application.

[0050] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentages are volume / volume percentages; when the present invention relates to the percentage between liquids and solids, the percentages are volume / weight percentages; when the present invention relates to the percentage between solids and liquids, the percentages are weight / volume percentages; and the rest are weight / weight percentages.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] 1. The present invention provides a method for preparing a compound of formula III, wherein the compound of formula III is used to induce cells or MSCs to produce a protein with a repair effect, wherein the repair function is selected from any one of cell repair, joint repair, hair follicle repair, cartilage repair, skin damage cell repair, nerve damage cell repair, organ damage cell repair, pulmonary fibrosis repair, liver damage repair, kidney damage repair, ovarian repair, Crohn's disease repair, sub-health repair, and aging repair, or a combination thereof.

[0053] 2. The preparation method of the present invention has the advantages of simple operation, improved resource utilization, environmental protection, and suitability for industrial production. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0055] Preparation Example 1 Preparation of compound 3

[0056] 2-Amino-3-methoxybenzoic acid (1.67 g, 10 mmol) and phloroglucinol (1.26 g, 10 mmol) were dissolved in 50 mL of n-hexanol. p-Toluenesulfonic acid (0.17 mg, 1 mmol) was added and refluxed for 16 h. The reaction solution was cooled to room temperature, 300 mL of n-hexane and 200 mL of dichloromethane were added, and stirred for 1 h to precipitate a pale yellow solid. After drying, the product was recrystallized from ethanol and water to obtain 3 (2.2 g, 86%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6):14.26(1H,sbr),11.23(1H,s),10.45(1H,sbr),7.71(1H,d,J=8.0 Hz),7.30(1H,d,J=8.0Hz),7.17(1H,t,J=8.0Hz),6.68(1H,s),6.00(1H,s),4.01(3H,s); 13 C(100MHz,DMSO-d6):179.8,163.9,163.4,147.3,143.1,131.7,120.7,119.4,116.1,112.7,103.4,95.8,92.0,56.2; ESI-MS: 258.1[M+H] + .

[0057] Preparation Example 2 Preparation of compound 4

[0058] Compound 3 (2.2 g, 8.56 mmol) and isopentanal (2.16 g, 25.68 mmol) were dissolved in 10 mL of toluene and cooled to -78°C under argon. Tetraisobutoxytitanium (11.0 g, 34.24 mmol) was slowly added dropwise. The mixture was allowed to warm to room temperature over 4 hours and stirred at room temperature for 20 hours. 100 mL of dichloromethane and 100 mL of water were added and the layers separated. The organic layer was washed with saturated brine and purified by column chromatography to yield 4 (2.07 g, 75%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6):14.53(1H,s),9.71(1H,s),7.73(1H,dd,J=8.1Hz,1.1Hz),7.37(1H,dd,J=7.8Hz,1.0Hz ),7.25(1H,t,J=8.0Hz),7.00(1H,d,J=10.0Hz),6.07(1H,s),5.70(1H,d,J=10.0Hz),4.02(3H,s),1.42(6H,s); 13 C(125MHz,DMSO-d6):180.5,163.3,159.1,147.3,136.8,131.2,125.6,121.7,119 .5,115.9,115.8,113.2,104.1,98.3,96.5,77.1,56.3,27.4; ESI-MS: 324.1[M+H] + .

[0059] Preparation Example 3 Preparation of compound 5

[0060] Compound 4 (2.07 g, 6.41 mmol) was dissolved in 20 mL of 1,4-dioxane, and boron trifluoride etherate (2.4 mL, 19.23 mmol) and 2-methyl-3-butene-2-ol (3.4 mL, 32.05 mmol) were added. The mixture was microwaved at 120°C for 5 h. The mixture was diluted with 300 mL of dichloromethane and washed with 50 mL each of saturated sodium bicarbonate aqueous solution, saturated ammonium chloride aqueous solution, and saturated brine. The mixture was then dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound 5 (1.18 g, 47%). ESI-MS: 392.4 [M+H] + .

[0061] Preparation Example 4 Preparation of compound 6

[0062] Compound 5 (0.5 g, 1.28 mmol) was dissolved in 10 mL of anhydrous DMF. NaH (230 mg, 60%, 5.76 mmol, 4.5 eq.) was added at 0°C and stirred for 10 minutes before adding iodomethane (0.55 g, 3.84 mmol, 3 eq.). The reaction was allowed to proceed for 10 hours at room temperature in the dark. Thin-layer chromatography indicated the disappearance of the starting material. DMF was evaporated under reduced pressure, and 100 mL of ethyl acetate and 50 mL of water were added. The layers were separated, and the aqueous phase was extracted with ethyl acetate (50 mL x 2). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration and column chromatography, a yellow solid was obtained, which was compound 6 (0.49 g, 91%). ESI-MS: 420.2 [M+H] + .

[0063] Preparation Example 5 Preparation of compound of formula III

[0064] Compound 6 (0.49 g, 1.17 mmol) was dissolved in 10 mL of anhydrous dichloromethane. A dichloromethane solution of boron tribromide (1.8 mL, 1.5 eq., 1 M) was added dropwise under argon in an ice bath. The mixture was allowed to stand at room temperature overnight (14 h). The reaction mixture was diluted with 50 mL of dichloromethane and quenched with 20 mL of saturated sodium bicarbonate solution. The layers were separated and the organic layer was dried over anhydrous sodium sulfate. After concentration and column chromatography, the compound of formula III (0.27 g, 59%) was obtained. ESI-MS: 392.2 [M+H] + .

[0065] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a compound of formula III, wherein compound 6 is demethylated in the presence of a demethylating agent to obtain a compound of formula III, wherein: The demethylation agent is selected from any one of boron tribromide, hydrobromic acid, sulfuric acid, sodium ethanethiolate, sodium amide, and trimethylsilyl iodide, or a combination thereof.

2. The method according to claim 1, wherein compound 6 is prepared by reacting compound 5 with an alkylating agent in the presence of a base, wherein The base is selected from any one of NaH, KH, sodium methoxide, sodium ethoxide, NaOH, KOH, and cesium carbonate, or a combination thereof; the alkylating agent is selected from any one of iodomethane, dimethyl sulfate, dimethyl carbonate, methyl trifluoromethanesulfonate, methyl fluorosulfonate, methyl methanesulfonate, 2,2-dimethoxypropane, dimethyl pyrocarbonate, N-methyl-N-nitrosourea, and dicumyl peroxide, or a combination thereof; 3. The method according to any one of claims 1 to 2, wherein the compound 5 is prepared by reacting the compound 4 with 2-methyl-3-butene-2-ol under Lewis acid catalysis conditions, wherein, The Lewis acid is selected from any one of boron trifluoride etherate, boron trifluoride acetonitrile, boron trifluoride, aluminum trichloride or a combination thereof, 4. The method according to any one of claims 1 to 3, wherein the compound 4 is prepared by reacting the compound 3 with isopentanal in the presence of a titanium catalyst, wherein: The titanium catalyst is selected from any one of tetraisobutoxytitanium and titanium tetrachloride or a combination thereof, 5. The method according to any one of claims 1 to 4, wherein the compound 3 is prepared by reacting 2-amino-3-methoxybenzoic acid and phloroglucinol under catalytic conditions of an activator, wherein, The activator is selected from any one of p-toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid or a combination thereof, 6. A method for preparing a compound of formula III, comprising the following steps: 1) 2-amino-3-methoxybenzoic acid and phloroglucinol react under catalytic conditions of an activator, Compound 3 was obtained; 2) Compound 3 reacts with isopentenal in the presence of a titanium catalyst to produce compound 4; 3) Compound 4 and 2-methyl-3-butene-2-ol react under Lewis acid catalysis conditions, Compound 5 was obtained; 4) Compound 5 reacts with an alkylating agent in the presence of a base to obtain compound 6; 5) Compound 6 is demethylated in the presence of a demethylating agent to obtain a compound of formula III.

7. The method according to claim 6, wherein in step 1), the reaction temperature is from 80°C to reflux temperature.

8. The method according to any one of claims 6 to 7, wherein in step 2), the titanium catalyst is added dropwise to the reaction system, wherein The dropping temperature is -78°C to -50°C, preferably -78°C to -60°C.

9. The method according to any one of claims 6 to 8, wherein in step 3), the reaction is carried out under a microwave environment at a reaction temperature of 100-150°C, preferably 120-130°C.

10. Use of the compound of formula III for stress-induced cells to produce proteins with repair efficacy.

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