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

Abstract

The invention relates to a preparation method of a compound as shown in a formula II, which comprises the following step of: demethylating a compound 5 under the catalytic condition of Lewis acid to prepare the compound as shown in the formula II. The compound as shown in the formula II 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 field of biomedical technologies, and particularly to a preparation method of a compound of formula II and its application in stress-induced cells. Background Art

[0002] Cells are the basic units of life activities and the foundation of the body's health. When the body's redox balance is disrupted, it will cause the interruption of redox signals and control, trigger oxidative stress damage, and lead to various diseases. Cerebral ischemia and hypoxia (such as stroke, cerebral hemorrhage, long-term plateau work, occlusion and / or stenosis of the internal carotid artery and vertebral artery, etc.) and oxidative stress damage to nerve cells can trigger 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 (caused by stenosis or occlusion of the cerebral blood supply artery resulting in blood not flowing into the brain) and cerebral hemorrhage (sudden rupture of blood vessels in the brain) are common central nervous system diseases. Due to insufficient cerebral blood supply, it causes ischemic and hypoxic damage to brain tissue and shows corresponding neurological deficit symptoms, including balance problems, hemiplegia, loss of sensation and vibration sensation, numbness, weakened reflexes, ptosis, visual field defects, aphasia, and apraxia, etc. Oxidative damage caused by ischemia, hypoxia, reperfusion, excessive free radicals, etc. is the main cause of the aggravation of ischemic reperfusion injury in early acute ischemic stroke.

[0004] Ataxia is mostly caused by dysfunction of the cerebellum, proprioception, and vestibular function, resulting in clumsy and uncoordinated movements. When it affects the trunk, limbs, and pharyngeal muscles, it can cause body balance, posture, gait, and speech disorders, including cerebellar ataxia, cerebral ataxia, sensory ataxia, vestibular ataxia, etc. The causes of ataxia are complex and are often the combined manifestations of multiple diseases, including acute cerebral ischemia, acute cerebral swelling, acute cerebral hemorrhage, etc. caused by infarction, edema, or hemorrhage, etc.

[0005] Brain trauma is mostly caused by external objects hitting the head, often causing permanent dysfunction of varying degrees, including abnormal focal symptoms in areas such as movement, sensation, speech, vision, and hearing. Peripheral nerve injury is a common clinical disease mainly caused by factors such as trauma, tumors, and metabolic diseases (such as diabetes and its complications), etc. It often leads to partial or complete loss of motor, sensory, and autonomic functions in the affected segments of the body, and even intractable neuralgia, seriously affecting the quality of life of patients.

[0006] Amyotrophic lateral sclerosis is a disease in which, after the upper and lower motor neurons are damaged, the muscles in the bulbar region, limbs, trunk, chest, and abdomen gradually become weak and atrophied. On May 11, 2018, five departments including the National Health Commission jointly formulated the Catalog of the First Batch of Rare Diseases, and amyotrophic lateral sclerosis was included in it.

[0007] When the blood supply and nerve innervation of articular cartilage malfunction, it is likely to cause joint damage, which in turn affects the joint repair ability. If joint damage and cartilage damage are not repaired in a timely manner, irreversible pathological changes may occur, and even joint degenerative changes may occur, affecting the quality of life of patients.

[0008] Arthritis is known as the "cancer that never dies", with a very high disability rate and seriously affecting the quality of life of patients. Joint injuries (such as joint traumatic lesions, degenerative osteoarthropathy, etc.) are common clinical conditions, including intractable wound lesions, knee osteoarthrosis, knee cartilage wear, cartilage damage, etc.

[0009] The 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, engineered cartilage. Physical therapy and nutrition represented by traditional Chinese medicine physical therapy and glucosamine, lubrication and relief represented by chitosan and hyaluronic acid, anti - inflammatory treatment represented by PRP, and joint replacement represented by arthroscope and artificial joint. Except for joint replacement, they are mainly aimed at relieving symptoms and delaying the disease course.

[0010] Articular cartilage has no blood vessels, nerves, or lymphatic system distribution, contains a small amount of chondrocytes, and has limited migration and proliferation ability. Articular cartilage injuries are difficult to heal on their own. When the blood supply and nerve innervation of articular cartilage malfunction, it is likely to cause joint damage, which in turn affects the joint repair ability. If timely and effective treatment and repair of the injury are not carried out, the injury will continue to worsen and cause osteoarthritic lesions, resulting in joint pain, joint swelling, or movement disorders, and even irreversible pathological changes and joint degenerative changes, affecting the patient's mobility and causing disability.

[0011] Mesenchymal stem cells (MSCs) have the potential for self-renewal and multi-directional differentiation. They are widely present in tissues and sites such as bone marrow, adipose tissue, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amniotic membrane, peripheral blood, muscle, urine, etc. They have the characteristics of wide source, no need for matching, low infection rate, strong differentiation potential, strong proliferation ability, convenient collection, etc. 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), interferon (IFN), etc., and participate in regulating processes such as cell growth, apoptosis, 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 harsh production and storage conditions (such as refrigeration) need to be adopted in the production, storage, transportation, and application links, etc., and their cell viability remains ≤12h, resulting in limited effective treatment radius and clinical application, etc.

[0012] When cells and microorganisms are stress-induced or stress-stimulated by external and exogenous stressors (including cold, heat, acid, alkali, high pressure, ultraviolet radiation, chemical substances, etc.), stress proteins will be induced in cells and microorganisms due to the stress response. Literature 1 (New limonophyllines A-C from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2 cell lines, Arch. Pharm. Res. (2018) 41:431–437) discloses that the compounds and their analogs isolated from the extract of the Rutaceae plant Atalantia monophylla have the activity of inhibiting the growth of tumor cells.

[0013] Injury repair is a long-term, progressive, and slow process. Timely repair of damaged cells or damaged organs can significantly improve, prevent, and treat related lesions and their complications. The cytokines secreted by stem cells are used to repair various injuries. Therefore, it is necessary to screen the types and dosages of exogenous stressors and the culture environment of cells and microorganisms to stress-induce the production of stress proteins with repair efficacy and safety and effectiveness to meet the urgent clinical needs such as injury repair. Summary of the Invention

[0014] The object of the present invention is to provide a preparation method of a compound of formula II. Compound 5 is demethylated in the presence of a demethylating agent to obtain the compound of formula II, wherein the demethylating agent is selected from any one or a combination of boron tribromide, hydrobromic acid, sulfuric acid, sodium ethyl mercaptide, sodium amide, trimethylsilyl iodide.

[0015]

[0016] In a preferred technical solution of the present invention, compound 5 is prepared by reacting compound 4 with 2-methyl-3-buten-2-ol under the catalysis of a Lewis acid. Among them, the Lewis acid is selected from any one or a combination of boron trifluoride diethyl ether, boron trifluoride acetonitrile, boron trifluoride, and aluminum trichloride.

[0017]

[0018] In a preferred technical solution of the present invention, compound 4 is prepared by reacting compound 3 with isopentenal in the presence of a titanium catalyst. Among them, the titanium catalyst is selected from any one or a combination of tetra-isobutoxy titanium and titanium tetrachloride.

[0019]

[0020] In a preferred technical solution of the present invention, compound 3 is prepared by reacting 2-amino-3-methoxybenzoic acid with phloroglucinol under the catalysis of an activator. Among them, the activator is selected from any one or a combination of p-toluenesulfonic acid, methanesulfonic acid, and benzenesulfonic acid.

[0021]

[0022] Another object of the present invention is to provide a method for preparing a compound of formula II, comprising the following steps:

[0023]

[0024] 1) React 2-amino-3-methoxybenzoic acid with phloroglucinol under the catalysis of an activator to obtain compound 3;

[0025] 2) React compound 3 with isopentenal in the presence of a titanium catalyst to obtain compound 4;

[0026] 3) React compound 4 with 2-methyl-3-buten-2-ol under the catalysis of a Lewis acid to obtain compound 5;

[0027] 4) Demethylate compound 5 in the presence of a demethylating agent to obtain the compound of formula II.

[0028] In a preferred technical solution of the present invention, in step 1), the activator is selected from any one or a combination of p-toluenesulfonic acid, methanesulfonic acid, and benzenesulfonic acid.

[0029] In a preferred technical solution of the present invention, in step 1), the reaction temperature is 80 °C to the reflux temperature.

[0030] In the preferred technical solution of the present invention, in step 2), the titanium catalyst is selected from any one of or a combination of tetra-isobutoxy titanium and titanium tetrachloride.

[0031] In the 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 from -78°C to -50°C, preferably from -78°C to -60°C.

[0032] In the preferred technical solution of the present invention, in step 2), after the dropping is completed, the temperature is raised to 0°C to room temperature for reaction.

[0033] In the preferred technical solution of the present invention, in step 2), the reaction is carried out under the protection of an inert gas, wherein the inert gas is selected from any one of or a combination of argon and nitrogen.

[0034] In the preferred technical solution of the present invention, in step 3), the reaction is carried out in a microwave environment, and the reaction temperature is 100 - 150°C, preferably 120 - 130°C.

[0035] In the preferred technical solution of the present invention, in step 3), the Lewis acid is selected from any one of or a combination of boron trifluoride diethyl etherate, boron trifluoride acetonitrile, boron trifluoride, and aluminum trichloride.

[0036] In the preferred technical solution of the present invention, in step 4), the demethylation reagent is added dropwise to the reaction system, wherein the dropping temperature is from -10°C to 5°C, preferably from -5°C to 0°C.

[0037] In the preferred technical solution of the present invention, in step 4), after the dropping is completed, the temperature is raised to 20°C to 35°C for reaction, preferably 25°C to 30°C.

[0038] In the preferred technical solution of the present invention, in step 4), the reaction is carried out under the protection of an inert gas, wherein the inert gas is selected from any one of or a combination of argon and nitrogen.

[0039] In the preferred technical solution of the present invention, in step 4), the demethylation reagent is selected from any one of or a combination of boron tribromide, hydrobromic acid, sulfuric acid, sodium ethanethiolate, sodium amide, and trimethylsilyl iodide.

[0040] The purpose of the present invention is to provide an application of a compound of formula II for inducing cells to produce proteins with repair efficacy under stress.

[0041] In the preferred technical solution of the present invention, the repair includes any one of or a combination of cell repair, joint repair, hair follicle repair, cartilage repair, skin injury cell repair, nerve injury cell repair, organ injury cell repair, pulmonary fibrosis repair, liver injury repair, kidney injury repair, ovarian repair, Crohn's disease repair, sub-health repair, and aging repair.

[0042] The prior patent applications (CN202310042911X, CN2023100429105, CN2023100429139, CN2023100429143, PCTCN2023073582, PCTCN2023073566, PCTCN2023073590, PCTCN2023073596) are used as essential technical content and technical references for this application.

[0043] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between a liquid and a solid, the percentage is volume / weight percentage; when the present invention relates to the percentage between a solid and a liquid, the percentage is weight / volume percentage; and the rest are weight / weight percentages.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1. The present invention provides a method for preparing a compound of formula II, which is used to induce stress in cells or MSCs to produce proteins with repair efficacy, and the repair includes any one or a combination of cell repair, joint repair, hair follicle repair, cartilage repair, skin injury cell repair, nerve injury cell repair, organ injury cell repair, pulmonary fibrosis repair, liver injury repair, kidney injury repair, ovarian repair, Crohn's disease repair, sub-health repair, and aging repair.

[0046] 2. The preparation method of the present invention has the advantages of simple operation, improved resource utilization rate, environmental friendliness, and suitability for industrial production. Detailed Embodiments

[0047] The technical solutions of the present invention are further introduced in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0048] Preparation Example 1 Preparation of Compound 3

[0049] Dissolve 2-amino-3-methoxybenzoic acid (1.67 g, 10 mmol) and phloroglucinol (1.26 g, 10 mmol) in 50 mL of n-hexanol, add p-toluenesulfonic acid (0.17 mg, 1 mmol), and reflux for 16 h. Cool the reaction solution to room temperature, add 300 mL of n-hexane and 200 mL of dichloromethane, and stir for 1 h to precipitate a pale yellow solid. After drying, recrystallize with ethanol and water to obtain yellow solid 3 (2.2 g, 86%). 11H NMR (400 MHz, 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 = 8.0 Hz), 7.17 (1H, t, J = 8.0 Hz), 6.68 (1H, s), 6.00 (1H, s), 4.01 (3H, s); 13 13C (100 MHz, 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] + 。

[0050] Preparation Example 2 Preparation of Compound 4

[0051] Dissolve Compound 3 (2.2 g, 8.56 mmol) and isopentenal (2.16 g, 25.68 mmol) in 10 mL of toluene. Cool to -78 °C under argon protection and slowly add tetra-isobutoxytitanium (11.0 g, 34.24 mmol). Allow the temperature to rise to room temperature naturally over 4 hours and continue stirring at room temperature for 20 h. Add 100 mL of dichloromethane and 100 mL of water, and separate the layers. Wash the organic layer with saturated brine and obtain yellow solid 4 (2.07 g, 75%) by column chromatography. 1 1H NMR (400 MHz, DMSO-d6): 14.53 (1H, s), 9.71 (1H, s), 7.73 (1H, dd, J = 8.1 Hz, 1.1 Hz), 7.37 (1H, dd, J = 7.8 Hz, 1.0 Hz), 7.25 (1H, t, J = 8.0 Hz), 7.00 (1H, d, J = 10.0 Hz), 6.07 (1H, s), 5.70 (1H, d, J = 10.0 Hz), 4.02 (3H, s), 1.42 (6H, s); 13 13C (125 MHz, 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] + 。

[0052] Preparation Example 3 Preparation of Compound 5

[0053] Compound 4 (2.07 g, 6.41 mmol) was dissolved in 20 mL of 1,4-dioxane. Boron trifluoride diethyl etherate (2.4 mL, 19.23 mmol) and 2-methyl-3-buten-2-ol (3.4 mL, 32.05 mmol) were added, and the reaction was carried out under microwave irradiation at 120 °C for 5 h. The reaction mixture was diluted with 300 mL of dichloromethane, washed successively with 50 mL of saturated aqueous sodium bicarbonate, 50 mL of saturated aqueous ammonium chloride, and 50 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to give compound 5 (1.18 g, 47%). ESI-MS: 392.4 [M+H] + 。

[0054] Preparation Example 4 Preparation of Compound of Formula II

[0055] Compound 5 (0.59 g, 1.51 mmol) was dissolved in 5 mL of anhydrous dichloromethane. A solution of boron tribromide (0.3 mL, 3.02 mmol) in dichloromethane (5 mL) was added dropwise to the above solution at -78 °C under argon protection. After the addition was complete, the reaction mixture was stirred at room temperature for 8 h. TLC analysis showed that the starting material had disappeared. The pH of the reaction mixture was adjusted to 7 by slowly adding saturated aqueous sodium bicarbonate solution, and the mixture was extracted and concentrated. The residue was purified by column chromatography to give the compound of formula II as a yellow solid (0.27 g, 47%). 1 H NMR (400 MHz, CDCl3 + DMSO-d6): 7.70 (dd, J = 8.1 Hz, 1.1 Hz), 6.98 (t, J = 8.1 Hz), 7.10 (dd, J = 1.1 Hz, 8.1 Hz), 10.05 (1H, s), 8.68 (1H, brs), 14.62 (1H, s), 3.32 (d, J = 7.2 Hz), 5.24 (1H, t, J = 7.2 Hz), 6.59 (1H, d, J = 10.0 Hz), 5.59 (1H, d, J = 10.0 Hz), 1.67 (3H, s), 1.81 (3H, s), 1.48 (6H, s); ESI-MS: 378.3 [M+H] + 。

[0056] 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 according to the present invention. As long as they do not depart from the spirit of the present invention, they shall fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a compound of formula II, wherein the compound 5 is demethylated in the presence of a demethylating agent to obtain the compound of formula II, wherein, The demethylating agent is selected from any one or a combination of boron tribromide, hydrobromic acid, sulfuric acid, sodium ethanethiolate, sodium amide, and trimethylsilyl iodide, 2. The method according to claim 1, wherein the compound 5 is prepared by reacting the compound 4 with 2-methyl-3-buten-2-ol under the catalysis of a Lewis acid, wherein, wherein, The Lewis acid is selected from any one or a combination of boron trifluoride diethyl etherate, boron trifluoride acetonitrile, boron trifluoride, and aluminum trichloride, 3. The method according to any one of claims 1-2, wherein 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 or a combination of tetra(isobutoxy)titanium and titanium tetrachloride, 4. The method according to any one of claims 1-3, wherein the compound 3 is prepared by reacting 2-amino-3-methoxybenzoic acid with phloroglucinol under the catalysis of an activator, wherein, The activator is selected from any one or a combination of p-toluenesulfonic acid, methanesulfonic acid, and benzenesulfonic acid, 5. A method for preparing a compound of formula II, comprising the following steps, 1) 2-Amino-3-methoxybenzoic acid and phloroglucinol are reacted under the catalysis of an activator, to obtain compound 3; 2) Compound 3 and isopentenal are reacted in the presence of a titanium catalyst to obtain compound 4; 3) Compound 4 and 2-methyl-3-buten-2-ol are reacted under the catalysis of a Lewis acid, to obtain compound 5; 4) Compound 5 is demethylated in the presence of a demethylating agent to obtain the compound of formula II.

6. According to the method of claim 5, in step 1), the reaction temperature is from 80 °C to the reflux temperature.

7. For the method according to any one of claims 5-6, in step 2), the reaction is carried out under the protection of an inert gas, wherein, The inert gas is selected from any one of argon and nitrogen, or a combination thereof.

8. According to the method of any one of claims 5-7, in step 3), the reaction is carried out in a microwave environment, the reaction temperature is 100-150 °C, preferably 120-130 °C.

9. For the method according to any one of claims 5-8, in step 4), the demethylating reagent is added dropwise to the reaction system, wherein, The dropping temperature is from -10 °C to 5 °C, preferably from -5 °C to 0 °C.

10. Use of the compound of formula II for inducing cells to produce proteins with repair efficacy under stress.

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