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

Abstract

The invention relates to a preparation method of a compound as shown in a formula IV, which comprises the following step of: performing demethylation on a compound 6 in the presence of a demethylation reagent to prepare the compound as shown in the formula IV. The compound as shown in the formula IV 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 IV 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. 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 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, amyotrophic lateral sclerosis), etc.

[0003] Stroke (narrowing or occlusion of the cerebral blood supply artery resulting in the inability of blood to flow 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, ischemic and hypoxic damage to brain tissue occurs, and corresponding neurological deficit symptoms are manifested, 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 disorders of the cerebellum, proprioception, and vestibular function, resulting in clumsy and uncoordinated movements. When it involves 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 brain 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). 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 the upper and lower motor neurons are damaged, resulting in the gradual weakness and atrophy of the muscles including the bulbar region, limbs, trunk, chest, and abdomen.

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

[0008] Arthritis is known as the "cancer that does not kill", with a very high disability rate and seriously affecting the quality of life of patients. Joint injuries (such as traumatic joint lesions, degenerative osteoarthropathy, etc.) are common clinical conditions, including intractable wound lesions, knee osteoarthrosis, knee cartilage wear, cartilage injury, etc.

[0009] Treatment or repair methods for joint injuries and articular cartilage injuries include anti-inflammatory, analgesic (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 mainly focus on relieving symptoms and delaying the disease process.

[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 innervation of articular cartilage malfunction, it is likely to cause joint injuries, which in turn affect the joint repair ability. If the injury is not treated and repaired effectively in a timely manner, the injury will continue to worsen and cause osteoarticular 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-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, amnion, 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, 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), interferon (IFN), etc., and participate in regulating cell growth, cell apoptosis, cell differentiation, antiviral, immune maturation and other processes. They can be used for immune regulation, tissue repair and 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 at ≤12h, 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.), the stress response will induce the cells and microorganisms to produce stress proteins. Document 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 Atalantia monophylla, a Rutaceae plant, and their analogs have the activity of inhibiting tumor cell growth.

[0013] Injury repair is a long-term, gradual and slow process. Timely repair of damaged cells or damaged 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 types and dosages of exogenous stressors, the culture environment of cells and microorganisms, and induce stress to produce stress proteins that have repair effects and are safe and effective, so as 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 IV, wherein compound 6 is demethylated in the presence of a demethylating agent to obtain a compound of formula IV, 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 methyl iodide, 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, compound 5 is prepared by a ring-closing reaction of compound 4 in the presence of a dehydrogenating agent.

[0019]

[0020] In a preferred technical solution of the present invention, compound 4 is prepared by reacting compound 3 with 2-methyl-3-buten-2-ol under the catalysis of a Lewis acid, 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.

[0021]

[0022] 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, wherein the activator is selected from any one or a combination of p-toluenesulfonic acid, methanesulfonic acid, and benzenesulfonic acid.

[0023]

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

[0025]

[0026] 1) React 2-amino-3-methoxybenzoic acid with phloroglucinol under the catalysis of an activator to obtain compound 3;

[0027] 2) React compound 3 with 2-methyl-3-buten-2-ol under the catalysis of a Lewis acid to obtain compound 4;

[0028] 3) Perform ring-closing of compound 4 in the presence of a dehydrogenating agent to obtain 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 the compound of Formula IV.

[0031] 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.

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

[0033] In a preferred technical solution of the present invention, in step 2), the Lewis acid is selected from any one or a combination of boron trifluoride diethyl etherate, boron trifluoride acetonitrile, boron trifluoride, and aluminum trichloride.

[0034] In a preferred technical solution of the present invention, in step 2), the reaction is carried out in a microwave environment, and the reaction temperature is 100 - 150 °C, preferably 120 - 130 °C.

[0035] In a preferred technical solution of the present invention, in step 3), the dehydrogenating agent is an oxidative dehydrogenating agent. Preferably, the oxidative dehydrogenating agent is selected from any one or a combination of polyphosphoric acid and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

[0036] In a preferred technical solution of the present invention, in step 4), the base is selected from any one or a combination of NaH, KH, sodium methoxide, sodium ethoxide, NaOH, KOH, and cesium carbonate.

[0037] In a preferred technical solution of the present invention, in step 4), the alkylating agent is selected from any one or a combination of methyl iodide, dimethyl sulfate, dimethyl carbonate, methyl trifluoromethanesulfonate, methyl fluorosulfonate, methyl methanesulfonate, 2,2-dimethoxypropane, dimethyl pyrocarbonate, N-methyl-N-nitrosourea, and dicumyl peroxide.

[0038] In a preferred technical solution of the present invention, in step 4), the molar ratio of Compound 5 to the base is 1:1 - 2, preferably 1:1.2 - 1.5.

[0039] In a preferred technical solution of the present invention, in step 4), the molar ratio of Compound 5 to the alkylating agent is 1:1 - 3, preferably 1:2 - 3.

[0040] In a preferred technical solution of the present invention, in step 5), the demethylating agent is selected from any one or a combination of boron tribromide, hydrobromic acid, sulfuric acid, sodium ethyl mercaptide, sodium amide, and trimethylsilyl iodide.

[0041] In a preferred technical solution of the present invention, in step 5), the demethylating agent 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.

[0042] In a preferred technical solution of the present invention, in step 5), after the dropping is completed, the temperature is raised to 20°C to 35°C for reaction, preferably 25°C to 30°C.

[0043] In a preferred technical solution of the present invention, in step 5), the reaction is carried out under the protection of an inert gas, wherein the inert gas is selected from any one or a combination of argon and nitrogen.

[0044] Another object of the present invention is to provide an intermediate having the structure shown in Compound 4.

[0045]

[0046] The object of the present invention is to provide an application of a compound of Formula IV in inducing cells to produce proteins with repair efficacy under stress.

[0047] In a preferred technical solution of the present invention, 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.

[0048] The prior patent applications (CN202310042911X, CN2023100429105, CN2023100429139, CN2023100429143, PCTCN2023073582, PCTCN2023073566, PCTCN2023073590, PCTCN2023073596) are essential technical contents and technical references for this application.

[0049] 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.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1. The present invention provides a method for preparing a compound of formula IV, which is used to induce stress-induced cells or MSCs to produce proteins with repair effects, 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.

[0052] 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 implementation mode

[0053] The technical solutions of the present invention will be further introduced in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0054] Preparation Example 1 Preparation of Compound 3

[0055] Dissolve 2-amino-3-methoxybenzoic acid (2.00 g, 12 mmol) and phloroglucinol (1.51 g, 12 mmol) in 50 mL of n-hexanol, add p-toluenesulfonic acid (0.20 mg, 1.2 mmol), and reflux for 16 h. Cool the reaction solution to room temperature, add 300 mL of n-hexane and 200 mL of dichloromethane, stir for 1 h to precipitate a pale yellow solid. After drying, recrystallize with ethanol and water to obtain yellow solid 3 (2.76 g, 90%).

[0056] 1 H 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 C (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] + .

[0057] Preparation Example 2 Preparation of Compound 4

[0058] Compound 3 (2.57 g, 10 mmol) was dissolved in 20 mL of 1,4-dioxane. Boron trifluoride diethyl etherate (4.9 mL, 40 mmol) and 2-methyl-3-buten-2-ol (8.62 g, 100 mmol) were added, and the reaction was carried out at 120 °C under microwave irradiation for 5 h. It was diluted with 300 mL of dichloromethane, washed successively with 50 mL each of saturated aqueous sodium bicarbonate, saturated aqueous ammonium chloride, and saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound 4 (2.9 g, 74%). ESI-MS: 394.2 [M+H] + , 416.4 [M+Na] + .

[0059] Preparation Example 3 Preparation of Compound 5

[0060] Compound 4 (1.45 g, 3.69 mmol) was dissolved in 15 mL of anhydrous toluene. 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (1.26 g, 5.54 mmol) was added, and the mixture was refluxed for 3 h. It was diluted with 100 mL of dichloromethane, washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound 5 (1.18 g, 82%).

[0061] 1 1H-NMR (400 MHz, DMSO-d 6 ): 14.57 (1H, s), 9.06 (1H, s), 7.73 (1H, d, J = 8.0 Hz), 7.38 (1H, d, J = 7.7 Hz), 7.25 (1H, t, J = 8.0 Hz), 6.67 (1H, d, J = 9.9 Hz), 5.74 (1H, d, J = 9.9 Hz), 5.07 (1H, t, J = 7.0 Hz), 4.03 (3H, s), 3.56 (2H, d, J = 7.0 Hz), 1.94 (3H, s), 1.73 (3H, s), 1.44 (6H, s). ESI-MS: 392.2 [M+H] + , 414.4 [M+Na] + .

[0062] Preparation Example 4 Preparation of Compound 6

[0063] Compound 5 (0.59 g, 1.51 mmol) was dissolved in 6 mL of anhydrous DMF. At 0 °C, NaH (90 mg, 60%, 2.27 mmol, 1.5 eq.) was added. After stirring for 10 minutes, methyl iodide (0.64 g, 4.53 mmol, 3 eq.) was added. The reaction was carried out at room temperature in the dark for 2 h. TLC detection showed that the raw material had disappeared. DMF was removed by rotary evaporation under reduced pressure. 50 mL of ethyl acetate and 20 mL of water were added. The layers were separated, and the aqueous phase was extracted with ethyl acetate (50 mL×2). The organic phases were combined and dried over anhydrous sodium sulfate. After concentration and column chromatography, a yellow solid, compound 6 (0.57 g, 93%), was obtained. 1 1H-NMR (400 MHz, CDCl3): 14.39 (1H, s), 7.90 (1H, d, J = 7.8 Hz), 7.22 (1H, d, J = 7.9 Hz), 7.16 (1H, d, J = 7.2 Hz), 6.78 (1H, d, J = 9.9 Hz), 5.57 (1H, d, J = 9.9 Hz), 5.29 (1H, t, J = 5.9 Hz), 3.98 (3H, s), 3.60 (3H, s), 3.47 (2H, d, J = 5.9 Hz), 1.79 (3H, s), 1.71 (3H, s), 1.46 (6H, s). ESI-MS: 406.2 [M+H] + .

[0064] Preparation Example 5 Preparation of Compound of Formula IV

[0065] Compound 6 (0.57 g, 1.40 mmol) was dissolved in 14 mL of anhydrous dichloromethane. Under ice bath and argon protection, a dichloromethane solution of boron tribromide (2.1 mL, 1.5 eq., 1 M) was added dropwise. The reaction was carried out at room temperature overnight (14 h). 60 mL of dichloromethane was added to dilute the reaction solution, and the reaction was 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 IV (0.35 g, 64%) was obtained. 1 1H-NMR (400 MHz, Acetone-d6): 14.74 (1H, s), 9.82 (1H, brs), 9.05 (1H, brs), 7.76 (1H, d, J = 7.8 Hz), 7.25 (1H, d, J = 7.8 Hz), 7.12 (1H, t, J = 7.8 Hz), 6.77 (1H, d, J = 9.8 Hz), 5.71 (1H, d, J = 9.8 Hz), 5.17 (1H, t, J = 7.2 Hz), 3.61 (2H, d, J = 7.2 Hz), 1.99 (3H, s), 1.76 (3H, s), 1.49 (6H, s). ESI-MS: 392.2 [M+H] + .

[0066] 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 deformations 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 IV, wherein the compound 6 is demethylated in the presence of a demethylating agent to obtain the compound of formula IV, 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, 2. The preparation method according to claim 1, wherein compound 6 is prepared by reacting compound 5 with an alkylating agent in the presence of a base, where The base is selected from any one or a combination of NaH, KH, sodium methoxide, sodium ethoxide, NaOH, KOH, cesium carbonate, and the alkylating agent is selected from any one or a combination of methyl iodide, dimethyl sulfate, dimethyl carbonate, methyl trifluoromethanesulfonate, methyl fluorosulfonate, methyl methanesulfonate, 2,2-dimethoxypropane, dimethyl pyrocarbonate, N-methyl-N-nitrosourea, dicumyl peroxide.

3. The preparation method according to any one of claims 1-2, wherein the compound 5 is prepared by a ring-closing reaction of the compound 4 in the presence of a dehydrogenating agent.

4. The preparation method according to any one of claims 1-3, wherein the compound 4 is prepared by reacting the compound 3 with 2-methyl-3-buten-2-ol under the catalysis of a Lewis acid, wherein, The Lewis acid is selected from any one or a combination of boron trifluoride diethyl ether, boron trifluoride acetonitrile, boron trifluoride, aluminum trichloride.

5. The preparation method according to any one of claims 1-4, wherein the compound 3 is prepared by reacting 2-amino-3-methoxybenzoic acid with phloroglucinol under the catalysis of an activator, where The activator is selected from any one or a combination of p-toluenesulfonic acid, methanesulfonic acid, and benzenesulfonic acid, 6. A preparation method of a compound of formula IV, comprising the following steps: 1) 2-Amino-3-methoxybenzoic acid and phloroglucinol react under the catalysis of an activator. to obtain compound 3; 2) Compound 3 and 2-methyl-3-buten-2-ol react under the catalysis of a Lewis acid. to obtain compound 4; 3) Compound 4 undergoes ring-closing in the presence of a dehydrogenating agent to obtain compound 5; 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 the compound of formula IV.

7. The preparation method according to claim 6, in step 2), the reaction is carried out in a microwave environment, and the reaction temperature is 100-150 °C, preferably 120-130 °C.

8. The preparation method according to any one of claims 6-7, in step 3), the dehydrogenating agent is an oxidative dehydrogenating agent, preferably, the oxidative dehydrogenating agent is selected from any one or a combination of polyphosphoric acid, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

9. An intermediate with the structure shown in Compound 4 10. Use of the compound of formula IV for inducing cells to produce proteins with repair efficacy under stress.

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