A protein phosphatase agonist and uses thereof

Abstract

The application discloses a benzopyrazoloxazine derivative or a pharmaceutically acceptable salt thereof, which is shown as formula I, R is selected from hydrogen, halogen, alkyl and alkoxy; m is an integer from 1 to 3; X is selected from oxygen or sulfur; R1 is selected from hydrogen, alkyl and acyl. The application also discloses application of the benzopyrazoloxazine derivative in preparation of a medicine for preventing and treating diseases caused by absence of PP2A activity or reduction of PP2A function. The application discloses application of the benzopyrazoloxazine derivative in preparation of a protein phosphatase agonist.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and relates to a protein phosphatase agonist and its application, specifically to a benzopyrazole-oxazine protein phosphatase agonist and its application. Background Technology

[0002] Protein phosphatase 2A (PP2A), a crucial member of the protein phosphatase family, exhibits high homology between its catalytic subunit and the catalytic domains of other members in the family. PP2A mediates the entire spectrum of cellular life processes, including cell signaling, cell differentiation, and apoptosis.

[0003] The loss or reduction of PP2A activity disrupts important downstream pathways, including MAPK and ERK, leading to cellular regulatory disorder and potentially causing carcinogenesis. Loss of PP2A activity in T cells can reduce their clearance capacity and may also be a significant factor in the occurrence and development of carcinogenesis.

[0004] The release of neurotransmitters and the opening and closing of ion channels accompany numerous protein phosphorylation and dephosphorylation processes, forming a crucial link in intracellular signal transmission. Under the combined action of adenosine triphosphate (ATP) and protein phosphorylases (protein kinases, PK), protein phosphorylation is catalyzed, altering the protein structure and transforming it into an active protein. The activated protein then activates downstream target proteins, thereby activating the entire cascade reaction and completing signal transduction. PP2A is a key factor in protein dephosphorylation, ensuring the timely closure of signaling pathways and preparing for the next neural activity.

[0005] Alzheimer's disease (AD), also known as senile dementia, is a neurological disorder characterized by memory, cognition, language, and behavioral impairments caused by chronic, progressive degenerative changes in the central nervous system.

[0006] According to the "China Alzheimer's Disease Report 2021" led by Ruijin Hospital in Shanghai, there were 13,243,950 people living with Alzheimer's disease and other dementias in China in 2019. The prevalence and mortality rates are slightly higher than the global average, and the relevant data are higher in women than in men. Specifically, the prevalence rate (1188.9 / 100,000) and mortality rate (30.8 / 100,000) in women are higher than those in men (669.3 / 100,000) and (14.6 / 100,000), respectively.

[0007] Currently, two classes of drugs are commonly used in clinical treatment of Alzheimer's disease (AD): cholinesterase inhibitors such as donepezil and N-methyl-D-aspartate (NMDA) receptor antagonists such as memantine. Donepezil works by inhibiting acetylcholinesterase, maintaining or increasing acetylcholine levels to a certain extent, thereby increasing cholinergic nerve signals and improving the patient's cognitive function. Memantine, on the other hand, improves cognitive function by inhibiting NMDA receptor activity, reducing the neurotoxic effects of excitatory amino acids. However, these drugs only provide symptomatic treatment and can improve cognitive and memory impairments to some extent; currently, no drug can stop or slow the progression of AD.

[0008] We believe that the occurrence and development of AD are closely related to the decline in PP2A function. Reduced PP2A activity prevents the protein from being dephosphorylated and inactivated, allowing the postsynaptic membrane to continuously receive neurotransmitters released from the presynaptic membrane. This prevents the signal pathway from closing in time, leading to delayed neural conduction and memory impairment.

[0009] Amyloid precursor protein (APP) is a widely distributed and essential bioactive substance in the body. β-amyloid (Aβ) is produced from APP through hydrolysis by β-secretase and γ-secretase. In vitro tests have shown that Aβ inhibits the biological activity of protein phosphatase (PP), and the intensity of this inhibition is closely related to Aβ levels. Normal Aβ levels have limited effect on PP activity; only when Aβ levels reach a certain level does it inhibit PP activity, and this inhibitory effect increases with increasing Aβ concentration. Therefore, abnormally elevated Aβ levels in the body inhibit PP activity and are a crucial factor in inducing Alzheimer's disease (AD).

[0010] The biological function of tau protein (P-tau) is to induce tubulin polymerization into microtubules and prevent their depolymerization, thereby maintaining the morphology and function of nerve cells. To restore PP biological activity, the body sacrifices P-tau by tangling with Aβ to form P-tau / Aβ complexes, which are deposited in nerve cells to reduce Aβ levels and restore PP activity. This fluctuation in PP biological activity caused by changes in Aβ levels manifests in Alzheimer's disease (AD) patients as alternating periods of good and bad memory. Over time, the P-tau / Aβ complex gradually overwhelms nerve cells, leading to irreversible organic neurological damage in AD patients. PET scans of the brains of AD patients have confirmed that P-tau / Aβ tangles (plaques) cause brain atrophy and nerve cell death.

[0011] PP agonists enhance PP bioactivity, catalyzing timely protein dephosphorylation to maintain normal neural activity. Furthermore, increased PP activity also promotes the dephosphorylation of phosphorylated tau protein (P-tau-Pi), thereby preventing neurotoxicity and neuronal death mediated by excessive tau protein phosphorylation.

[0012] Therefore, the development of PP2A agonists could bring hope for the recovery of patients with diseases caused by reduced PP2A activity, such as cancer patients and AD patients. Summary of the Invention

[0013] The purpose of this invention is to provide a class of benzopyrazine compounds that can act as protein phosphatase agonists, exhibiting a strong excitatory effect on impaired PP2A activity and significantly increasing the biological activity of PP2A.

[0014] Benzopyrazolidine derivatives or their pharmaceutically acceptable salts, as shown in Formula I, are as follows:

[0015]

[0016] in:

[0017] R is selected from hydrogen, halogen, alkyl, and alkoxy.

[0018] m is an integer from 1 to 3;

[0019] X is selected from oxygen or sulfur;

[0020] R1 is selected from hydrogen, alkyl, or acyl groups.

[0021] Preferably, R is selected from hydrogen, halogen, C1-C2 alkyl, and C1-C2 alkoxy.

[0022] m is an integer from 1 to 3;

[0023] X is selected from oxygen or sulfur;

[0024] R1 is selected from hydrogen, C1-C3 alkyl, formyl (-CHO), and acetyl (-COCH3).

[0025] Halogens are specifically selected from F and Cl; alkyl groups can be specifically selected from methyl, ethyl, n-propyl, etc.; alkoxy groups can be specifically selected from methoxy, ethoxy, etc.

[0026] The benzopyrazoloxazine derivatives are preferably derived from the following compounds, but are not limited to them:

[0027] (5-Imine-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-2-yl)methanol;

[0028] 2-(methoxymethyl)-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0029] 2-(ethoxymethyl)-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0030] 2-(propoxymethyl)-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0031] Methyl formate (5-imino-5H-benzo[d]pyrazole[5,1-b][1,3]oxazin-2-yl);

[0032] Methyl acetate (5-imino-5H-benzo[d]pyrazole[5,1-b][1,3]oxazin-2-yl);

[0033] 2-(methylthiomethyl)-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0034] 2-(methoxymethyl)-8-methyl-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0035] 2-(methoxymethyl)-8-ethyl-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0036] 2-(methoxymethyl)-8-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0037] 2-(methoxymethyl)-7-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0038] 2-(methoxymethyl)-7-chloro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0039] 2-(methoxymethyl)-7-methoxy-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0040] 2-(methoxymethyl)-7-ethoxy-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0041] 2-(ethoxymethyl)-7-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0042] 2-(propoxymethyl)-7-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0043] 2-(methoxyethyl)-8-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine;

[0044] 2-(methoxypropyl)-8-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine.

[0045] The pharmaceutically acceptable salts refer to salts that are produced by the reaction of the above-mentioned benzopyrazine derivatives with commonly used pharmaceutical acids or bases, and are free of side effects and can increase the physicochemical properties (such as water solubility) of the compounds; the acids are selected from hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, succinic acid, maleic acid, fumaric acid, acetic acid, citric acid, tartaric acid, benzoic acid, benzenesulfonic acid, and naphthalenesulfonic acid; the bases are selected from sodium hydroxide, sodium carbonate, sodium bicarbonate, or potassium hydroxide, etc.

[0046] Another object of the present invention is to provide a method for preparing benzopyrazoloid derivatives with structures as shown in Formula I, wherein when R1 is selected from hydrogen or alkyl groups, the reaction route is as follows:

[0047]

[0048] When R1 is selected from an acyl group, the reaction route is as follows:

[0049]

[0050] Compound II can be prepared from compound IV via a diazotization and reduction reaction, as follows:

[0051]

[0052] Compound II can also be prepared by reacting compound V with hydrazine solutions of different concentrations, hydrazine salts, or hydrazine derivatives with different substituents, via the following reaction route:

[0053]

[0054] Among them, Y includes, but is not limited to, F, Cl, Br, I, sulfonic acid groups, etc.

[0055] Unless otherwise specified, when preparing the benzopyrazoloid derivatives of Formula I of the present invention, the compound used as a starting material is a commercially available known compound, or can be prepared from a known compound by a known method, a similar method of a known method, or a similar method to the method described in the examples.

[0056] The benzopyrazolone derivatives shown in Formula I have a strong excitatory effect on impaired PP2A activity, significantly increasing PP2A bioactivity, significantly inhibiting okadaic acid-induced cellular Tau protein phosphorylation, and significantly increasing okadaic acid and Aβ levels. 1-42 Survival rate of damaged cells.

[0057] Another object of the present invention is to provide the use of the benzopyrazoloxazine derivatives of Formula I in the preparation of medicaments for the prevention and treatment of diseases caused by the loss of PP2A activity or the reduction of PP2A function.

[0058] Preferably, the disease caused by the loss of PP2A activity or the reduction of PP2A function is a tumor, and the benzopyrazoloid derivatives shown in Formula I can be used to prevent and treat the occurrence and development of tumors.

[0059] Preferably, the disease caused by the loss of PP2A activity or the reduction of PP2A function is a neurodegenerative disease, which includes but is not limited to Alzheimer's disease (AD). The benzopyrazine derivatives shown in Formula I can be used for the prevention and treatment of Alzheimer's disease.

[0060] Another object of the present invention is to provide the use of the benzopyrazoloxazine derivatives of Formula I in the preparation of protein phosphatase agonists, preferably in the preparation of protein phosphatase 2A agonists. Detailed Implementation

[0061] Experimental instruments

[0062] Rotary evaporator RE-52AA (Shanghai Yarong Biochemical Instrument Factory); SHB-III circulating water multi-purpose vacuum pump (Nanjing Wen'er Instrument Equipment Co., Ltd.); JJ-1 precision booster stirrer (Jintan Chengdong Xinrui Instrument Factory); 85-1 magnetic stirrer (Nanjing Wen'er Instrument Equipment Co., Ltd.); ZF-I three-way UV analyzer; X-4 digital melting point apparatus (Beijing Fukai Instrument Co., Ltd.); Bruker AV-300 nuclear magnetic resonance spectrometer (TMS as internal standard, DMSO-d6 as solvent); mass spectrometry was performed using an Agilent Technologies 6520B Accurate-Mass A-TOF LC / MS mass spectrometer; compound purity was determined using a Shimadzu LC-20AT high-performance liquid chromatograph. 18 The chromatographic column was equipped with an SAP-10 UV detector, a detection wavelength of 220 nm, and a mobile phase of methanol-water (70:30 V / V).

[0063] Material

[0064] Thin-layer chromatography silica gel plates (silica gel GF254, Yantai Dexin Biotechnology Co., Ltd.); DMF used Molecular sieve drying, ethanol use Molecular sieves were dried, and other reagents were commercially available analytical grade or chemically pure; most of the reagents were sourced from Aladdin Reagent Co., Ltd.

[0065] Example 1

[0066] Preparation of (5-imino-benzo[d][1,3]oxazine[5,1-b]pyrazol-2-yl)methanol (compound 1)

[0067] Preparation of 1,2-cyanophenylhydrazine

[0068] 2-Cyanophenylhydrazine was prepared according to the method in Example 1 of Chinese Patent CN 101508696A, “Preparation of 2-hydrazinophenyl nitrile hydrochloride”.

[0069] 2. Preparation of (5-imino-benzo[d][1,3]oxazine[5,1-b]pyrazol-2-yl)methanol

[0070] 1.3 g of crude 2-cyanophenylhydrazine and 15 mL of saturated hydrogen chloride ethanol solution were added to a reaction flask. 2.0 g of methyl 4-hydroxy-3-oxobutyrate was added with stirring. The reaction was allowed to proceed overnight at room temperature. 30 mL of ice water was added, and the mixture was stirred for 1 hour. The mixture was filtered, and the filter cake was washed several times with water. After drying, 1.7 g of crude compound 1 hydrochloride was obtained. Recrystallization from 10% ethanol yielded 1.3 g of purified compound 1 hydrochloride (white solid).

[0071] The purified hydrochloride product of compound 1 was added to a reaction flask, 20 mL of water was added, the temperature was controlled below 15 °C, sodium hydroxide solution was added dropwise to adjust the pH to 7-8, stirring was continued for 1 hour, filtered, washed with water several times, and dried to obtain 0.6 g of compound 1.

[0072] ESI-MS: m / z 216.2 [M+1] + .

[0073] Example 2

[0074] Preparation of 2-(methoxymethyl)-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 2)

[0075] Compound 2 was obtained by replacing methyl 4-hydroxy-3-oxobutyrate with methyl 4-methoxy-3-oxobutyrate according to the method of Example 1.

[0076] ESI-MS: m / z 230.2 [M+1] + .

[0077] Example 3

[0078] Preparation of 2-(ethoxymethyl)-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 3)

[0079] Compound 3 was obtained by replacing methyl 4-hydroxy-3-oxobutyrate with methyl 4-ethoxy-3-oxobutyrate according to the method of Example 1.

[0080] ESI-MS: m / z 243.3 [M+1] + .

[0081] Example 4

[0082] Preparation of 2-(propoxymethyl)-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 4)

[0083] Compound 4 was obtained by replacing methyl 4-hydroxy-3-oxobutyrate with methyl 4-propoxy-3-oxobutyrate according to the method of Example 1.

[0084] ESI-MS: m / z 258.3 [M+1] + .

[0085] Example 5

[0086] Preparation of methyl formate (5-imino-5H-benzo[d]pyrazole[5,1-b][1,3]oxazin-2-yl) (compound 5)

[0087] Add 10 mL of formic acid to the reaction flask, cool to -10 °C in an ice bath, add 2 mL of acetic anhydride, keep warm and stir for 0.5 hours, add 0.5 g of compound 1 and 0.1 g of DMAP, raise to room temperature and react for 2 hours, add 50 mL of ice water, filter, wash with water until the droplet is neutral, and then recrystallize from ethyl acetate to obtain compound 5.

[0088] ESI-MS: m / z 244.2 [M+1] + .

[0089] Example 6

[0090] Preparation of methyl acetate (5-imino-5H-benzo[d]pyrazole[5,1-b][1,3]oxazin-2-yl) (compound 6)

[0091] Compound 6 was obtained by replacing formic acid with acetic acid, following the method of Example 5.

[0092] ESI-MS: m / z 258.3 [M+1] + .

[0093] Example 7

[0094] Preparation of 2-(methylthiomethyl)-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 7)

[0095] 1. Preparation of methyl 4-(methylthio)-3-oxobutyrate

[0096] Add 500 mL of DMF, 108.5 g of methyl chloroacetate, and 72.1 g of sodium methanethiol to the reaction flask in sequence. React at 105 °C for 5 hours, then stop heating and cool to room temperature. Add 2.5 L of water to the reaction solution and extract the aqueous phase with dichloromethane (3 × 500 mL). Combine the organic phases and wash them successively with water (5 × 300 mL) and saturated brine (300 mL). Dry the organic phase with anhydrous sodium sulfate and concentrate under reduced pressure to obtain 89.2 g of oil, which is methyl 2-methylthioacetate. It is used directly in the next reaction without purification.

[0097] Dissolve methyl 2-methylthioacetate and 118.9 g of diethyl malonate in 300 mL of dry DMF and place the solution in a constant pressure dropping funnel for later use.

[0098] Add 300 mL of dry DMF to a dry three-necked flask. Under ice-salt bath cooling, add 44.5 g of 60% sodium hydroxide in portions, controlling the temperature at 0 ± 5 °C. Add the above solution dropwise. After the addition is complete, keep the reaction at this temperature for 1 hour, and add crushed ice to terminate the reaction. Pour the reaction solution into a separatory funnel, add 3 L of water, and extract with dichloromethane (3 × 500 mL). Combine the organic phases and wash them successively with water (5 × 300 mL) and saturated brine (300 mL). Dry the organic phase with anhydrous sodium sulfate and concentrate under reduced pressure to obtain 143.3 g of oil, which is diethyl 2-(2-(methylthio)acetyl)malonate.

[0099] Diethyl 2-(2-(methylthio)acetyl)malonate was added to 1 L of ethanol, and 50.8 g of sodium hydroxide was added while stirring. The mixture was stirred overnight at room temperature, filtered, and the filter cake was washed with an appropriate amount of ethanol to obtain disodium 2-(2-(methylthio)acetyl)malonate. Then, the disodium 2-(2-(methylthio)acetyl)malonate was added to 1.5 L of ethanol, and 150 mL of concentrated hydrochloric acid was added. The mixture was stirred for 1 hour, filtered, and the filter cake was washed with an appropriate amount of ethanol. The filtrate was concentrated to obtain 67.6 g of 2-(2-(methylthio)acetyl)malonate.

[0100] 2-(2-(methylthio)acetyl)malonic acid was added to a reaction flask, followed by 1 L of xylene. After reflux for 2 hours, the xylene was distilled off. 1 L of saturated hydrogen chloride in ethanol was added to the residue, and the mixture was stirred overnight at room temperature. The solvent was then removed by vacuum distillation. The residue was dissolved in 500 mL of ethyl acetate, washed with saturated sodium bicarbonate solution, and concentrated under reduced pressure to obtain 34.6 g of methyl 4-(methylthio)-3-oxobutyrate. This was used directly in the next reaction without purification.

[0101] 2. Preparation of Compound 7

[0102] Compound 7 was obtained by replacing methyl 4-hydroxy-3-oxobutyrate with methyl 4-methylthio-3-oxobutyrate according to the method of Example 1.

[0103] ESI-MS: m / z 246.3 [M+1] + .

[0104] Example 8

[0105] Preparation of 2-(methoxymethyl)-8-methyl-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 8)

[0106] 1. Preparation of 5-methyl-2-cyanophenylhydrazine

[0107] 67.6 g of 2-fluoro-4-methylbenzonitrile, 500 mL of tetrahydrofuran, and 500 mL of hydrazine hydrate were added to the reaction flask. The mixture was stirred at room temperature for 24 hours. The aqueous phase was removed, and the organic phase was concentrated to dryness under reduced pressure to obtain crude 2-cyano-5-methylphenylhydrazine, which was used directly in the next reaction without purification.

[0108] 2. Preparation of Compound 8

[0109] Compound 8 was obtained by replacing 2-cyanophenylhydrazine with 2-cyanophenylhydrazine and methyl 4-hydroxy-3-oxobutyrate with methyl 4-methoxy-3-oxobutyrate, following the method of Example 1.

[0110] ESI-MS: m / z 244.3 [M+1] + .

[0111] Example 9

[0112] Preparation of 2-(methoxymethyl)-8-ethyl-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 9)

[0113] 1. Preparation of 5-ethyl-2-cyanophenylhydrazine

[0114] 31.1 g of 2-fluoro-4-chlorobenzonitrile, 3.5 g of iron triacetylacetone, and 300 mL of THF were added to a reaction flask fitted with a rubber stopper and a vent tube. After three purgings with N2, 150 mL of 2 mol / L EtMgBr-THF solution was injected through a syringe via the rubber stopper. The mixture was stirred at room temperature for 0.5 hours. The reaction solution was then poured into 1.5 L of dilute hydrochloric acid and extracted with 500 mL of ethyl acetate. The organic phase was washed successively with 3 × 100 mL of water and 100 mL of saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was subjected to rapid column chromatography to give 17.3 g of 2-fluoro-4-ethylbenzonitrile.

[0115] 17.3 g of 2-fluoro-4-ethylbenzonitrile, 300 mL of tetrahydrofuran, and 300 mL of hydrazine hydrate were added to a reaction flask and stirred at room temperature for 24 hours. The aqueous phase was removed, and the organic phase was concentrated to dryness under reduced pressure to obtain crude 2-cyano-5-ethylphenylhydrazine, which was used directly in the next step of the reaction without purification.

[0116] 2. Preparation of Compound 9

[0117] Compound 9 was obtained by replacing 2-cyanophenylhydrazine with 5-ethyl-2-cyanophenylhydrazine and methyl 4-hydroxy-3-oxobutyrate with methyl 4-methoxy-3-oxobutyrate, following the method of Example 1.

[0118] ESI-MS: m / z 258.3 [M+1] + .

[0119] Example 10

[0120] Preparation of 2-(methoxymethyl)-8-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 10)

[0121] 1. Preparation of 2-cyano-5-fluorophenylhydrazine

[0122] 2-Cyano-5-fluorophenylhydrazine was prepared by replacing 2-aminobenzonitrile with 2-amino-4-fluorobenzonitrile, following the method described in Example 1 of Chinese Patent CN 101508696A.

[0123] 2. Preparation of Compound 10

[0124] Compound 10 was obtained by replacing 2-cyanophenylhydrazine with 2-cyano-5-fluorophenylhydrazine and methyl 4-hydroxy-3-oxobutyrate with methyl 4-methoxy-3-oxobutyrate, following the method of Example 1.

[0125] ESI-MS: m / z 248.2 [M+1] + .

[0126] Example 11

[0127] Preparation of 2-(methoxymethyl)-7-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 11)

[0128] 1. Preparation of 2-cyano-4-fluorophenylhydrazine

[0129] 2-Cyano-4-fluorophenylhydrazine was prepared by replacing 2-aminobenzonitrile with 2-amino-5-fluorobenzonitrile, following the method described in Example 1 of Chinese Patent CN 101508696A.

[0130] 2. Preparation of Compound 11

[0131] Compound 11 was obtained by replacing 2-cyanophenylhydrazine with 2-cyano-4-fluorophenylhydrazine and methyl 4-methoxy-3-oxobutyrate with methyl 4-hydroxy-3-oxobutyrate, following the method of Example 1.

[0132] ESI-MS: m / z 248.2 [M+1] + .

[0133] Example 12

[0134] Preparation of 2-(methoxymethyl)-7-chloro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 12)

[0135] 1. Preparation of 2-cyano-4-chlorophenylhydrazine

[0136] 2-Cyano-4-chlorophenylhydrazine was prepared by replacing 2-aminobenzonitrile with 2-amino-5-chlorobenzonitrile, following the method described in Example 1 of Chinese Patent CN 101508696A.

[0137] 2. Preparation of Compound 12

[0138] Compound 12 was obtained by replacing 2-cyanophenylhydrazine with 2-cyanophenylhydrazine and methyl 4-methoxy-3-oxobutyrate with methyl 4-hydroxy-3-oxobutyrate, following the method of Example 1. (ESI-MS: m / z 264.7 [M+1]) + ).

[0139] Example 13

[0140] Preparation of 2-(methoxymethyl)-7-methoxy-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 13)

[0141] 1. Preparation of 2-cyano-4-methoxyphenylhydrazine

[0142] 2-Cyano-4-methoxyphenylhydrazine was prepared by replacing 2-aminobenzonitrile with 2-amino-5-methoxybenzonitrile, following the method described in Example 1 of Chinese Patent CN 101508696A.

[0143] 2. Preparation of Compound 13

[0144] Compound 13 was obtained by replacing 2-cyanophenylhydrazine with 2-cyano-4-methoxyphenylhydrazine and methyl 4-methoxy-3-oxobutyrate with methyl 4-hydroxy-3-oxobutyrate, following the method of Example 1.

[0145] ESI-MS: m / z 260.3 [M+1] + .

[0146] Example 14

[0147] Preparation of 2-(methoxymethyl)-7-ethoxy-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 14)

[0148] 1. Preparation of 2-cyano-4-ethoxyphenylhydrazine

[0149] 2-Cyano-4-ethoxyphenylhydrazine was prepared by replacing 2-amino-5-ethoxybenzonitrile with the method described in Chinese Patent CN 101508696A.

[0150] 2. Preparation of Compound 14

[0151] Compound 14 was obtained by replacing 2-cyanophenylhydrazine with 2-cyano-4-ethoxyphenylhydrazine and methyl 4-methoxy-3-oxobutyrate with methyl 4-hydroxy-3-oxobutyrate, following the method of Example 1.

[0152] ESI-MS: m / z 274.3 [M+1] + .

[0153] Example 15

[0154] Preparation of 2-(ethoxymethyl)-7-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 15)

[0155] Compound 15 was obtained by replacing 2-cyanophenylhydrazine with 2-cyano-4-fluorophenylhydrazine and methyl 4-ethoxy-3-oxobutyrate with methyl 4-hydroxy-3-oxobutyrate, following the method of Example 1.

[0156] ESI-MS: m / z 262.3 [M+1] + .

[0157] Example 16

[0158] Preparation of 2-(propoxymethyl)-7-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 16)

[0159] Compound 16 was obtained by replacing 2-cyanophenylhydrazine with 2-cyano-4-fluorophenylhydrazine and methyl 4-propoxy-3-oxobutyrate with methyl 4-hydroxy-3-oxobutyrate, following the method of Example 1.

[0160] ESI-MS: m / z 276.3 [M+1] + .

[0161] Example 17

[0162] Preparation of 2-(methoxyethyl)-8-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 17)

[0163] Compound 17 was obtained by replacing 2-cyanophenylhydrazine with 2-cyano-5-fluorophenylhydrazine and methyl 4-hydroxy-3-oxobutyrate with methyl 5-methoxy-3-oxovalerate, following the method of Example 1.

[0164] ESI-MS: m / z 262.3 [M+1] + .

[0165] Example 18

[0166] Preparation of 2-(methoxypropyl)-8-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine (compound 18)

[0167] 1. Preparation of methyl 6-methoxy-3-oxohexanoate

[0168] Methyl 6-methoxy-3-oxohexanoate was prepared by replacing methyl 2-methylthioacetate with methyl 4-methoxybutyrate according to the method of Example 7.

[0169] 2. Preparation of Compound 18

[0170] Compound 18 was obtained by replacing 2-cyanophenylhydrazine with 2-cyanophenylhydrazine and methyl 4-hydroxy-3-oxobutyrate with methyl 6-methoxy-3-oxohexanoate, following the method of Example 1. ESI-MS: m / z 276.3 [M+1] + .

[0171]

[0172] Table 1. Benzopyrazolidine derivatives

[0173] Compound numbering R m X <![CDATA[R1]]> 1 H 1 O H 2 H 1 O <![CDATA[CH3]]> 3 H 1 O <![CDATA[CH2CH3]]> 4 H 1 O <![CDATA[CH2CH2CH3]]> 5 H 1 O CHO 6 H 1 O <![CDATA[COCH3]]> 7 H 1 S <![CDATA[CH3]]> 8 <![CDATA[8-CH3]]> 1 O <![CDATA[CH3]]> 9 <![CDATA[8-CH2CH3]]> 1 O <![CDATA[CH3]]> 10 8-F 1 O <![CDATA[CH3]]> 11 7-F 1 O <![CDATA[CH3]]> 12 7-Cl 1 O <![CDATA[CH3]]> 13 <![CDATA[7-CH3O]]> 1 O <![CDATA[CH3]]> 14 <![CDATA[7-CH3CH2O]]> 1 O <![CDATA[CH3]]> 15 7-F 1 O <![CDATA[CH2CH3]]> 16 7-F 1 O <![CDATA[CH2CH2CH3 <!-- 9 -->]]> 17 8-F 2 O <![CDATA[CH3]]> 18 8-F 3 O <![CDATA[CH3]]>

[0174] Example 19 Pharmacological, pharmacodynamic and bioactivity tests

[0175] A. Effect of compound 2 on PP2A protein expression level in PC12 cells

[0176] PC12 cells were cultured in adherent culture, and Aβ at concentrations of 1, 5, 10, 20, and 40 μmol / L were added and incubated for 24 h. Total protein was then extracted. PP2A activity was detected by Western blot. The results are shown in Table 2. The results indicate that Aβ concentrations ≤10 μmol / L had little effect on PP2A expression in PC12 cells; Aβ concentrations >10 μmol / L significantly inhibited PP2A expression in PC12 cells; and Aβ concentrations between 10 and 40 μmol / L showed a dose-dependent inhibition of PP2A activity.

[0177] PC12 cells were cultured in adherent culture, and five doses of compound 2 (0.1, 0.5, 1, 5, and 10 μmol / L) were added and incubated for 2 h. Then, 40 μmol / L of Aβ was added, and the cells were incubated for 24 h. Whole protein was then extracted. PP2A activity was detected by Western blot. The results are shown in Table 1. The results indicate that compound 2 at doses of 0.1, 0.5, 1, and 5 μmol / L all increased the expression level of PP2A protein in PC12 cells induced by Aβ inhibition, showing a dose-response relationship. A dose of 10 μmol / L of compound 2 significantly increased the expression level of PP2A protein (58.59%), showing almost no difference from the PP2A expression level in normal cells (58.95%).

[0178] Table 2: Effects of Aβ and compound 2 on PP2A protein expression levels in PC12 cells

[0179] sample 1 2 3 4 5 6 7 8 9 10 11 Aβ (μmol / L) 0 1 5 10 20 40 40 40 40 40 40 Compound 2 (μmol / L) 0 0 0 0 0 0 0.1 0.5 1 5 10 Protein (μg / μL) 3.15 2.89 2.67 2.91 2.78 2.80 3.06 2.60 2.84 2.62 2.73 PP2A 2069 1606.5 1628.1 1498.8 1018.9 914.16 1025.7 1129.7 1180.1 1284.7 2192.1 β-actin 3509.5 3563.5 3711.3 3533.8 3423.5 3354.1 3283.7 3404.8 3521.7 3607.8 3741.2 β-actin (%) 58.95 45.08 43.87 42.41 29.76 27.26 31.24 33.18 33.51 35.61 58.59

[0180] B. Effects of Compound 1 and Compound 2 on PP2A activity in PC12 cells

[0181] PC12 cells were seeded into T25 culture medium. After cell adhesion, compounds 1 and 2 were added and the cells were incubated for 2 hours, followed by incubation with 40 nmol / L okadaic acid for 24 hours. Protein was then extracted. Western blotting, SDS-PAGE electrophoresis and transfer, immunoblotting, color development, and G:BOX chemiXR5 imaging were used to detect the expression level of PP2A protein in PC12 cells.

[0182] The experimental results are shown in Table 3. They indicate that, compared with the negative control (only okadaic acid was added), the expression levels of PP2A were significantly increased at three doses of 1, 5 and 10 μmol / L for compounds 1 and 2, and the expression level of PP2A protein increased in a dose-dependent manner.

[0183] Table 3: Effects of Compound 1 and Compound 2 on PP2A protein expression levels in PC12 cells induced by okadaic acid

[0184]

[0185]

[0186] C. Effect of compound 2 on tau protein phosphorylation in human neuroblastoma cells (SH-SY5Y cells)

[0187] SH-SY5Y cells were cultured in vitro for 24 h using a medium containing different concentrations of compound 2, followed by treatment with 40 nmol / L okadaic acid for 24 h. Western blot was used to measure the p-tau content of SH-SY5Y cells. Ser396 level.

[0188] The experimental results are shown in Table 4. They indicate that, compared to the negative control group (containing only okadaic acid), compound 2 significantly inhibited okadaic acid-induced phosphorylation of cellular Tau protein. When the dosage of compound 2 was 1.0 and 3.0 μmol / L, the phosphorylation of p-tau protein in cells was significantly reduced. Ser396 The levels of all compounds decreased significantly (P<0.05); when compound 2 was administered at a dose of 10 μmol / L, the p-tau levels in cells decreased significantly. Ser396 The level decreased significantly (P<0.01).

[0189] Table 4. Effects of compound 2 on p-tau in okadaic acid-induced SH-SY5Y cells Ser396 Impact ( n=3)

[0190]

[0191] Compared with the negative control group, *P<0.05; **P<0.01.

[0192] D. Compounds related to Okada acid and Aβ 1-42 Effect of damage model on cell survival rate

[0193] 1. Experimental Methods

[0194] PC12 cells were digested, counted, and a cell suspension of 8.0 × 10⁸ cells was prepared. 4Cells / mL, 100 μL of cell suspension was added to each well of a 96-well cell culture plate; the 96-well cell culture plate was incubated at 37℃ in a 5% CO2 incubator for 24 hours; Experimental group: PC12 cells were pre-incubated for 2 hours in a medium containing the test compound (compound concentrations of 0.1, 0.5, 1, 5, and 10 μmol / L, compound concentrations set according to Tables 5, 6, 7, and 8); the test drug was not removed, and cells were treated with 50 nmol / L okadaic acid or 40 μmol / L Aβ1-42 for 24 hours. A negative control group (no test compound and okadaic acid added) and a model group (okadaic acid only) were also established; the 96-well cell culture plate was incubated at 37℃ in a 5% CO2 incubator for 24 hours; CCK-8 staining was performed on the 96-well plate, with 10 μL added to each well. CCK-8, continue incubation in an incubator for 1-2 hours; gently mix on a shaker for 10 minutes to remove air bubbles from the 96-well plate; λ = 450nm, read the OD value of each well with a microplate reader, and calculate the cell survival rate.

[0195] Formula for calculating cell viability:

[0196]

[0197] 2. Experimental Results

[0198] The effects of various compounds on okadaic acid and Aβ were determined using the CCK8 cell proliferation assay. 1-42 The effect of induced injury model on the survival rate of PC12 cells. The results are shown in Tables 5-8.

[0199] Table 5 shows that all compounds significantly improved the cell survival rate of the okadaic acid-induced injury model at a concentration of 5 μmol / L (P<0.01). Table 6 shows that compared with the okadaic acid model group, the cell survival rates treated with four doses of compound 1 (0.5, 1, 5, and 10 μmol / L) were significantly different (P<0.01), and the cell survival rate treated with a low dose of compound 1 (0.1 μmol / L) was significantly different (P<0.05). Table 7 shows that compared with the okadaic acid model group, the cell survival rates treated with three doses of compound 2 (1, 5, and 10 μmol / L) were significantly different (P<0.01), while the cell survival rate treated with low doses of compound 2 (0.1 and 0.5 μmol / L) showed no significant difference (P>0.05).

[0200] As shown in Table 8, with Aβ 1-42Compared with the model group, the cell survival rate of cells treated with three doses of compound 2 (1, 5, and 10 μmol / L) was statistically different (P<0.01), while the cell survival rate of cells treated with low doses of compound 2 (0.1 and 0.5 μmol / L) was not statistically different (P>0.05).

[0201] Table 5: Effects of different compounds (5 μmol / L) on cell survival rate in the Okada acid injury model

[0202] Group Mean±SD Survival rate% Negative control 1.299±0.023 100 Okaya acid (50 nmol / L) 0.643±0.014 49.50 Compound 1 0.916±0.017* 70.52 Compound 2 0.92±0.008* 70.82 Compound 3 0.858±0.009* 66.05 Compound 4 0.856±0.012* 65.90 Compound 5 0.852±0.004* 65.59 Compound 6 0.889±0.007* 68.44 Compound 7 0.919±0.008* 70.75 Compound 8 0.877±0.009* 67.51 Compound 9 0.852±0.010* 65.59

[0203] Compound 10 0.842±0.008* 64.82 Compound 11 0.857±0.007* 65.97 Compound 12 0.866±0.003* 66.67 Compound 13 0.879±0.009* 67.67 Compound 14 0.866±0.011* 66.67 Compound 15 0.872±0.012* 67.13 Compound 16 0.869±0.005* 66.90 Compound 17 0.882±0.005* 67.90 Compound 18 0.851±0.007* 65.55

[0204] Compared with Okada acid: *P<0.01.

[0205] Table 6: Effects of different concentrations of compound 1 on cell survival in the okadaic acid-induced injury model

[0206]

[0207] Compared with Okada acid: *P<0.05; **P<0.01.

[0208] Table 7: Effects of different concentrations of compound 2 on cell survival in the okadaic acid-induced injury model

[0209]

[0210] Compared with Okada acid: *P<0.01.

[0211] Table 8: Effects of different concentrations of compound 2 on cell survival in the Aβ1-42 injury model

[0212]

[0213] With Aβ 1-42 Comparison: *P < 0.01.

Claims

1. Benzopyrazoloxazine derivatives or pharmaceutically acceptable salts thereof with structures as shown in Formula I: ； in: R is selected from hydrogen, halogen, C1-C2 alkyl, and C1-C2 alkoxy. m is an integer from 1 to 3; X is selected from oxygen or sulfur; R1 is selected from hydrogen, C1-C3 alkyl, formyl, and acetyl.

2. The benzopyrazoloxazine derivative according to claim 1, characterized in that: Selected from the following compounds: (5-Imine-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-2-yl)methanol; 2-(methoxymethyl)-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; 2-(ethoxymethyl)-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; 2-(propoxymethyl)-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; Methyl formate (5-imino-5H-benzo[d]pyrazole[5,1-b][1,3]oxazin-2-yl); Methyl acetate (5-imino-5H-benzo[d]pyrazole[5,1-b][1,3]oxazin-2-yl); 2-(methylthiomethyl)-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; 2-(methoxymethyl)-8-methyl-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; 2-(methoxymethyl)-8-ethyl-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; 2-(methoxymethyl)-8-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; 2-(methoxymethyl)-7-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; 2-(methoxymethyl)-7-chloro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; 2-(methoxymethyl)-7-methoxy-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; 2-(methoxymethyl)-7-ethoxy-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; 2-(ethoxymethyl)-7-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; 2-(propoxymethyl)-7-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; 2-(methoxyethyl)-8-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine; 2-(methoxypropyl)-8-fluoro-5H-benzo[d]pyrazole[5,1-b][1,3]oxazine-5-imine.

3. The use of the benzopyrazoloid derivatives according to any one of claims 1-2 in the preparation of medicaments for the prevention and treatment of diseases caused by the loss of PP2A activity or the reduction of PP2A function.

4. The application according to claim 3, characterized in that: Diseases caused by the loss of PP2A activity or the reduction of PP2A function are tumors.

5. The application according to claim 3, characterized in that: Diseases caused by the loss of PP2A activity or the reduction of PP2A function are neurodegenerative diseases.

6. The application according to claim 5, characterized in that: The neurodegenerative disease mentioned is Alzheimer's disease.

7. The use of the benzopyrazoloid derivatives according to any one of claims 1-2 in the preparation of protein phosphatase agonists.

8. The use of the benzopyrazoloxazine derivatives according to any one of claims 1-2 in the preparation of protein phosphatase 2A agonists.

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