Protein phosphatase agonist and application

Abstract

The invention discloses benzopyrazoloxazine derivatives or pharmaceutically acceptable salts thereof, wherein the structure of the benzopyrazoloxazine derivatives is shown as a formula I; R is selected from hydrogen, halogen, alkyl and alkoxy; m is an integer of 1-3; x is selected from oxygen or sulfur; r1 is selected from hydrogen, alkyl and acyl. The invention also discloses an application of the benzopyrazoloxazine derivative in preparation of a medicine for preventing and treating diseases caused by PP2A activity deficiency or PP2A function reduction. The invention also discloses an application of the benzopyrazoloxazine derivative in preparation of a protein phosphatase agonist. # imgabs0 #

Description

Protein phosphatase agonist and application thereof Technical Field

[0001] The present invention belongs to the field of medicinal chemistry and relates to a protein phosphatase agonist and application thereof, and in particular to a benzopyrazoloxazine protein phosphatase agonist and application thereof. Background Art

[0002] Protein phosphatase 2A (PP2A) is a crucial member of the protein phosphatase family. The catalytic domain of its catalytic subunit shares high homology with that of other members of the family. PP2A mediates processes throughout cellular life, including cell signaling, cell differentiation, and apoptosis.

[0003] Loss of PP2A activity or reduced function disrupts important downstream pathways, including MAPK and ERK, leading to disrupted cell regulation and the development of cancer. Loss of PP2A activity in T cells can reduce the clearance capacity of T cells and may be a key factor in the development and progression of cell cancer.

[0004] The release of neurotransmitters and the opening and closing of ion channels, accompanied by numerous protein phosphorylation and dephosphorylation processes, are crucial components of information transmission within nerve cells. Under the combined action of adenosine triphosphate (ATP) and protein phosphatase (protein kinase, PK), protein phosphorylation is catalyzed, causing structural changes and the formation of active proteins. The activated proteins then activate downstream target proteins, thereby activating the entire cascade and completing signal transduction. PP2A is a key factor in protein dephosphorylation, ensuring the timely closure of signaling pathways and the subsequent onset of neural activity.

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

[0006] The "China Alzheimer's Disease Report 2021," published by Shanghai Ruijin Hospital, shows that there were 13,243,950 cases of AD and other dementias in China in 2019. The prevalence and mortality rates are slightly higher than the global average, and are higher in women than in men. The prevalence (1,188.9 per 100,000 population) and mortality (30.8 per 100,000 population) of women are higher than those of men (669.3 per 100,000 population) and 14.6 per 100,000 population), respectively.

[0007] Currently, two types of drugs are commonly used in the clinical treatment of AD: cholinesterase inhibitors such as donepezil and N-methyl-D-aspartate (NMDA) receptor antagonists such as memantine. Donepezil inhibits acetylcholinesterase, maintaining or increasing acetylcholine levels, thereby increasing cholinergic nerve signaling and improving patients' cognitive function. Memantine inhibits NMDA receptor activity, reducing the neurotoxic effects of excitatory amino acids and improving cognitive function. However, these drugs are only symptomatic treatments and can improve cognitive and memory impairments to a certain extent, but currently no drug can prevent or delay the progression of AD.

[0008] We believe that the onset and progression of AD is closely linked to decreased PP2A function. Reduced PP2A activity prevents the protein from being dephosphorylated and inactivated. Consequently, the postsynaptic membrane continues to receive neurotransmitters released from the presynaptic membrane, preventing the signaling pathway from closing in a timely manner. This leads to delayed nerve conduction and memory loss.

[0009] Amyloid precursor protein (APP) is a widely present and essential biologically active substance. β-amyloid (Aβ) is produced by the hydrolysis of APP by β-secretase and γ-secretase. In vitro tests have shown that Aβ has an inhibitory effect on the biological activity of protein phosphatase (PP), and the intensity of its activity inhibition is closely related to the Aβ level. Normal levels of Aβ have a limited effect on PP activity. Only when the Aβ level reaches a certain level will it inhibit PP activity, and its inhibitory effect on PP activity increases with the increase of Aβ concentration. Therefore, it can be seen that abnormally elevated Aβ levels in the body inhibit PP activity and are an extremely important factor in inducing the occurrence of AD.

[0010] The biological function of Tau protein (P-tau) is to induce tubulin to polymerize into microtubules and prevent its disassembly, thereby maintaining the morphology and function of nerve cells. To restore the biological activity of PP, the body sacrifices P-tau and tangles with Aβ to form P-tau / Aβ complexes, which are deposited in nerve cells to achieve the purpose of reducing Aβ levels and restoring PP activity. This fluctuation in PP biological activity caused by changes in Aβ levels manifests itself in AD patients as memory fluctuations. Over time, the P-tau / Aβ complex gradually overwhelms nerve cells, causing irreversible neurological organic lesions in AD patients. PET scans of the brains of AD patients have confirmed that P-tau / Aβ tangles (plaques) lead to brain atrophy and brain nerve cell death.

[0011] PP agonists enhance PP biological activity, catalyzing protein dephosphorylation in a timely manner to maintain normal neural activity. Furthermore, increased PP activity can also promote the dephosphorylation of phosphorylated tau protein (P-tau-Pi), thereby preventing neurotoxic effects and neuronal cell death mediated by tau hyperphosphorylation.

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

[0013] Summary of the Invention

[0014] The present invention aims to provide a class of benzopyrazoloxazine compounds, which can act as protein phosphatase agonists, have a strong excitatory effect on the activity-impaired PP2A, and can significantly increase the biological activity of PP2A.

[0015] The structure of the benzopyrazoloxazine derivative or a pharmaceutically acceptable salt thereof is shown in Formula I:

[0016]

[0017] in:

[0018] R is selected from hydrogen, halogen, alkyl, alkoxy;

[0019] m is an integer from 1 to 3;

[0020] X is selected from oxygen or sulfur;

[0021] R1 is selected from hydrogen, alkyl, and acyl.

[0022] Preferably, R is selected from hydrogen, halogen, C1-C2 alkyl, C1-C2 alkoxy;

[0023] m is an integer from 1 to 3;

[0024] X is selected from oxygen or sulfur;

[0025] R1 is selected from hydrogen, C1-C3 alkyl, formyl (-CHO), acetyl (-COCH3).

[0026] Halogen can be specifically selected from F and Cl; alkyl can be specifically selected from methyl, ethyl, n-propyl, etc.; alkoxy can be specifically selected from methoxy, ethoxy, etc.

[0027] The benzopyrazoloxazine derivatives are preferably selected from the following compounds, but are not limited to the following compounds:

[0028] (5-Imino-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-2-yl)methanol;

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

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

[0031] 2-(Propyloxymethyl)-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine;

[0032] (5-Imino-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-2-yl)methyl formate;

[0033] (5-Imino-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-2-yl)methyl acetate;

[0034] 2-(Methylthiomethyl)-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine;

[0035] 2-(Methoxymethyl)-8-methyl-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine;

[0036] 2-(Methoxymethyl)-8-ethyl-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine;

[0037] 2-(Methoxymethyl)-8-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine;

[0038] 2-(Methoxymethyl)-7-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine;

[0039] 2-(Methoxymethyl)-7-chloro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine;

[0040] 2-(Methoxymethyl)-7-methoxy-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine;

[0041] 2-(Methoxymethyl)-7-ethoxy-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine;

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

[0043] 2-(Propyloxymethyl)-7-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine;

[0044] 2-(Methoxyethyl)-8-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine;

[0045] 2-(Methoxypropyl)-8-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine.

[0046] The pharmaceutically acceptable salt refers to a salt in the general sense that is generated by the reaction of the above-mentioned benzopyrazoloxazine derivatives with an acid or base commonly used in pharmacy, which has no side effects and can increase the physicochemical properties (such as water solubility, etc.) of the compound; the acid is 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 base is selected from sodium hydroxide, sodium carbonate, sodium bicarbonate or potassium hydroxide, etc.

[0047] Another object of the present invention is to provide a method for preparing a benzopyrazoloxazine derivative having a structure as shown in Formula I. When R1 is selected from hydrogen or an alkyl group, the reaction scheme is:

[0048]

[0049] When R1 is selected from acyl, the reaction scheme is:

[0050]

[0051] Compound II can be prepared by sequentially subjecting compound IV to diazotization and reduction reactions, and the reaction route is:

[0052]

[0053] Compound II can also be prepared by reacting compound V with hydrazine solutions of varying concentrations, hydrazine salts, or hydrazine derivatives with varying substituents. The reaction scheme is as follows:

[0054]

[0055] Wherein: Y includes but is not limited to F, Cl, Br, I, sulfonic acid group, etc.

[0056] Unless otherwise specified, when preparing the benzopyrazoloxazine derivatives represented by Formula I of the present invention, the compounds used as starting materials are known compounds that are commercially available, or can be prepared from known compounds by known methods, methods analogous to known methods, or methods analogous to the methods described in the Examples.

[0057] The benzopyrazoloxazine derivatives shown in formula I have a strong excitatory effect on the impaired activity of PP2A, can significantly increase the biological activity of PP2A, can significantly inhibit the phosphorylation of cellular Tau protein induced by okadaic acid, and can significantly increase the activity of okadaic acid and Aβ 1-42 Survival rate of damaged cells.

[0058] Another object of the present invention is to provide the use of benzopyrazoloxazine derivatives represented by Formula I in the preparation of drugs for preventing and treating diseases caused by loss of PP2A activity or decreased PP2A function.

[0059] Preferably, the disease caused by the loss of PP2A activity or the reduction of PP2A function is a tumor, and the benzopyrazoloxazine derivatives shown in Formula I can be used to prevent and treat the occurrence and development of tumors.

[0060] Preferably, the disease caused by the loss of PP2A activity or reduced PP2A function is a brain degenerative disease, including but not limited to Alzheimer's disease (AD). The benzopyrazoloxazine derivatives represented by Formula I can be used to prevent and treat Alzheimer's disease.

[0061] Another object of the present invention is to provide the use of the benzopyrazoloxazine derivatives shown in Formula I in the preparation of protein phosphatase agonists, preferably in the preparation of protein phosphatase 2A agonists. DETAILED DESCRIPTION

[0062] Experimental instruments

[0063] A rotary evaporator RE-52AA was used (Shanghai Yarong Biochemical Instrument Factory); an SHB-III circulating water multi-purpose vacuum pump was used (Nanjing Wener Instrument Equipment Co., Ltd.); a JJ-1 precision force-boosting stirrer was used (Jintan Chengdong Xinrui Instrument Factory); an 85-1 magnetic stirrer was used (Nanjing Wener Instrument Equipment Co., Ltd.); a ZF-I three-function UV analyzer; an X-4 digital melting point analyzer was used (Beijing Fukai Instrument Co., Ltd.); and a Bruker AV-300 nuclear magnetic resonance spectrometer (TMS as the internal standard and DMSO-d6 as the solvent). Mass spectrometry was performed using an Agilent Technologies 6520B Accurate-Mass A-TOF LC / MS mass spectrometer. The purity of the compounds was determined using a Shimadzu LC-20AT high-performance liquid chromatograph. 18 The chromatographic column was SAP-10, the detection wavelength was 220 nM, and the mobile phase was methanol-water (70:30 V / V).

[0064] Material

[0065] Thin layer chromatography silica gel plates (Silica gel GF254, Yantai Dexin Biotechnology Co., Ltd.); DMF was used Molecular sieve drying, ethanol use The mixture was dried with molecular sieves, and other reagents were commercially available of analytical or chemical grade; most of the reagents were sourced from Aladdin Reagent Co., Ltd.

[0066] Example 1

[0067] Preparation of (5-Imino-benzo[d][1,3]oxazin[5,1-b]pyrazol-2-yl)methanol (Compound 1)

[0068] 1. Preparation of 2-cyanophenylhydrazine

[0069] 2-Cyanophenylhydrazine was prepared by referring to the method in Example 1 "Preparation of 2-hydrazinophenylnitrile hydrochloride" of Chinese patent CN 101508696A.

[0070] 2. Preparation of (5-imino-benzo[d][1,3]oxazine[5,1-b]pyrazol-2-yl)methanol

[0071] To a reaction flask, add 1.3 g of crude 2-cyanophenylhydrazine and 15 mL of saturated ethanolic hydrogen chloride solution. Add 2.0 g of methyl 4-hydroxy-3-oxobutanoate with stirring. Allow to react at room temperature overnight. Add 30 mL of ice water, stir for 1 hour, filter, and wash the filter cake several times with water. Dry to obtain 1.7 g of crude Compound 1 hydrochloride, which is recrystallized from 10% ethanol to obtain 1.3 g of refined Compound 1 hydrochloride (white solid).

[0072] The refined hydrochloride 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.

[0073] ESI-MS: m / z 216.2[M+1] + .

[0074] Example 2

[0075] Preparation of 2-(methoxymethyl)-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 2)

[0076] Using 4-methoxy-3-oxobutanoic acid methyl ester instead of 4-hydroxy-3-oxobutanoic acid methyl ester, compound 2 was obtained according to the method of Example 1.

[0077] ESI-MS: m / z 230.2[M+1] + .

[0078] Example 3

[0079] Preparation of 2-(ethoxymethyl)-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 3)

[0080] Using 4-ethoxy-3-oxobutanoic acid methyl ester instead of 4-hydroxy-3-oxobutanoic acid methyl ester, compound 3 was obtained according to the method of Example 1.

[0081] ESI-MS: m / z 243.3[M+1] + .

[0082] Example 4

[0083] Preparation of 2-(propyloxymethyl)-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 4)

[0084] Using 4-propoxy-3-oxobutanoic acid methyl ester instead of 4-hydroxy-3-oxobutanoic acid methyl ester, compound 4 was obtained according to the method of Example 1.

[0085] ESI-MS: m / z 258.3[M+1] + .

[0086] Example 5

[0087] Preparation of (5-imino-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-2-yl)methyl formate (Compound 5)

[0088] To the reaction flask, 10 mL of formic acid was added, and the temperature was cooled to -10°C in an ice bath. 2 mL of acetic anhydride was added dropwise, and the mixture was stirred for 0.5 h. 0.5 g of compound 1 and 0.1 g of DMAP were added, and the mixture was heated to room temperature and reacted for 2 h. 50 mL of ice water was added, and the mixture was filtered and washed with water until the droplet was neutral. The mixture was then recrystallized from ethyl acetate to obtain compound 5.

[0089] ESI-MS: m / z 244.2[M+1] + .

[0090] Example 6

[0091] Preparation of (5-imino-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-2-yl)methyl acetate (Compound 6)

[0092] Using acetic acid instead of formic acid, compound 6 was obtained according to the method of Example 5.

[0093] ESI-MS: m / z 258.3[M+1] + .

[0094] Example 7

[0095] Preparation of 2-(methylthiomethyl)-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 7)

[0096] 1. Preparation of methyl 4-(methylthio)-3-oxobutanoate

[0097] To the reaction flask, 500 mL of DMF, 108.5 g of methyl chloroacetate, and 72.1 g of sodium methyl mercaptan were added sequentially, and the reaction was carried out at 105° C. for 5 hours. The heating was stopped and the mixture was cooled to room temperature. 2.5 L of water was added to the reaction solution, and the aqueous phase was extracted with dichloromethane (3×500 mL). The organic phases were combined and washed sequentially with water (5×300 mL) and saturated brine (300 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 89.2 g of an oily product, namely methyl 2-methylthioacetate, which was used directly in the next step without purification.

[0098] Methyl 2-methylthioacetate and 118.9 g of diethyl malonate were dissolved in 300 mL of dry DMF, placed in a constant pressure dropping funnel and set aside.

[0099] To a dry three-necked flask was added 300 mL of dry DMF, and 44.5 g of 60% sodium hydrogen sulfide was added in portions under ice-salt cooling. The temperature was controlled at 0±5°C and the above solution was added dropwise. After the addition was complete, the reaction was kept warm for 1 hour and crushed ice was added to terminate the reaction. The reaction solution was poured into a separatory funnel, 3 L of water was added, and the mixture was extracted with dichloromethane (3×500 mL). The organic phases were combined and washed sequentially with water (5×300 mL) and saturated brine (300 mL). The organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 143.3 g of an oily substance, which is diethyl 2-(2-(methylthio)acetyl)malonate.

[0100] Diethyl 2-(2-(methylthio)acetyl)malonate was added to 1 L of ethanol, and 50.8 g of sodium hydroxide was added with stirring. The mixture was stirred at room temperature overnight, filtered, and the filter cake was washed with an appropriate amount of ethanol to obtain disodium 2-(2-(methylthio)acetyl)malonate. Disodium 2-(2-(methylthio)acetyl)malonate was then 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.

[0101] 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 evaporated. 1 L of saturated ethanolic hydrogen chloride solution was added to the residue, and the mixture was stirred at room temperature overnight. The solvent was then evaporated under reduced pressure. The residue was dissolved in 500 mL of ethyl acetate, washed with saturated sodium bicarbonate solution, and concentrated under reduced pressure to yield 34.6 g of methyl 4-(methylthio)-3-oxobutanoate. This was used directly in the next reaction without purification.

[0102] 2. Preparation of Compound 7

[0103] Substituting 4-methylthio-3-oxobutanoic acid methyl ester for 4-hydroxy-3-oxobutanoic acid methyl ester, compound 7 was obtained according to the method of Example 1.

[0104] ESI-MS: m / z 246.3[M+1] + .

[0105] Example 8

[0106] Preparation of 2-(methoxymethyl)-8-methyl-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 8)

[0107] 1. Preparation of 5-methyl-2-cyanophenylhydrazine

[0108] To the reaction flask, 67.6 g of 2-fluoro-4-methylbenzonitrile, 500 mL of tetrahydrofuran, and 500 mL of hydrazine hydrate were added 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-methylphenylhydrazine, which was used directly in the next reaction without purification.

[0109] 2. Preparation of Compound 8

[0110] Compound 8 was obtained according to the method of Example 1 by replacing 2-cyanophenylhydrazine with 2-cyano-5-methylphenylhydrazine and replacing 4-hydroxy-3-oxobutyric acid methyl ester with 4-methoxy-3-oxobutyric acid methyl ester.

[0111] ESI-MS: m / z 244.3[M+1] + .

[0112] Example 9

[0113] Preparation of 2-(methoxymethyl)-8-ethyl-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 9)

[0114] 1. Preparation of 5-ethyl-2-cyanophenylhydrazine

[0115] To a reaction flask fitted with a rubber stopper and a vent tube, 31.1 g of 2-fluoro-4-chlorobenzonitrile, 3.5 g of ferric triacetylacetonate, and 300 mL of THF were added. After nitrogen displacement three times, 150 mL of a 2 mol / L EtMgBr-THF solution was injected through the rubber stopper using a syringe. The mixture was stirred at room temperature for 0.5 hour. The reaction solution was poured into 1.5 L of dilute hydrochloric acid and extracted with 500 mL of ethyl acetate. The organic phase was washed sequentially with 3 × 100 mL of water, followed by 100 mL of saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was subjected to flash column chromatography to yield 17.3 g of 2-fluoro-4-ethylbenzonitrile.

[0116] 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 reaction without purification.

[0117] 2. Preparation of Compound 9

[0118] Compound 9 was obtained according to the method of Example 1 by replacing 2-cyanophenylhydrazine with 5-ethyl-2-cyanophenylhydrazine and replacing 4-hydroxy-3-oxobutyric acid methyl ester with 4-methoxy-3-oxobutyric acid methyl ester.

[0119] ESI-MS: m / z 258.3[M+1] + .

[0120] Example 10

[0121] Preparation of 2-(methoxymethyl)-8-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 10)

[0122] 1. Preparation of 2-cyano-5-fluorophenylhydrazine

[0123] 2-Cyano-5-fluorophenylhydrazine was prepared by using 2-amino-4-fluorobenzonitrile instead of 2-aminobenzonitrile according to the method in Example 1 of Chinese Patent CN 101508696A.

[0124] 2. Preparation of Compound 10

[0125] Compound 10 was obtained according to the method of Example 1 by replacing 2-cyanophenylhydrazine with 2-cyano-5-fluorophenylhydrazine and replacing 4-hydroxy-3-oxobutyric acid methyl ester with 4-methoxy-3-oxobutyric acid methyl ester.

[0126] ESI-MS: m / z 248.2[M+1] + .

[0127] Example 11

[0128] Preparation of 2-(methoxymethyl)-7-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 11)

[0129] 1. Preparation of 2-cyano-4-fluorophenylhydrazine

[0130] 2-Cyano-4-fluorophenylhydrazine was prepared by using 2-amino-5-fluorobenzonitrile instead of 2-aminobenzonitrile according to the method in Example 1 of Chinese Patent CN 101508696A.

[0131] 2. Preparation of Compound 11

[0132] Compound 11 was obtained according to the method of Example 1 by replacing 2-cyanophenylhydrazine with 2-cyano-4-fluorophenylhydrazine and replacing 4-hydroxy-3-oxobutyric acid methyl ester with 4-methoxy-3-oxobutyric acid methyl ester.

[0133] ESI-MS: m / z 248.2[M+1] + .

[0134] Example 12

[0135] Preparation of 2-(methoxymethyl)-7-chloro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 12)

[0136] 1. Preparation of 2-cyano-4-chlorophenylhydrazine

[0137] 2-Cyano-4-chlorophenylhydrazine was prepared by using 2-amino-5-chlorobenzonitrile instead of 2-aminobenzonitrile according to the method in Example 1 of Chinese Patent CN 101508696A.

[0138] 2. Preparation of Compound 12

[0139] Substituting 2-cyanophenylhydrazine with 2-cyano-4-chlorophenylhydrazine and 4-methoxy-3-oxobutyric acid methyl ester for 4-hydroxy-3-oxobutyric acid methyl ester, compound 12 was obtained according to the method of Example 1. (ESI-MS: m / z 264.7 [M+1] + ).

[0140] Example 13

[0141] Preparation of 2-(methoxymethyl)-7-methoxy-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 13)

[0142] 1. Preparation of 2-cyano-4-methoxyphenylhydrazine

[0143] 2-Cyano-4-methoxyphenylhydrazine was prepared by using 2-amino-5-methoxybenzonitrile instead of 2-aminobenzonitrile according to the method in Example 1 of Chinese Patent CN 101508696A.

[0144] 2. Preparation of Compound 13

[0145] Compound 13 was obtained by the method of Example 1, using 2-cyano-4-methoxyphenylhydrazine instead of 2-cyanophenylhydrazine and 4-methoxy-3-oxobutyric acid methyl ester instead of 4-hydroxy-3-oxobutyric acid methyl ester.

[0146] ESI-MS: m / z 260.3[M+1] + .

[0147] Example 14

[0148] Preparation of 2-(methoxymethyl)-7-ethoxy-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 14)

[0149] 1. Preparation of 2-cyano-4-ethoxyphenylhydrazine

[0150] 2-Cyano-4-ethoxyphenylhydrazine was prepared by using 2-amino-5-ethoxybenzonitrile instead of 2-aminobenzonitrile according to the method in Chinese patent CN 101508696A.

[0151] 2. Preparation of Compound 14

[0152] Compound 14 was obtained by the method of Example 1, using 2-cyano-4-ethoxyphenylhydrazine instead of 2-cyanophenylhydrazine and 4-methoxy-3-oxobutyric acid methyl ester instead of 4-hydroxy-3-oxobutyric acid methyl ester.

[0153] ESI-MS: m / z 274.3[M+1] + .

[0154] Example 15

[0155] Preparation of 2-(ethoxymethyl)-7-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 15)

[0156] Compound 15 was obtained according to the method of Example 1 by replacing 2-cyanophenylhydrazine with 2-cyano-4-fluorophenylhydrazine and replacing 4-hydroxy-3-oxobutyric acid methyl ester with 4-ethoxy-3-oxobutyric acid methyl ester.

[0157] ESI-MS: m / z 262.3[M+1] + .

[0158] Example 16

[0159] Preparation of 2-(propyloxymethyl)-7-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 16)

[0160] Compound 16 was obtained according to the method of Example 1 by replacing 2-cyanophenylhydrazine with 2-cyano-4-fluorophenylhydrazine and replacing 4-hydroxy-3-oxobutyric acid methyl ester with 4-propoxy-3-oxobutyric acid methyl ester.

[0161] ESI-MS: m / z 276.3[M+1] + .

[0162] Example 17

[0163] Preparation of 2-(methoxyethyl)-8-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 17)

[0164] Compound 17 was obtained according to the method of Example 1 by replacing 2-cyanophenylhydrazine with 2-cyano-5-fluorophenylhydrazine and replacing 4-hydroxy-3-oxobutanoic acid methyl ester with 5-methoxy-3-oxopentanoate.

[0165] ESI-MS: m / z 262.3[M+1] + .

[0166] Example 18

[0167] Preparation of 2-(methoxypropyl)-8-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine (Compound 18)

[0168] 1. Preparation of methyl 6-methoxy-3-oxohexanoate

[0169] Methyl 6-methoxy-3-oxohexanoate was prepared according to the method of Example 7, using methyl 4-methoxybutyrate instead of methyl 2-methylthioacetate.

[0170] 2. Preparation of Compound 18

[0171] Compound 18 was obtained by the method of Example 1, using 2-cyano-5-fluorophenylhydrazine instead of 2-cyanophenylhydrazine and 6-methoxy-3-oxohexanoic acid methyl ester instead of 4-hydroxy-3-oxobutanoic acid methyl ester. ESI-MS: m / z 276.3 [M+1] + .

[0172]

[0173] Table 1. Benzopyrazoloxazine derivatives

[0174] Compound No. RmXR11H1OH2H1OCH33H1OCH2CH34H1OCH2CH2CH35H1OCHO6H1OCOCH37H1SCH388-CH31OCH398-CH2CH31OCH3108-F1OCH3117-F1OCH3127-Cl1OCH3137-CH3O1OCH3147-CH3CH2O1OCH3157-F1OCH2CH3167-F1OCH2CH2CH3178-F2OCH3188-F3OCH3

[0175] Example 19 Pharmacology, efficacy and biological activity test

[0176] A. Effect of compound 2 on PP2A protein expression in PC12 cells

[0177] Adherent PC12 cells were treated with Aβ at concentrations of 1, 5, 10, 20, and 40 μmol / L for 24 hours, and total protein was extracted. PP2A activity was assayed by Western blot. The results, shown in Table 2, show that Aβ concentrations ≤10 μmol / L had little effect on PP2A expression in PC12 cells. However, Aβ concentrations >10 μmol / L significantly inhibited PP2A expression in PC12 cells. Within the Aβ concentration range of 10 to 40 μmol / L, Aβ inhibited PP2A activity in a dose-dependent manner.

[0178] PC12 cells were cultured on the wall and five doses of compound 2 were added, namely 0.1, 0.5, 1, 5 and 10 μmol / L, respectively. The cells were incubated for 2 hours, and then 40 μmol / L of Aβ was added and incubated for 24 hours. The whole protein was extracted. PP2A activity was detected by Western blot. The experimental results are shown in Table 1, which show that compound 2 at doses of 0.1, 0.5, 1 and 5 μmol / L can increase the expression level of PP2A protein in PC12 cells induced by Aβ, and show a certain dose-effect relationship. At a dose of 10 μmol / L, compound 2 can significantly increase the expression level of PP2A protein (58.59%), which is almost the same as the PP2A expression level of normal cells (58.95%).

[0179] Table 2: Effects of Aβ and Compound 2 on PP2A protein expression in PC12 cells

[0180] Sample 1 2 3 4 5 6 7 8 9 1 0 1 1 Aβ (μmol / L) 0 1 5 1 0 2 0 4 0 4 0 4 0 4 0 Compound 2 (μmol / L) 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 PP2 A2 0 6 9 1 6 0 6.5 1 6 2 8.1 1 4 9 8.8 1 0 1 8.99 14.1610 25.71129.71180.11284.72192.1β-actin3509.53563.53711.33533.83423.53354.13283.73404.8 3521.73607.83741.2β-actin(%)58.9545.0843.8742.4129.7627.2631.2433.1833.5135.6158.59

[0181] B. Effects of Compounds 1 and 2 on PP2A activity in PC12 cells

[0182] PC12 cells were seeded into T25 culture medium. After cell attachment, compounds 1 and 2 were added and incubated for 2 hours. 40 nmol / L okadaic acid was then added for 24 hours for protein extraction. PP2A protein expression in PC12 cells was determined using Western blotting, SDS-PAGE electrophoresis, transfer, immunoblotting, color development, and G:BOX chemiXR5 imaging.

[0183] The experimental results are shown in Table 3, which show 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 compound 1 and compound 2, and the expression level of PP2A protein increased in a dose-dependent manner.

[0184] Table 3: Effects of Compound 1 and Compound 2 on PP2A protein expression in PC12 cells induced by okadaic acid

[0185]

[0186]

[0187] C. Effect of compound 2 on tau protein phosphorylation in human neuroblastoma cells (SH-SY5Y cells)

[0188] SH-SY5Y cells were cultured in vitro with medium containing different concentrations of compound 2 for 24 hours, and then treated with 40 nmol / L okadaic acid for 24 hours. p-tau expression in SH-SY5Y cells was determined by Western Blot. Ser396 level.

[0189] The test results are shown in Table 4, which show that compared with the negative control group (only okadaic acid was added), compound 2 can significantly inhibit the phosphorylation of cellular Tau protein induced by okadaic acid. When the doses of compound 2 were 1.0 and 3.0 μmol / L, the phosphorylation of cellular p-tau protein was significantly inhibited. Ser396 The levels of p-tau in cells decreased significantly (P<0.05). When the dose of compound 2 was 10 μmol / L, the Ser396 The level decreased significantly (P<0.01).

[0190] Table 4. Effect of compound 2 on okadaic acid-induced p-tau in SH-SY5Y cells Ser396 The impact of n=3)

[0191]

[0192] Compared with the negative control group, *P<0.05; **P<0.01.

[0193] D. Effects of compounds on okadaic acid and Aβ 1-42 Effect of injury model on cell survival rate

[0194] 1. Experimental methods

[0195] PC12 cells were digested and counted, and a cell suspension of 8.0×10 4 / 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 placed in a 37°C, 5%, CO2 incubator for 24 hours; experimental group: PC12 cells were incubated in advance for 2 hours in a medium containing the test compound (the compound concentrations were 0.1, 0.5, 1, 5, and 10 μmol / L, and the compound concentrations were set according to the concentrations in Tables 5, 6, 7, and 8); the test drug was not removed, and 50 nmol / L of okadaic acid or 40 μmol / L of Aβ1-42 was added for 24 hours. At the same time, a negative control group (Negative control, no test compound and okadaic acid were added) and a model group (only okadaic acid was added) were set up; the 96-well cell culture plate was placed in a 37°C, 5%, CO2 incubator for 24 hours; the 96-well plate was stained with CCK-8, and 10 μL was added to each well. CCK-8 was cultured in an incubator for 1-2 hours; the cells were gently mixed on a shaker for 10 minutes to remove air bubbles in the 96-well plate; the OD value of each well was read using a microplate reader at λ = 450 nm to calculate the cell survival rate.

[0196] The formula for calculating cell viability is:

[0197]

[0198] 2. Experimental results

[0199] CCK8 cell proliferation assay was used to determine the effects of various compounds on okadaic acid and Aβ 1-42 Effects of induced injury model on the survival rate of PC12 cells. The results are shown in Tables 5-8.

[0200] As shown in Table 5, each compound significantly improved the survival rate of cells in the okadaic acid injury model at 5 μmol / L (P<0.01). As shown in Table 6, compared with the cell survival rate of the okadaic acid model group, the survival rates of cells treated with four doses of compound 1 (0.5, 1, 5 and 10 μmol / L) were significantly different (P<0.01), and the survival rate of cells treated with a low dose of compound 1 (0.1 μmol / L) was statistically different (P<0.05); as shown in Table 7, compared with the cell survival rate of the okadaic acid model group, the survival rates of cells treated with three doses of compound 2 (1, 5 and 10 μmol / L) were significantly different (P<0.01), and the survival rates of cells treated with a low dose of compound 2 (0.1, 0.5 μmol / L) were not statistically different (P>0.05).

[0201] As shown in Table 8, 1-42 Compared with the cell survival rate of the model group, the cell survival rates treated with three doses of compound 2 (1, 5 and 10 μmol / L) showed statistical differences (P<0.01), while the cell survival rates treated with low doses of compound 2 (0.1 and 0.5 μmol / L) showed no statistical differences (P>0.05).

[0202] Table 5: Effects of different compounds (5 μmol / L) on cell survival in the okadaic acid injury model

[0203] Group Mean±SDSurvival rate%Negative control 1.299±0.023100Okadaic acid (50nmol / L) 0.643±0.01449.50Compound 1 0.916±0.017*70.52Compound 2 0.92±0.008*70.82Compound 3 0.858±0.009*66.05Compound 4 0.856±0.012*65.90Compound 5 0.852±0.004*65.59Compound 6 0.889±0.007*68.44Compound 7 0.919±0.008*70.75Compound 8 0.877±0.009*67.51Compound 9 0.852±0.010*65.59

[0204] 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

[0205] Compared with okadaic acid: *P<0.01.

[0206] Table 6: Effects of different concentrations of compound 1 on cell survival in the okadaic acid injury model

[0207]

[0208] Compared with okadaic acid: *P<0.05; **P<0.01.

[0209] Table 7: Effects of different concentrations of compound 2 on cell survival in the okadaic acid injury model

[0210]

[0211] Compared with okadaic acid: *P<0.01.

[0212] Table 8: Effects of different concentrations of compound 2 on the survival rate of cells in the Aβ1-42 injury model

[0213]

[0214] and Aβ 1-42 Compared with: *P<0.01.

Claims

1. A benzopyrazoloxazine derivative having a structure as shown in Formula I or a pharmaceutically acceptable salt thereof: in: R is selected from hydrogen, halogen, alkyl, alkoxy; m is an integer from 1 to 3; X is selected from oxygen or sulfur; R1 is selected from hydrogen, alkyl, and acyl.

2. The benzopyrazoloxazine derivative according to claim 1, characterized in that: R is selected from hydrogen, halogen, C1-C2 alkyl, 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.

3. The benzopyrazoloxazine derivative according to claim 1, characterized in that: Selected from the following compounds: (5-Imino-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-2-yl)methanol; 2-(Methoxymethyl)-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; 2-(Ethoxymethyl)-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; 2-(Propyloxymethyl)-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; (5-Imino-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-2-yl)methyl formate; (5-Imino-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazin-2-yl)methyl acetate; 2-(Methylthiomethyl)-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; 2-(Methoxymethyl)-8-methyl-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; 2-(Methoxymethyl)-8-ethyl-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; 2-(Methoxymethyl)-8-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; 2-(Methoxymethyl)-7-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; 2-(Methoxymethyl)-7-chloro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; 2-(Methoxymethyl)-7-methoxy-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; 2-(Methoxymethyl)-7-ethoxy-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; 2-(Ethoxymethyl)-7-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; 2-(Propyloxymethyl)-7-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; 2-(Methoxyethyl)-8-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine; 2-(Methoxypropyl)-8-fluoro-5H-benzo[d]pyrazolo[5,1-b][1,3]oxazine-5-imine.

4. Use of the benzopyrazoloxazine derivative according to any one of claims 1 to 3 in the preparation of a medicament for preventing and treating diseases caused by loss of PP2A activity or decreased PP2A function.

5. The use according to claim 4, characterized in that: Diseases caused by loss of PP2A activity or decreased PP2A function are tumors.

6. The use according to claim 4, characterized in that: Diseases caused by loss of PP2A activity or decreased PP2A function are brain degenerative diseases.

7. The use according to claim 6, characterized in that: The brain degenerative disease is Alzheimer's disease.

8. Use of the benzopyrazoloxazine derivative according to any one of claims 1 to 3 in the preparation of a protein phosphatase agonist.

9. Use of the benzopyrazoloxazine derivative according to any one of claims 1 to 3 in the preparation of a protein phosphatase 2A agonist.

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