WO2022160138A1

WO2022160138A1 - Benzoxazine-4-one compound, preparation method therefor, and medical use thereof

Info

Publication number: WO2022160138A1
Application number: PCT/CN2021/074002
Authority: WO
Inventors: 张丽颖
Original assignee: 承德医学院
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2022-08-04

Abstract

The present invention relates to a benzoxazine-4-one compound represented by formula (I) that acts as a glycogen phosphorylase inhibitor, a preparation method therefor, and a use thereof in a drug for the treatment and/or prevention of diseases associated with glycogen metabolism abnormalities.

Description

Benzoxazin-4-one compound, its preparation method and medical use

technical field

The invention relates to the field of medicinal chemistry, in particular to a benzoxazin-4-one compound as a glycogen phosphorylase inhibitor, a preparation method and medical use thereof.

Background technique

The liver is an important organ that regulates blood sugar in the fasted state. It is estimated that after an overnight fast, 74% of fasting blood glucose is derived from hepatic glycogenolysis and the remainder from hepatic gluconeogenesis. In patients with type 2 diabetes, the rate of hepatic glucose production is significantly increased, and hepatic glucose production is on the high side. Therefore, inhibition of hepatic glucose production has become one of the important targets for the development of new antidiabetic drugs.

Currently, in clinical practice, metformin, the clinically preferred hypoglycemic drug, is believed to reduce blood sugar mainly by inhibiting hepatic gluconeogenesis; however, there is no effective drug in the field of glycogen degradation. For those patients with type 2 diabetes with fasting blood glucose >14 mg/dl (7.8 mM), inhibition of hepatic glycogen degradation, thereby reducing their hepatic glucose output, helps to reduce their fasting blood glucose.

Glycogen phosphorylase is a key enzyme that catalyzes glycogenolysis, which catalyzes the phosphorylation of glycogen, and the generated glucose-1-phosphate is converted into glucose- 6-phosphate, which is catalyzed by glucose-6-phosphatase to generate glucose, and blood sugar rises. There remains a need for drugs that inhibit glycogen phosphorylase, thereby inhibiting hepatic glycogen degradation.

CN103497181A discloses a benzazepine

Ketones have good inhibitory activity on glycogen phosphorylase, but the problem is that their half-life in the body is too short, they will be metabolized by the body soon after taking the drug, and the bioavailability is low, which causes serious problems. affect the efficacy.

SUMMARY OF THE INVENTION

The present invention provides a benzoxazinone compound represented by formula (I) with glycogen phosphorylase inhibitory activity, a preparation method and medical use thereof. Since the compound of formula (I) of the present invention can inhibit glycogen phosphorylase, it can be used for the prevention and/or treatment of diseases related to abnormal glycogen metabolism. In particular, the compounds of the present invention have a long half-life in vivo, high bioavailability, and improved curative effect.

According to one aspect of the present invention, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof as follows:

in:

X ₁ , X ₂ , X ₃ and X ₄ are all C or one of X ₁ , X ₂ , X ₃ and X ₄ is N and the other must be C;

R ₁ and R ₁ ' are each independently H, halogen, hydroxy, cyano, C _1-4 alkyl, C _1-4 alkoxy, fluoromethyl, difluoromethyl, trifluoromethyl, vinyl , ethynyl;

R ₂ and R ₂ ' are each independently H, halogen, hydroxy, cyano, C _1-4 alkyl, C _1-4 alkoxy, fluoromethyl, difluoromethyl, trifluoromethyl, vinyl , ethynyl;

R ₃ is H, unsubstituted or X-substituted linear or branched alkyl of 1-20 carbons, unsubstituted or X-substituted linear or branched alkenyl of 2-20 carbons, 2-20 unsubstituted or X-substituted straight or branched chain alkynyl, unsubstituted or X-substituted aryl, unsubstituted or X-substituted heteroaryl;

R ₄ and R ₅ are each independently H, unsubstituted or X-substituted linear or branched alkyl of 1-20 carbons, unsubstituted or X-substituted linear or branched alkene of 2-20 carbons base, 2-20 carbon unsubstituted or X-substituted linear or branched alkynyl, R ₄ and R ₅ can optionally form a ring;

Y is CHR ₆ , NH, O, S;

R ₆ is H, unsubstituted or X-substituted linear or branched alkyl of 1-20 carbons, unsubstituted or X-substituted linear or branched alkene of 2-20 carbons, 2-20 unsubstituted or X-substituted straight or branched chain alkynyl, phenyl, benzyl, naphthyl, nitrile groups of carbon atoms;

X is F, _Cl , Br, I, CN, NO2, _NH2 , _CF3 , SH, OH, _OCH3 , _OC2H5 , COOH, straight or branched chain alkyl of 1-10 carbons, ₂ - Linear or branched alkenyl of 10 carbons, linear or branched alkynyl of 2-10 carbons, aryl, heteroaryl.

Preferably, the structure of the compound of formula (I) is shown in the following formula (II):

Further preferably, in the compounds of formula (I) and formula (II):

X ₁ , X ₂ , X ₃ and X ₄ are all C or one of X ₂ and X ₃ is N and the other must be C;

R ₁ and R ₁ ' are each independently H, halogen, cyano, C _1-4 alkoxy;

R ₂ and R ₂ ' are each independently H;

R ₃ is H, unsubstituted or X-substituted linear or branched alkyl of 1-20 carbons, unsubstituted or X-substituted C _6-14 aryl, unsubstituted or X-substituted C _5-10 hetero Aryl;

R ₄ and R ₅ are each independently H, unsubstituted or X-substituted linear or branched alkyl of 1-20 carbons, and R ₄ and R ₅ may optionally form a ring;

Y is CH ₂ , NH, O;

X is F, _Cl , Br, I, CN, NO2, _NH2 , _CF3 , SH, OH, _OCH3 , _OC2H5 , COOH, straight or branched chain alkyl of 1-10 carbons, ₂ - Linear or branched alkenyl of 10 carbons, linear or branched alkynyl of 2 to 10 carbons, _C6-14 aryl, _C5-10 heteroaryl.

Further preferably, in the compounds of formula (I) and formula (II):

R ₁ and R ₁ ' are each independently H, F, Cl, Br, cyano, methoxy;

R ₂ and R ₂ ' are each independently H;

_R is H, unsubstituted or X-substituted straight or branched alkyl of 1-6 carbons;

R ₄ and R ₅ are each independently H, unsubstituted or X-substituted straight or branched chain alkyl of 1-6 carbons, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, Sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, etc., R ₄ and R ₅ can optionally form a ring, such as a five-membered ring (such as cyclopentyl), a six-membered ring (eg cyclohexyl), seven-membered ring (eg cycloheptyl), etc.;

Y is O;

X is F, _Cl , Br, I, CN, NO2, _NH2 , _CF3 , SH, OH, _OCH3 , _OC2H5 , _COOH , straight or branched chain alkyl of 1-6 carbons.

In the present invention, "halogen" refers to fluorine, chlorine, bromine and iodine. "C _1-4 alkyl" refers to straight or branched chain alkyl groups having 1-4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertiary Butyl. "C _1-4 alkoxy" refers to a straight or branched chain alkoxy having 1-4 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy base, isobutoxy, tert-butoxy. "Aryl" is eg phenyl, naphthyl, phenanthryl, anthracenyl and the like. "Heteroaryl" has 1, 2 or 3 heteroatoms selected from S, O, N, eg, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, thienyl, furyl, pyrrolyl, pyridyl azolyl, imidazolyl, thiazolyl,

azolyl, iso

azolyl, indolyl, benzo[b]thienyl, benzo[b]furanyl, quinolinyl, isoquinolinyl, quinazolinyl and the like.

Further preferably, the compounds of formula (I) and formula (II) are selected from the following compounds:

Those skilled in the art will understand that pharmaceutically acceptable salts such as salts formed by compounds of formula (I) or formula (II) with inorganic or organic acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, nitric acid, etc., Organic acids such as formic acid, acetic acid, propionic acid, valeric acid, diethylacetic acid, trifluoroacetic acid, maleic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, lactic acid, tartaric acid, malic acid, citric acid , gluconic acid, ascorbic acid, niacin, isonicotinic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenedisulfonic acid, etc.

According to the second aspect of the present invention, the present invention provides the preparation method of the above-mentioned compound, comprising the following steps:

a) will

Dissolve in ammonia water or dissolve in organic solvent and add ammonia water, preferably under the protection of inert gas, preferably nitrogen, react for 1-72 hours, preferably 24-48 hours, and the temperature is 0 ° C to reflux to obtain

Wherein, R ₃ ' is an organic group, preferably methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, benzyl, etc.; preferably, the organic solvent is selected from Dioxane, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, chloroform, toluene, n-hexane, cyclohexane, tert-butyl methyl ether, pyridine and mixtures of two or more thereof, more preferably dichloromethane oxane, tetrahydrofuran or mixtures thereof;

b) will

Dissolved in organic solvent, add

and catalyst PPTS, preferably under the protection of an inert gas, preferably nitrogen, and react for 1-72 hours at a temperature of 0 °C to reflux to obtain

Preferably, the organic solvent is selected from benzene, toluene, xylene, dioxane, DMF, DMSO, acetonitrile and mixtures of two or more thereof, more preferably dioxane, toluene, xylene and two or more thereof a mixture of one or more;

c) will

Dissolve in an organic solvent, add a hydrogen source, use a metal catalyst to catalyze the reduction of the nitro group on the benzene ring, and the temperature is 0 °C to reflux to obtain

Preferably, the metal catalyst is selected from palladium carbon, Raney nickel, iron powder, zinc powder, and stannous chloride; preferably, the hydrogen source is selected from hydrogen, hydrazine hydrate, amine formate, formic acid, ammonium chloride, cyclohexene; preferably, the organic solvent is selected from methanol, ethanol, n-butanol, tert-butanol, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, chloroform, toluene, n-hexane, cyclohexane , tert-butyl methyl ether and a mixture of two or more thereof, more preferably methanol, ethanol or a mixture thereof;

d) Substituted indole carboxylic acid or pyrrolopyridine-2-carboxylic acid

and

Dissolve in an organic solvent, add a condensation reagent and an organic amine or an inorganic base, and react for 1-72 hours at a temperature of 0°C to 45°C; preferably, the organic solvent is an inert solvent, more preferably an aprotic solvent , and further preferably the organic solvent is selected from acetonitrile, chloroform, dichloromethane, 1,2-dichloroethane, N,N-dimethylformamide, toluene, n-hexane, cyclohexane, tetrahydrofuran, tert-butyl methyl ether and Wherein the mixture of two or more, further preferably the organic solvent is selected from dichloromethane, 1,2-dichloroethane or, N,N-dimethylformamide and the mixture of two or more thereof; preferably , the condensation reagent is an amidation condensation reagent, more preferably 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), N,N'-dicyclohexylcarbodiimide Imine (DCC), O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid (TBTU), 2-(7-azobenzotriazole)-N , N,N',N'-tetramethylurea hexafluorophosphate (HATU), 1-propylphosphoric tricyclic anhydride (T ₃ P); preferably, the inorganic base is selected from sodium carbonate, sodium bicarbonate , potassium carbonate, potassium bicarbonate and mixtures of two or more thereof; preferably, the organic amine is N,N-diisopropylethylamine, triethylamine or a mixture thereof.

Preferably, it further comprises the following steps:

β-hydroxy acid

Dissolve in organic alcohol R ₃ 'OH, add organic acid or inorganic acid as catalyst, preferably under the protection of inert gas, preferably nitrogen, react for 1-72 hours, preferably 24-48 hours, the temperature is 0 ℃ to reflux, to obtain

Preferably, R ₃ ' in the organic alcohol R ₃ 'OH is an organic group, further preferably, the organic alcohol R ₃ 'OH is selected from methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, benzyl alcohol and mixtures of two or more thereof, further preferably methanol, ethanol, isopropanol, tert-butanol and mixtures of two or more thereof; Fluoroacetic acid, methanesulfonic acid and mixtures of two or more thereof; preferably, the inorganic acid is selected from hydrochloric acid, sulfuric acid and mixtures thereof.

According to the third aspect of the present invention, the present invention also provides a pharmaceutical composition comprising a compound of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

Those skilled in the art can understand that various commonly used adjuvants in the art can be used in the present invention, including but not limited to fillers, diluents, disintegrants, lubricants, binders, dispersants, wetting agents, solvents, pH adjusters, flavors, preservatives, antioxidants, and the like.

The dosage forms of the pharmaceutical composition of the present invention include, but are not limited to, tablets, capsules, pills, suppositories, soft capsules, oral liquids, suspensions, injections and other commonly used pharmaceutical forms.

It will be understood by those skilled in the art that various dosage forms of the pharmaceutical composition of the present invention can be prepared according to methods well known in the art.

According to a fourth aspect of the present invention, the present invention provides a method for preventing and/or treating a disorder associated with abnormal glycogen metabolism, comprising administering to an individual in need thereof an effective amount of formula (I) or formula (II) ) compound or a pharmaceutically acceptable salt thereof.

It will be understood by those skilled in the art that the dosage of a compound of formula (I) or formula (II), or a pharmaceutically acceptable salt thereof, will vary from formulation to formulation. Generally, in amounts that have proven beneficial, to achieve the desired results, the total amount of the compound of formula (I) or formula (II) administered per kilogram per 24 hours is about 0.01-800 mg, preferably a total amount of 0.1-100 mg /kg. If necessary, it is administered in several single doses. However, it is also possible to deviate from the above-mentioned amounts if necessary, i.e. it depends on the type and weight of the subject to be treated, the behavior of the individual with the drug, the nature and severity of the disease, the type of formulation and administration, and the time of administration and interval.

According to a fifth aspect of the present invention, the present invention provides a compound of formula (I) or formula (II) of the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the present invention in the preparation for use in the treatment and/or prevention of and Use in medicine for disorders associated with abnormal glycogen metabolism.

According to the present invention, diseases related to abnormal glycogen metabolism include diabetes (especially type 2 diabetes) or its complications (such as diabetic nephropathy, diabetic foot, diabetic neuropathy, cardiovascular and cerebrovascular diseases complicated by diabetes, etc.), hyperlipidemia disease, obesity, ischemic cardiovascular and cerebrovascular diseases (especially myocardial infarction, angina pectoris, myocardial ischemia, myocardial ischemia-reperfusion, arrhythmia, coronary heart disease, cerebral ischemia, stroke, cerebral infarction or ischemic neurodegeneration disease, etc.), hyperinsulinemia, insulin resistance, fasting hyperglycemia, hypertension or its complications, atherosclerosis, metabolic syndrome or tumors.

According to the sixth aspect of the present invention, the present invention provides a compound of formula (I) or formula (II) of the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the present invention in the preparation of a glycogen phosphorylase inhibitor the use of.

Description of drawings

Figure 1 shows the preparation process of some compounds of the present invention.

In FIG. 1 , X ₁ , X ₂ , X ₃ , X ₄ , R ₁ , R ₁ ′, R ₂ , R ₂ ′, R ₃ , R ₄ , R ₅ , R ₆ , X and Y are defined as above As defined in formula (I), R ₃ ' is an organic group, preferably methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, benzyl and the like.

Figure 2 is a mouse echocardiographic evaluation of the effect of compound treatment of Example 8 on myocardial ischemia-reperfusion injury

In Figure 2, (A) is a representative image of small animal M-mode ultrasound; (B) is a statistical map of small animal ultrasound EF, FS, EDV and ESV (*p<0.05)

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the contents described in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the limited scope of the present invention.

Example 1

Methyl 2-hydroxy-5-nitrobenzoate

5-Nitrosalicylic acid (4.0 g, 21.84 mmol) was dissolved in methanol (70 mL), concentrated sulfuric acid (1.5 mL) was added dropwise with stirring, and the reaction system was protected with nitrogen after the addition was complete. The reaction was heated to reflux for 2 days. After the reaction, the methanol in the reaction solution was removed, K ₂ CO ₃ was added until no more bubbles were generated, pure water (20 mL) was added, extracted with ethyl acetate (3×50 mL), and the organic layers were combined with anhydrous sodium sulfate. Dry for 2-3 hours, filter and concentrate, and perform silica gel column chromatography (petroleum ether/ethyl acetate=15%-20%) to obtain a white solid (4.0 g, 93%). mp112-114°C.

ESI-MS m/z: 239.2(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 4.04 (s, 3H), 7.09 (d, J=8.0 Hz, 1H), 8.34 (dd, J=8.0, 4.0 Hz, 1H), 8.80 (d, J= 2.8 Hz, 1H), 11.43 (s, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 169.3, 166.2, 140.0, 130.6, 126.7, 118.7, 112.1, 53.1.

2-Hydroxy-5-nitrobenzamide

To the ammonia water (70 mL) solution, methyl 5-nitrosalicylate (4.0 g, 20.29 mmol) was added, the reaction system was protected with nitrogen, heated to 50° C. and stirred for 2 days. After the reaction was completed, the reaction solution was evaporated to dryness under reduced pressure, pure water (30 mL) was added to the residue, and 2 mol/L hydrochloric acid solution was used to make it nearly acidic. Extraction with ethyl acetate (3×50 mL), the combined organic layers were dried over anhydrous sodium sulfate for 2-3 hours, filtered and concentrated, and subjected to silica gel column chromatography (petroleum ether/ethyl acetate=30%-50%) to obtain a pale yellow solid ( 2.70 g, 73%). m.p.130-132°C.

ESI-MS m/z: 180.7(MH) ^- .

¹ H-NMR (400 MHz, d ₆ -DMSO): 7.10 (d, J=8.0 Hz, 1H), 8.29 (dd, J=8.0, 4.0 Hz, 2H), 8.81 (s, 1H), 8.90 (d, J=2.4Hz, 1H), 14.16 (s, 1H). ¹³ C-NMR (100MHz, d6-DMSO): 170.6, 166.8, 139.5, 129.6, 125.5, 119.0, 115.1.

2-Methyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one

5-Nitrosalicylic amide (0.1 g, 0.549 mmol) was dissolved in toluene (6 mL) and acetaldehyde (8 mL), PPTS (0.14 _g , 0.557 mmol) was added, the reaction system was protected by N, and the reaction was stirred at 80 °C for 16 h , cooled to room temperature, the organic phase was washed successively with saturated NaHCO ₃ (15 mL×4) and saturated NaCl (15 mL×1), and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and silica gel column chromatography (petroleum ether/ethyl acetate=91/9-2/1) gave a pale yellow solid (0.0815 g, 71.37%). mp216-218°C.

ESI-MS m/z: 208.8(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.73 (d, J=5.6 Hz, 3H), 5.53-5.57 (m, 1H), 7.03 (s, 1H), 7.11 (d, J=8.8 Hz, 1H) , 8.35 (dd, J=9.2, 2.8Hz, 1H), 8.65 (d, J=2.8Hz, 1H). ¹³ C-NMR (100MHz, CDCl ₃ ): 162.2, 162.0, 136.1, 129.6, 124.7, 118.1, 117.9, 82.0, 20.2.

2-Methyl-6-amino-2H-benzo[e][1,3]azin-4(3H)-one

2-Methyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one (0.2874 g, 1.382 mmol) (crude) was dissolved in absolute ethanol (30 mL) , add HCOONH ₄ (0.74g, 11.7mmol), Pd/C (0.051g, 0.4806mmol), stir at room temperature for 3h, filter, concentrate, silica gel column chromatography (petroleum ether/ethyl acetate=3/1-1/1 ) to give a pale yellow solid (0.2262 g, 91.95%). mp180-182℃.

ESI-MS m/z: 179.0(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.61 (d, J=6.0 Hz, 3H), 3.61 (s, 2H), 5.33-5.38 (m, 1H), 6.33 (s, 1H), 6.81 (s, 2H), 7.22 (t, J=0.8Hz, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 164.1, 150.8, 141.5, 121.9, 118.5, 117.3, 113.1, 80.8, 20.3.

N-(2-Methyl-4-oxo-3,4-dihydro-2H-benzo[e][1,3]azin-6-yl)-5-chloro-1H-indole-2- formamide

5-Chloroindole-2-carboxylic acid (0.29 g, 1.48 mmol) was dissolved in dry DMF (7.4 mL), and HATU (0.38 g, 1 mmol, 0.4 mol/L of dry DMF) was slowly added under stirring. solution), triethylamine (3mmol, 2mol/L dry DMF solution), after stirring for 10min at room temperature, 2-methyl-6-amino-2H-benzo[e][1,3]azine-4( 3H)-ketone (0.1762 g, 0.9899 mmol, 5 mL of a dry DMF solution of about 0.2 mol/L), stirred at 45 °C for 4.5 h, cooled to room temperature, washed with saturated NaCl (15 mL × 3), and the organic phase was extracted with ethyl acetate, Dry over anhydrous _NaSO4 overnight. Filtration, concentration, and recrystallization from ethanol/water gave a light green solid (0.1786 g, 50.68%). mp302-304°C.

ESI-MS m/z: 354.1(MH) ^- .

¹ H-NMR (400 MHz, d ₆ -DMSO): 1.99 (s, 3H), 5.42 (d, J=4.4 Hz, 1H), 7.06 (d, J=8.4 Hz, 1H), 7.23 (d, J= 7.2Hz, 1H), 7.41(s, 1H), 7.48(d, J=8Hz, 1H), 7.78(s, 1H), 7.94(d, J=7.6Hz, 1H), 8.23(s, 1H), 8.70 (s, 1H), 10.38 (s, 1H), 11.95 (s, 1H). ¹³ C-NMR (100 MHz, d ₆ -DMSO): 163.1, 159.7, 154.1, 135.7, 133.8, 133.3, 128.5, 126.8, 124.9, 124.3, 121.3, 119.3, 118.8, 117.1, 114.5, 103.8, 81.6, 20.2.

Example 2

2-Ethyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one

5-Nitrosalicylamide (0.6 g, 3.29 mmol) was dissolved in toluene (10 mL) and propionaldehyde (10 mL), PPTS (0.83 g, 3.3 mmol) was added, the reaction system was protected by N, and the reaction was stirred at 80 °C for ₁₃ h , cooled to room temperature, the organic phase was washed successively with saturated NaHCO ₃ (15 mL×4) and saturated NaCl (15 mL×1), and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and silica gel column chromatography (petroleum ether/ethyl acetate=22/3-3/1) gave a white solid (0.4903 g, 67.13%). mp205-207°C.

ESI-MS m/z: 222.8(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.20 (t, J=7.6 Hz, 3H), 2.02-2.11 (m, 2H), 5.40 (t, J=5.2 Hz, 1H), 7.14 (d, J= 8.8Hz, 1H), 8.17 (s, 1H), 8.36 (dd, J=9.2, 2Hz, 1H), 8.84 (d, J=2Hz, 1H). ¹³ C-NMR (100MHz, CDCl ₃ ): 162.7, 162.4, 142.7, 129.5, 124.6, 118.2, 117.9, 86.1, 27.0, 7.7.

2-Ethyl-6-amino-2H-benzo[e][1,3]azin-4(3H)-one

2-Ethyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one (0.554 g, 2.485 mmol) was dissolved in 70 mL of absolute ethanol, and HCOONH ₄ (1.28 g, 20.298 mmol), Pd/C (0.079 g, 0.745 mmol), stirred at room temperature, TLC monitored the reaction, the reaction was completed after 3 h, and recrystallized from ethanol/petroleum ether to obtain a white solid (0.1478 g, 30.98%). mp128-131℃.

ESI-MS m/z: 193.0(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.12 (t, J=7.6 Hz, 3H), 1.85-2.01 (m, 2H), 3.59 (br s, 1H), 5.17 (t, J=5.2 Hz, 1H) ), 6.82 (d, J=7.2 Hz, 1H), 6.83 (s, 2H), 7.24 (s, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 164.4, 150.8, 141.2, 121.9, 118.6, 117.3 , 113.1, 84.9, 27.1, 8.0.

N-(2-Ethyl-4-oxo-3,4-dihydro-2H-benzo[e][1,3]azin-6-yl)-5-chloro-1H-indole-2- formamide

5-Chloroindole-2-carboxylic acid (0.14 g, 0.72 mmol) was dissolved in dry DMF (3.6 mL), and HATU (0.253 g, 0.665 mmol, 0.4 mol/L) was slowly added under stirring. DMF solution), triethylamine (2.009 mmol, 2 mol/L dry DMF solution), after stirring for 10 min at room temperature, 2-ethyl-6-amino-2H-benzo[e][1,3]azine- 4(3H)-one (0.1277g, 0.665mmol, 3.33mL of a dry DMF solution of about 0.2mol/L), stirred at 45°C for 22h, cooled to room temperature, washed with saturated NaCl (15mL×3), and the organic phase was washed with ethyl acetate. Extracted and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and recrystallization from ethanol/water gave a pale yellow solid (0.0989 g, 40.30%). mp308-310℃.

ESI-MS m/z: 367.9(MH) ^- .

¹ H-NMR (400 MHz, d ₆ -DMSO): 1.04 (t, J=5.6 Hz, 3H), 1.76-1.92 (m, 2H), 5.20-5.30 (m, 1H), 7.07 (d, J=8.4 Hz, 1H), 7.24(d, J=8Hz, 1H), 7.42(s, 1H), 7.49(d, J=8.4Hz, 1H), 7.79(s, 1H), 7.95(d, J=8Hz, 1H), 8.23 (s, 1H), 8.70 (s, 1H), 10.39 (s, 1H), 11.96 (s, 1H). ¹³ C-NMR (100 MHz, d ₆ -DMSO): 163.1, 159.7, 154.0, 135.7, 133.7, 133.3, 128.5, 126.8, 124.9, 124.3, 121.3, 119.3, 118.9, 117.2, 114.5, 103.8, 85.4, 26.7, 8.3.

Example 3

2-propyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one

5-Nitrosalicylamide (0.6 g, 3.29 mmol) was dissolved in toluene (10 mL) and butyraldehyde (10 mL), PPTS (0.83 _g , 3.3 mmol) was added, the reaction system was protected by N, and the reaction was stirred at 80 °C for 20.5 h, cooled to room temperature, the organic phase was washed successively with saturated NaHCO ₃ (15 mL×4) and saturated NaCl (15 mL×1), and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and silica gel column chromatography (petroleum ether/ethyl acetate=47/3-1/1) gave a white solid (0.4417 g, 56.89%). mp191-193°C.

ESI-MS m/z: 236.9(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.06 (t, J=7.4 Hz, 3H), 1.58-1.70 (m, 2H), 1.89-2.08 (m, 2H), 5.42 (t, J=4.8 Hz, 1H), 7.11 (d, J=9.0Hz, 1H), 7.46 (s, 1H), 8.34 (dd, J=9.0, 2.8Hz, 1H), 8.84 (d, J=2.8Hz, 1H). ¹³ C -NMR (100MHz, _CDCl3 ): 162.3, 142.8, 129.5, 124.7, 118.2, 117.9, 85.0, 35.8, 16.9, 13.7.

2-propyl-6-amino-2H-benzo[e][1,3]azin-4(3H)-one

2-Propyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one (0.397 g, 1.682 mmol) was dissolved in absolute ethanol (70 mL) and HCOONH was added ₄ (0.85g, 13.48mmol), Pd/C (0.053g, 0.5mmol), stirred at room temperature for 3h, filtered, concentrated, silica gel column chromatography (petroleum ether/ethyl acetate=4/1-7/3), A white solid was obtained (0.2638 g, 76.13%). mp114-116°C.

ESI-MS m/z: 207.0(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.01 (t, J=7.4 Hz, 3H), 1.50-1.65 (m, 2H), 1.76-1.98 (m, 2H), 3.61 (br s, 2H), 5.20 (t, J=4.9Hz, 1H), 6.80 (s, 2H), 7.04 (s, 1H), 7.23 (s, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 164.5, 150.9, 141.3, 121.9 , 118.7, 117.3, 113.1, 83.9, 35.9, 17.1, 13.8.

N-(2-propyl-4-oxo-3,4-dihydro-2H-benzo[e][1,3]azin-6-yl)-5-chloro-1H-indole-2- formamide

5-Chloroindole-2-carboxylic acid (0.302 g, 1.549 mmol) was dissolved in dry DMF (7.75 mL), and HATU (0.4 g, 1.052 mmol, 0.4 mol/L) was slowly added with stirring. dry DMF solution), triethylamine (3.16 mmol, 2 mol/L dry DMF solution), after stirring for 10 min at room temperature, 2-propyl-6-amino-2H-benzo[e][1,3]azine was added -4(3H)-one (0.2126 g, 1.032 mmol, 5.16 mL of a dry DMF solution of about 0.2 mol/L), stirred at 45°C for 25 h, cooled to room temperature, washed with saturated NaCl (15 mL×3), and the organic phase was washed with ethyl acetate. Ester extraction, dried over anhydrous _NaSO4 overnight. Filtration, concentration, and recrystallization from ethanol/water gave an off-white solid (0.1561 g, 39.49%). mp301-303°C.

ESI-MS m/z: 382.0(MH) ^- .

¹ H-NMR (400 MHz, d ₆ -DMSO): 0.95 (t, J=7.4 Hz, 3H), 1.49-1.57 (m, 2H), 1.77-1.82 (m, 2H), 5.29 (t, J=4.9 Hz, 1H), 7.06 (d, J=8.8Hz, 1H), 7.23 (dd, J=8.7, 2.0Hz, 1H), 7.42 (d, J=1.5Hz, 1H), 7.49 (d, J=8.7 Hz, 1H), 7.78 (d, J=1.8Hz, 1H), 7.94 (dd, J=8.9, 2.6Hz, 1H), 8.22 (d, J=2.6Hz, 1H), 8.68 (s, 1H), 10.37 (s, 1H), 11.94 (s, 1H). ¹³ C-NMR (100 MHz, d ₆ -DMSO): 163.0, 159.7, 154.0, 135.7, 133.7, 133.3, 128.6, 126.8, 124.9, 124.3, 121.3, 119.3 , 118.9, 117.1, 114.5, 103.8, 84.5, 35.6, 17.0, 14.1.

Example 4

2-Butyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one

5-Nitrosalicylamide (0.6 g, 3.29 mmol) was dissolved in toluene (10 mL) and n-valeraldehyde (10 mL), PPTS (0.83 _g , 3.3 mmol) was added, the reaction system was protected by N, and the reaction was stirred at 80 °C for 16 h , cooled to room temperature, the organic phase was washed successively with saturated NaHCO ₃ (15 mL×4) and saturated NaCl (15 mL×1), and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and silica gel column chromatography (petroleum ether/ethyl acetate=47/3-7/3) gave a white solid (0.5885 g, 71.55%). mp178-180℃.

ESI-MS m/z: 250.8(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 0.99 (t, J=7.2 Hz, 3H), 1.44-1.62 (m, 4H), 1.92-2.10 (m, 2H), 5.41 (t, J=5.1 Hz, 1H), 7.11 (d, J=9.0Hz, 1H), 7.86 (s, 1H), 8.34 (dd, J=9.0, 2.8Hz, 1H), 8.83 (d, J=2.7Hz, 1H). ¹³ C -NMR (100MHz, _CDCl3 ): 162.5, 162.3, 142.8, 129.5, 124.6, 118.2, 117.9, 85.3, 33.5, 25.5, 22.3, 13.9.

2-Butyl-6-amino-2H-benzo[e][1,3]azin-4(3H)-one

2-Butyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one (0.419 g, 1.676 mmol) was dissolved in absolute ethanol (50 mL) and HCOONH was added ₄ (0.85g, 13.48mmol), Pd/C (0.053g, 0.5mmol), stirred at room temperature for 3h, filtered, concentrated, and subjected to silica gel column chromatography (petroleum ether/ethyl acetate=17/3-2/1), An off-white solid was obtained (0.1512 g, 41.01%). HPLC analysis: 87.5%.mp 128-130°C.

ESI-MS m/z: 220.9(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 0.95 (t, J=7.2 Hz, 3H), 1.38-1.35 (m, 4H), 1.78-1.99 (m, 2H), 3.60 (br s, 2H), 5.19 (t, J=4.9Hz, 1H), 6.80 (s, 2H), 6.89 (s, 1H), 7.22 (s, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 164.4, 150.9, 141.4, 121.9 , 118.7, 117.3, 113.1, 84.1, 33.6, 25.8, 22.4, 13.9.

N-(2-Butyl-4-oxo-3,4-dihydro-2H-benzo[e][1,3]azin-6-yl)-5-chloro-1H-indole-2- formamide

5-Chloroindole-2-carboxylic acid (0.131 g, 0.668 mmol) was dissolved in dry DMF (3.34 mL), and HATU (0.17 g, 0.447 mmol, 0.4 mol/L) was slowly added with stirring. dry DMF solution), triethylamine (1.36 mmol, 2 mol/L dry DMF solution), after stirring for 10 min at room temperature, 2-butyl-6-amino-2H-benzo[e][1,3]azine was added -4(3H)-one (0.098g, 0.445mmol, 2.23mL of a dry DMF solution of about 0.2mol/L), stirred at 45°C for 24h, cooled to room temperature, washed with saturated NaCl (15mL×3), and the organic phase was washed with ethyl acetate. Ester extraction, dried over anhydrous _NaSO4 overnight. Filtration, concentration, and recrystallization from ethanol/water gave an off-white solid (0.0556 g, 31.47%). mp299-301°C.

ESI-MS m/z: 396.0(MH) ^- .

¹ H-NMR (400 MHz, d ₆ -DMSO): 0.91 (t, J=7.3 Hz, 3H), 1.33-1.50 (m, 4H), 1.78-1.82 (m, 2H), 5.28 (t, J=5.2 Hz, 1H), 7.06 (d, J=8.8Hz, 1H), 7.23 (dd, J=8.7, 2.0Hz, 1H), 7.41 (d, J=1.4Hz, 1H), 7.48 (d, J=8.7 Hz, 1H), 7.78 (d, J=1.8Hz, 1H), 7.94 (dd, J=8.9, 2.6Hz, 1H), 8.22 (d, J=2.6Hz, 1H), 8.66 (s, 1H), 10.36 (s, 1H), 11.93 (s, 1H). ¹³ C-NMR (100 MHz, d ₆ -DMSO): 163.0, 159.7, 154.0, 135.7, 133.7, 133.3, 128.6, 126.8, 124.9, 124.3, 121.3, 119.3 , 118.9, 117.1, 114.5, 103.8, 84.7, 33.3, 25.7, 22.3, 14.3.

Example 5

2-Pentyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one

5-Nitrosalicylamide (0.5 g, 2.747 mmol) was dissolved in toluene (10 mL) and n _- hexanal (10 mL), PPTS (0.69 g, 2.745 mmol) was added, the reaction system was protected by N, and the reaction was stirred at 80 °C for 16 h , cooled to room temperature, the organic phase was washed successively with saturated NaHCO ₃ (15 mL×4) and saturated NaCl (15 mL×1), and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and silica gel column chromatography (petroleum ether/ethyl acetate=100/0-2/1) gave a white solid (0.4874 g, 67.21%). mp174-176°C.

ESI-MS m/z: 264.9(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 0.95 (t, J=6.9 Hz, 3H), 1.40-1.42 (m, 4H), 1.58-1.63 (m, 2H), 1.91-2.09 (m, 2H), 5.41(t, J=5.3Hz, 1H), 7.11(d, J=9.0Hz, 1H), 7.93(s, 1H), 8.34(dd, J=9.0, 2.7Hz, 1H), 8.82(d, J =2.7Hz, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 162.6, 162.3, 142.8, 129.5, 124.6, 118.2, 117.9, 85.3, 33.8, 31.3, 23.1, 22.4, 13.9.

2-Pentyl-6-amino-2H-benzo[e][1,3]azin-4(3H)-one

2-Pentyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one (0.44 g, 1.67 mmol) was dissolved in absolute ethanol (50 mL) and _HCOONH was added (0.841g, 13.34mmol), Pd/C (0.053g, 0.5mmol), the reaction was stirred at room temperature for 3h, filtered, concentrated, and subjected to silica gel column chromatography (petroleum ether/ethyl acetate=17/3-2/1), Obtained as a white solid (0.2727 g, 69.78%). mp139-141°C.

ESI-MS m/z: 234.9(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 0.92 (t, J=6.7 Hz, 3H), 1.35-1.36 (m, 4H), 1.49-1.57 (m, 2H), 1.77-1.98 (m, 2H), 3.61 (br s, 2H), 5.19 (t, J=5.1 Hz, 1H), 6.80 (s, 2H), 6.90 (s, 1H), 7.22 (s, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 164.4, 150.9, 141.4, 121.9, 118.7, 117.3, 113.1, 84.1, 33.9, 31.4, 23.4, 22.5, 13.9.

N-(2-Pentyl-4-oxo-3,4-dihydro-2H-benzo[e][1,3]azin-6-yl)-5-chloro-1H-indole-2- formamide

5-Chloroindole-2-carboxylic acid (0.189 g, 0.964 mmol) was dissolved in dry DMF (4.82 mL), and HATU (0.245 g, 0.644 mmol, 0.4 mol/L) was slowly added under stirring. dry DMF solution), triethylamine (1.937 mmol, 2 mol/L dry DMF solution), after stirring for 10 min at room temperature, 2-pentyl-6-amino-2H-benzo[e][1,3]azine was added -4(3H)-one (0.1505g, 0.643mmol, 3.3mL of a dry DMF solution of about 0.2mol/L), stirred at 45°C for 23.5h, cooled to room temperature, washed with saturated NaCl (15mL×3), and the organic phase was washed with acetic acid. Extracted with ethyl ester, dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and recrystallization from ethanol/water gave a white solid (0.2257 g, 85.40%).

ESI-MS m/z: 409.9(MH) ^- .

¹ H-NMR (400 MHz, d ₆ -DMSO): 0.89 (t, J=6.8 Hz, 3H), 1.29-1.33 (m, 4H), 1.46-1.55 (m, 2H), 1.77-1.81 (m, 2H) ), 5.28(t, J=4.8Hz, 1H), 7.05(d, J=8.8Hz, 1H), 7.22(dd, J=8.7, 2.1Hz, 1H), 7.45(d, J=1.5Hz, 1H) ), 7.48(d, J=8.8Hz, 1H), 7.76(d, J=1.9Hz, 1H), 7.98(dd, J=8.9, 2.7Hz, 1H), 8.26(d, J=2.6Hz, 1H) ), 8.67 (s, 1H), 10.56 (s, 1H), 12.09 (s, 1H). ¹³ C-NMR (100 MHz, d ₆ -DMSO): 163.0, 159.6, 153.9, 135.6, 133.8, 133.4, 128.5, 126.8, 124.8, 124.3, 121.2, 119.3, 118.9, 117.1, 114.4, 104.3, 84.7, 33.5, 31.4, 23.2, 22.4, 14.3.

Example 6

2,2-Dimethyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one

5-Nitrosalicylic amide (0.8 g, 4.395 mmol) was dissolved in toluene (10 mL) and acetone (18 mL), PPTS (1.1 _g , 4.377 mmol) was added, the reaction system was protected by N, and the reaction was stirred at 80 °C for 53 h, After cooling to room temperature, the organic phase was washed successively with saturated NaHCO ₃ (15 mL×4) and saturated NaCl (15 mL×1), and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and silica gel column chromatography (petroleum ether/ethyl acetate=4/1-1/1) gave a white solid (0.3293 g, 32.3%). mp199-201°C.

ESI-MS m/z: 222.8(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.74 (s, 6H), 7.06 (d, J=8.8 Hz, 1H), 8.34 (d, J=9.2 Hz, 2H), 8.83 (s, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 161.3, 160.7, 142.4, 129.7, 124.3, 118.3, 116.8, 89.3, 27.9.

2,2-Dimethyl-6-amino-2H-benzo[e][1,3]azin-4(3H)-one

2,2-Dimethyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one (0.3293 g, 1.483 mmol) was dissolved in absolute ethanol (40 mL) , add HCOONH ₄ (0.749g, 11.87mmol), Pd/C (0.047g, 0.445mmol), stir at room temperature for 3h, filter, concentrate, silica gel column chromatography (petroleum ether/ethyl acetate=2/1), get purple Brown solid (0.2175 g, 76.54%). mp148-150℃.

ESI-MS m/z: 192.9(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.62 (s, 6H), 3.58 (s, 2H), 6.74 (d, J=8.4 Hz, 1H), 6.81 (dd, J=8.4, 2.4 Hz, 1H) , 7.23 (d, J=2.4Hz, 1H), 7.32 (s, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 163.1, 148.7, 140.9, 122.0, 117.8, 117.4, 113.0, 87.0, 27.5.

N-(2,2-Dimethyl-4-oxo-3,4-dihydro-2H-benzo[e][1,3]azin-6-yl)-5-chloro-1H-indole -2-Carboxamide

5-Chloroindole-2-carboxylic acid (0.255 g, 1.307 mmol) was dissolved in dry DMF (6.5 mL), and HATU (0.435 g, 1.144 mmol, 0.4 mol/L) was slowly added under stirring. dry DMF solution), triethylamine (3.432 mmol, 2 mol/L dry DMF solution), after stirring at room temperature for 10 min, add 2,2-dimethyl-6-amino-2H-benzo[e][1,3 ]oxazin-4(3H)-one (0.2196g, 1.144mmol, 5.65mL of a dry DMF solution of about 0.2mol/L), stirred at 45°C for 3h, cooled to room temperature, washed with saturated NaCl (15mL×3), and the organic phase, Extracted with ethyl acetate, dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and silica gel column chromatography (petroleum ether/ethyl acetate=4/1-1/1) gave off-white solid (0.0406 g, 9.59%). mp314-316°C.

ESI-MS m/z: 368.0(MH) ^- .

¹ H-NMR (400 MHz, d ₆ -DMSO): 1.54 (s, 6H), 7.00 (d, J=8.8 Hz, 1H), 7.22 (d, J=8.8 Hz, 1H), 7.40 (s, 1H) , 7.43(d, J=8.8Hz, 1H), 7.78(s, 1H), 7.93(dd, J=8.8, 2.4Hz, 1H), 8.21(d, J=2Hz, 1H), 8.67(s, 1H) _The ^_ , 118.9, 117.6, 117.5, 114.5, 103.8, 87.9, 27.7.

Example 7

2,2-Diethyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one

5-Nitrosalicylic amide (0.55 g, 3.022 mmol) was dissolved in toluene (10 mL) and 3-pentanone (7 mL), PPTS (0.87 _g , 3.466 mmol) was added, the reaction system was protected by N, and stirred at 80 °C The reaction was carried out for 48 h, cooled to room temperature, and the organic phase was washed successively with saturated NaHCO ₃ (15 mL×4) and saturated NaCl (15 mL×1), and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and silica gel column chromatography (petroleum ether/ethyl acetate=91/9-4/1) gave a light green solid (0.113 g, 14.96%). mp176-178°C.

ESI-MS m/z: 250.8(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.03 (t, J=7.6 Hz, 6H), 1.91-2.02 (m, 4H), 7.06 (d, J=9.2 Hz, 1H), 8.28 (s, 1H) , 8.33 (dd, J=8.8, 2.8Hz, 1H), 8.81 (d, J=2.8Hz, 1H). ¹³ C-NMR (100MHz, CDCl ₃ ): 161.5, 161.0, 142.2, 129.7, 124.2, 118.0, 116.8, 94.0, 31.0, 7.6.

2,2-Diethyl-6-amino-2H-benzo[e][1,3]azin-4(3H)-one

2,2-Diethyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one (0.2347 g, 0.9388 mmol) was dissolved in absolute ethanol (50 mL) , add HCOONH4 (0.593g, 9.4mmol), Pd/C (0.03g, 0.28mmol), stir at room temperature for 3h, filter, concentrate, silica gel column chromatography (petroleum ether/ethyl acetate=2/1), get gray solid (0.138 g, 66.82%). m.p.187-189°C.

ESI-MS m/z: 220.9(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 0.96 (t, J=7.6 Hz, 6H), 1.83-1.89 (m, 4H), 3.58 (br s, 2H), 6.75 (d, J=8.8 Hz, 1H) ), 6.82 (dd, J=8.4, 2.8Hz, 1H), 6.90 (s, 1H), 7.23 (d, J=2.4Hz, 1H). ¹³ C-NMR (100MHz, CDCl ₃ ): 163.1, 148.8, 140.5, 122.1, 117.7, 117.5, 113.1, 91.4, 30.2, 7.7.

N-(2,2-Diethyl-4-oxo-3,4-dihydro-2H-benzo[e][1,3]azin-6-yl)-5-chloro-1H-indole -2-Carboxamide

5-Chloroindole-2-carboxylic acid (0.1 g, 0.513 mmol) was dissolved in dry DMF (2.56 mL), and HATU (0.147 g, 0.387 mmol, 0.4 mol/L) was slowly added under stirring. DMF solution), triethylamine (1.148mmol, 2mol/L dry DMF solution), after stirring for 10min at room temperature, 2,2-diethyl-6-amino-2H-benzo[e][1,3 ]oxazin-4(3H)-one (0.085g, 0.386mmol, 1.93mL of a dry DMF solution of about 0.2mol/L), stirred at 45°C for 20h, cooled to room temperature, washed with saturated NaCl (15mL×3) organic phase, Extracted with ethyl acetate, dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and recrystallization from ethanol/water gave an off-white solid (0.0543 g, 35.43%). mp314-316°C.

ESI-MS m/z: 396.2(MH) ^- .

¹ H-NMR (400 MHz, d ₆ -DMSO): 0.92 (t, J=6.4 Hz, 6H), 1.73-1.86 (m, 4H), 7.01 (d, J=8.4 Hz, 1H), 7.23 (d, J=8.4Hz, 1H), 7.41(s, 1H), 7.48(d, J=8.4Hz, 1H), 7.78(s, 1H), 7.92(d, J=8.4Hz, 1H), 8.20(s, 1H), 8.63 (s, 1H), 10.36 (s, 1H), 11.95 (s, 1H). ¹³ C-NMR (100 MHz, d ₆ -DMSO): 161.6, 159.6, 152.1, 135.6, 133.3, 133.2, 128.5 , 127.0, 124.9, 124.3, 121.3, 118.9, 117.6, 117.4, 114.5, 103.8, 92.0, 30.4, 8.1.

Example 8

2,2-Dipropyl-6-nitro-2H-benzo[e][1,3]azin-4(3H)-one

5-Nitrosalicylamide (0.8 g, 4.395 mmol) was dissolved in toluene (20 mL) and 4-heptanone (20 mL), PPTS (1.1 _g , 4.377 mmol) was added, the reaction system was protected by N, and stirred at 90 °C The reaction was carried out for 65 h, cooled to room temperature, and the organic phase was washed successively with saturated NaHCO ₃ (15 mL×4) and saturated NaCl (15 mL×1), and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and silica gel column chromatography (petroleum ether/ethyl acetate=91/9-22/3) gave a white solid (0.0558 g, 4.57%). mp158-160℃.

ESI-MS m/z: 278.8(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 0.95 (t, J=7.3 Hz, 6H), 1.44-1.54 (m, 4H), 1.83-1.96 (m, 4H), 7.03 (d, J=9.0 Hz, 1H), 8.28 (s, 1H), 8.33 (dd, J=9.0, 2.7Hz, 1H), 8.81 (d, J=2.6Hz, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 161.3, 160.9 , 142.2, 129.7, 124.2, 117.9, 116.8, 93.4, 40.7, 16.7, 14.0.

2,2-Dipropyl-6-amino-2H-benzo[e][1,3]azin-4(3H)-one

6-Nitro-2,2-dipropyl-2H-benzo[e][1,3]azin-4(3H)-one (0.1552 g, 0.558 mmol) was dissolved in absolute ethanol (20 mL) , add HCOONH4 (0.282g, 4.47mmol), Pd/C (0.012g, 0.1132mmol), stir at room temperature for 4h, filter, concentrate, silica gel column chromatography (petroleum ether/ethyl acetate=17/3-2/1) , an off-white solid (0.0875 g, 63.23%) was obtained. m.p.119-121°C.

ESI-MS m/z: 248.9(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 0.90 (t, J=7.3 Hz, 6H), 1.39-1.48 (m, 4H), 1.73-1.85 (m, 4H), 3.56 (br s, 2H), 6.72 (d, J=8.6Hz, 2H), 6.80 (dd, J=8.6, 2.6Hz, 1H), 7.23 (d, J=2.6Hz, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 162.9, 148.8, 140.6, 122.0, 117.6, 117.5, 113.1, 90.8, 40.1, 16.8, 14.1.

N-(2,2-Dipropyl-4-oxo-3,4-dihydro-2H-benzo[e][1,3]azin-6-yl)-5-chloro-1H-indole -2-Carboxamide

5-Chloroindole-2-carboxylic acid (0.063 g, 0.323 mmol) was dissolved in dry DMF (1.65 mL), and HATU (0.083 g, 0.218 mmol, 0.4 mol/L) was slowly added under stirring. dry DMF solution), triethylamine (0.717mmol, 2mol/L dry DMF solution), after stirring at room temperature for 10min, add 2,2-dipropyl-6-amino-2H-benzo[e][1, 3] Azin-4(3H)-one (0.0537 g, 0.217 mmol, 1.1 mL of a dry DMF solution of about 0.2 mol/L), stirred at 45 °C for 9 h, cooled to room temperature, and washed with saturated NaCl (15 mL×3) for the organic phase , extracted with ethyl acetate, and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and recrystallization from ethanol/water gave an off-white solid (0.0285 g, 30.90%). mp293-295°C.

ESI-MS m/z: 424.0(MH) ^- .

¹ H-NMR (400 MHz, d ₆ -DMSO): 0.87 (t, J=7.3 Hz, 6H), 1.39-1.43 (m, 4H), 1.70-1.77 (m, 4H), 6.98 (d, J=8.8 Hz, 1H), 7.23 (dd, J=8.7, 2.0Hz, 1H), 7.40 (d, J=1.2Hz, 1H), 7.48 (d, J=8.7Hz, 1H), 7.78 (d, J=1.6 Hz, 1H), 7.91 (dd, J=8.8, 2.6Hz, 1H), 8.19 (d, J=2.6Hz, 1H), 8.61 (s, 1H), 10.33 (s, 1H), 11.92 (s, 1H) ). ¹³ C-NMR (100MHz, d ₆ -DMSO): 161.5, 159.6, 152.1, 135.7, 133.4, 133.1, 128.6, 127.0, 124.9, 124.3, 121.2, 118.9, 117.6, 117.4, 114.5, 103.8, 91.5, 16.8 , 14.4.

Example 9

N-(2,2-Dipropyl-4-oxo-3,4-dihydro-2H-benzo[e][1,3]azin-6-yl)-5-chloro-1H-pyrrolo [2,3-c]pyridine-2-carboxamide

5-Chloro-1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid (0.10 g, 0.51 mmol) and 2,2-dipropyl-6-amino-2H-benzo[e][1 , 3]oxazin-4(3H)-one (0.15 g, 0.62 mmol) was dissolved in ethyl acetate (5.0 mL), pyridine (2.5 mL) was added, the reaction system was placed in an ice bath, and T was slowly added to it ₃ P in ethyl acetate solution (1.0 mL, 50 wt%) was reacted under nitrogen protection for 20 h. After the reaction was completed, 0.5M hydrochloric acid solution (8.0 mL) was added thereto, and the mixture was stirred at room temperature for 2 h. The obtained suspension was filtered and washed with pure water three times. Filtration, concentration, and recrystallization from ethanol/water gave a white solid (40 mg, 18.3%).

ESI-MS m/z: 427.2(M+H) ⁺ .

Example 10

6-Nitrospiro[benzo[e][1,3]oxazine-2,1'-cyclopentane]-4(3H)-one (22i)

5-Nitrosalicylamide (0.8 g, 4.395 mmol) was dissolved in toluene (20 mL) and cyclopentanone (20 mL), PPTS (1.1 _g , 4.377 mmol) was added, the reaction system was protected by N, and the reaction was stirred at 80 °C 49h, cooled to room temperature, washed the organic phase with saturated NaHCO 3 (15 mL×4) and saturated NaCl (15 mL×1) successively, and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and recrystallization from ethanol/water gave a white solid (0.1702 g, 15.62%). mp195-197°C.

ESI-MS m/z: 249.0(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.90-2.02 (m, 6H), 2.27-2.33 (m, 2H), 7.08 (d, J=9.2 Hz, 1H), 8.35 (dd, J=9.2, 2.8 Hz, 1H), 8.39 (s, 1H), 8.84 (d, J=2.8 Hz, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 161.9, 161.0, 142.5, 129.6, 124.4, 118.3, 117.6, 99.2 , 38.7, 22.7.

6-Aminospiro[benzo[e][1,3]oxazin-2,1'-cyclopentane]-4(3H)-one

6-Nitrospiro[benzo[e][1,3]oxazin-2,1'-cyclopentane]-4(3H)-one (0.1073 g, 0.4322 mmol) was dissolved in absolute ethanol (25 mL) HCOONH ₄ (0.234 g, 3.707 mmol), Pd/C (0.02 g, 0.188 mmol) were added to the mixture, stirred at room temperature for 1 h, filtered, concentrated, and subjected to silica gel column chromatography (petroleum ether/ethyl acetate=2/1) to obtain Brown solid (0.055 g, 58.37%). mp149-151°C.

ESI-MS m/z: 218.9(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.77-1.88 (m, 6H), 2.20-2.28 (m, 2H), 3.62 (br s, 2H), 6.77 (d, J=8.4Hz, 1H), 6.81 (dd, J=8.4, 2.4Hz, 1H), 7.17 (s, 1H), 7.24 (d, J=2.4Hz, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 163.8, 149.1, 141.0, 121.8 , 118.2, 117.9, 113.1, 97.1, 38.1, 22.7.

N-(4-oxo-3,4-dihydrospiro[benzo[e][1,3]oxazine-2,1'-cyclopentane]-6-yl)-5-chloro-1H-indole Indol-2-carboxamide

5-Chloroindole-2-carboxylic acid (0.067 g, 0.342 mmol) was dissolved in dry DMF (1.71 mL), and HATU (0.087 g, 0.2288 mmol, 0.4 mol/L) was slowly added under stirring. dry DMF solution), triethylamine (0.717 mmol, 2 mol/L dry DMF solution), after stirring for 10 min at room temperature, 6-aminospiro[benzo[e][1,3]oxazine-2,1'- Cyclopentane]-4(3H)-one (0.05g, 0.229mmol, 1.15mL of a dry DMF solution of about 0.2mol/L), stirred at 45°C for 26h, cooled to room temperature, washed with saturated NaCl (15mL×3) The phase was extracted with ethyl acetate and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and recrystallization from ethanol/water gave an off-white solid (0.0427 g, 47.21%).

ESI-MS m/z: 394.0(MH) ^- .

¹ H-NMR (400MHz, d ₆ -DMSO): 1.68-1.79 (m, 4H), 1.80-1.88 (m, 2H), 2.06-2.10 (m, 2H), 7.01 (d, J=8.8Hz, 1H ), 7.23(d, J=8.6Hz, 1H), 7.41(s, 1H), 7.48(d, J=8.7Hz, 1H), 7.78(s, 1H), 7.93(dd, J=8.8, 2.5Hz , 1H), 8.21 (d, J=2.4Hz, 1H), 8.78 (s, 1H), 10.35 (s, 1H), 11.93 (s, 1H). ¹³ C-NMR (100MHz, d ₆ -DMSO): 162.1, 159.7, 152.3, 135.7, 133.5, 133.3, 128.6, 126.8, 124.9, 124.3, 121.3, 119.1, 118.3, 117.7, 114.5, 103.8, 98.0, 37.9, 22.9.

Example 11

6-Nitrospiro[benzo[e][1,3]oxazin-2,1'-cyclohexane]-4(3H)-one

5-Nitrosalicylamide (0.8 g, 4.395 mmol) was dissolved in toluene (15 mL) and cyclohexanone (15 mL), PPTS (1.1 _g , 4.377 mmol) was added, the reaction system was protected by N, and the reaction was stirred at 80 °C 48 h, cooled to room temperature, washed the organic phase with saturated NaHCO ₃ (15 mL×4) and saturated NaCl (15 mL×1) successively, and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and silica gel column chromatography (petroleum ether/ethyl acetate=4/1-1/1) gave a white solid (0.5209 g, 45.23%). mp249-251°C.

ESI-MS m/z: 262.8(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.45-1.82 (m, 8H), 2.15 (s, 2H), 7.11 (d, J=8.8 Hz, 1H), 7.80 (s, 1H), 8.36 (d, J=8.0 Hz, 1H), 8.84 (s, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 160.9, 160.4, 142.4, 129.7, 124.3, 118.2, 117.6, 89.9, 36.4, 24.3, 21.8.

6-Aminospiro[benzo[e][1,3]oxazin-2,1'-cyclohexane]-4(3H)-one

6-Nitrospiro[benzo[e][1,3]oxazin-2,1'-cyclohexane]-4(3H)-one (0.52 g, 1.98 mmol) was dissolved in absolute ethanol (50 mL) HCOONH ₄ (1 g, 15.86 mmol) and Pd/C (0.06 g, 0.566 mmol) were added to the mixture, stirred at room temperature for 3 h, filtered, concentrated, and subjected to silica gel column chromatography (petroleum ether/ethyl acetate=2/1) to obtain a Yellow solid (0.2404 g, 52.33%). mp185-187°C.

ESI-MS m/z: 232.9(M+H) ⁺ .

¹ H-NMR (400 MHz, CDCl ₃ ): 1.33-1.71 (m, 8H), 2.11-2.08 (m, 2H), 3.55 (s, 2H), 6.76-6.83 (m, 2H), 7.00 (s, 1H) ), 7.23 (d, J=2.3 Hz, 1H). ¹³ C-NMR (100 MHz, CDCl ₃ ): 162.9, 148.4, 140.9, 121.9, 118.1, 117.8, 113.1, 87.5, 35.9, 24.7, 22.0.

N-(4-oxo-3,4-dihydrospiro[benzo[e][1,3]oxazine-2,1'-cyclohexane]-6-yl)-5-chloro-1H-indole Indol-2-carboxamide

5-Chloroindole-2-carboxylic acid (0.22 g, 1.12 mmol) was dissolved in dry DMF (5.6 mL), and HATU (0.394 g, 1.036 mmol, 0.4 mol/L) was slowly added under stirring. dry DMF solution), triethylamine (3.16 mmol, 2 mol/L dry DMF solution), after stirring for 10 min at room temperature, 6-aminospiro[benzo[e][1,3]azine-2,1'- Cyclohexane]-4(3H)-one (0.24g, 1.0345mmol, 5.2mL about 0.2mol/L dry DMF solution), stirred at 45°C for 27h, cooled to room temperature, washed with saturated NaCl (15mL×3) The phase was extracted with ethyl acetate and dried over anhydrous NaSO ₄ overnight. Filtration, concentration, and recrystallization from ethanol/water gave an off-white solid (0.1244 g, 29.33%). mp332-334°C.

ESI-MS m/z: 409.4(MH) ^- .

¹ H-NMR (400MHz, d ₆ -DMSO): 1.18-1.68 (m, 8H), 2.00-2.03 (m, 2H), 7.04 (d, J=8.8Hz, 1H), 7.23 (dd, J=8.8 , 1.6Hz, 1H), 7.41 (s, 1H), 7.48 (d, J=8.8Hz, 1H), 7.78 (d, J=0.8Hz, 1H), 7.94 (dd, J=8.8, 2.4Hz, 1H) ), 8.20(d, J=2.4Hz, 1H), 8.67(s, 1H), 10.36(s, 1H), 11.95(s, 1H). ¹³ C-NMR (100MHz, d ₆ -DMSO): 161.5, 159.6, 151.5, 135.7, 133.4, 133.3, 128.5, 126.9, 124.9, 124.3, 121.3, 119.0, 118.3, 117.6, 114.5, 103.8, 88.1, 35.9, 24.7.

Example 12 In vitro glycogen phosphorylase inhibitory activity test

Preparation of reagents: 1) Preparation of color developing solution: Weigh 5g of ammonium molybdate, dissolve it in 500ml of 1M HCl, stir with a stirrer, add 190mg of malachite green after it is completely dissolved, continue to stir until it is completely dissolved, and use tin foil Protect from light; 2) Preparation of buffer solution: ① Precisely weigh Hepes 0.5958g, dissolve it in 5ml H ₂ O, adjust the pH to 7.2 with 10M NaOH, and prepare Hepes with a final concentration of 0.5M; ② Precisely weigh KCl 0.3728 g, dissolved in 5ml H ₂ O to prepare KCl with a final concentration of 1M; ③Precisely weigh 0.0255g of MgCl ₃ , dissolve in 1ml H ₂ O, and prepare a final concentration of 125mM MgCl ₂ ; ④Precisely weigh EGTA 0.0476g, dissolve in 5ml H ₂ O, adjust the pH to 7.0 with 10M NaOH, and prepare EGTA with a final concentration of 25mM; ⑤ Precisely weigh G-1-P 0.0152g, dissolve in 10ml H ₂ O, prepare G-1-P with a final concentration of 5 mM; ⑥ Precisely weigh 10 mg of glycogen, dissolve it in 1 ml of H ₂ O, and prepare a glycogen with a final concentration of 10 mg/ml; 3) Preparation of positive drug caffeine solution: dissolve caffeine Prepare 0.5, 5, 50 and 500 μM solutions in 10 ml H ₂ O; 4) Prepare GPa solution: add 1 μl of GPa to the 100 μl reaction system, the final concentration is 250 ng/100 μl; 5) Preparation of the test compound solution: the The compound to be tested was dissolved in DMSO to prepare a solution with a concentration of 10 mM, and an appropriate amount of the compound solution was added to the reaction system to different final concentrations.

Determination of the dose-response curve of rabbit muscle glycogen phosphorylase activity: by reading the OD value at 655nm after adding different concentrations of GPa to the chromogenic solution, the dose-response curve was measured. The amount of GPa can be selected as 250ng from the dose-response curve.

Experimental steps: 1) Design PC (positive control), Blank (blank control), and positive drug (caffeine); 2) Add 52 μl of reaction buffer; 3) Add test compound to the final concentration; 4) Add 1 μl of enzyme, the final concentration is 250 ng /100μl; 5) Add 150μl of color developing solution; 6) Reaction at 30 degrees Celsius for 20 minutes; 7) Colorimetry at a wavelength of 655 nm; 8) Data reading and calculation of inhibition rate: inhibition rate = [positive control- Sample to be tested]/[positive control-blank control].

The test results show that most of the compounds in the examples have IC ₅₀ <1 μM, which are proved to be effective, as shown in Table 1 below. The pharmacological data show that the compound of the general formula (I) of the present invention has an inhibitory effect on glycogen phosphorylase, and the activity is similar to that of the known compound (the compound of Example 1 of CN103497181A).

Table 1 Inhibitory activity of compound of formula (I) on rabbit muscle glycogen phosphorylase

It can be seen from the above test that the compound of formula (I) of the present invention has the activity of inhibiting glycogen phosphorylase, so it can be used to treat various diseases related to abnormal glycogen metabolism.

Example 13 Pharmacokinetic test

Take 24 adult rats, after fasting for 12 hours, they were randomly divided into 2 groups, 12 rats in each group. According to previous research experience, 50mg/kg was administered by intragastric administration. One group was given the compound of Example 8 of the present invention, and the other group was given the present invention. For the technical compound (the compound in Example 1 of CN103497181A), blood was collected by jugular vein puncture at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, and 12 h after administration, respectively. Plasma sample 0.2mL, add 0.4mL acetonitrile solution, vortex for 5min, centrifuge at 10000r·min ^-1 for 10min, take the supernatant, centrifuge at 10000r·min ^-1 for 10min, then take the supernatant, and use HPLC to determine the drug concentration in plasma . The pharmacokinetic parameters are listed in Table 2:

Table 2

It can be seen from the above experiments that the compound of the present invention has a longer half-life, higher _Cmax and better bioavailability than the prior art compound (the compound in Example 1 of CN103497181A), thereby improving the curative effect.

Example 14 Hypoglycemic experiment on high-fat diet-induced type 2 diabetic obese mice (DIO)

After 4-week-old male C57 BL/6J mice were adaptively fed for 5 days, 10 mice were randomly selected as normal control group, fed with low-fat diet, and the remaining mice were fed with high-fat diet. Mice were housed in a cage of 5 mice with free access to food and water, a light-dark cycle of 12 hours, and a room temperature of 22°C to 26°C. After the mice were continuously fed for 12 weeks, fasting and glucose tolerance were measured to evaluate insulin resistance, which showed that the modeling was successful. According to body weight, the high-fat feeding group was randomly divided into the following experimental groups according to 10 animals/group: model control group, metformin group (400 mg/kg, gavage) and Example 8 compound group (high, medium and low dose groups, respectively For 50, 25, 12.5 mg/kg, gavage, once a day). According to the experimental group, the patients were given continuous administration for 4 weeks, and they fasted overnight before the last administration.

The blood glucose levels of the model control group on the 0, 7, 14, 21, and 27 days of administration were significantly higher than those of the normal control group ( ^## p<0.01). On the 7th, 21st, and 27th days of administration, the metformin 400mg/kg group showed significant differences in blood glucose, which were lower than those in the model control group ( ^** p<0.01); the compound of Example 8 of the present invention 50mg/kg group showed significant differences on the 7th, 21st, and 27th days of administration. The blood glucose values on the 14th, 21st, and 27th days were significantly lower than those in the model control group ( ^* p<0.05, ^** p<0.01), and the blood glucose values in the 25mg/kg and 12.5mg/kg groups were significantly lower on the 21st and 27th days of administration compared with the model control group ( ^** p<0.01).

Table 3 The effect of the compound of Example 8 of the present invention on the blood glucose of the C57BL/6 hyperglycemia mouse model induced by high-fat diet

#p<0.05, ##p<0.01 vs. normal control group; ^* p<0.05, ^** p<0.01 vs. model control group, (Mean±SEM, n=10).

Example 15 Protective effect on myocardial ischemia/reperfusion (I/R) in mice

8-week-old SPF grade C57 BL/6J mice, weighing 20-25g, were randomly divided into the following experimental groups, 10 mice/group: sham operation group (Sham), I/R model group (I.R Model) and Example 8 Compound group high dose group (HD 100mg/kg), middle dose group (MD 50mg/kg) and low dose group (LD 30mg/kg). The mice were anesthetized with isoflurane, and after observing that they had no righting response, they were fixed in the supine position on the operation board. The surgical site on the front of the mouse chest was disinfected with 75% alcohol, and an incision of about 1 cm was cut along the third and fourth intercostal space. The pectoralis major and minor muscles were bluntly separated, and a curved hemostat was used to penetrate the intercostal muscle. Deep into the chest cavity, with its power, quickly squeeze the heart out of the intercostal space. The left anterior descending coronary artery was ligated with a suture needle, causing myocardial ischemia in the mouse, and the anterior wall of the left ventricle was observed to turn pale. After ligation, the heart was quickly returned to the thoracic cavity for repositioning, the end of the ligation slip-knot was exposed outside the body, and the skin incision was sutured with a suture needle. The timing of the ligation was started. After 30 minutes of ischemia, the ligature was gently pulled by hand to loosen the knot to restore blood supply to the heart. The mice were placed on a 37°C insulation pad until they recovered their automatic crawling ability. The I/R model was established 24 hours after the blood supply to the heart was restored. The high, medium and low dose groups of the compound of Example 8 were administered by tail vein injection after I/R modeling, once a day, for 7 consecutive days.

The chest of the mouse was exposed, and a medical cotton swab was dipped in an appropriate amount of depilatory cream for experimental animals, which was evenly applied to the skin of the chest and neck of the mouse, and the mouse hair was wiped off with a moist paper towel to expose the cardiac ultrasound site. Select the special ultrasound probe for mice (MS-400), anesthetize the mouse, fix it on the operation board with its chest facing up, apply ultrasound special couplant evenly on the chest, place the probe flat in the middle of the mouse thorax, fine-tune the probe, about Rotate 30° counterclockwise, observe the parasternal long-axis section of the clear heart in B-mode mode, and save the picture. Then switch to the M-mode mode and record the image at the maximum left ventricular diameter. The data were processed by analysis software to calculate the ejection fraction (EF), short-axis shortening (FS), end-diastolic volume (EDV) and end-systolic volume (ESV) of the mouse left ventricle. The results found that the compound of Example 8 significantly improved the ejection fraction of the heart of I/R mice, shortened the axial shortening rate, restored the systolic and end-diastolic volumes of the left ventricle, and significantly improved the heart of the mice at a dose of 50-100 mg/kg. Function.

Note: LVEF: left ventricular ejection fraction (Left Ventricular Ejection Fractions), refers to: the percentage of stroke volume in ventricular end-diastolic volume. When the ventricle contracts, all the blood in the ventricle cannot be ejected into the artery. Under normal adult resting state, the ventricular diastolic volume is about 125mL for the left ventricle, about 137mL for the right ventricle, and the stroke volume is 60-80mL, that is, ejection of blood. There is still a certain amount of residual blood in the ventricle at the end of the process. The percentage of the stroke volume in the diastolic volume of the ventricle is called the ejection fraction. Generally, more than 50% belongs to the normal range. The ejection fraction of the human body is about 55% to 65% at rest. The ejection fraction is related to the contractility of the myocardium. The stronger the myocardial contractility, the greater the stroke volume and the greater the ejection fraction. Under normal circumstances, the left ventricular ejection fraction is ≥50%; the right ventricular ejection fraction is ≥40%. If it is less than this value, it means cardiac insufficiency.

LVFS: Left Ventricular Fractional shortening (Left Ventricular Fractional shortening), refers to: the left ventricular end-diastolic diameter minus the left ventricular end-systolic diameter, the percentage of the left ventricular end-diastolic diameter, this value reflects the contraction of the heart and diastolic function.

LVEDV: Left Ventricular End-diastolic volume, literally, refers to the volume within the left ventricle at end-diastolic, used to calculate LVEF and LVFS.

LVESV: Left Ventricular End-systolic volume, literally, refers to the volume in the ventricle at the end of systolic left ventricle, used to calculate LVEF and LVFS.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

The following formula (I) compound or its pharmaceutically acceptable salt

in:

X 1 , X 2 , X 3 and X 4 are all C or one of X 1 , X 2 , X 3 and X 4 is N and the other must be C;

R 1 and R 1 ' are each independently H, halogen, hydroxy, cyano, C 1-4 alkyl, C 1-4 alkoxy, fluoromethyl, difluoromethyl, trifluoromethyl, vinyl , ethynyl;

R 2 and R 2 ' are each independently H, halogen, hydroxy, cyano, C 1-4 alkyl, C 1-4 alkoxy, fluoromethyl, difluoromethyl, trifluoromethyl, vinyl , ethynyl;

R 3 is H, unsubstituted or X-substituted linear or branched alkyl of 1-20 carbons, unsubstituted or X-substituted linear or branched alkenyl of 2-20 carbons, 2-20 unsubstituted or X-substituted straight or branched chain alkynyl, unsubstituted or X-substituted aryl, unsubstituted or X-substituted heteroaryl;

R 4 and R 5 are each independently H, unsubstituted or X-substituted linear or branched alkyl of 1-20 carbons, unsubstituted or X-substituted linear or branched alkene of 2-20 carbons base, 2-20 carbon unsubstituted or X-substituted linear or branched alkynyl, R 4 and R 5 can optionally form a ring;

Y is CHR 6 , NH, O, S;

R 6 is H, unsubstituted or X-substituted linear or branched alkyl of 1-20 carbons, unsubstituted or X-substituted linear or branched alkene of 2-20 carbons, 2-20 unsubstituted or X-substituted straight or branched chain alkynyl, phenyl, benzyl, naphthyl, nitrile groups of carbon atoms;

X is F, Cl , Br, I, CN, NO2, NH2 , CF3 , SH, OH, OCH3 , OC2H5 , COOH, straight or branched chain alkyl of 1-10 carbons, 2 - Linear or branched alkenyl of 10 carbons, linear or branched alkynyl of 2-10 carbons, aryl, heteroaryl.
The compound of claim 1, wherein the structure of the compound of formula (I) is shown in the following formula (II)
The compound of claim 1 or 2, wherein in the compounds of formula (I) and formula (II):

X 1 , X 2 , X 3 and X 4 are all C or one of X 2 and X 3 is N and the other must be C;

R 1 and R 1 ' are each independently H, halogen, cyano, C 1-4 alkoxy;

R 2 and R 2 ' are each independently H;

R 3 is H, unsubstituted or X-substituted linear or branched alkyl of 1-20 carbons, unsubstituted or X-substituted C 6-14 aryl, unsubstituted or X-substituted C 5-10 hetero Aryl;

R 4 and R 5 are each independently H, unsubstituted or X-substituted linear or branched alkyl of 1-20 carbons, and R 4 and R 5 may optionally form a ring;

Y is CH 2 , NH, O;

X is F, Cl , Br, I, CN, NO2, NH2 , CF3 , SH, OH, OCH3 , OC2H5 , COOH, straight or branched chain alkyl of 1-10 carbons, 2 - Linear or branched alkenyl of 10 carbons, linear or branched alkynyl of 2 to 10 carbons, C6-14 aryl, C5-10 heteroaryl.

Preferably:

X 1 , X 2 , X 3 and X 4 are all C or one of X 2 and X 3 is N and the other must be C;

R 1 and R 1 ' are each independently H, F, Cl, Br, cyano, methoxy;

R 2 and R 2 ' are each independently H;

R is H, unsubstituted or X-substituted straight or branched alkyl of 1-6 carbons;

R 4 and R 5 are each independently H, unsubstituted or X-substituted straight or branched chain alkyl of 1-6 carbons, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, Sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, etc., R 4 and R 5 can optionally form a ring, such as a five-membered ring (such as cyclopentyl), a six-membered ring (eg cyclohexyl), seven-membered ring (eg cycloheptyl), etc.;

Y is O;

X is F, Cl , Br, I, CN, NO2, NH2 , CF3 , SH, OH, OCH3 , OC2H5 , COOH , straight or branched chain alkyl of 1-6 carbons.
The compound of any one of claims 1-3, wherein the compounds of formula (I) and formula (II) are selected from the group consisting of the following compounds:
The preparation method of the compound according to any one of claims 1-4, is characterized in that, comprises the steps:

a) will
Dissolve in ammonia water or dissolve in organic solvent and add ammonia water, preferably under the protection of inert gas, preferably nitrogen, react for 1-72 hours, preferably 24-48 hours, and the temperature is 0 ° C to reflux to obtain
Wherein, R 3 ' is an organic group, preferably methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, benzyl, etc.; preferably, the organic solvent is selected from Dioxane, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, chloroform, toluene, n-hexane, cyclohexane, tert-butyl methyl ether, pyridine and mixtures of two or more thereof, more preferably dioxane oxane, tetrahydrofuran or mixtures thereof;

b) will
Dissolved in organic solvent, add
and catalyst PPTS, preferably under the protection of an inert gas, preferably nitrogen, and react for 1-72 hours at a temperature of 0 °C to reflux to obtain
Preferably, the organic solvent is selected from benzene, toluene, xylene, dioxane, DMF, DMSO, acetonitrile and a mixture of two or more thereof, more preferably dioxane, toluene, xylene and two or more thereof a mixture of one or more;

c) will
Dissolve in an organic solvent, add a hydrogen source, use a metal catalyst to catalyze the reduction of the nitro group on the benzene ring, and the temperature is 0 °C to reflux to obtain
Preferably, the metal catalyst is selected from palladium carbon, Raney nickel, iron powder, zinc powder, and stannous chloride; preferably, the hydrogen source is selected from hydrogen, hydrazine hydrate, amine formate, formic acid, ammonium chloride, cyclohexene; preferably, the organic solvent is selected from methanol, ethanol, n-butanol, tert-butanol, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, chloroform, toluene, n-hexane, cyclohexane , tert-butyl methyl ether and a mixture of two or more thereof, more preferably methanol, ethanol or a mixture thereof;

d) Substituted indole carboxylic acid or pyrrolopyridine-2-carboxylic acid
and
Dissolve in an organic solvent, add a condensation reagent and an organic amine or an inorganic base, and react for 1-72 hours at a temperature of 0°C to 45°C; preferably, the organic solvent is an inert solvent, more preferably an aprotic solvent , and further preferably the organic solvent is selected from acetonitrile, chloroform, dichloromethane, 1,2-dichloroethane, N,N-dimethylformamide, toluene, n-hexane, cyclohexane, tetrahydrofuran, tert-butyl methyl ether and Wherein the mixture of two or more, further preferably the organic solvent is selected from dichloromethane, 1,2-dichloroethane or, N,N-dimethylformamide and the mixture of two or more thereof; preferably , the condensation reagent is an amidation condensation reagent, more preferably 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), N,N'-dicyclohexylcarbodiimide Imine (DCC), O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboric acid (TBTU), 2-(7-azobenzotriazole)-N , N,N',N'-tetramethylurea hexafluorophosphate (HATU), 1-propylphosphoric tricyclic anhydride (T 3 P); preferably, the inorganic base is selected from sodium carbonate, sodium bicarbonate , potassium carbonate, potassium bicarbonate and mixtures of two or more thereof; preferably, the organic amine is N,N-diisopropylethylamine, triethylamine or a mixture thereof.

Preferably, it further comprises the following steps:

β-hydroxy acid
Dissolve in organic alcohol R 3 'OH, add organic acid or inorganic acid as catalyst, preferably under the protection of inert gas, preferably nitrogen, react for 1-72 hours, preferably 24-48 hours, the temperature is 0 ℃ to reflux, to obtain
Preferably, R 3 ' in the organic alcohol R 3 'OH is an organic group, further preferably, the organic alcohol R 3 'OH is selected from methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, tert-butanol, benzyl alcohol and mixtures of two or more thereof, further preferably methanol, ethanol, isopropanol, tert-butanol and mixtures of two or more thereof; Fluoroacetic acid, methanesulfonic acid and mixtures of two or more thereof; preferably, the inorganic acid is selected from hydrochloric acid, sulfuric acid and mixtures thereof.
A pharmaceutical composition, characterized in that it contains the compound according to any one of claims 1-4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable auxiliary material.
A method for preventing and/or treating a disease associated with abnormal glycogen metabolism, comprising administering to an individual in need an effective amount of a compound according to any one of claims 1-4 or a pharmaceutically acceptable salt thereof Or the pharmaceutical composition of claim 6.
The compound as claimed in any one of claims 1-4 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition as claimed in claim 6 is used for the treatment and/or prevention of diseases related to abnormal glycogen metabolism. use in medicine.
The method according to claim 7 or the use according to claim 8, wherein the diseases related to abnormal glycogen metabolism include diabetes (especially type 2 diabetes) or its complications (such as diabetic nephropathy, Diabetic foot, diabetic neuropathy, cardiovascular and cerebrovascular diseases complicated by diabetes, etc.), hyperlipidemia, obesity, ischemic cardiovascular and cerebrovascular diseases (especially myocardial infarction, angina pectoris, myocardial ischemia, myocardial ischemia-reperfusion, cardiac rhythm disorders, coronary heart disease, cerebral ischemia, stroke, cerebral infarction or ischemic neurodegenerative disease, etc.), hyperinsulinemia, insulin resistance, fasting hyperglycemia, hypertension or its complications, atherosclerosis, Metabolic syndrome or tumor.
Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 6 in the preparation of a glycogen phosphorylase inhibitor.