CN110511201B - 3' -aminoalkoxy-luteolin derivative and preparation method and application thereof - Google Patents

Abstract

The invention relates to preparation and application of a 3' -aminoalkoxy-luteolin derivative. Under the protection of inert gas, taking luteolin and active group substances with benzyl as raw materials, reacting under alkaline conditions to obtain dibenzyl substitutes, then reacting with active groups with amino under alkaline conditions, and removing benzyl of reaction products under the condition of metal catalysts to convert the reaction products into 3' -aminoalkoxy-luteolin derivatives. The compounds of the present invention may further form salts. The compound has the effects of antagonizing blood vessel injury of a rat against diabetes, antagonizing myocardial ischemia injury, cerebral ischemia injury and other multi-target effects of the rat, plays a relevant pharmacological efficacy effect by protecting blood vessels, and can be used for preparing medicines or health products for preventing and treating diabetic vascular complications, cardiovascular and cerebrovascular diseases and the like.

Description

3' -aminoalkoxy-luteolin derivative and preparation method and application thereof

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a 3' -aminoalkoxy-luteolin derivative, and a preparation method and application thereof.

Background

Diabetes Mellitus (DM) is a metabolic disease characterized by hyperglycemia, and the number of patients with Diabetes Mellitus worldwide increases year by year, currently 4.15 hundred million patients exist; yellow people are susceptible to high incidence, China has become the world with the most diabetes patients (the prevalence rate is up to 11.6%), and 90% of diabetes patients are type 2 diabetes. Long-term hyperglycemia damages large and micro blood vessels and seriously endangers the heart, brain, kidney, peripheral nerves, eyes, feet, etc., resulting in diabetes being the most complicated disease (up to 100). According to the American diabetes Association, the probability of the diabetic patients suffering from the complications for more than 3, 5 and 10 years is 46, 61 and 98 percent respectively. Diabetic vascular complications (DMVC) are the most serious complication of DM and the leading cause of death and disability in Diabetes, with 80% of diabetic patients dying from the complication. DMVC mainly comprises macrovascular complications (coronary heart disease, cerebral apoplexy, atherosclerosis, etc.) and microvascular complications (diabetic nephropathy, retinopathy, diabetic foot, peripheral neuropathy, etc.). The exact pathogenesis of DMVC is not completely clear, and hyperglycemia is the primary motive force for causing vascular diseases, and is mainly related to various comprehensive factors such as glucose metabolism, lipid metabolism disorder, hypertension and the like in diabetes mellitus to induce vascular injury (VD). VD involves vascular endothelial and vascular smooth muscle injuries, which are associated with vascular endothelial cells and smooth muscle cells, respectively, resulting in dysfunction of vasomotor function, endothelial secretion, barrier, etc. The pathological molecular mechanism mainly relates to oxidation, inflammatory stress reaction and the like.

At present, no special prevention and treatment medicine aiming at DMVC is clinically available, and the prevention and treatment mainly adopts medicines for regulating blood sugar, blood fat and blood pressure, resisting oxidation, inflammation and thrombus, and the like, and mainly comprises the following steps: rosiglitazone, metformin and sulfonylureas drugs which lower blood glucose and increase insulin sensitivity; statins (lovastatin, etc.) that regulate blood lipids; angiotensin Converting Enzyme Inhibitor (ACEI) drugs (captopril, etc.) for lowering blood pressure, and calcium antagonist hypotensor (nifedipine); aspirin for inhibiting thrombus and improving hemorheology. In addition, anti-inflammatory drugs such as JAK-STAT inhibitors, traditional Chinese medicines for promoting blood circulation and removing blood stasis, flavonoids and the like are being developed. The simple antioxidant has been used in clinical tests in foreign countries, but the results are not ideal. DMVC belongs to complicated diseases, and medicines which are used for interfering with blood fat and blood pressure by adopting a simple method and a single target point cannot effectively control and treat the diseases, and more importantly, the medicines have more side effects and higher toxicity and are not beneficial to long-term use. Therefore, the single-target drug is difficult to effectively treat and control the diabetic vascular complications caused by non-single genes or non-single targets, so the design and development of the multi-target drug can be a new effective method for relieving and treating the diabetic vascular complications.

Ischemic cardiovascular and cerebrovascular diseases (ICCVD) are the main pathogenic types of cardiovascular and cerebrovascular diseases, Ischemia Reperfusion (IR) injury is the main pathological process of ischemic cardiovascular and cerebrovascular diseases, and IR causes vascular injury, inflammatory reaction, oxidative stress, thrombosis and the like. In terms of pathogenesis, ICCVD belongs to complex diseases with relatively complex pathogenesis, and the pathological mechanism is related to a complex pathological network thereof, so that the clinical prevention and treatment of ICCVD aims at the pathological network mechanism and uses comprehensive intervention treatment methods, including measures of expanding blood vessels to relieve ischemia, resisting inflammation, resisting oxidative stress, resisting thrombosis, inducing ischemia pre-adaptation and the like, for prevention and treatment, so that multi-target intervention can be an effective method for clinically preventing and treating ICCVD.

Luteolin (LTD) is a natural flavone compound existing in various medicinal plants and vegetables, has a chemical name of 3', 4', 5, 7-tetrahydroxyflavone, and has pharmacological effects of resisting oxidation, resisting tumor, resisting inflammation, reducing blood sugar, antagonizing vascular complications and the like. However, luteolin has poor water solubility, poor drug action, low bioavailability and limited drug effect. The research and development of the luteolin derivative with strong activity, low toxic and side effects and novel structure is the research direction of the structure modification of the luteolin compound.

Disclosure of Invention

Aiming at the problems, the invention provides a 3' -aminoalkoxy-luteolin derivative by a chemical synthesis method, which plays roles of antagonizing diabetic vascular injury and myocardial ischemic injury and cerebral ischemic injury by protecting blood vessels.

The technical scheme of the invention is as follows:

a3' -aminoalkoxy-luteolin derivative and pharmaceutically acceptable salts thereof have the following structure:

wherein R represents- (CH)₂)nNR₁R₂、

Wherein R is₁And R₂The same or different, represents H or C1-C6 straight chain or branched chain alkyl, n represents an integer of 1-6, R₃Or R₄Represents H or C1-C6 straight chain or branched chain alkyl.

Preferably, said R is₁And R₂The same or different, represents H, methyl or ethyl, n represents an integer of 1 to 4, R₃Or R₄Represents H, methyl or ethyl.

More preferably, R represents- (CH)₂)₂N(CH₃)₂、–(CH₂)₂NHCH₃、–(CH₂)₂NH₂、–(CH₂)₃N(CH₃)₂、

The concrete structure is as follows:

the 3' -aminoalkoxy-luteolin derivative also comprises an inorganic acid salt or an organic acid salt of the derivative. The inorganic acid salt or organic acid salt is selected from sulfate, phosphate, hydrochloride, hydrobromide, acetate, oxalate, citrate, succinate, gluconate, tartrate, p-toluenesulfonate, benzenesulfonate, methanesulfonate, benzoate, lactate or maleate.

Another objective of the present invention is to provide a method for preparing the 3 '-aminoalkoxy-luteolin derivatives and pharmaceutically acceptable salts thereof, wherein luteolin and active substances having benzyl groups are used as raw materials, and the reaction is carried out under alkaline conditions to obtain luteolin dibenzyl substituent, and amino active groups are introduced into 3' -hydroxyl groups of the luteolin dibenzyl compound, and the protecting groups are removed to obtain the luteolin derivatives.

The synthetic route is as follows:

a preferred preparation of the invention comprises the following steps:

(1) under the protection of inert gas, preparing luteolin alkaline solution, and adding active substances with benzyl groups for reaction;

(2) adding a substance with an oxygen-based active group, triphenylphosphine and an anhydrous non-alcoholic organic solvent into the reaction product obtained in the step (1), stirring, and adding diethyl azodicarboxylate or diisopropyl azodicarboxylate for reaction;

(3) dissolving the reaction product of the step (2) with methanol, H₂And (3) adding a metal catalyst into a displacement reaction system, and removing a protecting group benzyl to obtain the 3' -aminoalkoxy-luteolin derivative.

In the preparation method, the inert gas in the step (1) is selected from one or more of nitrogen or argon, the active substance with benzyl group is selected from one or more of benzyl chloride, benzyl bromide and benzyl iodide, the used base is selected from diisopropylethylamine, triethylamine, pyridine or DBU, the solvent is a conventional organic solvent, and DMF is used as the solvent in a specific embodiment.

The substance having an amino active group in the step (2) is selected from HO (CH)₂)nNR₁R₂、

Wherein R is₁And R₂The same or different, represents H or C1-C6 straight chain or branched chain alkyl or tert-butyloxycarbonyl, n represents an integer of 1-6, R₃Or R₄Represents H or C1-C6 straight chain or branched chain alkyl or tert-butyloxycarbonyl. Preferably, said R is₁And R₂The same or different, represents H, methyl or ethyl, n represents an integer of 1 to 4, R₃Or R₄Represents H, methyl or ethyl. More preferably, it is selected from 2-dimethylaminoethanol, N-tert-butoxycarbonyl-N-methylaminoethanol, N- (tert-butoxycarbonyl) ethanolamine, 3-dimethylamino-1-propanol, N-tert-butoxycarbonyl-4-hydroxypiperidine or 1-tert-butoxycarbonyl-3-hydroxypyrrolidine.

Preferably, the reaction temperature in the step (2) is 15-20 ℃, and the reaction time is 24-48 h.

The metal catalyst in the step (3) is selected from Pd/C, Au-Pd/TiO₂Or Ag/ZnO.

In the above preparation method, the steps (1) to (3) further comprise a step of separating and purifying the reaction product, or a step (4) of further salifying the product with an acid.

A specific preparation scheme of the invention comprises the following steps:

(1) under the protection of inert gas, preparing luteolin alkaline solution, stirring for 10min, dripping benzyl active substance in ice water bath, stirring for 24 hr at 15-20 deg.C, and detecting by TLC.

(2) Concentrating the mixed solution obtained in step (1), adding 100mL of ethyl acetate and 200mL of H₂O, stirring for 30min, filtering, and concentrating the organic phase to obtain a crude product. The crude product was purified by silica gel column chromatography.

(3) And (3) placing the purified fraction obtained in the step (2) into a reflux condensing device, adding a substance with an amino active group, triphenylphosphine and anhydrous THF, stirring for 10min, dropwise adding DEAD in an ice-water bath, controlling the reaction temperature to be 15-20 ℃, detecting by LC-MS, concentrating the reaction solution, and purifying by silica gel column chromatography.

(4) Dissolving the concentrated fraction obtained in the step (3) by using methanol, replacing a reaction system by H2, adding a metal catalyst, obtaining a product by adopting one or more methods of washing, suction filtration, extraction and rotary evaporation, and then salifying the product with 1N hydrochloric acid.

A preferred purification scheme for step (2) is: the crude LTD-Bn2 was purified by silica gel column chromatography eluting: EA/PE is 1/5-1/3. The crude LTD2 was purified by silica gel column chromatography eluting with: EA/PE is 1/3-1/1.

The invention also aims to provide the application of the 3' -aminoalkoxy-luteolin derivative and the pharmaceutically acceptable salt thereof in preparing the medicines for preventing and treating ischemic injury. Preferably, the medicine is a medicine for preventing and treating diabetic vascular complications and ischemic cardiovascular and cerebrovascular diseases, wherein the diabetic complications comprise diabetic nephropathy, diabetic retinopathy, diabetic foot and diabetic vascular injury, and the ischemic cardiovascular and cerebrovascular diseases comprise myocardial ischemia injury and cerebral ischemia injury, such as coronary heart disease, cerebral apoplexy and the like.

The invention has the advantages that:

(1) the invention adopts a chemical synthesis method to obtain a multi-target compound 3' -aminoalkoxy-luteolin derivative and pharmaceutically-formable salts thereof, and compared with luteolin, the multi-target compound has good dissolubility and bioavailability and higher vasoprotective activity.

(2) The compound has various pharmacological actions of antagonizing diabetic vascular injury (but not influencing blood sugar), antagonizing myocardial ischemia injury and cerebral ischemia injury, has the characteristic of multi-target point of an action mechanism, and is suitable for preventing and treating cardiovascular and cerebrovascular diseases and complications caused by diabetes.

(3) At present, the main ICCVD prevention and treatment medicines in clinic mainly take single-target medicines, such as calcium ion antagonists and the like, and the invention researches and designs related new medicine candidate compounds from multiple targets such as blood vessel protection, anti-inflammation, antioxidation and the like, and belongs to the innovative new medicine research and development direction.

(4) Luteolin has low water solubility, so that the preparation difficulty is high, the bioavailability is low, and a series of derivatives are researched and designed to improve the water solubility of the medicine and improve or maintain the vascular activity of the medicine. In a series of derivatives studied in this invention, LTD3 was most water soluble but had little vasoactivity (see table 1, fig. 13), LTD10 was most vasoactive but poor water solubility (LTD was close), therefore LTD2 was preferred as the primary subject.

Drawings

FIG. 1 is a LCMS diagram of LTD2 according to the present invention.

FIG. 2 shows LTD2 of the compound of the invention¹H-NMR chart.

FIG. 3 is a LCMS diagram of LTD7 according to the present invention.

FIG. 4 shows LTD7 of the compound of the invention¹H-NMR chart.

FIG. 5 is a LCMS diagram of LTD8 according to the present invention.

FIG. 6 shows LTD8 of the compound of the present invention¹H-NMR chart.

FIG. 7 is a LCMS diagram of LTD9 according to the present invention.

FIG. 8 shows LTD9 of the compound of the present invention¹H-NMR chart.

FIG. 9 shows LCMS of LTD10 according to the present invention.

FIG. 10 shows LTD10 of the compound of the present invention¹H-NMR chart.

FIG. 11 is a LCMS diagram of LTD11 according to the invention.

FIG. 12 shows LTD11 of the compound of the present invention¹H-NMR chart.

FIG. 13 is a dose-response graph of compound LTD2 and other derivatives of the present invention dilating isolated thoracic aortic blood vessels in rats.

FIG. 14 is a laser speckle machine detection of the compound LTD2/LTD of the present invention increasing ZDF rat foot skin blood flow.

FIG. 15 is a statistical plot of the LTD2/LTD of the compound of the present invention increasing the blood flow in the feet of ZDF rats. (note: in comparison with the ZDF group,^*P<0.05,^**P<0.01.)。

FIG. 16 shows that in vitro incubation of the compound LTD2/LTD improves the isolated coronary vasodilation response of ZDF rats. (note: in comparison with the ZDF group,^**P<0.01,^***P<0.001.)。

FIG. 17 shows that intragastric administration of LTD2/LTD of the compound of the present invention enhances isolated coronary vasodilatory response in ZDF rats. (note: in comparison with the ZDF group,^*P<0.05,^***P<0.001.)。

FIG. 18 shows the effect of intragastric administration of LTD2 on the pathological morphology of kidney tissue in ZDF rats (A1-A3: fa/+ rat normal kidney tissue structure, B1-B3: ZDF rat kidney tissue structure, C1-C3: LTD2 on ZDF rat kidney tissue structure).

FIG. 19 shows the effect of intragastric administration of LTD2 of the compound of the present invention on the pathological tissue morphology of thoracic aorta in ZDF rats.

FIG. 20 is a typical cross-sectional view of ischemic myocardium from different treatment groups.

FIG. 21 shows the protective effect of LTD2 according to the present invention on myocardial ischemia-reperfusion in rats. (note: in comparison to the model set,^**P<0.01,^***P<0.001.)。

FIG. 22 is a typical cross-sectional view of ischemic brain tissue from different treatment groups.

Figure 23 corrects for ischemia volume percentage. (note: compared to the Model set (Model),^***P<0.001.)。

FIG. 24 percent ischemic area. (note: compared to the Model set (Model),^***P<0.001.)。

figure 25 neurobehavioral scores. (note: compared to the Model set (Model),^**P<0.01,^***P<0.001.)。

Detailed Description

The following examples illustrate specific steps of the present invention, but are not intended to limit the invention.

Terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified. The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that this example is intended to illustrate the invention and not to limit the scope of the invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

The present invention is further illustrated by the following specific examples.

Example 1: preparation of 3' - (dimethylamino) ethoxy-luteolin hydrochloride (1:1) (LTD2)

Under the protection of inert gas, 5.0g luteolin, 6.8g DIPEA and 30mL DMF are added into a 50mL three-necked flask, stirred for 10min, and added with 4.15mL benzyl chloride under ice water bath. Stirring at 15-20 deg.C for 24-48 hr, detecting by TLC, and allowing the raw materials to react completely. After the reaction mixture was concentrated in vacuo, 100mL ethyl acetate and 200mL H were added₂And O, stirring for 30min, filtering, and detecting a filter cake as a dibenzyl substituent. The filtrate is layered, the water phase is discarded, and the organic phase is concentrated to obtain a crude product. The crude product was purified by column chromatography on silica gel (eluent: EA/PE. RTM. 1/5-1/3), and the fractions were collected and dried to give LTD-Bn2 as an off-white solid, 2.0g, in 24.5% yield. Under inert gas atmosphere, 1.0g of LTD-Bn2, 0.23g of 2-dimethylaminoethanol, 1.12g of triphenylphosphine and 30mL of anhydrous THF were added into a 50L three-necked flask. Stirring for 10min, and dropping 680 μ L of LDEAD under ice-water bath. Stirring at 15-20 deg.C for 24-48 hr, and LC-MS detecting to obtain target molecular weight product with small amount of residual material. The reaction was stopped. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (eluent: EA/PE-1/3-1/1). The collected fractions were spin-dried to give 0.8g of LTD2 as a pale yellow solid. The yield thereof was found to be 69.5%. A250 mL three-necked flask was charged with 700mg of LTD2, 120.0mg of the metal catalyst and 100mL of methanol. H2 gas was used to replace the reaction system three times overnight at room temperature. The next day, the reaction was monitored by HPLC for completion. The reaction solution was filtered to remove the metal catalyst, and the filter cake was washed with THF. Mixing filtrates, and concentrating. Adding 2mL of LTHF and 25mL of ethanol into the residue, pulping for 2 hours at normal temperature, and filtering to obtain340mg of LTD2 as a solid. 10mL of 1N diluted hydrochloric acid is added into the solid, and the mixture is beaten for 20min under ultrasonic wave to obtain milky turbid liquid. The mixture was freeze dried to yield 380mg of LTD2 hydrochloride as a pale yellow solid. The yield thereof was found to be 74.1%.

LCMS[[M+H]⁺:358.14]As shown in fig. 1.

¹H NMR(400MHz,DMSO)7.63(1H,overlapped,H-2′),7.63(1H,overlapped,H-6′),6.99(1H,d,J＝2.63Hz,H-5′),6.93(1H,s,H-3),6.55(1H,d,J＝0.63Hz,H-8),6.23(1H,d,J＝0.63Hz,H-6),4.47(2H,m,H-1″),3.56(2H,m,H-2″)，2.856(N-CH₃,s,3H),2.853(N-CH₃S,3H) is shown in FIG. 2.

The chemical structure of the obtained LTD2 hydrochloride salt:

example 2: preparation of 3' - (monomethylamino) ethoxy-luteolin hydrochloride (1:1) (LTD7)

Under the protection of inert gas, 9.0g of LTD-Bn2, 2.64-2.64 g N-tert-butoxycarbonyl-N-methylaminoethanol and 4.87g of PPh3 were added to a 250mL three-necked flask, stirred for 10min, and 2.92mL of DEAD was added dropwise in an ice-water bath. After the dropwise addition, the reaction solution was returned to room temperature and stirred for 16 hours. LCMS detection reaction was complete. The reaction was concentrated in vacuo to give 20.0g of crude brown. Without further purification, LTD7a01 was used as starting material in the next reaction. 20.0g of crude LTD7a01 was dissolved in 50mL of dichloromethane under inert gas and 10mL of TFA was added slowly dropwise at room temperature. After the addition was complete, stirring was continued for 16h and LCMS monitored for reaction completion. The reaction was concentrated, 100mL of saturated NaHCO3 solution was added thereto, extracted with dichloromethane, the filtrate was spun dry, and column chromatography (DCM/MeOH 50/1-15/1) was performed to give 3.0g of the objective compound as yellow color. A500 mL single vial was charged with 2.0g of LTD7a02, 400.0mg of metal catalyst, 100mL of methanol and 100mL of THF, H2 gas-exchanged reaction three times at room temperature overnight. The next day, the reaction was monitored by HPLC for completion. The reaction solution was filtered to remove the metal catalyst, and the filter cake was washed with a mixed solution of THF and methanol. Mixing filtrates, and concentrating. Purification by semi-preparative HPLC afforded 450.0mg of LTD 7. 15mL of 0.1N HCl was added thereto and lyophilized to give 480.0mg of LTD7 hydrochloride. The yield was 35%.

LCMS[[M+H]⁺:343.98]As shown in fig. 3.

¹H NMR(400MHz,DMSO)7.63(1H,overlapped,H-2′),7.62(1H,overlapped,H-6′),6.99(1H,d,J＝2.75Hz,H-5′),6.94(1H,s,H-3),6.54(1H,d,J＝0.63Hz,H-8),6.22(1H,d,J＝0.63Hz,H-6),4.38(2H,m,H-1″),3.41(2H,m,H-2″)，2.67(N-CH₃S,3H) are shown in FIG. 4.

The chemical structural formula of the obtained compound LTD 7:

example 3: preparation of 3' -Aminoethoxy-luteolin hydrochloride (1:1) (LTD8)

Under the protection of inert gas, 6.0g of LTD-Bn2, 1.6mL of N- (tert-butoxycarbonyl) ethanolamine and 3.38g of PPh3 were added to a 250m L three-necked flask, stirred for 10min, and added dropwise to 1.91mL of DEAD in an ice-water bath. After the dropwise addition, the reaction solution was returned to room temperature and stirred for 16 hours. LCMS detection reaction was complete. The reaction was concentrated in vacuo and purified by column chromatography (DCM/MeOH 20/1-10/1) to yield 3.3g of crude product as a pale yellow solid. 3.3g of crude LTD8a01 are dissolved in 30mL of dichloromethane under inert gas and 6.0mL of TFA are slowly added dropwise at room temperature. Stirring is continued for 16h after the dropwise addition is completed. The reaction was monitored by HPLC for completion. The reaction was concentrated, to which was added 30.0mL of saturated NaHCO3 solution, extracted with dichloromethane, the filtrate was spun dry, and subjected to column chromatography (DCM/MeOH 20/1-10/1) to give 1.0g of the title compound as a yellow color. A500 mL single neck flask was charged with 1.0g of LTD8a02, 181.0mg of metal catalyst, 100mL of methanol and 100mL of THF, H₂The reaction system was air-replaced three times overnight at room temperature. The next day, the reaction was monitored by HPLC for completion. The reaction solution was filtered to remove the metal catalyst, 12.0mL of 1N HCl aqueous solution was added, and the filter cake was washed with a mixed solution of methanol and water. Mixing filtrates, and concentrating. Purification by semi-preparative HPLC afforded 280.0mg of LTD8 hydrochloride. The yield was 46.6%.

LCMS[[M+H]⁺:330.03]As shown in fig. 5.

¹H NMR(400MHz,DMSO)7.62(1H,overlapped,H-2′),7.62(1H,overlapped,H-6′),6.99(1H,d,J＝2.75Hz,H-5′),6.94(1H,s,H-3),6.54(1H,d,J＝0.63Hz,H-8),6.22(1H,d,J＝0.63Hz,H-6),4.38(2H,m,H-1″),3.41(2H,m,H-2″)，2.67(N-CH₃S,3H) is shown in fig. 6.

The chemical structural formula of the obtained compound LTD 8:

example 4: preparation of 3' - (dimethylamino) propoxy-luteolin hydrochloride (1:1) (LTD9)

Under the protection of inert gas, 14.0g of LTD-Bn2, 1.8mL of 3-dimethylamino-1-propanol and 4.84g of PPh3 were added to a 250m L three-necked flask, stirred for 10min, and added dropwise to 2.9mL of DEAD in an ice-water bath. After the dropwise addition, the reaction solution was returned to room temperature and stirred for 16 hours. LCMS detection reaction was complete. The reaction was concentrated in vacuo and the crude product obtained by column chromatography (DCM/MeOH-20/1-15/1) was further purified by semi-preparative HPLC to give 2.0g of crude product as a pale grey colour. Into a 500.0mL single neck flask was added 1.5g of LTD9a01, 150mg of the metal catalyst, 100mL of methanol and 100mL of THF, H₂The reaction system was air-replaced three times overnight at room temperature. The next day, the reaction was monitored by HPLC for completion. The reaction was filtered to remove the metal catalyst, concentrated, slurried with 100mL EtOAc, slurried with 2mL 1N aqueous HCl and lyophilized to provide 900.0mg LTD9 hydrochloride. The yield thereof was found to be 93.1%.

LCMS[[M+H]⁺:372.06]As shown in fig. 7.

¹H NMR(400MHz,DMSO)7.59(1H,overlapped,H-2′),7.59(1H,overlapped,H-6′),7.01(1H,d,J＝2.75Hz,H-5′),6.89(1H,s,H-3),6.55(1H,d,J＝0.63Hz,H-8),6.23(1H,d,J＝0.63Hz,H-6),4.18(2H,m,H-1″),3.29(2H,m,H-3″)，2.18(2H,m,H-2″)，2.80(N-CH₃,s,3H)，2.78(N-CH₃S,3H) is shown in FIG. 8.

The chemical structural formula of the obtained compound LTD 9:

example 5: preparation of 3' - (4 "-piperidinyloxy) -luteolin hydrochloride (1:1) (LTD10)

Under the protection of inert gas, 14.0g of LTD-Bn2, 1.6mL of N-tert-butoxycarbonyl-4-hydroxypiperidine and 4.79g of PPh3 were put into a 250m L three-necked flask, stirred for 10min, and 2.87mL of DEAD was added dropwise in an ice-water bath. After the dropwise addition, the reaction solution was returned to room temperature and stirred for 16 hours. LCMS detection reaction was complete. The reaction was concentrated in vacuo to give 30.0g of crude pale yellow. The crude product was used as the starting material for the next step without purification. Under inert gas, 1.6g of crude LTD10a01 was dissolved in 100mL of dichloromethane and 30mL of TFA was added slowly dropwise at room temperature. Stirring is continued for 16h after the dropwise addition is completed. LCMS monitor reaction completion. The reaction was concentrated, 100mL of saturated NaHCO3 solution was added, extracted with dichloromethane, the filtrate was spun dry and purified by column chromatography (DCM/MeOH ═ 50/1-15/1) and semi-preparative HPLC to give 1.6g of white LTD10a 02. A500 mL single neck flask was charged with 1.6g of LTD10a02, 160mg of metal catalyst, 100mL of methanol and 100mL of THF, H₂The reaction system was air-replaced three times overnight at room temperature. The next day, LCMS monitored the reaction completion. The reaction solution was filtered to remove the metal catalyst, and the filter cake was washed with a mixed solution of methanol and THF. Mixing filtrates, concentrating, adding 100mL acetone, ultrasonic pulping, filtering to obtain pure product, adding 2mL 1N HCl aqueous solution, and lyophilizing to obtain 377.0mg LTD10 hydrochloride. The yield thereof was found to be 37%.

LCMS[[M+H]⁺:369.99]As shown in fig. 9.

¹H NMR (400MHz, DMSO)7.68(1H, s, H-2 '), 7.61(1H, d, J ═ 2.75Hz, H-6 '), 7.03(1H, d, J ═ 2.75Hz, H-5 '), 6.92(1H, s, H-3),6.54(1H, d, J ═ 0.63Hz, H-8),6.22(1H, d, J ═ 0.63Hz, H-6),4.01(1H, m, H-4 "), 3.34(2H, m, H-2" and H-6 "), 3.07(2H, m, H-2" and H-6 "), 2.05(2H, m, H-3" and H-5 "), 1.76(2H, m, H-3" and H-5 ", as shown in fig. 10").

The chemical structural formula of the obtained compound LTD 10:

example 6: preparation of 3' - (3 "-pyrrolidinyloxy) -luteolin hydrochloride (1:1) (LTD11)

Under the protection of inert gas, 14.0g of LTD-Bn2, 2.78mL of 1-tert-butoxycarbonyl-3-hydroxypyrrolidine and 4.79g of PPh3 were added to a 250mL three-necked flask, stirred for 10min, and 2.87mL of DEAD was added dropwise in an ice-water bath. After the dropwise addition, the reaction solution was returned to room temperature and stirred for 16 hours. LCMS detection reaction was complete. The reaction was concentrated in vacuo to give 30.0g of crude LTD11a01 as an orange yellow color. The crude product was used as the starting material for the next step without purification. Under inert gas, 30.0g of crude LTD11a01 was dissolved in 100mL of dichloromethane and 30mL of TFA was added slowly and dropwise at room temperature. Stirring is continued for 16h after the dropwise addition is completed.

The next day, LCMS monitored the reaction completion. The reaction was concentrated, 100mL of saturated NaHCO3 solution was added, extracted with dichloromethane, the filtrate was spun dry and purified by column chromatography (DCM/MeOH ═ 50/1-15/1) and semi-preparative HPLC to give 1.2g of the white compound LTD10a 02. Into a 500mL single neck flask was added 1.2g of LTD10a02(2.24mmol), 120mg of 10% metal catalyst, 100mL of methanol and 100mL of THF, H₂The reaction system was air-replaced three times overnight at room temperature. The next day, LCMS monitored the reaction completion. The reaction solution was filtered to remove the metal catalyst, and the filter cake was washed with a mixed solution of methanol and THF. The filtrates were combined, concentrated, added with 100mL of acetone, ultrasonically beaten, filtered to give a pure product, added with 2mL of 1N HCl aqueous solution, and lyophilized to give 430.0mg of LTD11 hydrochloride. The yield thereof was found to be 51.3%.

LCMS[[M+H]⁺:355.96]As shown in fig. 11.

¹H NMR (400MHz, DMSO)7.69(1H, s, H-2 '), 7.65(1H, d, J ═ 2.63Hz, H-6 '), 7.03(1H, d, J ═ 2.63Hz, H-5 '), 6.94(1H, s, H-3),6.54(1H, d, J ═ 0.63Hz, H-8),6.23(1H, d, J ═ 0.63Hz, H-6),4.03(1H, m, H-3 "), 3.34(2H, m, H-2"), 2.89,2.73(2H, m, H-5 "), 2.16(2H, m, H-4") is shown in fig. 12.

The chemical structural formula of the obtained compound LTD 11:

the following analogs were also prepared with reference to the above procedure:

LTD3：

LTD4：

and LTD6 a:

example 7: the invention discloses a method for detecting water solubility of 3' -aminoalkoxy-luteolin derivatives

The solubility of the sample in pure water was determined as follows:

TABLE 1 Water solubility of LTD and its derivatives

Note: the water solubility detection of LTD and the derivative thereof mainly detects the complete dissolving amount of a sample in pure water.

Example 8: the influence of the 3' -aminoalkoxy-luteolin derivative on isolated blood vessels of rats

1 materials of the experiment

1.1 Experimental animals: adult healthy male SPrague-Da' wley (SD) rats (university of Kunming medical laboratory animal center, license number: SYXK (Dian) 2005-.

1.2 Experimental drugs and reagent configuration

Sample preparation: sample LTD was purchased from Dian Asahi Brilliant Biotech; the 3' -aminoalkoxy-luteolin derivatives obtained in examples 1-6, LTD2, LTD7, LTD8, LTD9, LTD10 and LTD11 were all yellow powders.

Medicine preparation: phenylephrine (PE), Sigma USA (batch number P1240000)

Reagent: chloral hydrate, Qingdao sand Kouchi plant (batch No. 20100401);

2. experimental methods

2.1 Effect of LTD and its derivatives on Ex vivo vasodilatory function in rats

2.1.1 preparation and equilibration of vascular rings

Rat isolated Coronary Artery (CA) and cerebral Basilar Artery (BA) vascular rings preparation: rats were sacrificed by intra-abdominal injection of chloral hydrate (3.5mg/kg) anesthesia, and the heart and brain were rapidly removed and placed in ice MOPS-PSS solution. Under a body type dissecting microscope, cardiac Coronary Artery (CA) and cerebral Basilar Artery (BA) vessels are separated by microsurgical instruments, cut into about 1mm arterial rings, and the endothelium-removed and endothelium-removed complete in vitro vessel rings are prepared by a mechanical injury method. Two 40 μ M wires were threaded through the vascular ring and hung in a pair of metal anchors in opposite directions in a DMT bath. 5ml of MOPS-PSS working solution is contained in the bath tank, the temperature is kept at 37 ℃ and O is continuously introduced₂. After the vascular rings were fully placed on DMT, the isolated vessels were preloaded with 1 mN. The MOPS-PSS working solution is changed every 20min, and after balancing for 90min, the MOPS-KPSS working solution (containing 60mM K) is changed⁺) And detecting the activity of the vascular ring. Activity qualification (>0.5mN), and two contraction amplitudes<10% was used for the experiment.

Preparing an isolated thoracic aorta vascular ring of a rat: after the animals are anesthetized and killed, the thoracic aorta is taken out and placed in MOPS-PSS solution at 4 ℃. Under a dissecting microscope, surrounding tissues of blood vessels are removed, the thoracic aorta is cut into 8 sections of blood vessel rings with the length of about 3mm to 5mm, the blood vessels are respectively hung in a bath tank with preset 5ml of MOPS-PSS buffer solution in a looped mode, one end of the blood vessel ring is connected with a Powerlab data recording system through a tension transducer, and the other end of the blood vessel ring is connected with a fine adjustment device for adjusting the preload of the blood vessel rings. At a constant temperature of 37 ℃ and with continuous introduction of 95% O₂And 5% CO₂The base tension of the fine adjustment device was 1.5 g. The MOPS-PSS working solution is changed every 20min, and after balancing for 90min, the MOPS-KPSS working solution (containing 60mM K) is changed⁺) After balancing, the mixture is washed 3 times by MOPS-PSS solution and then by MOPS-KPSS working solution (containing 60mM K)⁺) Preshrinking once, flushing 3 times with MOPS-PSS solution after reaching balance, and repeating the stepsThe activity of the blood vessel and the activity of the blood vessel ring are detected. If the tension value change value reaches twice of the basic tension, the activity is qualified, and the two-time contraction amplitude<10% was used for the experiment.

2.1.2 Ex vivo vascular Ring tensiometry

After isolated CA/BA vessel ring bath was supplemented with U46619 (1. mu.M) for vasoconstriction to reach maximum and maintain equilibrium, LTD and LTD2 were added in cumulative concentrations, respectively, and their effects on vessel ring tone were observed.

PE (1 mu M) is added into an isolated thoracic aorta vessel bath to constrict vessels, after the constriction reaches the maximum value and the vessels are kept in equilibrium, samples with different series of concentrations of LTD2, LTD3, LTD4, LTD6a, LTD7, LTD8, LTD9, LTD10 and LTD11 are added respectively, the vessel tension is recorded, and the dose response is observed.

2.2 data processing

The experimental results are all expressed as (means +/-SE), and data are analyzed and processed by adopting SigmaStat 10.0 statistical analysis software to calculate EC₅₀And drawing a dose-effect curve graph by using SigmaPlut.

3 results of the experiment

3.1 Effect of LTD/LTD2 on Ex vivo CA/BA vessels

LTD has low water solubility, which makes the preparation difficult and the bioavailability low, so that the derivative LTD2 is designed to improve the water solubility and improve or maintain the vasoactivity.

The results are shown in tables 2 and 3, and LTD/LTD2(0.3-100 μ M) was added by the cumulative dose method, and both of them can relax intact endothelium or de-endothelialized CA/BA vessels in isolated rats. LTD2 vasodilated EC on endothelial intact BA vessels₅₀Values significantly lower than LTD; LTD2 vasodilated EC on de-endothelialized (incomplete endothelial) CA/BA vessels₅₀The values are also significantly lower than LTD. The experimental results suggest that LTD2 has higher vasodilatory activity than LTD.

TABLE 2 EC of LTD/LTD2 relaxation of rat endothelial intact vessels BA₅₀And Emax

ANOVA on Ranks,vs LTD2，^*P<0.05

TABLE 3 EC of LTD/LTD2 relaxation of rat de-endothelial CA/BA₅₀And Emax

ANOVA on Ranks,vs LTD2，^＊P<0.05

3.2 Effect of LTD2 and derivatives on isolated rat thoracic aortic vessels

The dose-response curves of LTD2, LTD3, LTD4, LTD6a, LTD7, LTD8, LTD9, LTD10 and LTD11 for dilating isolated thoracic aortic blood vessels of rats are shown in FIG. 13, and the results show that: compared with LTD2 (200-1000 mu M), LTD10 (200-1000 mu M) has the most obvious effect on the isolated thoracic aorta of SD rats; when the cumulative concentration is 200-400 μ M, LTD8, LTD9, and LTD11 all have better activity than LTD2, and can relax thoracic aorta blood vessels and affect the relaxation function of blood vessels. The results show that the compounds LTD2, LTD7, LTD8, LTD9, LTD10 and LTD11 can play a role in the diastolic function of isolated rat thoracic aorta blood vessels, and the effect of LTD10 is more obvious.

Of these derivatives, LTD3 was most water soluble but had little vasoactivity (see table 1, fig. 13), LTD10 was most vasoactive but poor in water solubility (LTD was close), and LTD2 was experimentally demonstrated to be highly water soluble and vasoactive, so LTD2 was subsequently selected as the primary target compound for studies.

Example 9: vascular injury test of LTD2/LTD antagonistic antidiabetic rats

1.1 Experimental materials experimental animals: male diabetic ZDF rats and normal male fa/+ rats (normal controls), with ZDF rats weighing 388 ± 6.95g and fa/+ rats weighing 332 ± 8.67 g. Purchased from Experimental animals technology, Inc. of Weitonglihua, Beijing at 8 weeks of age under license number SCXK (Beijing) 2016-0011. ZDF rats and normal control rats were fed Purina 5008 diet (PMI Nutrition International, Inc.) and the test was started by feeding the rats to 32 weeks of age.

1.2 Experimental methods

1.2.1 Effect on diabetic ZDF rat microcirculation blood flow.

Measuring the blood flow of foot skin of 33-week-old diabetic ZDF rats and fa/+ rats, comparing the blood flow difference between the ZDF rats and observing the VD condition of the foot skin of the ZDF rats. At 36 weeks of age, ZDF rats were gavaged with LTD2 (45mg/kg) for 3 weeks, and the foot skin blood flow was measured and compared to the pre-gavage (33 weeks of age) blood flow values to observe the effect of LTD2 on the foot skin blood flow of ZDF rats.

The specific detection method comprises the following steps: after the ZDF rats were anesthetized by isoflurane inhalation, the left hind limb hairs were removed by depilatory cream, and then the skin blood flow of the left hind limb feet of the ZDF and fa/+ rats was measured by laser speckle blood flow imager. And uniformly selecting two parts in the middle of the middle toe and the instep as observation and data acquisition parts.

1.2.2 Effect on vasomotor function in diabetic ZDF rats

Starting from 33 weeks old, the experimental rats adopt a microvascular tonometry system to detect the endothelium-dependent relaxation response and the endothelium-independent relaxation response of the ZDF and fa/+ rats; the test drugs LTD2, LTD gavage were administered to ZDF rats, gavage was started at 36 weeks of age, and ex vivo vascular tone was measured 3 weeks later (39 weeks).

The specific detection method comprises the following steps:

after blood sampling, the rat heart was rapidly removed and placed in an ice MOPS-PSS solution. Under a dissecting microscope, CA was isolated with microsurgical instruments, cut into approximately 1mm arterial loops, and two 40 μ M wires were passed through the vascular loops and respectively hung on a pair of metal anchors in opposite directions in a DMT bath. 5ml of MOPS-PSS working solution is contained in the bath tank, the temperature is kept at 37 ℃ and O is continuously introduced₂. CA 1mN was preloaded after the vascular rings were all placed on DMT. The MOPS-PSS working solution is changed every 20min, and after balancing for 90min, the MOPS-KPSS working solution (containing 60mM K) is changed⁺) And detecting the activity of the vascular ring. Activity qualification (>0.5mN), and two contraction amplitudes<10% was used for the experiment.

(1) LTD2 effect of in vitro incubation of ZDF rat isolated vessels on VD: after the blood vessel is balanced and the activity is detected to be qualified, adding LTD2(10 mu M, 30 mu M) to pre-incubate the CA blood vessel ring for 30min, and adding U46619 (final concentration of 1 mu M) or 5ml60mM K⁺And (3) contracting the blood vessel, adding Ach or SNP cumulative concentration (0.001-100 mu M) after the contraction reaches the maximum value and keeps balance, and observing the dose-response curve of the dependent and independent relaxation reaction of the in vitro vascular endothelium.

(2) Effect of intragastric administration of LTD2 on in vitro vascular VD in ZDF rats: animals were sacrificed 3 weeks after continuous gavage (45mg/kg, ig) with LTD2 to prepare ZDF rats isolated vascular CA, and isolated vascular endothelium-dependent and independent vasodilation responses were observed by adding cumulative concentrations of Ach and SNP.

1.2.3 Effect of LTD2 on ZDF rat Kidney tissue injury and glomerulosclerosis

Rats were sacrificed after drug treatment and thoracic aorta and left kidney tissue were fixed in 4% paraformaldehyde solution. Dehydrating, embedding in paraffin, slicing, HE staining, and finally placing the slices under a microscope to observe pathological changes of thoracic aorta and kidney tissues.

1.2.4 data and statistical processing

The experimental results are all expressed as (means ± SE), and the data were statistically analyzed using the SigmaStat 10.0 statistical analysis software. The median Effective Concentration (EC) was calculated by simulation using SigmaPlut mapping₅₀). Blood flow value, EC₅₀Data which conform to normal distribution and are uniform in variance, Emax, etc. are subjected to a t-Test (t-Test), one-way ANOVA (one-way ANOVA), and a Rank-Sum Test (Mann-Whitney Rank Sum Test) for non-normal distribution data. The concentration response curves were analyzed by two-way analysis of variance (two-way ANOVA). P<A difference of 0.05 is statistically significant.

1.3 results of the experiment

1.3.0 the effect of LTD/LTD2 on blood sugar, blood lipid and liver and kidney function index of ZDF rat.

By adopting a blood biochemical detection method and checking relevant indexes such as blood sugar, blood fat, liver and kidney functions and the like of animals in groups, the results prove that after the administration of LTD/LTD2 through stomach irrigation (ig,45mg/kg) for 3 weeks, the blood sugar value of the animals is not obviously influenced, the blood fat is reduced, and the liver and kidney function indexes of the animals are not influenced (as shown in Table 4).

TABLE 4 influence of LTD/LTD2 on blood glucose and blood lipid of ZDF rats (means. + -. SE)

1.3.1 Effect of LTD/LTD2 on the microcirculation blood flow of ZDF rats.

The results of the experiment are shown in figures 14 and 15 and table 5: the blood flow of the foot skin of the ZDF rat is obviously lower than that of the normal control rat (P <0.01), which indicates that the blood flow of the foot skin of the ZDF rat is reduced and indicates that the skin blood vessel of the ZDF rat is damaged. After the LTD2 is administrated by gastric lavage, the blood flow of the foot skin of the ZDF rat is obviously increased, which indicates that the LTD2 has the function of improving the blood flow.

TABLE 5 Effect of LTD/LTD2 on the microcirculation blood flow of ZDF rats

Mann-Whitney Rank Sum Test,ZDF vs fa+，^*P＜0.05，^**P＜0.01

1.3.2 Effect of LTD2/LTD on vasomotor function in ZDF rats

(1) Effect of LTD2/LTD in vitro incubation on VD of isolated blood vessels of ZDF rats

The results are shown in tables 6 and 7 and FIG. 16. Compared with normal rat Control (fa/+), the Model group Model diabetes ZDF rat coronary artery CA blood vessel relaxation response is obviously weakened, including Ach induced vascular endothelium-dependent relaxation response and SNP induced non-endothelium-dependent relaxation response, the Model group ZDF rat SNP relaxation response is weaker, and the EC of the Model group ZDF rat is higher₅₀The values can not be detected, and the diabetic ZDF rat is proved to have obvious vascular endothelial and non-endothelial function damage.

After the in vitro incubation of the LTD2/LTD (10 mu M) on the blood vessel CA, the blood vessel CA has different degrees of enhancement effects on the endothelial dependent and independent vasodilation responses induced by Ach/SNP, particularly improves the endothelial relaxation response induced by Ach, and proves that the in vitro incubation of the LTD2/LTD can antagonize the vascular injury of the rat with diabetes mellitus ZDF, wherein the activity of LTD2 is stronger.

TABLE 6 Effect of LTD2/LTD (10. mu.M) incubation in vitro on Ach relaxation of isolated vascular CA in ZDF rats (means. + -. SE)

ANOVA on Ranks,Student-Newman-Keuls Method,^**P<0.01，^***P<0.001vs Model(ZDF)

TABLE 7 Effect of LTD2/LTD (10. mu.M) in vitro incubation on SNP relaxation of isolated vascular CA in ZDF rats (means. + -. SE)

One Way ANOVA，^***P<0.001vs Model(ZDF).

(2) Effect of stomach administration of LTD2 on VD in vitro blood vessels of ZDF rats

The results are shown in table 8 and fig. 17. Compared with normal rat Control (fa/+), the Model group Model diabetes ZDF rat has obviously weakened endothelium-dependent relaxation response induced by Ach of coronary artery CA blood vessel and EC thereof₅₀The value can not be detected, and the diabetic ZDF rat is proved to have obvious vascular endothelial function damage.

After administration of LTD2/LTD by gavage (ig.45mg/kg) to rats for 3 weeks, the vascular CA vessels enhanced to varying degrees the Ach-induced endothelium-dependent vasodilation response, demonstrating that administration of LTD2/LTD by gavage antagonizes vascular damage in antidiabetic ZDF rats.

TABLE 8 Effect of intragastric administration of LTD2 on Ach relaxation of isolated vascular CA in ZDF rats (means. + -. SE)

ANOVA on Ranks,Dunn′s method,^*P<0.05，^***P<0.001vs Model(ZDF)

1.3.3 Effect of LTD2 on ZDF rat Kidney tissue injury and glomerulosclerosis

The results are shown in FIGS. 18-19, where ZDF rat kidney tissue volume was increased, glomerular capillary walls were thickened, and nodular vitreous deposits were found in the mesangium, compared to fa/+ rat kidney tissue. LTD2 can obviously improve the glomerulosclerosis lesion of ZDF rats and improve the vascular lesion in renal tissues. The HE stained section also shows that the thoracic aorta of the ZDF rat is homogenized, red stained and transparent denatured like ground glass, which indicates that the thoracic aorta of the ZDF rat is arteriosclerosis and that the endothelial cells of the thoracic aorta of the ZDF rat have no obvious structural change. LTD2 significantly improved the vitreogenesis of the thoracic aorta. And (4) prompting: LTD2 has improving effect on diabetic ZDF rat renal tissue injury and glomerulosclerosis.

And (4) conclusion: in the rats with diabetes and hyperlipemia ZDF, obvious vascular injury conditions appear, including microvascular blood flow reduction, vasodilation function weakening, glomerular angiosclerosis and the like, while LTD2 obviously improves the vasomotor function of ZDF rats, improves the functions of vascular endothelium and smooth muscle, increases the blood flow of the foot skin of rats, improves the vascular injury of renal tissues and the glomerular angiosclerosis and the like, has protective effect on the vascular injury induced by diabetes, and can be used for preparing medicines for preventing and treating diabetic vascular complications.

Example 10: LTD2 antagonistic rat myocardial ischemia injury experiment

1.1 Experimental materials

Experimental animals, grouping: healthy SD rats (Experimental animal center of Kunming medical university, license number: SYXK (Dian) 2005-.

1.2 Experimental methods

The animals were divided into 6 treatment groups at random, and SD rats were divided into 6 treatment groups at random, i.e., IR Sham (Sham), Model (Model), low (12.5mg/kg, i.g.) and high (50mg/kg, i.g.) dose groups of LTD2, each group being n-10.

Dosing and modeling: samples were weighed at high (50mg/kg, i.g.) and low (12.5mg/kg, i.g.) doses and SD rat body weight, respectively, and dissolved in 5% glucose injection. The model group was given 2 mL/mouse of 5% glucose injection. The administration is performed by intragastric administration (2ml/100g) three days before operation, 5% glucose is administered in the same way in a model group and a sham operation group, once a day, once again in the fourth day, and the operation is performed immediately thereafter. The specific method comprises the following steps: the Left Anterior Descending (LAD) heart of the rat was ligated, and 40min later the ligation was cut off and the perfusion was performed for 120min, resulting in an IR model.

Measurement of ischemic area: after the molding is finished, the thoracic cavity is opened, the heart is taken out, the heart is placed in precooled normal saline to wash away bloodstains, and the atrium, the right ventricle and fascia tissues are removed. The heart was placed in a refrigerator at-20 ℃ for 5min and cut evenly into slices with a thickness of 1mm along the cross section with a blade. Placing the heart slices into 1% TTC solution preheated at 37 deg.C, placing in a water bath constant temperature oscillator, oscillating and dyeing at 37 deg.C for 10-15min, fixing with 4% formaldehyde solution, and observing the dyeing result to obtain red non-infarcted myocardial tissue and pale ischemic necrotic myocardial tissue. The stained heart sections were placed in saline, a ruler was set with a ruler, and then a photograph was taken, and the ischemic area was calculated with Image-Pro Plus (IPP) Image processing software, and the percentage of the myocardial ischemic area in the ventricular area was calculated.

Data and statistical analysis: the experimental results are all as follows

Show that two-by-two comparisons between groups were performed using the student-Newman-keuls (snk) test of one-way ANOVA with a 0.05 test level in the SigmaStat 10.0 statistical analysis software. The generation of the correlation pattern was performed by using SigmaPlot 10.0 pattern generation software.

1.3 results of the experiment

The ischemic area percentage results are shown in table 9 and fig. 20 and 21, and compared among the values of each group, single-factor analysis of variance (Student-Newman Keuls test) is adopted, so that compared with the model group, the ischemic area percentage of the LTD2 low and high dose (12.5 and 50mg/kg) treatment group is obviously reduced (P <0.01 and P <0.001), which indicates that LTD2 has obvious protective effect on rat cardiac IR injury through intragastric administration, and the ischemic area percentage of the Lab03 low and high dose (12.5 and 50mg/kg) treatment group is obviously reduced (P <0.001 and P <0.01) compared with the model group, which indicates that Lab03 has obvious protective effect on rat cardiac IR injury through intragastric administration. Compared with the LTD2 low dose (12.5mg/kg), the ratio of myocardial ischemia area after rat myocardial ischemia reperfusion is obviously reduced by Lab03 low dose (12.5mg/kg) treatment group (P is less than 0.05), and the effect of Lab03(12.5mg/kg) after intragastric administration is better than that of LTD2(12.5 mg/kg).

TABLE 9 protective Effect of LTD2 on myocardial ischemia reperfusion in rats

And (4) conclusion: the LTD2 has obvious protective effect on the IR injury of the myocardium of rats at low and high doses. Can be used for preparing medicine for preventing and treating coronary heart disease and angina pectoris.

Example 11: experiment for LTD2 antagonizing cerebral ischemic injury of rat

1.1 Experimental materials

Experimental animals, grouping: healthy SD rats (Experimental animal center of Kunming medical university, license number: SYXK (Dian) 2005-.

1.2 Experimental methods

The animals were divided into 6 treatment groups at random, and SD rats were divided into 6 treatment groups at random, i.e., IR Sham (Sham), Model (Model), low (12.5mg/kg, i.g.) and high (50mg/kg, i.g.) dose groups of LTD2, each group being n-10.

Dosing and modeling: samples were weighed at high (50mg/kg, i.g.) and low (12.5mg/kg, i.g.) doses and SD rat body weight, respectively, and dissolved in 5% glucose injection. The model group was given 2 mL/mouse of 5% glucose injection. The medicine is administrated 4 days before operation, and is administrated by gastric lavage, 2 mL/patient. A rat cerebral central artery ischemia (MCAO)/reperfusion model was established by methods referred to Longa and Shen, etc. Reperfusion was performed after 1h of embolization, and right MCAO was performed for each animal.

Observation indexes are as follows: and (3) after modeling, injecting 10% chloral hydrate into the abdominal cavity for anesthesia, cutting the head to take out the brain, freezing the brain in a low-temperature refrigerator at the temperature of-20 ℃, taking out the brain after about 15min, uniformly cutting the brain into 6 pieces from the head to the tail, putting the brain pieces into 1% TTC solution, carrying out constant-temperature oscillation dyeing on the brain in water bath at the temperature of 37 ℃ for 10min, and then fixing the brain pieces in 4% paraformaldehyde solution for 1-2 h. The observation indexes include the sign score of the nerve functional deficiency, the ischemic area and the corrected ischemic volume.

Data and statistical analysis: the brain picture was processed with Image-Pro Plus17.0 Image analysis software to calculate the ischemic area and correct the ischemic volume. Results of the experiment to

Data were analyzed using Sigma Stat 10.0 statistical analysis software. Single-way ANOVA (one-way ANOVA) is adopted among multiple groups of indexes, and t-test is adopted between two groups.

1.3 results of the experiment

(1) Effect of LTD2 on corrected ischemic volume percentage of IR rats:

compared with the Model group (Model), the LTD2 high dose group had a significant reduction in the corrected ischemic volume percentage (P < 0.05); the results are shown in Table 10 and FIGS. 22 and 23.

TABLE 10 corrected ischemic volume percent

(2) Effect of LTD2 on the percentage of ischemic area in IR rats:

compared with the Model group (Model), the percentage of ischemic area in the LTD2 high dose group was significantly reduced (P < 0.05); the results are shown in Table 11 and FIG. 24.

TABLE 11 percent ischemic area

(3) Effect of LTD2 on neural function of IR rats:

compared with the Model group (Model), the nerve function of the LTD2 high-dose group is obviously improved (P < 0.05); the results are shown in Table 12 and FIG. 25.

TABLE 12 neurobehavioral scores

Results and conclusions: LTD2 was high (50mg/kg, i.g.) with a significant reduction in the percent ischemic volume, percent ischemic area, and neurological score in the treated groups compared to the model group. LTD2 has obvious protective effect on IR injury of rat brain, and can be used for preparing medicine for preventing and treating cerebral apoplexy and cerebral ischemia.

Claims (7)

1. A3' -aminoalkoxy-luteolin derivative and pharmaceutically-formable salts thereof are characterized by having the following structure:

2. 3' -aminoalkoxy-luteolin derivatives and pharmaceutically acceptable salts thereof according to claim 1, wherein the pharmaceutically acceptable salts are inorganic or organic acid salts.

3. 3' -aminoalkoxy-luteolin derivatives and pharmaceutically acceptable salts thereof according to claim 2, characterized in that the inorganic or organic acid salts are selected from sulfate, phosphate, hydrochloride, hydrobromide, acetate, oxalate, citrate, succinate, gluconate, tartrate, p-toluenesulfonate, benzenesulfonate, methanesulfonate, benzoate, lactate or maleate.

4. A process for producing a3 '-aminoalkoxy-luteolin derivative and pharmaceutically acceptable salts thereof according to any one of claims 1 to 3, wherein luteolin and an active substance having a benzyl group are used as raw materials and reacted under an alkaline condition to obtain a luteolin dibenzyl substituent, an active group having an amino group is introduced into the 3' -hydroxyl group of the luteolin dibenzyl compound, and the protecting group is removed from the benzyl group to produce the luteolin derivative, which comprises the following steps:

said R represents- (CH)₂)₂N(CH₃)₂、-(CH₂)₂NH₂、-(CH₂)₃N(CH₃)₂、

5. Use of the luteolin derivative and pharmaceutically acceptable salts thereof according to any one of claims 1-3 in the preparation of a medicament for the prevention and treatment of ischemic injury.

6. Use of the luteolin derivative and pharmaceutically acceptable salts thereof according to any one of claims 1-3 in the preparation of medicaments for preventing and treating diabetic vascular complications and ischemic cardiovascular and cerebrovascular diseases.

7. The use according to claim 6, characterized in that the diabetic vascular complications comprise diabetic nephropathy, diabetic retinopathy, diabetic feet, and the ischemic cardiovascular and cerebrovascular diseases comprise myocardial ischemia injury and cerebral ischemia injury.

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