CN114796214B - Application of DNJ and derivatives thereof in preparation of medicines for preventing and/or treating pulmonary arterial hypertension - Google Patents

Application of DNJ and derivatives thereof in preparation of medicines for preventing and/or treating pulmonary arterial hypertension Download PDF

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CN114796214B
CN114796214B CN202210433992.1A CN202210433992A CN114796214B CN 114796214 B CN114796214 B CN 114796214B CN 202210433992 A CN202210433992 A CN 202210433992A CN 114796214 B CN114796214 B CN 114796214B
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CN114796214A (en
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李晓晖
邓旭
殷明慧
冯雪祥
王森林
李莹
周应军
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Central South University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

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Abstract

The invention discloses an application of DNJ and a derivative thereof in preparing medicines for preventing and/or treating pulmonary arterial hypertension, wherein the DNJ and the derivative thereof up-regulate the levels of BMPR2, p-smad 1/5/8 and ID proteins by inhibiting lysosomes from degrading BMPR2, reduce pulmonary vascular injury, play a role in treating PAH, provide a new thought for treating PAH, and expand the application fields of DNJ and the derivative thereof.

Description

Application of DNJ and derivatives thereof in preparation of medicines for preventing and/or treating pulmonary arterial hypertension
Technical Field
The invention relates to the technical field of medicines, in particular to application of DNJ and derivatives thereof in preparing medicines for preventing and/or treating pulmonary arterial hypertension.
Background
Pulmonary arterial hypertension (pulmonary arterial hypertension, PAH) is a type of disease characterized by a sustained increase in pulmonary arterial pressure, which ultimately leads to right heart hypertrophy, heart failure and even death. There is no specific therapeutic drug clinically at present, the prognosis of patients is extremely poor, and the annual mortality rate of patients is still up to 15% under the condition of receiving treatment. Vascular endothelium is a barrier that maintains normal structure and function of blood vessels, protecting blood vessels from damage by dangerous factors. Pulmonary artery endothelial injury (endothelial injury) is a key pathological mechanism that induces pulmonary artery hyperbaric vascular lesions, where excessive apoptosis, migration of pulmonary artery endothelial cells (Pulmonary arterial endothelial cells, PAECs) can cause an increase in endothelial permeability, resulting in disruption of its barrier function, which is the initiating link for pulmonary vascular injury.
The bone morphogenic protein type 2 receptor (bone morphogenetic protein receptor type, bmpr 2) is a serine/threonine kinase receptor that is expressed primarily in pulmonary artery endothelial cells. Several studies show that various pathogenic factors such as BMPR2 gene mutation, inflammation, oxidative stress and the like can lead to significant reduction of BMPR2 expression, and cause down regulation of BMPR2 signaling pathway, so that the BMPR2 is one of key pathological mechanisms for inducing pulmonary arterial hypertension. It has been found that lysosomal degradation is a key factor in the loss of endothelial BMPR 2.
Lysosomes (lysosomes) are an organelle in eukaryotic cells that contain various hydrolytic enzymes for the breakdown of various exogenous and endogenous macromolecular substances and play an important role in the degradation and recovery of cellular waste, cell signaling, and energy metabolism. Lysosomes are relatively closely related to human diseases, and lysosomal abnormalities can cause a variety of diseases.
DNJ is a natural alkaloid found in mulberry. The Chinese name is 1-deoxynojirimycin and the molecular weight is 163.7.DNJ is taken as an alpha glycosidase inhibitor, can obviously delay the degradation process of polysaccharide, has research directions of reducing blood sugar, resisting virus and tumor metastasis, does not damage liver and kidney health, and has no gastrointestinal side effect. Based on DNJ, two commercially available drugs have been developed: miglitol for the treatment of gaucher's disease, a lysosomal storage disease, and miglitol for the treatment of type 2 diabetes. Wherein, the Gaucher disease of lysosomal storage disease is also called cerebroside deposition disease, which is an autosomal recessive genetic disease caused by mutation of acid beta-glucocerebrosidase gene, and the enzyme activity after mutation is still partially maintained, but most of enzyme is subjected to endoplasmic gateway joint degradation and can not be operated to lysosome due to abnormal structure, so that the substrate glucocerebroside can not be normally degraded, but is stored in the lysosome. However, in the related art, there is no application of DNJ in medicines for preventing and/or treating pulmonary hypertension, and there is no research on related mechanisms of DNJ in medicines for preventing and/or treating pulmonary hypertension.
Based on the background, an application of DNJ and a derivative thereof in preparing medicaments for preventing and/or treating pulmonary arterial hypertension is developed.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: the application of DNJ and its derivatives in preparing medicines for preventing and/or treating pulmonary arterial hypertension is provided.
In order to solve the technical problems, the technical scheme provided by the invention is as follows: the application of DNJ and its derivatives in preparing medicines for preventing and/or treating pulmonary arterial hypertension is provided.
According to some embodiments of the invention, the DNJ and its derivatives comprise at least one of a compound of general formula (I):
wherein R is selected from one of H, hydrocarbyl, hydroxyalkyl and alkoxy;
R 1 is selected from one of H or hydroxyl.
In the general formula (I), 1 to 6 refer to carbon positions.
According to some embodiments of the invention, the stereochemical combination of the substituents at the C2, C3, C4, C5 positions in formula (I) is selected from one of (2 r,3s,4r,5 r), (2 r,3s,4r,5 s), (2 r,3s,4s,5 r), (2 r,3r,4r,5 s), (2 r,3s,4s,5 s).
DNJ has a function of stabilizing lysosomes, and DNJ up-regulates BMPR2 expression, so that the pulmonary arterial hypertension process is relieved.
BMPR2 plays an important role in PAH occurrence and development, and the process of lysosome degradation of BMPR2 is interfered, so that PAH treatment is realized.
According to some embodiments of the invention, the hydrocarbyl group is selected from alkyl, alkenyl, aryl, or substituted aryl.
According to some embodiments of the invention, the alkyl group comprises a C 1～C12 alkyl group.
According to some embodiments of the invention, the alkyl group comprises at least one of propyl, n-butyl, n-nonyl, and adamantyl.
According to some embodiments of the invention, the alkenyl group comprises a C 1～C12 alkenyl group.
According to some embodiments of the invention, the alkenyl group comprises at least one of vinyl, propenyl, allyl.
According to some embodiments of the invention, the alkoxy group comprises an alkoxy group comprising 3 to 30 atoms.
According to some embodiments of the invention, the alkoxy group comprises at least one of ethylene glycol group, di-polyethylene glycol group, tri-polyethylene glycol group.
According to some embodiments of the invention, the hydroxyalkyl group comprises a hydroxyalkyl group of 3 to 30 atoms.
According to some embodiments of the invention, the hydroxyalkyl group comprises at least one of hydroxymethyl, hydroxyethyl, hydroxypropyl, and hydroxybutyl.
According to some embodiments of the invention, the aryl group comprises an aryl group containing 6 to 30 atoms.
According to some embodiments of the invention, the aryl group comprises at least one of benzyl, naphthyl, and biphenyl.
According to some embodiments of the invention, the substituted aryl group comprises a substituted aryl group containing 6 to 30 atoms.
According to some embodiments of the invention, the substituted aryl group comprises at least one of phenethyl, phenylpropyl, 4-methoxyphenylpropyl.
According to some embodiments of the invention, the DNJ and its derivatives comprise at least one of the compounds DL-1 to DL-15 shown in the following formula:
According to some embodiments of the invention, the pharmaceutical preparation further comprises a pharmaceutically acceptable carrier.
According to some embodiments of the invention, the pharmaceutically acceptable carrier is a pharmaceutical carrier conventional in the pharmaceutical arts.
According to some embodiments of the invention, the pharmaceutically acceptable carrier comprises at least one of diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, sweeteners and flavoring agents.
According to some embodiments of the invention, the excipient comprises water.
According to some embodiments of the invention, the filler comprises at least one of starch and sucrose.
According to some embodiments of the invention, the binder comprises at least one of cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone.
According to some embodiments of the invention, the humectant comprises glycerin.
According to some embodiments of the invention, the disintegrant comprises at least one of agar, calcium carbonate, and sodium bicarbonate.
According to some embodiments of the invention, the absorption enhancer comprises a quaternary ammonium compound.
According to some embodiments of the invention, the surfactant comprises cetyl alcohol.
According to some embodiments of the invention, the adsorption carrier comprises at least one of kaolin clay and soap clay.
According to some embodiments of the invention, the lubricant comprises at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.
According to some embodiments of the invention, the pharmacologically acceptable salts of the present invention include salts with inorganic acids, organic acids, alkali metals, alkaline earth metals and basic amino acids.
According to some embodiments of the invention, the inorganic acid comprises at least one of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, hydrobromic acid.
According to some embodiments of the invention, the organic acid comprises at least one of maleic acid, fumaric acid, tartaric acid, lactic acid, citric acid, acetic acid, methanesulfonic acid, p-toluenesulfonic acid, adipic acid, palmitic acid, and tannic acid.
According to some embodiments of the invention, the alkali metal comprises at least one of lithium, sodium and potassium.
According to some embodiments of the invention, the alkaline earth metal comprises at least one of calcium and magnesium.
According to some embodiments of the invention, the basic amino acid comprises lysine.
According to some embodiments of the present invention, the pharmaceutical dosage form is various dosage forms conventional in the art, preferably solid, semi-solid or liquid form, and may be an aqueous solution, non-aqueous solution or suspension, more preferably a tablet, capsule, soft capsule, granule, pill, oral liquid, dry suspension, drop pill, dry extract, injection or infusion. According to some embodiments of the invention, the mode of administration of the drug may be conventional in the art, including but not limited to injection or oral administration. The injection administration can be intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.
According to some embodiments of the present invention, the preparation method of the DNJ and the derivative thereof comprises the following steps:
s1, reduction:
Adding the compound 1 into a reducing agent and an organic solvent I, controlling the temperature for reaction, carrying out solid-liquid separation, and collecting a solid phase to obtain a compound 2;
s2, swern oxidation/reduction amination:
S21, adding the compound 2 into acyl halide and an organic solvent II, controlling the temperature for reaction, and then adding organic amine for reaction to prepare an intermediate solution;
s22, adding the intermediate solution into a boron reducer, ammonium salt and an organic solvent III for reaction, carrying out solid-liquid separation, and collecting a solid phase to obtain a compound 3;
s3, reductive amination:
adding the compound 3 into aldehyde, a palladium catalyst I, organic carboxylic acid and an organic solvent IV for reaction to prepare a compound 4;
s4, catalytic hydrogenation debenzylation:
And adding the compound 4 into an ether solvent, and then adding inorganic acid and a palladium catalyst II to react to obtain DNJ or DNJ derivatives.
According to some embodiments of the invention, the reducing agent comprises lithium aluminum hydride.
According to some embodiments of the invention, the organic solvent i comprises Tetrahydrofuran (THF).
According to some embodiments of the invention, the molar ratio of the reducing agent to compound 1 is 1:3 to 4.
According to some embodiments of the invention, the reaction temperature in step S1 is-5 ℃ to 5 ℃.
According to some embodiments of the invention, the acid halide comprises an acid chloride.
According to some embodiments of the invention, the acid chloride retains oxalic acid chloride.
According to some embodiments of the invention, the organic solvent ii comprises at least one of halogenated hydrocarbon and sulfone solvents.
According to some embodiments of the invention, the halogenated hydrocarbon comprises Dichloromethane (DCM).
According to some embodiments of the invention, the sulfone-based solvent comprises dimethyl sulfoxide (DMSO).
According to some embodiments of the invention, the organic amine comprises at least one of Triethanolamine (TEA) and diethanolamine.
According to some embodiments of the invention, the molar ratio of compound 2, acyl halide and organic amine is 1:1.5 to 2.5:2.5 to 3.5.
According to some embodiments of the invention, the boron reducing agent comprises NaBH 3 CN.
According to some embodiments of the invention, the ammonium salt is at least one of ammonium acetate and ammonium formate.
According to some embodiments of the invention, the molar ratio of the ammonium salt to the boron reducing agent is 1:1.9 to 2.1.
According to some embodiments of the invention, the organic solvent III comprises an alcoholic solvent I.
According to some embodiments of the invention, the alcoholic solvent i comprises at least one of methanol, ethanol and propanol.
According to some embodiments of the invention, the reaction temperature in step S21 is-90 ℃ to-70 ℃.
According to some embodiments of the invention, the reaction temperature in step S22 is 20 ℃ to 25 ℃.
According to some embodiments of the invention, the aldehyde comprises at least one of an alkyl aldehyde, an aryl aldehyde, and an alkoxy aldehyde.
According to some embodiments of the invention, the molar ratio of aldehyde to compound 3 is 2:3-4.
According to some embodiments of the invention, the palladium catalyst i comprises a palladium on carbon catalyst.
According to some embodiments of the invention, the organic carboxylic acid comprises at least one of formic acid, acetic acid, and propionic acid.
According to some embodiments of the invention, the organic solvent iv comprises an alcoholic solvent ii.
According to some embodiments of the invention, the alcoholic solvent ii comprises at least one of methanol, ethanol and propanol.
According to some embodiments of the invention, the ether-based solvent comprises at least one of methyl ether, diethyl ether, and methyl tert-butyl ether (MTBE).
According to some embodiments of the invention, the inorganic acid comprises at least one of sulfuric acid, hydrogen chloride, and hydrogen bromide.
According to some embodiments of the invention, the molar ratio of the mineral acid to compound 4 is 1:5 to 10.
According to some embodiments of the invention, the palladium catalyst ii comprises a palladium on carbon catalyst.
The invention also provides another technical scheme as follows: a medicament for preventing and/or treating pulmonary hypertension, the active ingredient of the medicament comprising DNJ and a derivative thereof.
According to some embodiments of the invention, the mass content of DNJ and its derivatives in the medicament for preventing and/or treating pulmonary hypertension is 0.1% -99%.
According to some embodiments of the invention, the mass content of DNJ and its derivatives in the medicament for preventing and/or treating pulmonary hypertension is 0.5-95%.
The invention also provides another technical scheme as follows: a pulmonary hypertension treatment kit comprising the medicament described above.
According to some embodiments of the invention, the administration amount of the drug for preventing and/or treating pulmonary arterial hypertension is as follows: DNJ and its derivatives are 0.1 mg/day to 1000 mg/day.
The term "administered dose" as used herein is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. May depend on the particular disease being treated, as well as other factors including age, weight, health, severity of symptoms, route of administration, frequency of treatment, and whether additional medications are concomitantly used during the treatment.
The term "treatment" as used herein refers to reducing the extent of colorectal cancer and its complications, or curing colorectal cancer and its complications to normalize it, or slowing the progression of colorectal cancer and its complications.
The application of the medicine prepared according to the embodiment of the invention has at least the following beneficial effects: DNJ and its derivatives can inhibit lysosome degradation BMPR2, improve pulmonary vascular injury, and treat PAH. The invention not only provides a new way for treating PAH, but also expands the application range of DNJ and derivatives thereof.
Drawings
FIG. 1 is a graph showing the right ventricular systolic pressure of the DL-1 group (different concentrations), the hypoxia group and the control group according to the present invention.
FIG. 2 is a graph showing the ratio of the mass of the right ventricle to the total mass of the left ventricle and the upper chamber interval of the DL-1 group (different concentrations), the hypoxia group and the control group in comparison with the embodiment of the present invention.
FIG. 3 is a cross-sectional view of the treated pulmonary artery HE after treatment with DL-1 group (at various concentrations), hypoxia group and control group in accordance with the present invention.
FIG. 4 is a graph showing the comparison of the cell viability of the DL-1 group (different concentrations) and the control group in the example of the present invention.
FIG. 5 is a graph showing the comparison of the results of the scratch test after treatment of the DL-1 group and the control group (magnification: 20 times) in the example of the present invention.
FIG. 6 is a graph showing mobility comparisons between DL-1 groups (at different concentrations) and control groups in accordance with an embodiment of the present invention.
FIG. 7 is a graph showing the results of gel electrophoresis of different proteins in the DL-1 group (different concentrations) and the control group in the example of the present invention.
FIG. 8 is a graph showing comparison of the expression levels of BMPR2 proteins in the DL-1 group (different concentrations) and the control group in the example of the present invention.
FIG. 9 is a graph showing comparison of the expression levels of p-Smad1/5/8 proteins in the DL-1 group (different concentrations) and the control group in the example of the present invention.
FIG. 10 is a graph showing comparison of the expression level of the ID protein of the DL-1 group (different concentrations) and the control group in the example of the present invention.
FIG. 11 is a graph showing the results of gel electrophoresis of different proteins in the DL-1 group (different concentrations), the hypoxia group and the control group (same conditions were used) in the example of the present invention.
FIG. 12 is a graph showing comparison of the expression levels of BMPR2 proteins in the DL-1 group (different concentrations), the hypoxia group and the control group according to the embodiment of the present invention.
FIG. 13 is a graph showing comparison of the expression levels of p-Smad1/5/8 proteins in the DL-1 group (different concentrations), the hypoxia group and the control group in the examples of the present invention.
FIG. 14 is a graph showing comparison of the levels of ID protein expression in the DL-1 group (different concentrations), the hypoxia group and the control group in the example of the present invention.
FIG. 15 is a graph showing the results of gel electrophoresis of different proteins in the DL-2 group (different concentrations) and the control group in the example of the present invention.
FIG. 16 is a graph showing comparison between the expression levels of BMPR2 proteins in the DL-2 group (different concentrations) and the control group in the example of the present invention.
FIG. 17 is a graph showing comparison of the expression levels of p-Smad1/5/8 proteins in the DL-2 group (different concentrations) and the control group in the example of the present invention.
FIG. 18 is a graph showing comparison of the levels of expression of ID proteins in the DL-2 group (different concentrations) and the control group in the example of the present invention.
FIG. 19 is a graph showing cell viability of DL-3 group (at different concentrations), tnf group and control group in accordance with the present invention.
FIG. 20 is a graph showing the comparison of the cell viability of DL-3 groups (at different concentrations) and control groups in accordance with the present invention.
FIG. 21 is a graph showing cell viability of DL-4 (at different concentrations), tnf and control groups according to the present invention.
FIG. 22 is a graph showing the comparison of the cell viability of the DL-4 group (at different concentrations) and the control group in the example of the present invention.
FIG. 23 is a graph showing cell viability of DL-5 (at different concentrations), tnf and control groups according to the present invention.
FIG. 24 is a graph showing cell viability of DL-5 groups (at different concentrations) compared with that of a control group in the example of the present invention.
FIG. 25 is a graph showing cell viability of DL-9 group (at different concentrations), control group and hypoxia group in accordance with the present invention.
FIG. 26 is a graph showing cell viability of DL-10 groups (at different concentrations), control groups and hypoxia groups in accordance with an embodiment of the present invention.
FIG. 27 is a graph showing cell viability of DL-11 group (at different concentrations), control group and hypoxia group in accordance with the present invention.
FIG. 28 is a graph showing cell viability of DL-12 groups (at different concentrations), control groups and hypoxia groups in accordance with an embodiment of the present invention.
FIG. 29 is a graph showing cell viability of DL-13 groups (at different concentrations), control groups and hypoxia groups in accordance with an embodiment of the present invention.
FIG. 30 is a graph showing cell viability of DL-14 groups (at different concentrations), control groups and hypoxia groups in accordance with the present invention.
FIG. 31 is a graph showing cell viability of DL-15 groups (at different concentrations), control groups and hypoxia groups in accordance with the present invention.
Marking:
*：P<0.05；**：P<0.01；***：P<0.001。
#：P<0.05；##：P<0.01；###：P<0.001。
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
For a better understanding of the essence of the present invention, the following description will further explain the essence of the present invention by referring to the drawings, but the present invention is not limited thereto. Modifications of the invention in accordance with the essence of the invention are within the scope of the invention.
The first embodiment of the invention is as follows: the preparation method of DNJ is shown in the following formula, and comprises the following steps:
s1, reduction
To 25mL of THF (tetrahydrofuran) suspension of lithium aluminum hydride (LAH, 2.28g,60 mmol) at 0 ℃ in ice bath, 50mL of THF solution of compound 1 (10.82 g,20 mmol) was slowly added dropwise, TLC monitored the progress of the reaction, after completion of the reaction, sodium sulfate decahydrate was added to quench LAH, followed by celite addition to assist filtration, spin-drying of the filtrate, and flash column chromatography purification (DCM: meoh=100:1) to give milky product compound 2 (10.4 g, yield 95%).
S2, swern oxidation/reduction amination
To a solution of (COCl) 2 (10.24 mL,120 mmoL) in anhydrous DCM (dichloromethane, 50 mL) was added dropwise a solution of DMSO (dimethyl sulfoxide, 11.35mL,160 mmoL) in DCM (10 mL) at-78deg.C, and after the addition was stirred for half an hour, a solution of compound 2 in DCM (50 mL) was added dropwise thereto, after 2 hours TEA (triethanolamine, 27.67mL,200 mmoL) was added dropwise thereto, and after the addition was completed, the reaction was slowly warmed to room temperature and monitored by TLC.
After the raw materials are consumed, slowly dripping the reaction solution into MeOH (methanol, 30 mL) solution of NH 4OAc(4.62g,60mmoL)、NaBH3 CN (7.56 g,120 mmoL) precooled to zero ℃ to react at room temperature, adding ice water to quench the reaction after TLC monitoring, adding NaOH aqueous solution to adjust the pH to be neutral, extracting with DCM, washing with saturated salt water, and performing crude product flash column chromatography (PE: EA=30:1) to obtain the compound 3 as a yellowish solid 5.6g(54％).1H NMR(500MHz,Chloroform-d)δ7.43–7.28(m,18H),7.28–7.23(m,2H),5.03(d,J＝10.9Hz,1H),4.90(t,J＝11.4Hz,2H),4.78–4.69(m,2H),4.58–4.50(m,2H),4.48(d,J＝11.8Hz,1H),3.72(dd,J＝9.0,2.6Hz,1H),3.61(t,J＝8.9Hz,1H),3.60–3.50(m,2H),3.40(t,J＝9.3Hz,1H),3.30(dd,J＝12.3,5.0Hz,1H),2.77(ddd,J＝9.1,5.9,2.6Hz,1H),2.56(dd,J＝12.3,10.3Hz,1H),1.89(s,1H).13C NMR(126MHz,CDCl3)δ138.96,138.56,138.46,138.03,128.47,128.45,128.43,128.41,128.06,128.00,127.91,127.83,127.81,127.73,127.71,127.57,87.39,80.72,80.16,75.72,75.23,73.44,72.83,70.33,59.80,48.19.
S3, catalytic hydrogenation debenzylation
To a solution of Compound 3 (4.28 mmol) in MTBE (methyl tert-butyl ether, 50 mL), an aqueous HCl solution (2.0M, 12.3 mL) was added dropwise, pd/C (321 mg) was added, nitrogen was bubbled for half an hour, and then bubbling was changed to hydrogen, and the reaction was carried out under vigorous stirring for 12 hours, after the completion of TLC monitoring, celite was filtered and concentrated to obtain DNJ (hereinafter abbreviated as DL-1,612mg, 86%).
1H NMR(400MHz,Deuterium Oxide)δ3.70(dd,J＝11.6,3.0Hz,1H),3.49(dd,J＝11.7,6.3Hz,1H),3.36(ddd,J＝10.8,9.1,5.1Hz,1H),3.18(t,J＝9.1Hz,1H),3.10(t,J＝9.4Hz,1H),2.98(dd,J＝12.3,5.2Hz,1H),2.41(ddd,J＝9.5,6.2,2.9Hz,1H),2.32(dd,J＝12.3,10.8Hz,1H).13C NMR(126MHz,D2O)δ78.31,71.44,70.81,61.30,60.43,48.60.
The second embodiment of the invention is as follows: preparing DL-2; the difference from the first embodiment is that:
s4, reductive amination
To a solution of CH 3(CH2)8 CHO (18 mmol) and Pd/C (650 mg) in ethanol (65 mL) were added a solution of compound 3 (12 mmol) in ethanol (65 mL) and acetic acid (6.7 mL) in this order, nitrogen was bubbled for 15 minutes, then H 2 was replaced, the reaction was carried out at room temperature for 12 hours, after completion of the TLC detection, celite was filtered and concentrated, and the crude product (compound 7) was directly added as the next step.
S5, catalytic hydrogenation debenzylation
To a solution of compound 7 (18 mmol) in MTBE (methyl tert-butyl ether, 100 mL), aqueous HCl (2.0 m,25 mL) was added dropwise, pd/C (642 mg) was added, nitrogen was bubbled for half an hour, then hydrogen bubbling was changed, and the reaction was carried out for 12 hours under vigorous stirring, after TLC monitoring the reaction was completed, celite was filtered and concentrated to obtain the product of the following formula:
Hereinafter, this is abbreviated as DL-2 (4.96 g, 91%).
1H NMR(400MHz,Methanol-d4)δ3.92–3.80(m,2H),3.49(ddd,J＝10.5,9.1,4.9Hz,1H),3.36(t,J＝9.3Hz,1H),3.14(t,J＝9.1Hz,1H),3.00(dd,J＝11.1,4.9Hz,1H),2.86–2.75(m,1H),2.66–2.53(m,1H),2.18(t,J＝10.9Hz,1H),2.12(dt,J＝9.5,2.8Hz,1H),1.51(p,J＝7.3Hz,2H),1.39–1.26(m,14H),0.96–0.88(t,3H).13C NMR(101MHz,MeOD)δ79.19,70.64,69.36,65.97,58.04,56.31,52.41,31.65,29.33,29.28,29.01,27.24,23.78,22.33,13.03.
The third embodiment of the invention is as follows: preparing DL-3; the difference from the second embodiment is that:
Step S4 the reductive amination is replaced by CH 3(CH2)8 CHO with BnCHO.
The structure of the product prepared in the step S5 of catalytic hydrogenation debenzylation is shown as the following formula:
hereinafter abbreviated as DL-3 (DL-3.20 g,95% from 5.0mmol of Compound 3)
1H NMR(400MHz,Deuterium Oxide)δ7.34–7.21(m,5H),4.08–3.97(m,2H),3.85(dd,J＝12.9,2.7Hz,1H),3.38–3.29(m,3H),3.11(t,J＝9.3Hz,1H),2.78(dd,J＝11.5,4.9Hz,1H),2.13(dt,J＝10.1,2.6Hz,1H),1.91(t,J＝11.1Hz,1H).13C NMR(101MHz,D2O)δ130.32,128.46,127.77,78.15,69.82,68.43,66.23,57.42,56.17,54.83.
The fourth embodiment of the invention is as follows: preparing DL-4; the difference from the second embodiment is that:
Step S4 reductive amination is performed with CH 3(CH2)8 CHO replaced by CH 3(CH2)2 CHO.
The structure of the final product obtained in the step S5 of catalytic hydrogenation debenzylation is shown as the following formula:
Hereinafter, this is abbreviated as DL-4 (DL-4 898.0mg,82% from 5.0mmol of Compound 3).
1H NMR(400MHz,Deuterium Oxide)δ3.91–3.75(m,2H),3.50(ddd,J＝10.7,9.2,4.9Hz,1H),3.34(t,J＝9.5Hz,1H),3.22(t,J＝9.2Hz,1H),2.99(dd,J＝11.5,5.0Hz,1H),2.70(ddd,J＝13.5,10.2,6.3Hz,1H),2.57(ddd,J＝13.3,10.2,6.2Hz,1H),2.32–2.20(m,2H),1.41(tdd,J＝12.4,7.5,3.7Hz,2H),1.23(h,J＝7.2Hz,2H),0.85(t,J＝7.3Hz,3H).13C NMR(101MHz,D2O)δ78.26,70.03,68.85,65.00,57.36,55.18,51.83,25.02,20.18,13.32.
The fifth embodiment of the invention is as follows: preparing DL-5; the difference from the second embodiment is that:
Step S4 reductive amination is performed with CH 3(CH2)8 CHO replaced by CH 3CH2 CHO.
The structure of the product prepared in the step S5 of catalytic hydrogenation debenzylation is shown as the following formula:
Hereinafter, DL-5 (obtained from 5.0mmol of Compound 3, DL-5 973mg, 96%) is referred to simply as "DL-5".
1H NMR(500MHz,CD3OD)δ3.84(d,J＝28.1Hz,2H),3.56–3.47(m,1H),3.39(t,J＝9.2Hz,1H),3.18(t,J＝9.0Hz,1H),3.01(dd,J＝10.9,4.2Hz,1H),2.81–2.69(m,1H),2.62–2.51(m,1H),2.20(t,J＝10.8Hz,1H),2.13(d,J＝9.4Hz,1H),1.52(d,J＝5.4Hz,2H),0.91(t,J＝7.2Hz,3H).13C NMR(126MHz,CD3OD)δ79.14,70.63,69.37,65.91,58.07,56.33,54.33,16.94,10.83.
The sixth embodiment of the invention is: preparing DL-6; the difference from the second embodiment is that:
Step S4 the reductive amination is replaced by CH 3(CH2)8 CHO with BnOCH 2 CHO.
The structure of the product prepared in the step S5 of catalytic hydrogenation debenzylation is shown as the following formula:
hereinafter abbreviated as DL-6 (DL-6,765 mg,74% from 5.0mmol of Compound 3).
The seventh embodiment of the invention is as follows: preparing DL-7; the difference from the second embodiment is that:
step S4 the reductive amination is replaced by CH 3(CH2)8 CHO with BnOCH 2CH2OCH2 CHO.
The structure of the product prepared in the step S5 of catalytic hydrogenation debenzylation is shown as the following formula:
hereinafter, this is abbreviated as DL-7 (DL-7,768 mg,56% was obtained from 5.0mmol of Compound 3).
The eighth embodiment of the invention is: preparing DL-8; the difference from the second embodiment is that:
Step S4 the reductive amination was replaced by BnO (CH 2CH2O)2CH2 CHO) for CH 3(CH2)8 CHO.
The structure of the product prepared in the step S5 of catalytic hydrogenation debenzylation is shown as the following formula:
hereinafter, DL-8 is abbreviated (DL-8501 mg,34% is obtained from 5.0mmol of Compound 3).
The ninth embodiment of the present invention is: the preparation of DL-9 comprises the following steps:
s1, oxidation and ammonification reaction:
Compound 1 (2.1626 g,4 mmol) was weighed into a 100mL round bottom flask, potassium carbonate (1.1057 g,8 mmol), iodine (2.0304 g,8 mmol), methanol 40mL, and n-butylamine (1.2 mL,12 mmol) was measured by syringe and added to the reaction system. The reaction was completed by TLC monitoring sulfuric acid ethanol color development at 50℃for 4 hours. The excess elemental iodine was removed by addition of saturated sodium thiosulfate solution, most of the methanol solvent was removed by rotary evaporator, extracted with ethyl acetate (50 mL. Times.3), and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 2.3g of crude compound 14 as a pale yellow oil.
S2, swern oxidation:
A100 mL two-necked round bottom flask was subjected to water removal and oxygen removal treatment, 40mL of the re-steamed methylene dichloride was sucked by a syringe and added to the round bottom flask, the round bottom flask was placed in a low-temperature reactor at-78 ℃, oxalyl chloride (0.7 mL,8 mmol) was sucked by a syringe, and after 10 minutes, the re-steamed dimethyl sulfoxide (0.3 mL,16 mmol) was sucked by a syringe and slowly added to the reaction system for reaction at-78 ℃ for 2 hours. The dried compound 14 was dissolved in 10mL of redistilled methylene chloride and added to the reaction system by syringe and reacted at-78℃for 2 hours. The re-evaporated triethylamine (4.5 mL,32 mmol) was then added to the reaction system by syringe, and after the reaction was allowed to return to room temperature, the starting material was found to be completely reacted by TLC. Extraction with dichloromethane (50 mL. Times.3), drying of the organic layer with anhydrous sodium sulfate, concentration under reduced pressure, purification of the crude product with a silica gel column (petroleum ether: ethyl acetate 6:1 by volume) gave 1.9512g of a white powdery solid, which was detected by nuclear magnetism as compound 15, with a total yield of 80% in the two steps.
1H NMR(400MHz,CDCl3)δ7.50–7.17(m,20H),6.70(t,J＝5.8Hz,1H),4.76–4.70(m,1H),4.69–4.59(m,3H),4.59–4.53(m,1H),4.53–4.45(m,2H),4.39(dd,J＝14.1,6.1Hz,2H),4.35–4.12(m,4H),3.40–3.25(m,1H),3.25–3.10(m,1H),1.53–1.40(m,2H),1.36–1.29(m,2H),0.93(t,J＝7.3Hz,3H).13C NMR(101MHz,CDCl3)δ206.10,170.20,137.53,137.41,137.27,136.78,128.82,128.70,128.57,128.45,128.42,128.25,128.22,128.11,128.02,127.97,127.87,81.37,80.71,79.76,75.33,74.59,74.25,73.53,73.25,38.88,31.57,20.11,13.78.
S3, cyclizing:
A100 mL two-necked round bottom flask was subjected to dehydration and deoxygenation treatment, 20mL of redistilled acetonitrile was sucked by a syringe and added to the round bottom flask, 5mL of redistilled acetonitrile-dissolved compound 15 (1.8293 g,3 mmol) and trifluoromethanesulfonic acid (142.3 mg,0.3 mmol) were respectively sucked by a syringe and then added to a reaction flask, triethylsilane (1.4 mL,9 mmol) was sucked by a syringe and added to the reaction system, and the reaction was carried out at 60℃for 30 minutes by TLC monitoring the development of bismuth potassium iodide to find the complete reaction of the starting materials. After cooling to room temperature, the acetonitrile solvent was removed by a rotary evaporator, and the crude product was purified by a silica gel column (petroleum ether: ethyl acetate: volume ratio: 8:1) to give 908.5mg of a colorless transparent oily liquid, which was detected by nuclear magnetism as compound 16, in a yield of 51%.
1H NMR(400MHz,CDCl3)δ7.56–7.17(m,20H),5.19(d,J＝11.3Hz,1H),4.87(d,J＝11.2Hz,1H),4.74–4.63(m,3H),4.55(d,J＝11.7Hz,1H),4.51–4.40(m,2H),4.10(d,J＝9.1Hz,1H),3.95–3.89(m,1H),3.84(ddd,J＝17.1,9.7,5.3Hz,2H),3.62–3.50(m,2H),3.50–3.39(m,1H),3.00–2.88(m,1H),1.60–1.50(m,2H),1.36–1.29(m,2H),0.92(t,J＝7.3Hz,3H).13C NMR(101MHz,CDCl3)δ169.43,138.26,138.24,137.87,137.45,128.52,128.44,128.37,128.36,128.32,128.02,127.97,127.92,127.84,127.78,127.70,127.64,81.91,78.35,76.75,74.47,74.12,73.35,72.60,68.27,60.49,45.49,29.59,20.11,13.91.
S4, amide reduction:
A50 mL two-necked round bottom flask was subjected to dehydration and deoxygenation treatment, then lithium aluminum hydride (170.8 mg,4.5 mmol) was added to the flask, and after 3 times of air exchange under nitrogen atmosphere, the reaction system was placed in an ice-water bath. 10mL of the redistilled tetrahydrofuran was taken in the round bottom flask by syringe, and 5mL of redistilled tetrahydrofuran-dissolved compound 16 (890.76 mg,1.5 mmol) was taken in the reaction flask by syringe, and the reaction was carried out at 0℃for 10 minutes by TLC monitoring the development of bismuth potassium iodide to find the starting material was completely reacted. Then adding excessive sodium sulfate decahydrate into the reaction system at the temperature of 0 ℃ to quench the reaction, and continuing stirring until obvious layering phenomenon appears in the reaction. Suction filtration in a Buchner funnel and concentration of the organic layer under reduced pressure gave 910.2mg of crude compound 17 as a colorless transparent oil.
S5, removing benzyl protection
303.4Mg of the crude compound 8 was dissolved in a 50mL round bottom flask with 10mL of methanol, and formic acid (0.23 mL,6 mmol) and 303mg of 10% palladium hydroxide were added. Then placing the mixture in a high-pressure reaction kettle, replacing air in the reaction kettle for 3 times by using nitrogen, replacing nitrogen in the reaction kettle for 3 times by using hydrogen, and then filling 4MPa hydrogen into the reaction kettle for reaction for 10 hours at room temperature. TLC monitors the color development of the o-tolidine after the chlorine fumigation to find that the raw materials are completely reacted, a Buchner funnel is used for suction filtration, the filtrate is added with excessive potassium carbonate to react for 30 minutes at 50 ℃, the Buchner funnel is used for suction filtration, the filtrate is concentrated under reduced pressure, the crude product is purified by a reverse phase silica gel column (the volume ratio of methanol to water is 1:4), 61.4mg of colorless transparent oily liquid is obtained, the compound DL-9 is detected by nuclear magnetism, and the total yield of the two steps of reaction is 56%.
1H NMR(500MHz,CD3OD)δ4.13(d,J＝12.3Hz,1H),3.94(d,J＝12.3Hz,1H),3.75(s,1H),3.63(t,J＝9.2Hz,1H),3.49(d,J＝8.5Hz,1H),3.45–3.37(m,2H),3.24(s,1H),3.10(d,J＝9.6Hz,1H),3.02(t,J＝11.3Hz,1H),1.75(s,2H),1.46(s,2H),1.03(t,J＝6.9Hz,3H).13C NMR(126MHz,CD3OD)δ76.66,67.46,66.43,66.11,53.70,53.52,52.89,24.74,19.58,12.55.
The tenth embodiment of the invention is as follows: the preparation of DL-10 comprises the following steps:
The difference from the ninth embodiment is that: the n-BuNH 2 in the oxidative amination of step S1 is replaced by AllylNH 2 (allylamine), and the structure of the prepared product is shown as the following formula:
Hereinafter, this is abbreviated as DL-10 (DL-10 60mg,59% was obtained from 0.5mmol of Compound 20).
1H NMR(500MHz,CD3OD)δ5.89(ddt,J＝16.8,10.2,6.5Hz,1H),5.22(ddd,J＝13.7,10.9,1.1Hz,2H),3.91–3.84(m,2H),3.74(dd,J＝8.5,5.0Hz,1H),3.56(td,J＝8.5,4.9Hz,1H),3.44(dd,J＝14.6,6.4Hz,2H),3.35(dd,J＝11.8,4.7Hz,1H),3.05(dd,J＝10.8,5.2Hz,1H),2.84(dd,J＝12.5,4.8Hz,1H),2.61(dd,J＝12.4,9.2Hz,1H).13C NMR(126MHz,CD3OD)δ135.24,117.10,74.03,71.30,69.75,62.92,57.38,56.57,50.88.
An eleventh embodiment of the present invention is: the preparation of DL-11 comprises the following steps:
The difference from the ninth embodiment is that: the n-BuNH 2 in the oxidative amination in the step S1 is replaced by BnCH 2NH2, and the structure of the prepared product is shown as the following formula:
hereinafter, this is abbreviated as DL-11 (DL-11 27mg,59% is obtained from 0.3mmol of Compound 24).
1H NMR(400MHz,CD3OD)δ7.30–7.16(m,5H),3.95(dd,J＝11.9,2.5Hz,1H),3.90–3.84(m,1H),3.52(ddd,J＝10.4,9.3,4.9Hz,1H),3.36(t,J＝9.3Hz,1H),3.18(t,J＝9.1Hz,1H),3.09(dd,J＝11.1,4.9Hz,1H),3.02–2.89(m,2H),2.86–2.74(m,2H),2.37(dd,J＝21.3,10.5Hz,1H),2.32–2.22(m,1H).13C NMR(101MHz,CD3OD)δ140.25,128.37,128.07,125.65,79.18,70.64,69.44,65.32,58.17,56.44,54.47,30.04.
The twelfth embodiment of the invention is as follows: the preparation of DL-12 comprises the following steps:
the difference from the ninth embodiment is that: the n-BuNH 2 in the oxidative amination of the step S1 is replaced by (4-MeO) -PhCH 2CH2NH2, and the structure of the prepared product is shown as the following formula:
hereinafter, this is abbreviated as DL-12 (DL-12 51mg,58% from 0.3mmol of Compound 28).
1H NMR(500MHz,CD3OD)δ7.13(t,J＝6.8Hz,2H),6.91–6.79(m,2H),3.93(dt,J＝8.7,4.3Hz,1H),3.86(dd,J＝12.0,2.9Hz,1H),3.77(s,3H),3.53–3.47(m,1H),3.39–3.34(m,1H),3.19–3.15(m,1H),3.09(dd,J＝11.1,4.9Hz,1H),2.99–2.92(m,1H),2.91–2.85(m,1H),2.79–2.68(m,2H),2.36(t,J＝10.8Hz,1H),2.30–2.21(m,1H).13C NMR(126MHz,CD3OD)δ158.13,132.13,129.26,113.51,79.19,70.65,69.43,65.39,58.17,56.42,54.58,54.24,29.22.
The thirteenth embodiment of the invention is: the preparation of DL-13 comprises the following steps:
The difference from the ninth embodiment is that: the compound 1 in the oxidative amination of the step S1 is replaced by a compound 30, and the structure of the prepared product is shown as the following formula:
Hereinafter, this is abbreviated as DL-13 (DL-13 obtained from 0.3mmol of Compound 33 36mg, 58%).
1H NMR(500MHz,CD3OD)δ4.02–3.99(m,1H),3.88–3.75(m,3H),3.22(dt,J＝14.9,7.5Hz,1H),2.99(dd,J＝11.2,4.9Hz,1H),2.78–2.69(m,1H),2.56–2.45(m,1H),2.38(td,J＝5.4,1.6Hz,1H),2.14(dd,J＝18.6,7.9Hz,1H),1.53–1.45(m,2H),1.32(ddd,J＝15.0,7.1,2.7Hz,2H),0.98–0.94(m,3H).13C NMR(126MHz,CD3OD)δ75.90,70.81,67.62,63.75,60.89,56.71,52.40,25.82,20.36,12.97.
The fourteenth embodiment of the invention is as follows: the preparation of DL-14 comprises the following steps:
The difference from the ninth embodiment is that: the oxidative amination of step S1 was replaced with compound 35 and steps S2-S5 were identical to example nine, the structure of the resulting product was as follows:
hereinafter referred to as DL-14 (37 mg,57% of DL-14 was obtained from 0.3mmol of Compound 38).
1H NMR(500MHz,CD3OD)δ4.03–3.96(m,1H),3.94(dd,J＝4.5,2.4Hz,1H),3.83(ddd,J＝18.9,10.1,3.6Hz,2H),3.78(s,1H),2.77–2.70(m,2H),2.69(d,J＝4.8Hz,1H),2.60(dd,J＝19.6,9.4Hz,2H),1.50(dd,J＝18.7,11.2Hz,2H),1.32(dd,J＝14.7,7.3Hz,2H),0.96(t,J＝7.3Hz,3H).13C NMR(126MHz,CD3OD)δ71.52,70.81,66.10,60.19,59.17,53.21,51.71,26.24,20.42,13.02.
The fifteenth embodiment of the invention is: the preparation of DL-15 comprises the following steps:
The difference from the ninth embodiment is that: the oxidative amination of step S1 was replaced with compound 40 and steps S2-S5 were identical to example nine, the structure of the resulting product was as follows:
Hereinafter, DL-15 was obtained (37 mg,61% of DL-15 was obtained from 0.3mmol of Compound 43).
1H NMR(400MHz,CD3OD)δ3.67(ddt,J＝11.0,10.1,4.4Hz,1H),3.18(ddd,J＝11.2,8.9,4.5Hz,1H),3.00(ddd,J＝10.8,4.4,1.9Hz,1H),2.77–2.68(m,1H),2.55(ddd,J＝13.3,9.5,6.2Hz,1H),2.33–2.21(m,1H),2.08(t,J＝8.2Hz,1H),2.05–1.98(m,1H),1.52–1.43(m,2H),1.33(ddd,J＝9.0,7.3,2.9Hz,2H),1.21(t,J＝5.1Hz,3H),0.97(dd,J＝9.5,5.1Hz,3H).13C NMR(101MHz,CD3OD)δ70.42,64.49,61.64,58.65,52.52,42.18,25.67,20.36,13.89,12.94.
The beneficial effects of the present invention are further illustrated by the following pharmacodynamic tests of the compounds prepared in examples one to five and examples nine to fifteen.
Loss of the bone morphogenic protein type2 receptor (BMPR 2) is the initiating link to induce pulmonary arterial high pressure endothelial dysfunction. Lysosomal degradation is a key factor in the loss of endothelial BMPR 2. DNJ with different concentrations is established on a hypoxia-induced pulmonary artery high pressure rat model and a PAECs treated by TNF-alpha and 3% hypoxia, and the treatment effect of DNJ on pulmonary artery high pressure endothelial cell injury model is verified by using methods such as WB (Western blot method ), HE staining (hematoxylin-eosin staining method), scratch experiment and the like.
The content of the study is divided into the following two aspects.
(1) Both animals and cells observe the effect of DNJ on BMPR2 signaling pathway in pulmonary arterial hypertension.
(2) Functional experiment: the effect of DNJ on pulmonary artery endothelial cell proliferation, apoptosis, and migration was studied.
Grouping description:
The DL-1 group represents the pharmacodynamic experiments performed by selecting the compound prepared in example one.
DL-2 group represents the selection of the compound prepared in example two for pharmacodynamic experiments.
The DL-3 group represents the pharmacodynamic experiments using the compounds prepared in example III.
The DL-4 group represents the pharmacodynamic experiments using the compounds prepared in example four.
The DL-5 group represents the pharmacodynamic experiment using the compound prepared in example five.
The DL-9 group represents the pharmacodynamic experiments using the compound prepared in example nine.
The DL-10 group represents the pharmacodynamic experiments using the compounds prepared in example ten.
The DL-11 group represents the pharmacodynamic experiments using the compounds prepared in example eleven.
The DL-12 group represents the pharmacodynamic experiments using the compounds prepared in example twelve.
The DL-13 group represents the pharmacodynamic experiments using the compounds prepared in example thirteen.
The DL-14 group represents the compounds prepared in example fourteen for pharmacodynamic experiments.
The DL-15 group represents the compounds prepared in example fifteen were selected for pharmacodynamic experiments. the TNF group represents PAECs selected for TNF-. Alpha.treatment.
The hypoxia group represents PAECs selected for 3% hypoxia treatment.
1. Pharmacodynamic experiments
1.1 Animal experiments
The entire experiment was performed according to national institutes of health's guidelines for care and use of laboratory animals. The protocol was approved by the Committee for animal welfare, hunan medical college, university of south China. Male healthy Sprague-Dawley (SD) rats (100 g-150 g) were supplied by Hunan SJA laboratory animal Co., ltd. (number: SYXK (Xiang) 2015-0017). All animals are raised in an environment with controlled temperature (18 ℃ to 25 ℃) and humidity (50 to 60%), and are alternately subjected to 12 hours of light and shade circulation. Food and water are available at will. After one week of adaptive feeding, animals were weighed, randomly grouped and numbered. Two PH rat models were established separately, hypoxia-induced PH rat model: the molding time period was 4 weeks, which were divided into four groups of Control (Control group, abbreviated as Con group), hypoxia (hypoxia group, abbreviated as Hyp group), 25mg/kg DL-1 group, and 50mg/kg DL-1 group, each of which was 10. The first week was followed by placing Hypoxia, 25mg/kg DL-1 and 50mg/kg DL-1 into a hypoxia chamber (the oxygen concentration was set to 3%), feeding the cells into the hypoxia chamber for 4 weeks, and the third week was followed by feeding the two groups of 25mg/kg DL-1 and 50mg/kg DL-1 by the intragastric administration method for two weeks. The Control group does not intervene at all.
1.2 Hemodynamic measurements and morphological analysis
After the molding was completed, the rats were anesthetized (1% pentobarbital sodium intraperitoneal injection, 50 mg/kg). A PE catheter was inserted through the right jugular vein to the right atrium, and Right Ventricular Systolic Pressure (RVSP) was recorded. After detection of RVSP, lung tissue samples were collected. The Right Ventricle (RV), left ventricle plus ventricular septum (lv+s) and weighed separately to calculate RV/(lv+s) mass ratio. The rat hind limb tibia was taken and RV/Tibial length was calculated using the ulnar distance. The lower right lung was taken and placed in 4% paraformaldehyde solution for fixed storage. The fixed tissue was used for subsequent morphometric analysis, and the tissue sections were kept at 60 ℃ for 2 hours and then 30 minutes in xylene solution. Then put it into 100% ethanol solution, 95% ethanol solution and 70% ethanol solution in this order for 5 minutes each time. The samples were then washed 3 times in phosphate buffered saline (PBS, procell, china) for 5 minutes each, held for 5 minutes in high pressure antigen retrieval and 20 minutes in hydrogen peroxide. HE staining was used to observe the morphology of the blood vessels, the effect of the film was observed under a microscope, a photograph was taken at the appropriate position, and data analysis was performed.
1.3 Cell culture
Culture conditions and morphology of human Pulmonary Artery Endothelial Cells (PAECs) pulmonary artery endothelial cells PAECs were placed in an incubator, cultured with DMEM/F-12:1 (volume ratio) complete medium (diabody 1% and fetal bovine serum FBS 10%), and after cell attachment, the normal state of the cells was observed. Normally growing endothelial cells show shuttle, and the cells are tightly adhered and grow in an array. After cell culture, cells were observed once a day without much, if cells were observed to have grown as an immediate monolayer (indicating that cells had fused to 90% -95% or even higher), cells were digested with trypsin (pre-warmed at 37 ℃) and the digested pulmonary endothelial cells PAECs were collected, stopped with complete DMEM/F-12:1 (volume ratio) medium containing 10% fetal bovine serum and 1% diab, centrifuged in a centrifuge at 800 rpm/min for 4 minutes, the original medium was discarded, and fresh DMEM/F-12:1 (volume ratio) complete medium (diab 1% and fetal bovine serum FBS 10%) was added and passaged in 25cm 2 cell flasks at a ratio of 1:2.
1.4CCK8 detection of cell proliferation Capacity
The digested PAEC cells are blown by a 1mL gun head to prepare cell suspension, counted by a cell counter, inoculated in a 96-well plate, added with culture medium to make up 100 mu L of total volume in each well, inoculated with 1X 10 4 cells per well, and cultured in a cell culture incubator with constant temperature of 37 ℃,5% CO 2 and saturated humidity. After the cells are attached and the cell fusion degree reaches 80%, the cells are grouped (the compounds to be tested (DL-1-DL-5 and DL-9-DL-15) with different concentrations, namely, the compounds and the hypoxia treatment (the cells are placed in an environment with 3% oxygen concentration) are performed simultaneously), a control group (Con group, control group, without any intervention) and a Hyp group (hypoxia group, the cells are placed in an environment with 3% oxygen concentration)) and are correspondingly treated, and 5 compound wells are respectively arranged in each group. Wells not used in the 96-well plate were filled with PBS solution. Culturing in a 5% CO 2 incubator at 37deg.C for 1 day. To each well was added 10 mu LCCK-8 (Cell Counting Kit-8 cell counting reagent) solution. The plates were incubated for a further 2 hours at 37℃in a cell incubator with 5% CO 2 and saturated humidity. The absorbance at 450nm was measured with a microplate reader.
The calculation formula is as follows: cell viability= (treatment OD-blank OD)/(control OD-blank OD) ×100%, where OD value is absorbance.
1.5 Scratch assay to detect cell migration Capacity
Before inoculating the pulmonary artery endothelial cells on the S1 and 6-hole culture plates, 3 horizontal lines are drawn in parallel (at intervals of about 1 cm) on the back by using a marker pen as marks (so as to be beneficial to positioning the same visual field when photographing at different times).
S2, after the pulmonary artery endothelial cells PAECs are digested, adding a DMEM/F-12:1 complete culture medium (double antibody 1% and fetal bovine serum FBS 5%) to prepare a cell suspension, and inoculating the pulmonary artery endothelial cell suspension into a 6-hole plate, wherein the number is that the cells are completely fused after adherence, namely are in a completely close state, and are in a monolayer state without overlapping each other, and the monolayer cells are preferably in a monolayer state.
S3, after the pulmonary artery endothelial cells are fully paved on the bottom of the plate, a trace is gently scratched by using a tip gun head with the size of 1mL which is perpendicular to the hole of the 6-hole plate, and the cell scratches with the width of one gun head are manufactured, so that the widths of all scratches are ensured to be consistent as much as possible in principle, and large deviation is avoided.
S4, sucking cells and cell fragments generated by the previous scratches by using a Pasteur pipette, and washing for 3 times by using 1 XPBS, so that the 6-hole plate is completely cleaned, and floating cells and cell fragments are not required to remain to influence the photographing effect.
S5, adding DMEM/F-12:1 (volume ratio) complete culture medium (1% of double antibody and 5% of fetal bovine serum FBS), adding 2mL of each well, and photographing under a microscope. The plates were further placed in a cell incubator at 37 ℃,5% co 2 and saturated humidity.
S6, taking out the 6-hole plate from the cell incubator with 37 ℃ and 5% CO 2 and saturated humidity every 6 hours, and taking out the 6-hole plate for photographing until the end time is 24 hours. And (5) observing an experimental result according to the shot picture, and carrying out data analysis.
1.6Western blot detection of protein expression
Proteins were extracted from pulmonary arteries or HPASMCs (human pulmonary vascular smooth muscle cells) with RIPA buffer containing 0.1% pmsf (phenylmethylsulfonyl fluoride), and an equal amount of protein (30 μg) was separated by 10% sds-PAGE and transferred to polyvinylidene fluoride membranes. 1% BSA at room temperature for 1h. Then incubating the membrane with a primary antibody against BMPR2(Protein tech,USA)、p-Smad1/5(Cell Signaling Technology,USA)、ID1(Protein tech,USA)、β-actin(β-actin,USA) at 4 ℃ overnight, immersing all PVDF membranes in a biotin-labeled secondary antibody (the secondary antibody is goat anti-rabbit or goat anti-mouse, TBST is diluted 1:10000), shaking for 2h on a room temperature shaking table, and rinsing with TBST for 3 times each for 10min; the pipette sucks an equal amount of the luminous liquid A and the luminous liquid B, uniformly mixes the luminous liquid A and the luminous liquid B, uniformly drops the mixed liquid on the film as much as possible, and places the film into the imaging instrument for development, and the whole process is carried out under the light-proof condition. Image scanning was performed using Quantity one (Bio. Rad) software, semi-quantification was performed using image J software, and relative gray scale values (RELATIVE GRAY SCALE, RGS) of each protein and β -actin were calculated to represent the relative expression levels of each protein.
1.7 Data statistics and analysis
Data statistical analysis was performed using Graph pad software. All data are presented as mean ± standard deviation, and comparison of data between sets is analyzed using one-way ANOVA (analysis of variance) test. P <0.05 is statistically significant and P <0.01 is statistically significant.
As can be seen from FIG. 1, the shrinkage pressure of the DL-1 group was reduced compared with that of the hypoxia group, and at a concentration of 50mg/kg, the shrinkage pressure was much lower than that of the hypoxia group (P < 0.01).
From fig. 2, it is known that the ratio of the right ventricular mass to (total left ventricular to upper ventricular septum mass) of the DL-1 group is reduced compared to the hypoxia group.
From FIG. 3, it is seen that DL-1 group DNJ reduced the right heart hypertrophy and right ventricular pressure in hypoxia-induced pulmonary hypertension rats.
As can be seen from FIG. 4, the DL-1 group cells had higher viability, and the cell viability was enhanced with an increase in DNJ concentration.
From FIG. 5, it is clear that the result of the scratch test before and after the treatment of compound 1 (DL 1 group) at different concentrations and the control group is compared, and from the graph, DNJ has the effect of inhibiting the migration of normal PAECs after the treatment of DL-1.
As can be seen from FIG. 6, the DL-1 group significantly reduced cell mobility (P < 0.01).
As can be seen from FIG. 7, the expression level of each protein in the DL-1 group was higher than that in the control group.
As can be seen from FIG. 8, the expression level of BMPR2 protein in the DL-1 group is higher than that in the control group, and the expression level of BMPR2 protein is gradually increased at low dosage (1. Mu. Mol to 10. Mu. Mol) and reaches the maximum at 10. Mu. Mol (P < 0.01); at high doses (10. Mu. Mol to 30. Mu. Mol) the BMPR2 protein expression level gradually decreased but was still higher than that of the control group.
As can be seen from FIG. 9, the expression level of the P-Smad1/5/8 protein in the DL-1 group was higher than that in the control group (P < 0.05).
As can be seen from FIG. 10, the expression level of the DL-1 group ID protein was higher than that of the control group (P < 0.01).
FIG. 11 shows the results of gel electrophoresis of different proteins in the DL-1 group (different concentrations), the hypoxia group and the control group, wherein the expression level of each protein in the DL-1 group is higher than that in the hypoxia group, and the condition that DNJ up-regulates the expression of the BMPR2 and the P-smad proteins in lung tissues of mice with lung hypertension induced by low oxygen is shown.
As can be seen from FIG. 12, the BMPR2 protein expression level of the DL-1 group was much higher than that of the hypoxia group, and the expression level was slightly higher than that of the control group (P < 0.01) at a concentration of 25 mg/kg.
As can be seen from FIG. 13, the expression level of P-Smad1/5/8 protein in the DL-1 group was much higher than that in the hypoxia group, and the expression level was slightly lower than that in the control group (P < 0.01) at a concentration of 50 mg/kg.
As can be seen from FIG. 14, the expression level of the ID protein in the DL-1 group was much higher than that in the hypoxia group, and at a concentration of 50mg/kg, the expression level was slightly higher than that in the control group (P < 0.05).
FIG. 15 shows the results of gel electrophoresis of different proteins in the DL-2 group (different concentrations) and the control group of the present invention; the expression level of each protein was higher in the high dose DL-1 group (dose: 10. Mu. Mol) than in the control group.
FIGS. 16 to 18 are graphs showing comparison of the expression levels of different proteins (BMPR 2 protein, p-Smad1/5/8 protein and ID protein) in the DL-2 group (at different concentrations) of the present invention and the control group; from FIGS. 16-18, it is seen that the BMPR2 signal path in normal PAECs is up-regulated by DL-2.
FIGS. 19, 21 and 23 are graphs showing that the treatment of ordinary cells was directly performed with different concentrations of DL-3, DL-4 and DL-5 in order to find the concentration without cytotoxicity; FIGS. 20, 22 and 24 are graphs showing that the use of tnf stimulates cell proliferation based on the non-toxic concentrations shown in FIGS. 19, 21 and 23, respectively, and then treatment with different concentrations of DL-3, DL-4 and DL-5 was performed in order to obtain concentrations that could inhibit cell proliferation.
FIGS. 19 to 24 are graphs showing cell viability comparisons of the DL-3 group (different concentrations), the DL-4 group (different concentrations) and the DL-5 group (different concentrations) of the present invention with the control group; as can be seen from FIGS. 19 to 24, DL-3, DL-4 and DL-5 inhibited normal PAECs proliferation.
FIGS. 25 to 31 are graphs showing cell viability comparisons of DL-9 (different concentrations), DL-10 (different concentrations), DL-11 (different concentrations), DL-12 (different concentrations), DL-13 (different concentrations), DL-14 (different concentrations), DL-15 (different concentrations), control (Con) and hypoxia (Hyp) groups according to the present invention; as can be seen from FIGS. 25 to 31, DL-9, DL-10, DL-11, DL-12, DL-13, DL-14 and DL-15 inhibited PAECs proliferation compared to the hypoxia group.
From this, it is known that DL-6, DL-7, DL-8, DL-9, DL-10, DL-11, DL-12, DL-13, DL-14 and DL-15 in the sixth to fifteen embodiments of the present invention have the similar effects to those of the compounds in the first to fifteen embodiments of the present invention, namely, BMPR2, p-smad 1/5/8 and ID protein expression can be up-regulated, and pulmonary vascular injury can be reduced.
DNJ and its derivatives take lysosome degradation BMPR2 as target spot, up-regulate BMPR2, p-smad 1/5/8, ID protein expression, reduce pulmonary vascular injury, achieve the effect of treating PAH, and find new thought for PAH treatment.
In conclusion, the application of DNJ and the derivatives thereof in preparing medicaments for preventing and/or treating pulmonary arterial hypertension improves PAH vascular state by inhibiting lysosome degradation BMPR2, and expands the application of mulberry leaf extract DNJ and the derivatives thereof.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims (20)

  1. The application of DNJ and its derivatives in preparing medicines for treating pulmonary hypertension is characterized in that: the DNJ and the derivative thereof comprise at least one of a compound shown as a general formula (I) or pharmaceutically acceptable salt thereof:
    wherein R is selected from one of H, benzyl, propyl and n-butyl;
    r 1 is hydroxy.
  2. 2. The use according to claim 1, characterized in that: the stereochemical combination of the substituents at the C2, C3, C4, C5 positions in the general formula (I) is selected from one of (2 r,3s,4r,5 r), (2 r,3r,4r,5 r), and (2 r,3s,4s,5 s).
  3. 3. The use according to claim 1, characterized in that: the pharmacologically acceptable salts include salts with inorganic acids, organic acids, alkali metals, alkaline earth metals or basic amino acids.
  4. 4. A use according to claim 3, characterized in that: the inorganic acid includes at least one of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, and hydrobromic acid.
  5. 5. A use according to claim 3, characterized in that: the organic acid includes at least one of maleic acid, fumaric acid, tartaric acid, lactic acid, citric acid, acetic acid, methanesulfonic acid, p-toluenesulfonic acid, adipic acid, palmitic acid, and tannic acid.
  6. 6. A use according to claim 3, characterized in that: the alkali metal includes at least one of lithium, sodium and potassium.
  7. 7. A use according to claim 3, characterized in that: the alkaline earth metal includes at least one of calcium and magnesium.
  8. 8. A use according to claim 3, characterized in that: the basic amino acid includes lysine.
  9. The application of DNJ and its derivatives in preparing medicines for treating pulmonary hypertension is characterized in that: the DNJ and the derivative thereof comprise at least one of compounds DL-1, DL-3, DL-4, DL-5, DL-9, DL-12 and DL-13 shown in the following formula:
    、/>、/>、/>、/>、、/>。
  10. 10. Use according to claim 1 or 9, characterized in that: the preparation raw materials of the medicine for treating pulmonary arterial hypertension also comprise medicinal carriers.
  11. 11. The use according to claim 10, characterized in that: the pharmaceutical carrier comprises at least one of an excipient, a filler, an adhesive, a disintegrant, an absorption enhancer, an adsorption carrier, a lubricant, a sweetener, and a flavoring agent.
  12. 12. The use according to claim 11, characterized in that: the excipient comprises water.
  13. 13. The use according to claim 11, characterized in that: the filler includes at least one of starch and sucrose.
  14. 14. The use according to claim 11, characterized in that: the binder includes at least one of cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone.
  15. 15. The use according to claim 11, characterized in that: the disintegrant includes at least one of agar, calcium carbonate, and sodium bicarbonate.
  16. 16. The use according to claim 11, characterized in that: the absorption enhancer includes a quaternary ammonium compound.
  17. 17. The use according to claim 11, characterized in that: the absorption enhancer includes a surfactant.
  18. 18. The use according to claim 17, characterized in that: the surfactant comprises cetyl alcohol.
  19. 19. The use according to claim 11, characterized in that: the adsorption carrier comprises at least one of kaolin and soap clay.
  20. 20. The use according to claim 11, characterized in that: the lubricant comprises at least one of talc, calcium stearate, magnesium stearate and polyethylene glycol.
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