A preparation method and application of furano[2,3-b]quinoline derivative

Abstract

The present invention relates to the field of biomedicine, and specifically to the use of furano[2,3-b]quinoline derivatives, their tautomers, pharmaceutical salts, prodrugs or solvates in the preparation of drugs for treating chronic kidney disease, wherein the structure of the furano[2,3-b]quinoline derivatives is shown in formula (I): wherein R1 is a methoxy group; R2 is hydrogen, hydroxyl, or methoxyl group; R3 is hydrogen, hydroxyl, or methoxyl group; and R4 is hydrogen or methoxyl group. Experiments show that the compounds of the present invention have significant effects on treating chronic kidney disease. The compounds of the present invention have diverse preparation routes, are economical, and have the characteristics of clear activity, low toxicity, etc., and have broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of medicine and chemical industry, and specifically relates to a preparation method and application of a furano[2,3-b]quinoline derivative. Background Art

[0002] Chronic kidney disease (RF) is a chronic progressive fibrotic kidney disease associated with almost all chronic kidney disease (CKD) and progressive kidney disease. The exact pathogenesis of RF is still unclear. There are many factors that cause RF. After the kidney is affected by multiple factors such as injury, infection, hypertension and bad living habits, the kidney undergoes cell damage, fibrosis and sclerosis. This process is the process of renal fibrosis, and its pathological characteristics are injury, inflammation, myofibroblast activation and migration, and tubular epithelial cell-mesenchymal transformation, as well as extracellular matrix deposition and remodeling. The deposition of fibrous matrix after injury may initially help tissue repair, and the fibrous matrix can be absorbed during tissue repair after mild injury. However, after the chronic injury of CKD occurs, the damage is continuous, the fibrous matrix will not be completely absorbed, and the deposition of fibrous matrix is ​​not inhibited, which will eventually destroy the organ structure, reduce blood supply, and disrupt organ function. Fibrosis reduces the ability of tissue repair and eventually leads to renal failure.

[0003] According to current research, inhibiting myofibroblast activation and differentiation, reducing inflammatory responses, and clearing extracellular matrix deposition are the main ways to improve chronic kidney disease. Given the increasing prevalence of RF and the increasing related mortality, there is an urgent need to develop safe and effective drugs to treat chronic kidney disease.

[0004] Furanoquinoline compounds are widely found in plants, such as Dictamni, Stinkweed, Psoralea corylifolia and Bamboo-leaf Pepper. These compounds have certain biological activities. For example, dittanyline (in small doses) has an excitatory effect on isolated frog hearts, increases myocardial tension, has a significant contraction effect on isolated rabbit ear blood vessels, and has a strong contraction effect on rabbit and guinea pig uterine smooth muscle. In addition, it also has antibacterial and skin eczema and skin itching therapeutic effects. However, there has been no literature report on the evaluation of the anti-RF activity of furanoquinoline derivatives. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a furano[2,3-b]quinoline derivative and its application in preparing a drug for treating chronic kidney disease.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] Use of a furano[2,3-b]quinoline derivative, its tautomer, pharmaceutically acceptable salt, prodrug or solvate in the preparation of a drug for treating chronic kidney disease, wherein the structure of the furano[2,3-b]quinoline derivative is shown in formula (I):

[0008]

[0009] Among them, R1 is methoxy; R2 is hydrogen, hydroxyl, methoxy; R3 is hydrogen, hydroxyl, methoxy; R4 is hydrogen, methoxy.

[0010] Preferably, the structure and substituents of the furano[2,3-b]quinoline derivative are shown in Table 1:

[0011] Table 1 Substituents and structures of furan[2,3-b]quinoline alkaloid compounds of the present invention

[0012]

[0013]

[0014] Preferably, the drug further comprises a pharmaceutically acceptable carrier, adjuvant or excipient.

[0015] The pharmaceutically acceptable "carriers, adjuvants or excipients" mentioned above refer to conventional drug carriers in the pharmaceutical field, for example: diluents, excipients such as water, fillers such as starch sucrose, etc.; binders such as cellulose derivatives, gelatin, etc.; in addition, other excipients such as flavoring agents, sweeteners, etc. can also be added to the composition.

[0016] The compounds of the present invention may be in the form of a preferred compound or a crystalline solvate. The solvation method is well known in the art, and a suitable solvate is a pharmaceutically acceptable solvate. In a specific embodiment, the solvate is a hydrate.

[0017] Preferably, the chronic kidney disease is renal fibrosis.

[0018] A method for extracting and preparing a furano[2,3-b]quinoline derivative, wherein the furano[2,3-b]quinoline derivative has the structural formula:

[0019]

[0020] The extraction and preparation method of the furano[2,3-b]quinoline derivative comprises the following steps:

[0021] S1. Crush the branches and leaves of Rutaecarpa, add water for ultrasonic extraction, concentrate the extract, filter, adsorb the filtrate on a macroporous resin column, and elute with water, 50% ethanol and 90% ethanol in sequence, collect the eluate with 90% ethanol, concentrate, and obtain a fluid extract; apply the fluid extract to a normal phase silica gel column, elute with a dichloromethane-methanol solution gradient, combine similar fractions, and dry to obtain fractions YXC-1 to YXC-14;

[0022] S2, YXC-5 was loaded onto a gel column, eluted with petroleum ether-dichloromethane-methanol solution, similar fractions were combined and dried to obtain fractions YXC5-1 to YXC5-6;

[0023] S3, YXC5-2 was subjected to reverse phase semi-preparative HPLC to obtain the compound furano[2,3-b]quinoline derivative LYY-12 (t R =40.134min).

[0024] Preferably, the rue branches and leaves are ultrasonically extracted twice, each time for 1 hour, and the extracts are combined.

[0025] Preferably, the macroporous resin column is a D101 macroporous resin column.

[0026] Preferably, the gradient elution is carried out using a dichloromethane-methanol solution, with the volume ratio of dichloromethane to methanol ranging from 100:0 to 0:1.

[0027] During the elution process, wash and tap the thin layer plate until there is no substance in the eluted solvent, then change to the next gradient.

[0028] Preferably, the steps of combining similar fractions also include identifying the fractions by tracking the spot plate with thin layer chromatography.

[0029] Preferably, in the petroleum ether-dichloromethane-methanol solution, the volume ratio of petroleum ether-dichloromethane-methanol is (5-20):5:1, and the optimal ratio is 5:5:1.

[0030] The separation effect of petroleum ether-dichloromethane-methanol solution in this ratio is the best among the systems tried, and it can just separate the substances according to different polarities. However, when using dichloromethane and methanol systems (10:1, 5:1, 2:1) or petroleum ether and ethyl acetate systems (10:1, 5:1, 2:1), some compounds cannot be separated.

[0031] Preferably, the column of reverse phase semi-preparative HPLC is a phenyl column, and the mobile phase is acetonitrile-water with a volume ratio of 29:71. The separation effect (peak shape and retention time, resolution) of the phenyl column is better than that of C18 and C8 columns. According to multiple test results, in the mobile phase, the separation effect with a ratio of 29:71 is the best, which is significantly better than the acetonitrile-water solution with a ratio of (40-50): (50-60).

[0032] Preferably, the medicinal residue is added with 80% ethanol solution to submerge the medicinal material for ultrasonic extraction, the extract is concentrated, the filtrate is extracted three times with petroleum ether, the petroleum ether layer is combined and extracted three times with ethyl acetate, and the ethyl acetate extract is obtained after concentration and freeze-drying. The ethyl acetate extract is applied to a normal phase silica gel column, gradient eluted with a dichloromethane-methanol solution, similar fractions are combined, and dried to obtain fractions YXY-1 to YXY-18.

[0033] YXY-4 was loaded onto a normal phase silica gel column and eluted with a petroleum ether-ethyl acetate system as a gradient elution. Similar fractions were combined and dried to obtain fractions YXY4-1 to YXY4-10.

[0034] The precipitated crystals of YXY4-6 were identified as the furano[2,3-b]quinoline derivative LYY-12.

[0035] Preferably, the remaining soluble portion of YXY4-6 after crystallization is further dissolved in methanol and then applied to a Sephadex LH-20 gel column, eluted with a dichloromethane-methanol system (1:1), and similar fractions are combined and dried to obtain fractions YXY4-6-1 to YXY4-6-6; YXY-4-6-3 and YXY-4-6-5 are partially analyzed out of the crystals, which are the furano[2,3-b]quinoline derivative LYY-12.

[0036] Preferably, the residue is ultrasonically extracted twice, each time for 1 hour, and the extracts are combined.

[0037] Preferably, the petroleum ether-ethyl acetate solution is used for gradient elution, and the volume ratio of petroleum ether to ethyl acetate is from 4:1 to 1:2.

[0038] Preferably, in the extraction preparation method of the compound furano[2,3-b]quinoline derivative LYY-12, LYY-12 is mainly present in the alcohol extraction part of the drug residue. Preferably, the preparation method of the furano[2,3-b]quinoline derivative comprises the following steps:

[0039] (1) adding malonic acid diester, chloroacetyl chloride and a catalyst dropwise in a strong base to react to obtain compound 1;

[0040] The structure of the diester of malonate is:

[0041]

[0042] Wherein, R is selected from C1-C4 branched or straight chain alkyl, phenyl or substituted phenyl; the substituent of the substituted phenyl includes halogen, C1-C4 branched or straight chain alkyl, C1-C4 alkoxy;

[0043] (2) adding an arylamine dropwise to the compound 1 obtained in step (1), and after the reaction, adding an aqueous solution of ethyl acetate to precipitate, thereby obtaining a solid phase compound 2 and a solution;

[0044] The structure of the arylamine is:

[0045]

[0046] R2, R3 and R4 are independently selected from hydrogen, hydroxyl, halogen, C1-C4 straight or branched alkyl, C1-C4 alkoxy, benzyloxy;

[0047] (3) Compound 2 is added to an inert solvent, heated to 250-260° C., refluxed and stirred for 0.5 h-1 h, and then petroleum ether is added, filtered, and dried to obtain compound 3;

[0048] (4) adding a chlorination reagent to compound 3 for reaction, followed by low-temperature extraction with dichloromethane and concentration to obtain compound 4;

[0049] (5) Add a reducing agent to compound 4 under ice bath to react, then concentrate, extract with dichloromethane, wash with water, and dry to obtain compound 5;

[0050] (6) Potassium hydrogen sulfate or hydrochloric acid was added to compound 5, and the mixture was refluxed for 3 h to 5 h, and then extracted with ethyl acetate, neutralized with alkali, and dried to obtain compound 6;

[0051] (7) under the action of a base, a nucleophilic reagent is added to compound 6, and the mixture is refluxed for 2-3 days, and then extracted with ethyl acetate, washed with water, and dried to obtain a crude product, and the crude product is separated and purified by normal phase column chromatography to obtain the furano[2,3-b]quinoline derivative;

[0052] The structure of the furano[2,3-b]quinoline derivative is:

[0053]

[0054] Among them, R1 is methoxy; R2 is hydrogen, hydroxyl, methoxy; R3 is hydrogen, hydroxyl, methoxy; R4 is hydrogen, methoxy.

[0055] Preferably, in steps (1) and (2), the C1-C4 branched or straight chain alkyl group includes methyl, ethyl, isopropyl, n-propyl, n-butyl, and tert-butyl.

[0056] Preferably, in steps (1) and (2), the C1-C4 alkoxy group is selected from methoxy, ethoxy, and propoxy.

[0057] Preferably, in step (1), the strong base is one of sodium hydride, potassium tert-butoxide and sodium tert-butoxide.

[0058] Preferably, in step (1), the catalyst is selected from triethylamine, N,N-diisopropylethylamine or 1,8-diazacyclo[5,4,0]undecene-7, and triethylamine is further preferred. If no catalyst is added in this step, the yield of the product is extremely low.

[0059] Preferably, the reaction temperature of step (1) is 0-30°C.

[0060] Preferably, in the step (1), after adding chloroacetyl chloride to react for 1 to 2 hours, triethylamine is added and the reaction is continued for 2 to 4 hours.

[0061] Preferably, the solution in step (2) is separated into an organic phase and an aqueous phase, the organic phase is concentrated under reduced pressure, dispersed by stirring with ether such as diethyl ether, filtered to obtain a crude product, and the crude product is recrystallized with ethanol to obtain a pure product of compound 2.

[0062] Preferably, the inert solvent in step (3) is a solvent that does not contain active hydrogen and ester groups and does not react with the reactants at high temperatures. Preferably, the inert solvent is diphenyl ether.

[0063] Preferably, the chlorination agent in step (4) is selected from phosphorus oxychloride, phosphorus trichloride, phosphorus pentachloride, oxalyl chloride, thionyl chloride, sulfonyl chloride or chlorine, preferably phosphorus oxychloride. Phosphorus oxychloride has the best selectivity in the reaction system of the present invention.

[0064] Preferably, a surfactant is added to the chlorination reaction in step (4), preferably trioctylmethylammonium chloride. Under the promotion of the surfactant, phosphorus oxychloride further improves the selectivity of the reaction.

[0065] The extraction reagent in step (3-6) has been optimized many times and has the best effect, and can obtain selectivity that other organic solvents cannot achieve.

[0066] Preferably, the reducing agent in step (5) is selected from sodium borohydride, sodium borocyanide, lithium tetraborate, lithium tetrahydride aluminate or diisobutylaluminum hydride.

[0067] Preferably, the mobile phase of the normal phase column chromatography in step (7) is a mixed solution of petroleum ether / ethyl acetate in a volume ratio of 2 / 1.

[0068] Preferably, the base in step (7) includes sodium hydride, sodium hydroxide, sodium alkyl alcoholate or potassium alkyl alcoholate, and the sodium alkyl alcoholate includes sodium methoxide, sodium ethoxide and sodium tert-butoxide.

[0069] Preferably, the nucleophilic agent in step (7) is selected from alcohols, phenols, thiols, thiophenols, amines, and N-heterocyclic compounds.

[0070] Preferably, the synthetic route of the furano[2,3-b]quinoline derivative is as follows:

[0071]

[0072] The synthesis method of the present invention uses malonic acid diester and arylamine as starting materials, and synthesizes the furano[2,3-b]quinoline derivative through seven-step reaction. The chemical synthesis method has simple reaction, the extraction process is optimized, the intermediate steps only need to be subjected to simple extraction and filtration, the finished product processing operation is simple, and the furano[2,3-b]quinoline derivative pure product can be prepared through only one column chromatography separation, without the need for column separation in each step as in the prior art.

[0073] The present invention is further explained below:

[0074] The present invention discovered compound LYY12 from Rue, which was identified as kokusaginine. Through research, it was found for the first time that LYY12 has the kidney effect of improving chronic kidney disease. Kokusaginine, English name: 6,7-dimethoxydictamnine, is referred to as LYY12 in the present invention. The International Union of Pure and Applied Chemistry (IUPAC) named formula (I) 4,6,7-trimethoxyfuro[2,3-b]quinoline, and its Chinese name is 4,6,7-trimethoxyfuro[2,3-b]quinoline. Its molecular formula: C14H13NO4, molecular weight: 259.257. Chemical Abstracts Number (CAS): 484-08-2. It is an alkaloid extracted from Dictamnus dasycarpus Turcz. It can also be derived from rutagraveolens, orixajaponica, zanthoxylumbungeanummaxim., ormasp., acronychiasp., evodiasp., haplopayllumsp., and melicopesp. It has the effects of increasing norepinephrine and dopamine levels (Chem. Res. Toxicol, 2014, 27 (2), 219-239); phototoxicity (Planta Medica 1981, 41 (2), 136-42.); insecticidal (Tennen Yuki Kagobutsu Toronkai Koen Yoshishu 1999, 41st, 451-456.), antitrypanosomal activity (ChemMedChem 2022, 17(5), e202100784), anti-inflammatory (J. Ethnopharmacol. 2019, 238, 111827; Bio. Med. Chem. 2011, 19(21), 6340-6347; AJPTR 2017, 7(2), 315-324), anti-proliferative activity on human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480) (Nat. Pro. Res. 2022, 36, 379-384), inhibitory effect on human breast cancer cell growth and multidrug resistance (Bio. Med. Chem. Lett. 2018, 28(14), 2490-2492) and antispasmodic effect (AJPTR 2017, 7(2), 315-324), etc.

[0075] The present invention establishes a chronic kidney disease animal model to observe the therapeutic effect of LYY12 on chronic kidney disease in mice. The research results show that LYY12 has a good therapeutic effect on chronic kidney disease.

[0076] The present invention studies the chronic kidney disease inhibitory effect of different doses of LYY and HL series compounds through human renal cortical proximal tubule epithelial cells (HK-2) and rat renal fibroblasts (NRK-49F), detects the cell proliferation after incubation with different concentrations through CCK8, and screens out LYY12 for the next animal experiment.

[0077] Male, 7-week-old C57 mice were used as experimental animals. All mice were adaptively fed for one week before the experiment was officially carried out. All mice had free access to food and water, and a 12-h day and night cycle was adopted. The prepared LYY12 was dissolved in 0.5% CMC-Na, and the following dosage groups of different concentrations were prepared. The mice were randomly divided into a blank group (N), a model group (M), a LYY12 high-dose group (LYY12-H), a LYY12 medium-dose group (LYY12-M), and a LYY12 low-dose group (LYY12-L). Except for the blank group mice, the remaining groups of mice were required to establish a chronic kidney disease model.

[0078] The present invention induces a chronic kidney disease model by allowing C57BL / 6 mice to autonomously ingest a feed containing 0.2% adenine every day for 21 consecutive days, and starts to administer drugs at the same time as the adenine feed is ingested, and mice are given CMC-Na, LYY12 10mg / kg / day (low dose), 20mg / kg / day (medium dose), 40mg / kg / day (high dose) and allopurinol 20mg / kg / day by gavage at a volume of 10ml / kg, respectively, for 21 days. The results show that different doses of LYY12 have different degrees of improvement on chronic kidney disease. After induction with 0.2% adenine feed, the weight of mice decreased significantly, urea nitrogen and creatinine values ​​increased, the cortical tubules were severely dilated and atrophied, the cell casts and protein casts in the cortex increased, the interstitial inflammatory infiltration increased, and the collagen deposition worsened. After treatment with LYY12, the renal function of mice was significantly improved, the renal tissue structural damage was significantly improved, the collagen deposition was alleviated, and LYY12 could significantly reduce the inflammation of the mouse renal tissue. The above results fully demonstrated that LYY12 can effectively treat chronic kidney disease.

[0079] The LYY12 product used in the present invention is prepared by conventional natural product chemistry, separation and purification, and drug synthesis methods. This laboratory uses high performance liquid chromatography (HPLC) analysis and detection, and its purity is more than 99%, and it has been analyzed and identified by mass spectrometry and nuclear magnetic resonance, indicating that the chemical structure of the LYY12 product used in this laboratory is correct. This study shows that the purity and chemical structure of LYY12 meet the requirements for conducting in vivo and in vitro biological activity and pharmacological action research.

[0080] The beneficial effects of the present invention are:

[0081] The present invention first discovered that the furano[2,3-b]quinoline derivative has a therapeutic effect on chronic kidney disease; specifically, a chronic kidney disease model was induced by C57BL / 6 mice voluntarily ingesting a feed containing 0.2% adenine for 21 consecutive days. The administration was started at the same time as the adenine feed was ingested, and mice were gavaged with LYY12 10 mg / kg, 20 mg / kg and 40 mg / kg, respectively, for 21 days. The results showed that high doses of LYY12 can more significantly reduce the weight loss of mice and improve the renal function and renal structural damage of mice compared with allopurinol, indicating that LYY12 has a good therapeutic effect. The furano[2,3-b]quinoline derivative was synthesized by seven steps using diethyl malonate and 3,4-dimethoxyaniline as raw materials. The synthesis method is simple and easy to operate. The synthesis route provides an efficient synthesis method for vanilla wood Ning and provides a feasible route and scheme for its industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 is the HRESI-MS image of compound LYY12;

[0083] Figure 2 is the H NMR spectrum of compound LYY12;

[0084] Figure 3 is the carbon NMR spectrum of compound LYY12;

[0085] Figure 4 is the HRESI-MS spectrum of compound LYY38;

[0086] Figure 5 is the H NMR spectrum of compound LYY38;

[0087] Figure 6 is the carbon NMR spectrum of compound LYY38;

[0088] Figure 7 is the HRESI-MS graph of compound LYY40;

[0089] Figure 8 is the H NMR spectrum of compound LYY40;

[0090] Fig. 9 is the carbon NMR spectrum of compound LYY40;

[0091] Fig.10 is the HRESI-MS image of compound LYY66;

[0092] Fig.11 is the H NMR spectrum of compound LYY66;

[0093] Fig.12 is the carbon NMR spectrum of compound LYY66;

[0094] Fig.13 is the HRESI-MS spectrum of compound LYY68;

[0095] Fig.14 is the H NMR spectrum of compound LYY68;

[0096] Fig.15 is the HRESI-MS graph of compound LYY103;

[0097] Fig.16 is the H NMR spectrum of compound LYY103;

[0098] Fig.17 is the carbon NMR spectrum of compound LYY103;

[0099] Fig.18 is the HRESI-MS diagram of compound HL-14;

[0100] Fig.19 is the hydrogen nuclear magnetic resonance spectrum of compound HL-14;

[0101] Fig. 20 is the carbon NMR spectrum of compound HL-14;

[0102] Fig.21 is the HRESI-MS graph of compound HL-17;

[0103] Fig. 22 is the H NMR spectrum of compound HL-17;

[0104] Fig.23 This is the carbon NMR spectrum of compound HL-17

[0105] Fig.24 is the HRESI-MS diagram of compound HL-18;

[0106] Fig.25 is the hydrogen nuclear magnetic resonance spectrum of compound HL-18;

[0107] Fig.26 is the H NMR spectrum of compound 2a;

[0108] Fig. 27 is the H NMR spectrum of compound 3a;

[0109] Fig.28 This is the H NMR spectrum of vanilla wood nin;

[0110] Fig.29 The effects of LYY and HL series compounds on the proliferation of NRK-49F cells;

[0111] Fig.30 The effects of LYY and HL series compounds on HK-2 cell proliferation;

[0112] Fig.31 This is a diagram showing the results of modeling mice with chronic kidney disease induced by 0.2% adenine diet;

[0113] Fig.32 The effect of LYY12 on renal function in mice with chronic kidney disease;

[0114] Fig.33 Effects of LYY12 on renal tissue of mice with chronic kidney disease Specific implementation methods

[0115] The following examples may help those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0116] Example 1

[0117] Extraction of furano[2,3-b]quinoline derivatives

[0118] Take 10kg of Rutaecarpa branches and leaves and crush them, add water solution to cover the medicinal materials, and perform ultrasonic extraction twice, each time for 1 hour, combine the extracts, concentrate, filter to obtain the water extract, and reserve the residue. The water extract is adsorbed on a D101 macroporous resin column, eluted with water, 50% ethanol and 90% ethanol in turn, and the water, 50% ethanol and 90% ethanol eluates are collected respectively, concentrated, and fluid extracts of three elution parts are obtained. Among them, the 90% ethanol part fluid extract is about 14.3g, which is applied to a normal phase silica gel column, and gradient eluted with a dichloromethane-methanol system (the volume ratio of dichloromethane to methanol is from 100:0 to 0:1), and the thin layer chromatography is tracked on the spot plate, and similar fractions are combined, dried under reduced pressure, and combined to obtain fractions YXC-1 to YXC-14.

[0119] YXC-3 was subjected to Sephadex LH-20 gel column, petroleum ether-dichloromethane-methanol system (5:5:1), thin layer chromatography tracking spot plate, similar fractions were combined, dried under reduced pressure, and fractions YXC3-1 to YXC3-10 and YXC3-6 were obtained. After reverse phase semi-preparative HPLC, phenyl column, 29% acetonitrile-water as mobile phase, fraction YXC3-6-3 was obtained to obtain compound LYY-68 (6 mg) (t R =35.601min), to give compound LYY-66 (8mg) (t R =39.956min).

[0120] YXC-5 was subjected to Sephadex LH-20 gel column, petroleum ether-dichloromethane-methanol system (5:5:1), thin layer chromatography tracking spot plate, similar fractions were combined, dried under reduced pressure, and fractions YXC5-1 to YXC5-6 were obtained. YXC5-2 was subjected to reverse phase semi-preparative HPLC, phenyl column, and 29% acetonitrile-water as mobile phase to obtain compound LYY-12 (10 mg) (t R =40.134min).

[0121] YXC-6 was subjected to Sephadex LH-20 gel column, and thin layer chromatography was performed on the plate with petroleum ether-dichloromethane-methanol system (5:5:1). Similar fractions were combined and dried under reduced pressure to obtain fractions YXC6-1 to YXC6-11. YXC6-4 was subjected to reverse phase semi-preparative HPLC on a phenyl column with 20% acetonitrile-water as the mobile phase to obtain fraction YXC6-4-4. YXC6-4-4 was further subjected to reverse phase semi-preparative HPLC with 20% acetonitrile-water as the mobile phase to obtain compound LYY-38 (8 mg) (t R =37.353min).

[0122] YXC6-5 was subjected to reverse phase semi-preparative HPLC, phenyl column, and 30% acetonitrile-water as the mobile phase to obtain fraction YXC6-5-2. YXC6-5-2 was further subjected to reverse phase semi-preparative HPLC, and 30% acetonitrile-water as the mobile phase to obtain compound LYY-40 (12 mg) (t R =18.581min).

[0123] YXC6-2 was loaded onto a normal phase silica gel column and eluted with a petroleum ether-ethyl acetate system (2:1). The thin layer chromatography was followed by spot plates. Similar fractions were combined and dried under reduced pressure to obtain fractions YXC6-2-1 to YXC6-2-11. YXC6-2-9 was subjected to reverse phase semi-preparative HPLC on a C18 column with 30% acetonitrile-water as the mobile phase to obtain fraction YXC6-2-9-2. YXC6-2-9-2 was further subjected to reverse phase semi-preparative HPLC on a phenyl column with 59% acetonitrile-water as the mobile phase to obtain compound LYY-103 (10 mg) (t R =19.56min).

[0124] The residue was added with 80% ethanol solution to cover the medicinal material and ultrasonically extracted twice, each time for 1 hour, the extracts were combined, concentrated, and filtered to obtain an alcohol extract, which was extracted three times with petroleum ether, and the petroleum ether layer was combined and extracted three times with ethyl acetate, and concentrated and freeze-dried to obtain an ethyl acetate extract (80g). The ethyl acetate extract was loaded onto a normal phase silica gel column, and gradient eluted with a dichloromethane-methanol system (the volume ratio of dichloromethane to methanol was from 100:0 to 0:1), and the thin layer chromatography was followed by a spot plate, and similar fractions were combined, dried under reduced pressure, and combined to obtain fractions YXY-1 to YXY-18.

[0125] YXY-4 was subjected to a normal phase silica gel column and gradient eluted with a petroleum ether-ethyl acetate system (volume ratio of petroleum ether to ethyl acetate was 4:1, 2:1, 1:1, 1:2), followed by thin layer chromatography, similar fractions were combined, dried under reduced pressure, and fractions YXY4-1 to YXY4-10 were obtained. YXY4-6 was dissolved in methanol, and the soluble part was subjected to a Sephadex LH-20 gel column and eluted with a dichloromethane-methanol system (1:1), followed by thin layer chromatography, similar fractions were combined, dried under reduced pressure, and fractions YXY4-6-1 to YXY4-6-6 were obtained. Crystals from YXY-4-6-3 and YXY-4-6-5 were analyzed as LYY12. Crystals from the insoluble part were analyzed and identified as compound LYY12. YXY4-6-4 was separated by preparative thin layer chromatography using dichloromethane-methanol system (20:1) to obtain YXY4-6-4-1 to YXY4-6-4-4. YXY4-6-4-3 was separated by reverse phase semi-preparative HPLC using C18 column and 38% methanol-formic acid water as mobile phase to obtain compound HL-14 (2 mg) (t R =30.060min), to obtain compound HL-18 (2mg) (t R =46.783min). YXY4-6-4-3 was further subjected to reverse phase semi-preparative HPLC, phenyl column, and 38% methanol-formic acid water as the mobile phase to obtain fraction YXY4-6-4-3-6, which is compound HL-17 (2 mg) (t R =38.108min).

[0126] Results: After identification, the structures of the compounds are as follows:

[0127] The compound LYY-12 is a light yellow needle-shaped crystal with the chemical structure:

[0128]

[0129] Spectral data are as follows: ESI-MS m / z: 260.0924 [M+H] + (calcd for C 14 H 13 NO4). ESI-MS Figure 1 The hydrogen and carbon nuclear magnetic resonance spectra are shown in Figure 2 and Figure 3 shown. 1 H NMR (500MHz, Methanol-d4) δ: 7.72 (d, J = 3.0 Hz, 3H), 7.46 (d, J = 2.4 Hz, 3H), 7.28 (s, 3H), 7.17 (d, J = 2.4 Hz, 3H), 4.94 (s, 2H), 4 .60(s,1H),4.46(d,J=2.4Hz,9H),3.97(d,J=2.5Hz,9H),3.94(d,J=2.5Hz,9H),3.31(s,12H),1.32(s,1H),1.30–1.20(m,2H). 13 C NMR (126MHz, Methanol-d4) δ: 158.55, 155.39, 150.22, 144.77, 143.97, 114.95, 107.36, 107.06, 104.37, 102.57, 60.78, 57.24, 57.20.

[0130] The compound LYY-38 is a brown powder with the chemical structure:

[0131]

[0132] Spectral data are as follows: ESI-MS m / z: 246.0774 [M+H] + (calcd for C 13 H 11 NO4). ESI-MS Figure 4 The hydrogen and carbon nuclear magnetic resonance spectra are shown in Figure 5 and Figure 6 shown. 1H NMR (600MHz, Methanol-d4) δ: 8.54 (s, 6H), 7.97–7.89 (m, 2H), 7.74 (d, J = 3.0Hz, 2H), 7.33 (d, J=2.9Hz,2H),7.10(dd,J=9.1,2.1Hz,2H),4.62(s,7H),4.48(t,J=1.9Hz,7H),3.96(t,J=1.9 Hz,7H),3.70(s,2H),3.65(s,3H),3.34(d,J=1.9Hz,7H),3.15(p,J=6.5,5.6Hz,6H),2.53(t, J=6.6Hz,2H),1.62(d,J=11.7Hz,1H),1.36–1.26(m,16H),1.21(s,1H),0.89(d,J=7.9Hz,1H). 13 C NMR (151MHz, Methanol-d4) δ: 170.28, 151.92, 144.07, 142.46, 119.79, 117.48, 106.42, 61.68, 60.05, 49.89, 47.74, 21.63, 9.33.

[0133] The compound LYY-40 is a brown-red powder with the chemical structure:

[0134]

[0135] ESI-MS m / z:216.0682[M+H] + (calcd for C 12 H9NO4). ESI-MS Figure 7 The hydrogen and carbon nuclear magnetic resonance spectra are shown in Figure 8 and Fig. 9 shown. 1 H NMR (600MHz, Methanol-d4) δ: 7.81–7.70(m,2H),7.52(s,1H),7.36–7.26(m,2H),4.48(s,3H). 13 C NMR (151MHz, Methanol-d4) δ: 163.80, 157.49, 155.18, 145.06, 141.06, 128.82, 123.34, 120.86, 106.14, 104.89, 104.80, 59.99.

[0136] The compound LYY-66 is a white powder with the chemical structure:

[0137]

[0138] ESI-MS m / z:230.0839[M+H] + (calcd for C 13 H 11 NO3). ESI-MS Fig.10 The hydrogen and carbon nuclear magnetic resonance spectra are shown in Fig.11 and Fig.12 shown. 1 H NMR (600MHz, Methanol-d4) δ: 7.80 (d, J = 2.8 Hz, 1H), 7.77 (s, 1H), 7.58 (d, J = 2.9 Hz, 1H), 7.38 (d, J =2.9Hz,1H),7.36(t,J=2.9Hz,2H),4.63(d,J=7.5Hz,1H),4.52(s,3H),3.92(s,3H),3.34(s,4H). 13 C NMR (151MHz, Methanol-d4) δ: 164.07, 157.80, 157.75, 145.20, 141.82, 128.98, 123.83, 120.40, 106.21, 105.09, 101.36, 60.08, 56.01.

[0139] The compound LYY-68 is a white powder with the chemical structure:

[0140]

[0141] ESI-MS m / z:200.0699[M+H] + (calcd for C 12 H9NO2). ESI-MS Fig.13 As shown, the H NMR spectrum is as Fig.14 shown. 1 H NMR (600MHz, Methanol-d4) δ: 8.31 (dd, J=8.4, 1.9Hz, 1H), 7.88 (dt, J=8.5, 0.9Hz, 1H), 7.83 (d, J=2.8Hz, 1H), 7.72 (ddd, J=8.4,6.8,1.5Hz,1H),7.49(ddd,J=8.2,6.8,1.2Hz,1H),7.40(d,J=2.8Hz,1H),4.62(s,2H),4.53(s,3H),3.34(s,9H).

[0142] The compound LYY-103 is white needle-shaped and has the following chemical structure:

[0143] ESI-MS Fig.15 The hydrogen and carbon nuclear magnetic resonance spectra are shown in Fig.16 and Fig.17 shown. 1 H NMR (600MHz, Methanol-d4) δ: 8.03 (d, J=9.4Hz, 1H), 7.75 (d, J=2.8Hz, 1H), 7.35 (d, J= 9.4Hz,1H),7.31(d,J=2.8Hz,1H),4.48(s,3H),4.01(s,3H),3.97(s,3H),3.35(s,1H). 13 CNMR(151MHz,MeOD)δ166.10,159.29,153.92,144.48,143.99,142.65,142.04,119.70,115.89,113.46,106.53,106.37,106.2 8,103.57,103.38,101.74,61.76,60.11,59.98,57.23,56.44,56.39,49.90,49.48,49.33,49.19,49.05,48.91,48.77,48.62.

[0144]

[0145] Compound HL-14 is a light yellow powder with the chemical structure:

[0146] ESI-MS m / z:216.0685[M+H] + (calcd for C 12 H9NO3). ESI-MS Fig.18 The hydrogen and carbon nuclear magnetic resonance spectra are shown in Fig.19 and Fig. 20 shown. 1 H NMR (600MHz, Methanol-d4) δ: 7.76 (d, J = 2.8 Hz, 1H), 7.73 (d, J = 9.1 Hz, 1H), 7.52 (d, J = 2.7 Hz, 1H), 7.33–7.26 (m, 2H), 4.47 (s, 3H). 13C NMR (151MHz, MeOD) δ: 163.48, 157.16, 154.84, 144.74, 140.76, 128.51, 123.01, 120.55, 105 .80,104.58,104.49,64.13,59.67,49.29,49.14,49.00,48.86,48.72,48.58,48.43,48.29.

[0147]

[0148] Compound HL-17 is a white powder with the chemical structure:

[0149] ESI-MS m / z:246.0793[M+H] + (calcd for C 13 H 11 NO4). ESI-MS Fig.21 The hydrogen and carbon nuclear magnetic resonance spectra are shown in Fig. 22 and Fig.23 shown. 1 H NMR (600MHz, Methanol-d4) δ: 7.70 (d, J = 2.8 Hz, 1H), 7.53 (s, 1H), 7.29 (d, J = 2.8 Hz, 1H), 7.16 (s, 1H), 4.48 (s, 3H), 3.98 (s, 3H). 13 C NMR (151MHz, MeOD) δ: 164.17, 157.55, 152.03, 148.51, 143.39, 143.10, 113.54, 109.36, 105.92,102.75,101.28,59.61,56.05,49.14,49.00,48.86,48.72,48.57,48.43,48.29.

[0150]

[0151] Compound HL-18 is a yellow oil with the chemical structure:

[0152] ESI-MS m / z:246.0791[M+H] + (calcd for C 13 H 11 NO4). ESI-MS Fig.24 As shown, the H NMR spectrum is as Fig.25 shown. 1H NMR (600MHz, Methanol-d4) δ: 7.93 (d, J = 9.0Hz, 11H), 7.74 (d, J = 2.8Hz, 13H), 7.32 (s, 9H), 7.10 (d, J = 9.2Hz, 13H), 5 .34(s,1H),4.60(s,5H),4.47(s,37H),3.96(s,33H),3.89–3.82(m,4H),3.65(dd,J=6.7,4.3Hz,3H),3.58(t,J=5.6 Hz,3H),3.35(s,4H),3.21(t,J=7.3Hz,3H),3.16(t,J=6.6Hz,3H),2.35–2.30(m,3H),2.18(t,J=7.4Hz,3H),2.06–2 .03(m,2H),1.77(s,2H),1.64–1.58(m,6H),1.53–1.48(m,3H),1.37–1.25(m,25H),0.94–0.85(m,8H),0.10(s,2H).

[0153]

[0154] Example 2.

[0155] Chemical Synthesis of Vanillin

[0156]

[0157] Scheme A: Preparation of vanilla extract by single column chromatography

[0158] Synthesis of compound 2a:

[0159] Method 1: In a 100mL round-bottomed bottle, 4g of sodium hydride (60%) was rinsed with dry petroleum ether and added to 20mL of tetrahydrofuran for dispersion. Stir at room temperature and slowly add diethyl malonate (16g, 15mL) within 1h. After the addition is complete, set aside. Take another 500mL reaction bottle, dissolve chloroacetyl chloride (5.6g, 4mL) in 30mL of dry tetrahydrofuran, and slowly add the solution prepared in the previous step to the reaction bottle within 1h under an ice bath, and control the reaction temperature below 30℃. After the addition is completed, stir at room temperature for 1h, then add triethylamine (10g, 13.5mL) and continue stirring for 2h. Then, under an ice bath, slowly add a solution of 3,4-dimethoxyaniline (7.8g) in dry tetrahydrofuran (50mL) within 1h, control the temperature not to exceed 30℃, and stir at room temperature overnight after the addition is completed. After the reaction of the raw materials was complete, the mixture was concentrated under reduced pressure to remove tetrahydrofuran, and 300 mL of ethyl acetate and 100 mL of water were added for extraction. During the extraction, the solid was filtered to obtain 2.2 g of pure compound 2a. The organic phase of the filtrate was dried, concentrated under reduced pressure, and then dispersed with ether by stirring, and filtered to obtain a crude product. The crude product was added with 10 mL of ethanol for recrystallization to obtain 3.2 g of compound 2a, a grayish white solid, with a yield of 42%. The H NMR spectrum was as follows: Fig.26 As shown, the NMR data is 1 H NMR (500MHz, Chloroform-d) δ: 10.08 (s, 1H), 6.88 (dd, J = 8.6, 2.5Hz, 1H), 6.83–6.77 (m, 2H), 4.60 (s, 2H) ,4.32(q,J=7.0Hz,2H),3.83(d,J=2.2Hz,7H),1.34(t,J=7.1Hz,4H),1.24(s,1H),1.19(d,J=4.6Hz,2H).

[0160] Method 2: In a 25mL round-bottomed bottle, 0.3g of sodium hydride (60%) was rinsed with dry petroleum ether and then added to 2.1mL of tetrahydrofuran for dispersion. Stir at room temperature and slowly drop diethyl malonate (2.1g, 1.96mL) within 1h. After the addition is complete, set aside. Take another 100mL reaction bottle, dissolve chloroacetyl chloride (0.77g, 0.54mL) in 2.9mL of dry tetrahydrofuran, and slowly drop the solution prepared in the previous step into the reaction bottle within 1h under an ice bath, and control the reaction temperature to be below 30℃. After the drop is completed, stir at room temperature for 1h. Then, slowly drop a solution of 3,4-dimethoxyaniline (1g) in dry tetrahydrofuran (4.3mL) within 1h under an ice bath, and control the temperature not to exceed 30℃. After the drop is completed, stir at room temperature overnight. After the reaction of the raw materials was completed by thin layer chromatography (petroleum ether: ethyl acetate 2:1), tetrahydrofuran was removed under reduced pressure at 40°C, 30 mL of ethyl acetate and 10 mL of water were added for extraction, the ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. After silica gel column chromatography, 0.23 g of compound 2a was obtained with a yield of 16%.

[0161] Synthesis of compound 3a:

[0162] In a 100 mL reaction bottle, compound 2a (2.2 g) was added to 22 mL of diphenyl ether, and the temperature was raised to 250-260 ° C. Reflux stirring for 1 h. Thin layer chromatography (petroleum ether: ethyl acetate 1:1) showed that the raw material reaction was complete, and the mixture was cooled to room temperature. 200 mL of petroleum ether was slowly added dropwise to the reaction solution, the generated solid was filtered, and the filter cake was rinsed with petroleum ether to obtain 1.8 g of a brown solid crude compound 3a. The H NMR spectrum was as follows Fig. 27 As shown, the NMR data is 1 H NMR (600MHz, DMSO-d6) δ: 13.06 (s, 3H), 7.54 (s, 3H), 7.49–7.43 (m, 1H), 7.23–7 .18(m,1H),7.13–7.03(m,1H),6.99(s,3H),4.97(s,1H),4.85(s,6H),4.18(s,1 H),3.94(s,9H),3.90(s,9H),3.85–3.75(m,4H),3.23(s,1H),2.10–2.01(m,1H ),1.52(t,J=7.2Hz,1H),1.35(s,1H),1.32–1.28(m,5H),0.92(t,J=7.0Hz,1H).

[0163] Synthesis of compound 4a:

[0164] In a 25 mL reaction bottle, add 1.8 g of compound 3a from the previous step, followed by 4 mL of phosphorus oxychloride and 320 mg of trioctylmethylammonium chloride, and stir at room temperature for 48 h under nitrogen protection. Thin layer chromatography (dichloromethane: methanol 5:1 or petroleum ether: ethyl acetate 1:1) analysis showed that the raw materials reacted completely, and the reaction solution was poured into 20 mL of crushed ice, extracted with dichloromethane (3x50 mL), combined, dried, filtered, and concentrated to obtain a crude compound 4a.

[0165] Synthesis of compound 5a:

[0166] The compound 4a from the previous step was dissolved in 20 mL of a 1:1 dichloromethane / methanol mixed solvent, and 270 mg of sodium borohydride was added under ice bath, and the mixture was stirred at room temperature for 1 h. After the reaction of the raw material was complete, 5 mL of water was added to quench the sodium borohydride, and dichloromethane and methanol were removed under reduced pressure at 40°C, and the mixture was extracted with ethyl acetate (3x50 mL). The ethyl acetate phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to remove ethyl acetate to obtain a crude compound 5a.

[0167] Synthesis of compound 6a:

[0168] Compound 5a from the previous step was added to dry 1,4-dioxane (20 mL) and stirred, and 1 g of potassium hydrogen sulfate was added, and the temperature was raised to 110°C and refluxed and stirred for 3 h. Thin layer chromatography (petroleum ether: ethyl acetate 2:1) showed that the raw material reaction was complete, and dioxane was removed under reduced pressure, and 50 mL of ethyl acetate was added for extraction, and saturated sodium carbonate aqueous solution (3x20 mL) was used to wash until neutral, and the aqueous phase was extracted with ethyl acetate (3x50 mL), and the ethyl acetate phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude compound 6a.

[0169] Synthesis of vanillin:

[0170] The crude compound 6a in the previous step was added with methanol (5 mL) and stirred at room temperature, and 5 mL of sodium methoxide methanol solution (5.4 M) was added, and the mixture was heated at 80°C for 3 days under reflux. The reaction of the raw material was complete by thin layer chromatography (petroleum ether: ethyl acetate 1:1), and the methanol was removed under reduced pressure at 40°C, and 50 mL of ethyl acetate and 20 mL of water were added for extraction. The aqueous phase was extracted with ethyl acetate (3x50 mL), combined and dried, filtered and concentrated to obtain a crude product, which was separated by silica gel normal chromatography column (petroleum ether: ethyl acetate 3:1 to petroleum ether: ethyl acetate 1:1) to obtain 237 mg of vanilla wood nin, and the nuclear magnetic resonance hydrogen spectrum was as follows Fig.28 As shown, the NMR data is 1H NMR (600MHz, Chloroform-d) δ: 7.51 (d, J=2.8Hz, 3H), 7.42 (s, 3H), 7.27 (s, 3H), 7.19 (s, 1H), 6.98 (t, J= 2.8Hz,3H),4.38(d,J=2.9Hz,9H),3.96(d,J=2.0Hz,18H),2.15(t,J=7.7Hz,1H),1.99–1.92(m,2H),1.59 –1.54(m,1H),1.35(d,J=5.4Hz,1H),1.30(d,J=3.0Hz,1H),1.24(d,J=29.0Hz,6H),1.22(s,7H),1.18(s ,11H),1.16(s,3H),1.15(s,1H),1.04(s,1H),0.82(s,1H),0.83–0.74(m,2H),0.78(s,2H),0.36(s,1H).

[0171] Result: Vanilla was synthesized.

[0172] Scheme B: Preparation of vanilla by two column chromatography

[0173] Synthesis of compound 2a:

[0174] In a 25mL round-bottomed bottle, 900mg of sodium hydride (60%) was rinsed with dry petroleum ether and added to 6mL of tetrahydrofuran for dispersion. Stir at room temperature and slowly add diethyl malonate (2.1g, 5.9mL) within 1h. After the addition is completed, set aside. Take another 100mL reaction bottle, dissolve chloroacetyl chloride (2.3g, 1.6mL) in 9mL of dry tetrahydrofuran, and slowly add the solution prepared in the previous step to the reaction bottle within 1h under an ice bath, and control the reaction temperature below 30℃. After the addition is completed, stir at room temperature for 1h, then add triethylamine (3.8g, 5.2mL) and continue stirring for 2h. Then, under an ice bath, slowly add a solution of 3,4-dimethoxyaniline (3.0g) in dry tetrahydrofuran (13mL) within 1h, control the temperature not to exceed 30℃, and stir at room temperature overnight after the addition is completed. After the reaction of the raw materials was completed by thin layer chromatography (petroleum ether: ethyl acetate 2: 1), the tetrahydrofuran was removed by vacuum concentration, and 50 mL of ethyl acetate and 20 mL of water were added for extraction. During the extraction, solids were filtered to obtain 210 mg of pure compound 2a. The organic phase of the filtrate was dried, concentrated under reduced pressure, and then dispersed with ether by stirring, filtered to obtain a crude product, and the crude product was recrystallized by adding 2 mL of ethanol to obtain 600 mg of compound 2a, with a yield of 27%.

[0175] Synthesis of compound 3a:

[0176] In a 50 mL reaction bottle, add 810 mg of compound 2a to 5.4 mL of diphenyl ether, heat to 250-260 °C and reflux with stirring for 1 h. The reaction was completely cooled to room temperature. 20 mL of petroleum ether was slowly added dropwise to the reaction solution. After the solid precipitated, it was filtered and the filter cake was rinsed with petroleum ether to obtain 730 mg of brown solid compound 3a.

[0177] Synthesis of compound 4a:

[0178] The crude compound 3 from the previous step was added to a 25 mL reaction bottle, followed by 1.5 mL of phosphorus oxychloride and 117 mg of trioctylmethylammonium chloride, and stirred at room temperature for 48 h under nitrogen protection. After the reaction was complete, the reaction solution was poured into 20 mL of crushed ice, and dichloromethane (3*50 mL) was added for extraction. The organic phases were combined, dried, and concentrated to obtain a crude product. The crude product was subjected to column chromatography to obtain 275 mg of compound 4a, with a yield of 35%.

[0179] Synthesis of compound 5a:

[0180] The compound 4a from the previous step was dissolved in 5 mL of a 1:1 mixed solvent of dichloromethane / methanol, and 190 mg of sodium borohydride was added under ice bath, and the mixture was stirred at room temperature for 1 h. After the reaction was complete, 2 mL of water was added to quench the sodium borohydride, and dichloromethane and methanol were removed under reduced pressure at 40°C, and the mixture was extracted with ethyl acetate (3*50 mL), and the ethyl acetate phases were combined, filtered and concentrated to remove ethyl acetate to obtain 224 mg of crude compound 5a.

[0181] Synthesis of compound 6a:

[0182] Compound 5a from the previous step was added to dry 1,4-dioxane (11.2 mL) and stirred, and 1.1 g of potassium hydrogen sulfate was added, and the temperature was raised to 110°C and refluxed and stirred for 3 h. After the reaction was complete, dioxane was removed under reduced pressure, 30 mL of ethyl acetate was added to dilute, and saturated sodium carbonate aqueous solution (10 mL) was washed until neutral, and the aqueous phase was back-extracted with ethyl acetate (3*50 mL), and the ethyl acetate phases were combined, dried and concentrated to obtain 196 mg of crude compound 6a.

[0183] Synthesis of vanillin:

[0184] Compound 6a in the previous step was added with methanol (5 mL) and stirred at room temperature, and 1 mL of sodium methoxide methanol solution (5.4 M) was added, and the mixture was heated under reflux at 80°C for 3 days. After the reaction was completed, methanol was removed under reduced pressure, 50 mL of ethyl acetate and 20 mL of water were added for extraction, and the organic phase was dried and concentrated to obtain a crude product, which was separated by a silica gel normal chromatography column (petroleum ether: ethyl acetate 3:1 to petroleum ether: ethyl acetate 1:1) to obtain 113 mg of vanilla wood nin.

[0185] Result: Vanilla was synthesized.

[0186] Example 3

[0187] Inhibitory effects of furano[2,3-b]quinoline derivatives on NRK-49F cell proliferation

[0188] Methods: NRK-49F cells can be activated after being stimulated, and after further proliferation, a large amount of extracellular matrix is ​​abnormally deposited in the renal interstitium, causing renal interstitial fibrosis. NRK-49F cells were cultured in complete DMEM medium (containing 10% fetal bovine serum, 100μg / mL penicillin, 100μg / mL streptomycin) at 37 degrees Celsius and 5% CO2 saturated humidity. The medium was changed every 24 hours. Logarithmically growing cells were taken, digested with 0.25% trypsin, the cell concentration was diluted with complete medium, and inoculated into 96-well cell culture plates. After culturing overnight, different concentrations of LYY12, 38, 40, 66, 68, 103, HL-14, 17, 18 (0μM-320μM) were added to each well and cultured for 24 hours. The CCK8 detection kit was used to detect the inhibitory ability of NRK-49F cell proliferation, and this experiment was repeated three times.

[0189] Results: Fig.29 As shown, LYY-12 concentration>20μM, LYY-38>320μM, LYY-40>160μM, LYY-68 concentrations of 20μM and 320μM, LYY-103>10μM, and HL-14, 17, 18>10μM had a significant inhibitory effect on the proliferation of NRK-49F cells, preliminarily verifying that LYY-12, 38, 40, 68, 103 and HL-14, 17, 18 have the potential to treat chronic kidney disease in cells, and LYY-66 has a weaker potential for treating chronic kidney disease in cells.

[0190] Example 4

[0191] Inhibitory effects of furano[2,3-b]quinoline derivatives on HK-2 cell proliferation

[0192] Methods: HK-2 cells belong to the normal adult renal tubular epithelial cell line and are a layer of cells outside the renal tubules. HK-2 cells were cultured in complete DMEM medium (containing 10% fetal bovine serum, 100μg / mL penicillin, 100μg / mL streptomycin) at 37 degrees Celsius and 5% CO2 saturated humidity. The medium was changed every 24 hours. Logarithmically growing cells were taken, digested with 0.25% trypsin, the cell concentration was diluted with complete medium, and inoculated into 96-well cell culture plates. After culturing overnight, different concentrations of LYY12, 38, 40, 66, 68, 103, HL-14, 17, 18 (0μM-640μM) were added to each well and cultured for 24 hours. The CCK8 detection kit was used to detect the ability to inhibit HK-2 cell proliferation. This experiment was repeated three times.

[0193] Results: Fig.30 As shown, LYY-12, 66, 103>160μM have a significant inhibitory effect on the proliferation of HK-2 cells; LYY-40>40μM, HL-14, 18>20μM, HL-17>10μ have a significant inhibitory effect on the proliferation of HK-2 cells, LYY38, 68 have no significant inhibitory effect on the proliferation of HK-2 cells in the concentration range of 0μM-320μM, and do not affect the proliferation of normal cells (*P<0.05, **P<0.01, ***P<0.001). Experiments have shown that LYY12 has an inhibitory effect on the proliferation of NRK-49F cells at a concentration of 20μM to 80μM, but has no inhibitory effect on the proliferation of normal HK-2 cells, so LYY-12 is preferred for the following experiments.

[0194] Example 5

[0195] 0.2% adenine diet induced chronic kidney disease mouse model

[0196] Methods: A chronic kidney disease model was induced by feeding C57BL / 6 mice with a diet containing 0.2% adenine for 21 consecutive days. There were two groups of mice in the experiment, with 6 mice in each group. The mice in the normal group consumed normal diet, and the mice in the experimental group consumed a diet containing 0.2% adenine. They were killed on the 22nd day, and the eyeballs were removed to obtain blood from the mice. After standing at 37℃ for 30 minutes, the serum was centrifuged at 13000rpm for 10 minutes to obtain renal function test; the right kidney of the mouse was taken and fixed with 4% paraformaldehyde, and the pathological changes of renal tissue were detected by HE staining and Masson staining.

[0197] Results: Fig.31 As shown in the figure, the creatinine and urea nitrogen values ​​of the normal group were within the normal range, and the renal tissue pathology showed no pathological damage and no obvious collagen deposition. Compared with the normal group, the creatinine and urea nitrogen values ​​of the mice in the model group increased significantly, and the pathological results showed that the renal tubules in the cortex of the mice's renal tissue were severely dilated and atrophied, the cell casts and protein casts in the cortex increased, the interstitial inflammatory infiltration increased, and the collagen deposition was serious. This shows that the method was successful in modeling chronic kidney disease mice.

[0198] Example 6

[0199] Furo[2,3-b]quinoline derivatives improve renal function in mice with chronic kidney disease induced by 0.2% adenine diet

[0200] Methods: A chronic kidney disease model was induced by feeding C57BL / 6 mice with 0.2% adenine feed every day for 21 consecutive days. Drugs were administered at the same time as the intake of adenine feed. Mice were gavaged with LYY12 10 mg / kg, 20 mg / kg, 40 mg / kg and allopurinol 20 mg / kg for 21 days and killed on the 22nd day. Blood was collected from the normal group, model group, and mice gavaged with three doses of LYY12 and allopurinol after induction with 0.2% adenine feed. The blood was collected and placed at 37°C for 30 min. Then the mice were centrifuged at 13000 rpm for 10 min to obtain serum for renal function detection.

[0201] Results: Fig.32 As shown in the results, 10mg / kg, 20mg / kg and 40mg / kg LYY12 can reduce plasma creatinine and urea nitrogen values ​​to varying degrees and improve renal function. The improvement effect becomes better with increasing concentration. The effect of LYY-12 40mg / kg on improving the increase of creatinine value is equivalent to that of allopurinol, and the effect on improving the increase of urea nitrogen value is better than that of allopurinol (*P<0.05, **P<0.01, ***P<0.001).

[0202] Example 7

[0203] Furo[2,3-b]quinoline derivatives improve renal pathological damage in mice with chronic kidney disease induced by 0.2% adenine diet

[0204] Methods: A chronic kidney disease model was induced by allowing C57BL / 6 mice to voluntarily ingest 0.2% adenine feed daily for 21 consecutive days. Drugs were administered at the same time as the ingestion of adenine feed. Mice were gavaged with LYY12 10 mg / kg, 20 mg / kg, 40 mg / kg and allopurinol 20 mg / kg for 21 days and killed on the 22nd day. The right kidneys of mice in the normal group, model group, and the group induced by 0.2% adenine feed and gavaged with three doses of LYY12 and allopurinol were obtained and fixed with 4% paraformaldehyde. The pathological changes of renal tissues were detected by HE staining and Masson staining.

[0205] Results: Fig.33As shown in the figure, the HE staining scoring results showed that after 0.2% adenine feed induction, the renal tubules in the cortex of the mice were severely dilated and atrophied, the cell casts and protein casts in the cortex increased, and the interstitial inflammatory infiltration increased. After oral administration of LYY12 10mg / kg, 20mg / kg, 40mg / kg and allopurinol, the renal tissue showed different degrees of improvement. Compared with allopurinol, high-dose LYY12 can more significantly improve the pathological damage of the kidney in mice. The Masson staining scoring results showed that after 0.2% adenine feed induction, the renal tubular interstitial collagen deposition in mice was serious. After oral administration of LYY12 20mg / kg, 40mg / kg and allopurinol, the collagen deposition showed different degrees of improvement. Compared with allopurinol, high-dose LYY12 can more significantly improve the collagen deposition in mice (*P<0.05, **P<0.01, ***P<0.001).

[0206] The research results show that high-dose LYY12 can more significantly improve the renal function and renal structure damage of mice compared with allopurinol, indicating that LYY12 has a better therapeutic effect.

Claims

1. Use of a furano[2,3-b]quinoline derivative or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating chronic kidney disease, characterized in that: The structure of the furano[2,3-b]quinoline derivative is shown in formula (I): wherein R1 is methoxy; R4 is hydrogen or methoxy; When R2 is hydrogen or hydroxyl; R3 is hydrogen, hydroxyl or methoxy; When R2 is a methoxy group, R3 is a hydroxyl group or a methoxy group.

2. The use according to claim 1, characterized in that: The structure and substituents of the furano[2,3-b]quinoline derivatives are shown in the following table:

3. A method for extracting furano[2,3-b]quinoline derivatives, characterized in that: The following steps are involved: S1. Crush the branches and leaves of Rutaecarpa, add water for ultrasonic extraction, concentrate the extract, filter, and set aside the residue. Adsorb the filtrate on a macroporous resin column, and elute with water, 50% ethanol and 90% ethanol in turn. Collect the eluate with 90% ethanol and concentrate to obtain a fluid extract. Apply the fluid extract on a normal phase silica gel column and elute with a dichloromethane-methanol solution gradient. Combine similar fractions and dry to obtain fractions YXC-1 to YXC-14. S2, YXC-5 was loaded onto a gel column, eluted with petroleum ether-dichloromethane-methanol solution, similar fractions were combined and dried to obtain fractions YXC5-1 to YXC5-6; S3, subjecting YXC5-2 to reverse phase semi-preparative HPLC to obtain a furano[2,3-b]quinoline derivative; The structure of the furano[2,3-b]quinoline derivative is shown below: In the petroleum ether-dichloromethane-methanol solution, the volume ratio of petroleum ether-dichloromethane-methanol is (5-20):5:1; the column of the reversed-phase semi-preparative HPLC is a phenyl column, and the mobile phase is acetonitrile-water with a volume ratio of 29:

71.

4. The method for extracting furano[2,3-b]quinoline derivatives according to claim 3, characterized in that: The Rutaecarpa branches and leaves were ultrasonically extracted twice, each time for 1 hour, and the extracts were combined; the macroporous resin column was a D101 macroporous resin column.

5. The method for extracting furano[2,3-b]quinoline derivatives according to claim 3, characterized in that: The fractions were eluted with a gradient of dichloromethane-methanol solution, with the volume ratio of dichloromethane to methanol ranging from 100:0 to 0:1; before merging similar fractions, identification fractions were also included, and identification was performed by tracking the spot plate with thin layer chromatography.

6. The method for extracting furano[2,3-b]quinoline derivatives according to claim 3, characterized in that: The residue was ultrasonically extracted with 80% ethanol solution, the extract was concentrated and filtered, the filtrate was extracted with petroleum ether for three times, the petroleum ether layers were combined and extracted with ethyl acetate to obtain ethyl acetate extract; the ethyl acetate extract was loaded on a normal phase silica gel column and eluted with a dichloromethane-methanol solution gradient, similar fractions were combined and dried to obtain fractions YXY-1 to YXY-18; YXY-4 was loaded onto a normal phase silica gel column and eluted with a petroleum ether-ethyl acetate system gradient, similar fractions were combined and dried to obtain fractions YXY4-1 to YXY4-10; YXY4-6 precipitated into crystals, which was a furano[2,3-b]quinoline derivative.

7. The method for extracting furano[2,3-b]quinoline derivatives according to claim 6, characterized in that: The soluble part left after the crystallization of YXY4-6 was loaded on a gel column and eluted with a dichloromethane-methanol system. Similar fractions were combined and dried to obtain fractions YXY4-6-1 to YXY4-6-6. Crystals of YXY-4-6-3 and YXY-4-6-5 were analyzed, which were furano[2,3-b]quinoline derivatives.

8. The method for extracting furano[2,3-b]quinoline derivatives according to claim 6, characterized in that: The residue was ultrasonically extracted twice, each time for 1 h, and the extracts were combined.

9. The method for extracting furano[2,3-b]quinoline derivatives according to claim 6, characterized in that: During the gradient elution of petroleum ether-ethyl acetate solution, the volume ratio of petroleum ether to ethyl acetate ranged from 4:1 to 1:2; before merging similar fractions, identification of fractions was also included, and identification was performed by tracking spot plates with thin layer chromatography.

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