CN116284018A - Preparation method and application of furo [2,3-b ] quinoline derivative - Google Patents

Preparation method and application of furo [2,3-b ] quinoline derivative Download PDF

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CN116284018A
CN116284018A CN202310319881.2A CN202310319881A CN116284018A CN 116284018 A CN116284018 A CN 116284018A CN 202310319881 A CN202310319881 A CN 202310319881A CN 116284018 A CN116284018 A CN 116284018A
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furo
compound
ethyl acetate
quinoline derivative
extracting
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刘韶
蒋跃平
彭张哲
方静
陶立坚
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Xiangya Hospital of Central South University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C07D491/048—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being five-membered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to the field of biological medicine, in particular to furo [2,3-b ]]Use of quinoline derivatives, tautomers, pharmaceutically acceptable salts, prodrugs or solvates thereof for the preparation of a medicament for the treatment of chronic kidney disease, said furo [2,3-b ]]The quinoline derivative has a structure shown in a formula (I):
Figure DDA0004151307390000011
wherein R is 1 Is methoxy; r is R 2 Is hydrogen, hydroxy, methoxy; r is R 3 Is hydrogen, hydroxy, methoxy; r is R 4 Is hydrogen or methoxy. Experiments show that the compound has obvious effect of treating chronic kidney diseases. The compound of the invention has the characteristics of diversity of preparation routes, economy, definite activity, low toxicity and the like, and has wide application prospect.

Description

Preparation method and application of furo [2,3-b ] quinoline derivative
Technical Field
The invention belongs to the fields of medicine and chemical industry, and particularly relates to a preparation method and application of a furo [2,3-b ] quinoline derivative.
Background
Chronic kidney disease (RF) is a chronic progressive fibrotic kidney disease associated with almost all chronic kidney diseases (chronickidney disease, CKD) and progressive kidney disease. The clear pathogenesis of RF is currently unknown. There are many factors involved in RF pathogenesis, and after the kidney is affected by a variety of factors such as injury, infection, hypertension and bad lifestyle, the kidney undergoes cell damage, fibrosis and sclerosis, which is the process of kidney fibrosis, and its pathological features are injury, inflammation, myofibroblast activation and migration and tubular epithelial cell-mesenchymal transition, and extracellular matrix deposition and remodeling. Deposition of fibrous matrix after injury may initially aid in tissue repair, and fibrous matrix can be absorbed during tissue repair after mild injury. However, after chronic injury to CKD occurs, the injury continues, the fibrous matrix is not fully absorbed, fibrous matrix deposition is not inhibited, and eventually the organ structure is destroyed, blood supply is reduced, and organ function is disturbed. Fibrosis reduces the ability of tissue repair, ultimately leading to renal failure.
From the current research, inhibition of myofibroblast activation and differentiation, reduction of inflammatory response, removal of extracellular matrix deposition, etc. are major approaches to improve chronic kidney disease. In view of the increasing prevalence of RF occurrences and associated mortality, there is an urgent need to develop safe and effective medicaments for the treatment of chronic kidney disease.
Furanoquinoline compounds are widely used in plants, such as dittany, common sage herb, pinus koraiensis and zanthoxylum bungeanum, and have certain biological activity, such as dictamnine (small dose) has exciting effect on isolated frog heart, can increase myocardial tension, has obvious contraction effect on isolated rabbit ear blood vessels, and has powerful contraction effect on rabbit and guinea pig uterine smooth muscle. In addition, it has antibacterial and skin eczema and skin pruritus treating effects. However, no report has been made on the evaluation of the RF-resistant activity of furoquinoline derivatives.
Disclosure of Invention
The invention aims to provide a preparation method of a furo [2,3-b ] quinoline derivative and application thereof in preparing medicines for treating chronic kidney disease.
In order to achieve the above object, the technical scheme of the present invention is as follows:
use of a furo [2,3-b ] quinoline derivative, a tautomer, a pharmaceutically acceptable salt, a prodrug or a solvate thereof in the manufacture of a medicament for the treatment of chronic kidney disease, wherein the structure of the furo [2,3-b ] quinoline derivative is as shown in formula (i):
Figure BDA0004151307370000011
Wherein R is 1 Is methoxy; r is R 2 Is hydrogen, hydroxy, methoxy; r is R 3 Is hydrogen, hydroxy, methoxy; r is R 4 Is hydrogen or methoxy.
Preferably, the structure and substituents of the furo [2,3-b ] quinoline derivatives are as shown in table 1:
TABLE 1 substituents and structures of furan [2,3-b ] quinoline alkaloids of the invention
Figure BDA0004151307370000021
Figure BDA0004151307370000031
Preferably, the medicament further comprises a pharmaceutically acceptable carrier, adjuvant or excipient.
The above-described pharmaceutically acceptable "carrier, adjuvant or vehicle" refers to a pharmaceutical carrier conventional in the pharmaceutical arts, such as: diluents, excipients such as water, etc., fillers such as starch sucrose, etc.; binding agents such as cellulose derivatives, gelatin, etc.; other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.
The compounds of the present invention may be in crystalline form as advantageous compounds or as solvates. Methods of solvation are well known in the art, and suitable solvates are pharmaceutically acceptable solvates. In a specific embodiment, the solvate is a hydrate.
Preferably, the chronic kidney disease is renal fibrosis.
A method for extracting and preparing a furo [2,3-b ] quinoline derivative, wherein the structural formula of the furo [2,3-b ] quinoline derivative is as follows:
Figure BDA0004151307370000032
The extraction and preparation method of the furo [2,3-b ] quinoline derivative comprises the following steps:
s1, crushing branches and leaves of rutin, adding water for ultrasonic extraction, concentrating the extract, filtering, adsorbing the filtrate by a macroporous resin column, eluting with water, 50% ethanol and 90% ethanol in sequence, collecting an eluent of 90% ethanol, and concentrating to obtain a fluid extract; loading the fluid extract on a normal phase silica gel column, gradient eluting with dichloromethane-methanol solution, mixing similar fractions, and drying to obtain fractions YXC-1-YXC-14;
s2, loading the YXC-5 on a gel column, eluting with petroleum ether-dichloromethane-methanol solution, merging similar fractions, and drying to obtain fractions YXC 5-1-YXC 5-6;
s3, subjecting YXC5-2 to reverse phase semi-preparative HPLC to obtain the compound furo [2,3-b ]]Quinoline derivative LYY-12 (t) R ＝40.134min)。
Preferably, the branches and leaves of Ruta graveolens are extracted by ultrasound twice for 1h each time, and the extracts are combined.
Preferably, the macroporous resin column is a D101 macroporous resin column.
Preferably, the elution is performed with a gradient of dichloromethane-methanol solution, with a volume ratio of dichloromethane to methanol ranging from 100:0 to 0:1.
The thin layer plate is washed while the solvent is eluted until the solvent is free of substances, namely the next gradient is replaced.
Preferably, the identification fractions are also included before combining the similar fractions, and are identified by thin layer chromatography tracking spots.
Preferably, the volume ratio of petroleum ether to methylene chloride to methanol in the petroleum ether to methylene chloride to methanol solution is (5-20) to 5:1, and most preferably 5:5:1.
The separation effect of the petroleum ether-methylene chloride-methanol solution in this ratio is optimal in the system tried, just to separate the substances according to different polarities. With either the methylene chloride and methanol system (10:1, 5:1, 2:1) or the petroleum ether and ethyl acetate system (10:1, 5:1, 2:1), partial compound separation occurred.
Preferably, the column of the reverse phase semi-preparative HPLC is a phenyl column and the mobile phase is acetonitrile-water in a volume ratio of 29:71. The separation effect (peak shape and retention time, degree of separation) of the phenyl column was superior to that of the C18 and C8 columns. According to the results of multiple tests, the separation effect in the mobile phase in the ratio of 29:71 is the best, and the separation effect is obviously better than that of an acetonitrile-water solution in the ratio of (40-50): in the ratio of (50-60).
Preferably, the residue is added with 80% ethanol solution, the mixture is subjected to ultrasonic extraction, the extract is concentrated, the filtrate is extracted with petroleum ether for three times, the petroleum ether layer part is combined and extracted with ethyl acetate for three times, and the ethyl acetate extract is obtained after concentration and freeze-drying. And (3) loading the ethyl acetate extract on a normal phase silica gel column, gradient eluting with dichloromethane-methanol solution, combining similar fractions, and drying to obtain fractions YXY-1-YXY-18.
And (3) loading the mixture on a YXY-4 normal phase silica gel column, performing gradient elution by using a petroleum ether-ethyl acetate system, merging similar fractions, and drying to obtain fractions YXY 4-1-YXY 4-10.
YXY4-6 precipitated crystals were identified as the furo [2,3-b ] quinoline derivative LYY-12.
Preferably, the residual dissolved part after YXY4-6 crystallization is dissolved by methanol and then is put on a Sephadex LH-20 gel column, a methylene dichloride-methanol system (1:1) is used for eluting, similar fractions are combined and dried, and fractions YXY4-6-1 to YXY4-6-6 are obtained; the YXY-4-6-3 and YXY-4-6-5 are partially separated out to obtain crystals, namely the furo [2,3-b ] quinoline derivative LYY-12.
Preferably, the residue is sonicated twice, 1h each time, and the extracts are combined.
Preferably, the petroleum ether-ethyl acetate solution is eluted in a gradient, and the volume ratio of petroleum ether to ethyl acetate is from 4:1 to 1:2.
Preferably, in the extraction preparation method of the compound furo [2,3-b ] quinoline derivative LYY-12, LYY-12 is mainly in the alcohol extraction part of the medicine residue. Preferably, the preparation method of the furo [2,3-b ] quinoline derivative comprises the following steps:
(1) Sequentially dripping malonic acid diester, chloroacetyl chloride and a catalyst into strong alkali for reaction to obtain a compound 1;
the malonic diester has the structure that:
Figure BDA0004151307370000041
Wherein R is selected from C1-C4 branched or straight chain alkyl, phenyl or substituted phenyl; the substituent of the substituted phenyl group comprises halogen, C1-C4 branched or straight-chain alkyl and C1-C4 alkoxy;
(2) Dripping arylamine into the compound 1 prepared in the step (1), adding an aqueous solution of ethyl acetate for precipitation after reaction, and obtaining a solid-phase compound 2 and a solution;
the structure of the arylamine is as follows:
Figure BDA0004151307370000042
R 2 、R 3 and R is 4 Independently selected from hydrogen, hydroxy, halogen, C1-C4 linear or branched alkyl, C1-C4 alkoxy, benzyloxy;
(3) Adding the compound 2 into an inert solvent, heating to 250-260 ℃, refluxing and stirring for 0.5-1 h, adding petroleum ether, filtering and drying to obtain a compound 3;
(4) Adding a chloro reagent into the compound 3 for reaction, and then extracting and concentrating the mixture at a low temperature by using dichloromethane to obtain a compound 4;
(5) Adding a reducing agent into the compound 4 for reaction in ice bath, concentrating, extracting with dichloromethane, washing with water, and drying to obtain a compound 5;
(6) Adding potassium bisulfate or hydrochloric acid into the compound 5, carrying out reflux reaction for 3-5 h, extracting with ethyl acetate, neutralizing with alkali, and drying to obtain a compound 6;
(7) Adding a nucleophilic reagent into the compound 6 under the action of alkali, carrying out reflux reaction for 2-3d, extracting with ethyl acetate, washing with water, and drying to obtain a crude product, and separating and purifying the crude product by normal phase column chromatography to obtain the furo [2,3-b ] quinoline derivative;
The structure of the furo [2,3-b ] quinoline derivative is as follows:
Figure BDA0004151307370000051
wherein R is 1 Is methoxy; r is R 2 Is hydrogen, hydroxy, methoxy; r is R 3 Is hydrogen, hydroxy, methoxy; r is R 4 Is hydrogen or methoxy.
Preferably, in the steps (1) and (2), the C1-C4 branched or straight-chain alkyl group comprises methyl, ethyl, isopropyl, n-propyl, n-butyl and tert-butyl.
Preferably, in the steps (1) and (2), the C1-C4 alkoxy group is selected from methoxy, ethoxy and propoxy.
Preferably, in the step (1), the strong base is one of sodium hydride, potassium tert-butoxide and sodium tert-butoxide.
Preferably, in the step (1), the catalyst is selected from triethylamine, N-diisopropylethylamine or 1, 8-diaza [5,4,0] undecene-7; further preferred is triethylamine. The catalyst is not added in the step, and the yield of the product is extremely low.
Preferably, the reaction temperature of the step (1) is 0-30 ℃.
Preferably, in the step (1), after the chloroacetyl chloride is added for reaction for 1 to 2 hours, triethylamine is added, and the reaction is continued for 2 to 4 hours.
Preferably, the solution in the step (2) is divided into an organic phase and a water phase, the organic phase is concentrated under reduced pressure, and is stirred and dispersed by ether such as diethyl ether, and the crude product is obtained by filtration, and is recrystallized by ethanol to obtain a pure product of the compound 2.
Preferably, the inert solvent in the step (3) is a solvent which does not contain active hydrogen and ester groups and does not react with the reactant at high temperature. Preferably the inert solvent is diphenyl ether.
Preferably, the chlorinating agent in step (4) is selected from phosphorus oxychloride, phosphorus trichloride, phosphorus pentachloride, oxalyl chloride, thionyl chloride, sulfonyl chloride or chlorine gas, preferably phosphorus oxychloride. Phosphorus oxychloride has the best selectivity in the reaction system of the invention.
Preferably, the chlorination reaction of step (4) also incorporates a surfactant, preferably trioctylmethyl ammonium chloride. Under the promotion of the dissolution of the surfactant, the phosphorus oxychloride further improves the selectivity of the reaction.
The extraction reagent in the step (3-6) is optimized for a plurality of times, the effect is optimal, and the selectivity which cannot be achieved by other organic solvents can be obtained.
Preferably, the reducing agent of step (5) is selected from sodium borohydride, sodium borocyanide, lithium tetraborate, lithium tetrahydride aluminate or diisobutyl aluminum hydride.
Preferably, the mobile phase of the normal phase column chromatography in the step (7) is a mixed solution with the volume ratio of petroleum ether to ethyl acetate being 2/1.
Preferably, the base in the step (7) comprises sodium hydride, sodium hydroxide, sodium alkyl alkoxide or potassium alkyl alkoxide, and the sodium alkyl alkoxide comprises sodium methoxide, sodium ethoxide or sodium tert-butoxide.
Preferably, the nucleophile in step (7) is selected from alcohols, phenols, thiols, thiophenols, amines, N-heterocyclic compounds.
Preferably, the furo [2,3-b ] quinoline derivative is synthesized by the following route:
Figure BDA0004151307370000061
the synthesis method of the invention takes malonic diester and arylamine as starting materials, and synthesizes the furo [2,3-b ] quinoline derivative through seven steps of reactions. The chemical synthesis method has simple reaction, the extraction process is optimized, the intermediate step only needs simple extraction and filtration, the processing operation of the finished product is simple and convenient, the furo [2,3-b ] quinoline derivative pure product can be prepared only through one-time column chromatography separation, and each step does not need column separation like the prior art.
The invention is further explained below:
the invention discovers a compound LYY12 from Ruta graveolens, is identified as vanilloid, and discovers that LYY12 has the kidney effect of improving chronic kidney disease for the first time through research. Xiangguoning, english name kokuseagine, alias: 6, 7-dimethoxydiminune, which is abbreviated LYY12 in the present invention. The International Union of Pure and Applied Chemistry (IUPAC) names 4,6,7-trimethoxyfuro [2,3-b ] quinoline of formula (I), wherein the name is 4,6,7-trimethoxy furan [2,3-b ] quinoline. Its molecular formula: C14H13NO4, molecular weight: 259.257. the chemical abstract number (CAS) is: 484-08-2. Is an alkaloid extracted from plants of Dictamni (Dictamnus dasycarpus Turcz), and can be derived from plants of Cynanchum (rutagaveolens), cynanchum (orixajaponica), zanthoxylum bungeanum (zanthoxylum bungeanum maxim.), dictyophora (orimasp.), acronomum (acronychiasp.), evodia (evodia sp.), picea (halopropylalumsp.), and Melicope (melicope). It has increased norepinephrine and dopamine levels (chem. Res. Toxicol,2014,27 (2), 219-239); phototoxic effects (Planta medical 1981,41 (2), 136-42); insecticidal (Tennen Yuki Kagobutsu Toronkai Koen Yoshishu 1999,41st, 451-456), anticoccidial activity (chemmed chem 2022,17 (5), e 202100784), anti-inflammatory (j. Ethn opharmacol.2019,238,111827; bio. Med. Chem.2011,19 (21), 6340-6347;AJPTR 2017,7 (2), 315-324), antiproliferative activity against human cancer cell lines (HL-60, smmc-7721, a-549, mcf-7 and SW 480) (nat. Pro. Res.2022,36, 379-384), inhibition of human breast cancer cell growth and multidrug resistance (bio. Med. Chem. Lett.2018,28 (14), 2490-2492) and antispasmodic action (AJPTR 2017,7 (2), 315-324), and the like.
According to the invention, by establishing a chronic kidney disease animal model, the therapeutic effect of LYY12 on the chronic kidney disease of mice is observed. The research result shows that LYY12 has good therapeutic effect on chronic kidney disease.
According to the invention, the inhibition effect of LYY and HL series compounds on chronic kidney diseases is studied by human renal cortex proximal tubular epithelial cells (HK-2) and rat kidney fibroblasts (NRK-49F), and the proliferation of cells after incubation at different concentrations is detected by CCK8, so that LYY12 is screened out for the next animal experiment.
The experimental animals were male and 7-week-old C57 mice. All mice were fed with adaptability for one week before the experiments were formally developed, and all mice had free to ingest water with a 12h day-night period. LYY12 was dissolved in 0.5% CMC-Na, and the following doses were prepared at different concentrations in the following groups, and mice were randomly assigned to the blank group (N), model group (M), LYY12 high dose group (LYY 12-H), LYY12 medium dose group (LYY 12-M), and LYY12 low dose group (LYY 12-L). The mice in the other groups except the blank group need to be established with a chronic kidney disease model.
According to the invention, a chronic kidney disease model is built by independently taking 0.2% adenine feed daily for 21 continuous days in a C57BL/6 mouse, and administration is started while the adenine feed is taken, and CMC-Na, LYY12 mg/kg/day (low dose), 20mg/kg/day (medium dose), 40mg/kg/day (high dose) and allopurinol 20mg/kg/day are respectively administrated by lavage according to a volume of 10ml/kg for 21 days. The research results show that different doses of LYY12 have different degrees of improvement on chronic kidney disease. After 0.2% adenine feed induction, the weight of mice is obviously reduced, urea nitrogen and creatinine values are increased, the expansion and atrophy of cortical tubular is serious, the tubular and protein tubular type in cortex is increased, interstitial inflammatory infiltration is increased, collagen deposition is aggravated, and after LYY12 administration treatment, the renal function of the mice is obviously improved, the damage of renal tissue structures is obviously improved, the collagen deposition condition is relieved, and meanwhile LYY12 can obviously reduce the renal tissue inflammation of the mice, so the results fully indicate that LYY12 can effectively treat chronic kidney diseases.
LYY12 is prepared by conventional natural product chemistry, separation and purification and pharmaceutical synthesis methods. The laboratory adopts High Performance Liquid Chromatography (HPLC) analysis and detection, the purity of the LYY12 product reaches more than 99 percent, and the analysis and identification by mass spectrometry and nuclear magnetic resonance method show that the LYY12 product used in the laboratory has correct chemical structure. This study shows that LYY12 has purity and chemical structure in accordance with the requirement of in vivo and in vitro biological activity and pharmacological action.
The beneficial effects of the invention are as follows:
the invention discovers that the furo [2,3-b ] quinoline derivative has a therapeutic effect on chronic kidney disease for the first time; specifically, a chronic kidney disease model was established by inducing spontaneous daily intake of 0.2% adenine feed in C57BL/6 mice for 21 consecutive days, and administration was started while the adenine feed was taken, and LYY1210mg/kg, 20mg/kg and 40mg/kg were administered to the mice by gavage for 21 days, respectively. The research result shows that compared with allopurinol, the high-dose LYY12 can remarkably reduce the weight reduction of mice and improve the kidney function and the kidney structure injury of the mice, and the LYY12 has better treatment effect. Diethyl malonate and 3, 4-dimethoxy aniline are used as raw materials, the furo [2,3-b ] quinoline derivative is synthesized through seven steps of reactions, the synthesis method is simple, the operation is simple and convenient, the synthesis route provides an efficient synthesis method for vanillyl tannin, and a feasible route and scheme are provided for industrial production of the vanillyl tannin.
Drawings
FIG. 1 is a HRESI-MS of compound LYY 12;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of LYY 12;
FIG. 3 is a nuclear magnetic resonance carbon spectrum of LYY 12;
FIG. 4 is a HRESI-MS of compound LYY 38;
FIG. 5 shows a hydrogen nuclear magnetic resonance spectrum of LYY 38;
FIG. 6 is a nuclear magnetic resonance carbon spectrum of compound LYY 38;
FIG. 7 is a HRESI-MS of compound LYY 40;
FIG. 8 is a hydrogen nuclear magnetic resonance spectrum of LYY 40;
FIG. 9 is a nuclear magnetic resonance carbon spectrum of LYY 40;
FIG. 10 is a HRESI-MS of compound LYY 66;
FIG. 11 is a nuclear magnetic resonance hydrogen spectrum of LYY 66;
FIG. 12 is a nuclear magnetic resonance carbon spectrum of compound LYY 66;
FIG. 13 is a HRESI-MS of compound LYY 68;
FIG. 14 is a nuclear magnetic resonance hydrogen spectrum of LYY 68;
FIG. 15 is a HRESI-MS of compound LYY 103;
FIG. 16 is a nuclear magnetic resonance hydrogen spectrum of LYY 103;
FIG. 17 is a nuclear magnetic resonance carbon spectrum of compound LYY 103;
FIG. 18 is a HRESI-MS plot of compound HL-14;
FIG. 19 is a nuclear magnetic resonance hydrogen spectrum of compound HL-14;
FIG. 20 is a nuclear magnetic resonance carbon spectrum of compound HL-14;
FIG. 21 is a HRESI-MS plot of compound HL-17;
FIG. 22 is a nuclear magnetic resonance hydrogen spectrum of compound HL-17;
FIG. 23 is a nuclear magnetic resonance carbon spectrum of compound HL-17
FIG. 24 is a HRESI-MS plot of compound HL-18;
FIG. 25 is a nuclear magnetic resonance hydrogen spectrum of compound HL-18;
FIG. 26 is a nuclear magnetic resonance hydrogen spectrum of compound 2 a;
FIG. 27 is a nuclear magnetic resonance hydrogen spectrum of compound 3 a;
FIG. 28 is a nuclear magnetic resonance hydrogen spectrum of vanilloid;
FIG. 29 shows the effect of LYY, HL series of compounds on NRK-49F cell proliferation;
FIG. 30 shows the effect of LYY, HL series of compounds on HK-2 cell proliferation;
FIG. 31 is a graph showing the modeling results of 0.2% adenine feed-induced chronic kidney disease mice;
FIG. 32 shows the effect of LYY12 on kidney function in chronic kidney disease mice;
FIG. 33 shows the effect of LYY12 on kidney tissue of a chronic kidney disease mouse
Detailed description of the preferred embodiments
The following examples will assist those skilled in the art in a more complete understanding of the invention, but are not intended to limit the invention in any way.
Example 1
Extraction of furo [2,3-b ] quinoline derivatives
Pulverizing 10kg of Ruta graveolens leaves, adding water solution, performing ultrasonic extraction twice for 1h each time, mixing the extractive solutions, concentrating, filtering to obtain water extractive solution, standing residues, adsorbing with D101 macroporous resin column, sequentially eluting with water, 50% ethanol and 90% ethanol, collecting water, 50% ethanol and 90% ethanol eluents, and concentrating to obtain fluid extract of three elution parts. Wherein about 14.3g of 90% ethanol partial flow extract is put on a normal phase silica gel column, the mixture is eluted in a gradient way by a methylene dichloride-methanol system (the volume ratio of methylene dichloride to methanol is from 100:0 to 0:1), a point plate is tracked by thin layer chromatography, similar fractions are combined, and the mixture is dried under reduced pressure, and fractions YXC-1 to YXC-14 are obtained by combining.
Applying YXC-3 to Sephadex LH-20 gel column, tracking with petroleum ether-dichloromethane-methanol system (5:5:1), thin layer chromatography, mixing similar fractions, drying under reduced pressure, mixing to obtain fractions YXC 3-1-YXC 3-10, YXC3-6, subjecting to reversed-phase semi-preparative HPLC and phenyl column, and using 29% acetonitrile-water as mobile phase to obtain fraction YXC3-6-3 to obtain compound LYY-68 (6 mg) (t R = 35.601 min) to give LYY-66 (8 mg) (t) R ＝39.956min)。
Applying YXC-5 to Sephadex LH-20 gel column, tracking with petroleum ether-dichloromethane-methanol system (5:5:1), thin layer chromatography, mixing similar fractions, drying under reduced pressure, mixing to obtain fractions YXC 5-1-YXC 5-6, subjecting YXC5-2 to reverse phase semi-preparative HPLC, and phenyl column with 29% acetonitrile-water as mobile phase to obtain LYY-12 (10 mg) (t) R ＝40.134min)。
Applying YXC-6 to Sephadex LH-20 gel column, tracking with petroleum ether-dichloromethane-methanol system (5:5:1), thin layer chromatography, mixing similar fractions, drying under reduced pressure, mixing to obtain YXC 6-1-YXC 6-11, subjecting YXC6-4 to reversed-phase semi-preparative HPLC, subjecting to phenyl column with 20% acetonitrile-water as mobile phase to obtain YXC6-4-4, and subjecting YXC6-4-4 to reversed-phase semi-preparative HPLC with 20% acetonitrile-water as mobile phase to obtain LYY-38 (8 mg) (t) R ＝37.353min)。
YXC6-5 was subjected to reversed-phase semi-preparative HPLC using 30% acetonitrile-water as mobile phase to give a fraction YXC6-5-2, and YXC6-5-2 was further subjected to reversed-phase semi-preparative HPLC using 30% acetonitrile-water as mobile phase to give compound LYY-40 (12 mg) (t R ＝18.581min)。
And (3) loading the YXC6-2 on a normal phase silica gel column, eluting with a petroleum ether-ethyl acetate system (2:1), tracking a point plate by using a thin layer chromatography, merging similar fractions, drying under reduced pressure, and merging to obtain fractions YXC 6-2-1-YXC 6-2-11.YXC6-2-9 was subjected to reversed-phase semi-preparative HPLC, C18 column, and 30% acetonitrile-water as mobile phase to give a fraction YXC6-2-9-2, YXC6-2-9-2 was subjected to reversed phase semi-preparative HPLC, phenyl column, and 59% acetonitrile-water as mobile phase to give LYY-103 (10 mg) (t) R ＝19.56min)。
Adding 80% ethanol solution into the residue, ultrasonic extracting twice, each for 1 hr, mixing the extractive solutions, concentrating, filtering to obtain ethanol extractive solution, extracting the ethanol extractive solution with petroleum ether for three times, mixing the petroleum ether layer parts, extracting with ethyl acetate for three times, concentrating, and lyophilizing to obtain ethyl acetate extract (80 g). The ethyl acetate extract is put on a normal phase silica gel column and eluted with a dichloromethane-methanol system (the volume ratio of dichloromethane to methanol is from 100:0 to 0:1), a thin layer chromatography is used for tracking a point plate, similar fractions are combined, and the mixture is dried under reduced pressure, so that fractions YXY-1-YXY-18 are obtained.
And (3) loading the mixture on a YXY-4 normal-phase silica gel column, performing gradient elution by using a petroleum ether-ethyl acetate system (the volume ratio of petroleum ether to ethyl acetate is from 4:1, 2:1, 1:1 and 1:2), tracking a point plate by using a thin-layer chromatography, merging similar fractions, drying under reduced pressure, and merging to obtain fractions YXY4-1 to YXY4-10. Dissolving YXY4-6 with methanol, loading the dissolved part on Sephadex LH-20 gel column, tracking a dot plate by using methylene dichloride-methanol system (1:1), merging similar fractions, drying under reduced pressure, and merging to obtain fractions YXY 4-6-1-YXY 4-6, wherein part of YXY-4-6-3 and part of YXY-4-6-5 are precipitated crystals to be LYY12. Insoluble fraction precipitated crystals were identified as compound LYY12. Separating YXY4-6-4 by preparative thin layer chromatography with dichloromethane-methanol system (20:1) to obtain YXY 4-6-4-1-YXY 4-6-4-4.YXY4-6-4-3 was subjected to reversed-phase semi-preparative HPLC, C18 column, with 38% methanol-formic acid water as mobile phase to give compound HL-14 (2 mg) (t) R 30.060 min) to give compound HL-18 (2 mg) (t) R = 46.783 min). YXY4-6-4-3 was subjected to reverse phase semi-preparative HPLC with a phenyl column with 38% methanol-formic acid water as mobile phase to give YXY4-6-4-3-6 as compound HL-17 (2 mg) (t) R ＝38.108min)。
Results: the structure of each compound was identified as follows:
LYY-12 is light yellow needle crystal, and has a chemical structural formula:
Figure BDA0004151307370000091
The spectral data are as follows: ESI-MS m/z 260.0924[ M+H ]] + (calcd for C 14 H 13 NO 4 ). ESI-MS is shown in FIG. 1. The nuclear magnetic resonance hydrogen spectrum and the nuclear magnetic resonance carbon spectrum are shown in fig. 2 and 3, respectively. 1 H NMR(500MHz,Methanol-d 4 )δ：7.72(d,J＝3.0Hz,3H),7.46(d,J＝2.4Hz,3H),7.28(s,3H),7.17(d,J＝2.4Hz,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-d 4 )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.
Compound LYY-38 is brown powder with chemical structural formula:
Figure BDA0004151307370000092
the spectral data are as follows: ESI-MS m/z 246.0774[ M+H ]] + (calcd for C 13 H 11 NO 4 ). ESI-MS is shown in FIG. 4, and nuclear magnetic resonance hydrogen spectrogram and nuclear magnetic resonance carbon spectrogram are shown in FIG. 5 and FIG. 6, respectively. 1 H NMR(600MHz,Methanol-d 4 )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＝1.9Hz,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-d 4 )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.
LYY-40 is brownish red powder, and has the chemical structural formula:
Figure BDA0004151307370000101
ESI-MS m/z:216.0682[M+H] + (calcd for C 12 H 9 NO 4 ). The ESI-MS is shown in FIG. 7, and the nuclear magnetic resonance hydrogen spectrum and the nuclear magnetic resonance carbon spectrum are shown in FIG. 8 and FIG. 9, respectively. 1 H NMR(600MHz,Methanol-d 4 )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-d 4 )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.
LYY-66 is white powder, and has the chemical structural formula:
Figure BDA0004151307370000102
ESI-MS m/z:230.0839[M+H] + (calcd for C 13 H 11 NO 3 ). The ESI-MS is shown in FIG. 10, and the hydrogen nuclear magnetic resonance spectrum and the carbon nuclear magnetic resonance spectrum are shown in FIG. 11 and FIG. 12, respectively. 1 H NMR(600MHz,Methanol-d 4 )δ：7.80(d,J＝2.8Hz,1H),7.77(s,1H),7.58(d,J＝2.9Hz,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-d 4 )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.
LYY-68 is white powder, and has the chemical structural formula:
Figure BDA0004151307370000103
ESI-MS m/z:200.0699[M+H] + (calcd for C 12 H 9 NO 2 ). ESI-MS is shown in FIG. 13, and nuclear magnetic resonance hydrogen spectrum is shown in FIG. 14. 1 H NMR(600MHz,Methanol-d 4 )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).
The compound LYY-103 is white needle-shaped and has a chemical structural formula:
the ESI-MS is shown in FIG. 15, and the hydrogen nuclear magnetic resonance spectrum and the carbon nuclear magnetic resonance spectrum are shown in FIG. 16 and FIG. 17, respectively. 1 H NMR(600MHz,Methanol-d 4 )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 C NMR(151MHz,MeOD)δ166.10,159.29,153.92,144.48,143.99,142.65,142.04,119.70,115.89,113.46,106.53,106.37,106.28,103.57,103.38,101.74,61.76,60.11,59.98,57.23,56.44,56.39,49.90,49.48,49.33,49.19,49.05,48.91,48.77,48.62.
Figure BDA0004151307370000111
Compound HL-14 is light yellow powder, and has a chemical structural formula:
ESI-MS m/z:216.0685[M+H] + (calcd for C 12 H 9 NO 3 ). The ESI-MS is shown in FIG. 18, and the hydrogen nuclear magnetic resonance spectrum and the carbon nuclear magnetic resonance spectrum are shown in FIG. 19 and FIG. 20, respectively. 1 H NMR(600MHz,Methanol-d 4 )δ：7.76(d,J＝2.8Hz,1H),7.73(d,J＝9.1Hz,1H),7.52(d,J＝2.7Hz,1H),7.33–7.26(m,2H),4.47(s,3H). 13 C 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.
Figure BDA0004151307370000112
Compound HL-17 is white powder, and has a chemical structural formula:
ESI-MS m/z:246.0793[M+H] + (calcd for C 13 H 11 NO 4 ). The ESI-MS is shown in FIG. 21, and the hydrogen nuclear magnetic resonance spectrum and the carbon nuclear magnetic resonance spectrum are shown in FIG. 22 and FIG. 23, respectively. 1 H NMR(600MHz,Methanol-d 4 )δ：7.70(d,J＝2.8Hz,1H),7.53(s,1H),7.29(d,J＝2.8Hz,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.
Figure BDA0004151307370000113
Compound HL-18 is yellow oil with a chemical structural formula:
ESI-MS m/z:246.0791[M+H] + (calcd for C 13 H 11 NO 4 ). ESI-MS is shown in FIG. 24, and nuclear magnetic resonance hydrogen spectrum is shown in FIG. 25. 1 H NMR(600MHz,Methanol-d 4 )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＝5.6Hz,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).
Figure BDA0004151307370000121
Example 2.
Chemical synthesis of Xiangcaoning
Figure BDA0004151307370000122
Scheme a: preparation of moldavica dragonhead by single column chromatography
Synthesis of compound 2 a:
method 1: in a 100mL round bottom flask, 4g of sodium hydride (60%) was rinsed with dry petroleum ether and then added to 20mL of tetrahydrofuran for dispersion, stirred at room temperature, and diethyl malonate (16 g,15 mL) was slowly added dropwise over 1h, and the addition was ready for use. Another 500mL reaction flask was taken, and chloroacetyl chloride (5.6 g,4 mL) was dissolved in 30mL dry tetrahydrofuran, followed by ice bathThe solution prepared in the previous step is slowly added into the reaction bottle in a dropwise manner within 1h, and the reaction temperature is controlled to be lower than 30 ℃. After the completion of the dropping, the mixture was stirred at room temperature for 1 hour, followed by addition of triethylamine (10 g,13.5 mL) and further stirring for 2 hours. A solution of 3, 4-dimethoxyaniline (7.8 g) in dry tetrahydrofuran (50 mL) was then slowly added dropwise over 1h under ice-bath at a controlled temperature of no more than 30deg.C and stirred overnight at room temperature after completion of the dropwise addition. After the completion of the reaction of the starting materials was analyzed by thin layer chromatography (petroleum ether: ethyl acetate 2:1), tetrahydrofuran was removed by concentration under reduced pressure, 300mL of ethyl acetate and 100mL of water were added for extraction, and during this time, solid filtration was produced, whereby 2.2g of pure compound 2a was obtained. Drying the organic phase of the filtrate, concentrating under reduced pressure, stirring with diethyl ether, filtering to obtain crude product, adding 10mL of ethanol, recrystallizing to obtain 3.2g of compound 2a as off-white solid with yield of 42%, and nuclear magnetic resonance hydrogen spectrum as shown in figure 26, with nuclear magnetic resonance data of 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)。
Method 2: in a 25mL round bottom flask, 0.3g of sodium hydride (60%) was rinsed with dry petroleum ether and then added to 2.1mL of tetrahydrofuran for dispersion, stirred at room temperature, and diethyl malonate (2.1 g,1.96 mL) was slowly added dropwise over 1h, and the addition was completed for use. Another 100mL reaction flask was taken, chloroacetyl chloride (0.77 g,0.54 mL) was dissolved in 2.9mL dry tetrahydrofuran, and the solution prepared in the previous step was slowly added dropwise to the reaction flask in 1h under ice bath, and the reaction temperature was controlled to be lower than 30 ℃. After the dripping is finished, stirring for 1h at room temperature. Subsequently, a solution of 3, 4-dimethoxyaniline (1 g) in dry tetrahydrofuran (4.3 mL) was slowly added dropwise over 1h under ice-bath at a controlled temperature of not more than 30℃and stirred overnight at room temperature after completion of the dropwise addition. After the completion of the reaction of the starting materials was analyzed by thin layer chromatography (petroleum ether: ethyl acetate 2:1), tetrahydrofuran was removed under reduced pressure at 40℃and extracted with 30mL of ethyl acetate and 10mL of water, the ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give 0.23g of Compound 2a in 16% yield.
Synthesis of compound 3 a:
in a 100mL reaction flask, compound 2a (2.2 g) was added to 22mL diphenyl ether, heated to 250-260 ℃ and stirred under refluxStirring for 1h, analyzing the raw materials by thin layer chromatography (petroleum ether: ethyl acetate 1:1) to react completely, and cooling to room temperature; 200mL of petroleum ether was slowly added dropwise to the reaction solution, the resulting solid was filtered, and the cake was rinsed with petroleum ether to give 1.8g of crude brown solid compound 3a, whose nuclear magnetic resonance hydrogen spectrum is shown in FIG. 27, and whose nuclear magnetic data were 1 H NMR(600MHz,DMSO-d 6 )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,1H),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)。
Synthesis of compound 4 a:
in a 25mL reaction flask, 1.8g of the above compound 3a was added, followed by 4mL of phosphorus oxychloride and 320mg of trioctylmethyl ammonium chloride, and the mixture was stirred at room temperature under nitrogen for 48 hours. Thin layer chromatography (dichloromethane: methanol 5:1 or petroleum ether: ethyl acetate 1:1) analysis of the raw materials were completely reacted, the reaction solution was poured into 20mL crushed ice, dichloromethane (3X 50 mL) was added for extraction, and the crude product of the compound 4a was obtained by combining, drying, filtration and concentration.
Synthesis of compound 5 a:
compound 4a of the previous step was purified using 20mL of dichloromethane/methanol 1:1, dissolving the mixed solvent, adding 270mg of sodium borohydride in an ice bath, and stirring at room temperature for reaction for 1h. After the reaction of the starting materials was completely analyzed by thin layer chromatography (petroleum ether: ethyl acetate 1:1), 5mL of water was added to quench sodium borohydride, methylene chloride and methanol were removed under reduced pressure at 40℃and extracted with ethyl acetate (3X 50 mL), the ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and concentrated by filtration to remove ethyl acetate to give crude compound 5 a.
Synthesis of compound 6 a:
the compound 5a of the previous step was stirred by adding dried 1, 4-dioxane (20 mL), 1g of potassium hydrogen sulfate was added, and the mixture was heated to 110℃and stirred under reflux for 3 hours. Thin layer chromatography (petroleum ether: ethyl acetate 2:1) analysis of the starting material was complete, removal of dioxane under reduced pressure, extraction with 50mL ethyl acetate, washing to neutrality with saturated aqueous sodium carbonate (3×20 mL), extraction of the aqueous phase with ethyl acetate (3×50 mL), combining the ethyl acetate phases, drying over anhydrous sodium sulfate, filtration and concentration to give crude compound 6 a.
Synthesis of the vanillin:
the crude product of the above compound 6a was stirred at room temperature by adding methanol (5 mL), and then heated at 80℃for 3 days with 5mL of a sodium methoxide methanol solution (5.4M) under reflux. Analyzing the raw materials by thin layer chromatography (petroleum ether: ethyl acetate 1:1), removing methanol under reduced pressure at 40deg.C, adding 50mL ethyl acetate and 20mL water for extraction, extracting the water phase with ethyl acetate (3×50 mL), mixing, drying, filtering, concentrating to obtain crude product, separating the crude product by silica gel forward chromatographic column (petroleum ether: ethyl acetate 3:1 to petroleum ether: ethyl acetate 1:1), and obtaining vanilla 237mg, wherein nuclear magnetic resonance hydrogen spectrum is shown in figure 28, and nuclear magnetic resonance data is shown in figure 28 1 H 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)。
Results: synthesizing to obtain the vanillin.
Scheme B: preparation of moldavica dragonhead by twice column chromatography
Synthesis of compound 2 a:
in a 25mL round bottom flask, 900mg of sodium hydride (60%) was rinsed with dry petroleum ether and then added to 6mL of tetrahydrofuran for dispersion, stirred at room temperature, and diethyl malonate (2.1 g,5.9 mL) was slowly added dropwise over 1h, and the addition was completed for further use. Another 100mL reaction flask was taken, chloroacetyl chloride (2.3 g,1.6 mL) was dissolved in 9mL dry tetrahydrofuran, and the solution prepared in the previous step was slowly added dropwise to the reaction flask in an ice bath for 1h, and the reaction temperature was controlled to be lower than 30 ℃. After the completion of the dropping, the mixture was stirred at room temperature for 1 hour, followed by addition of triethylamine (3.8 g,5.2 mL) and further stirring for 2 hours. A solution of 3, 4-dimethoxyaniline (3.0 g) in dry tetrahydrofuran (13 mL) was then slowly added dropwise over 1h under ice-bath at a controlled temperature of no more than 30deg.C and stirred overnight at room temperature after completion of the dropwise addition. After the completion of the reaction of the starting materials was analyzed by thin layer chromatography (petroleum ether: ethyl acetate 2:1), tetrahydrofuran was removed by concentration under reduced pressure, 50mL of ethyl acetate and 20mL of water were added for extraction, and the resultant solid was filtered to obtain 210mg of pure compound 2 a. The organic phase of the filtrate was dried, concentrated under reduced pressure, dispersed with diethyl ether, filtered to give a crude product, which was recrystallized from 2mL of ethanol to give 600mg of compound 2a in 27% yield.
Synthesis of compound 3 a:
in a 50mL reaction bottle, 810mg of compound 2a is added into 5.4mL of diphenyl ether, the temperature is raised to 250-260 ℃ and the mixture is stirred for 1h under reflux, and the reaction is completely cooled to room temperature; 20mL of petroleum ether was slowly added dropwise to the reaction solution, and after precipitation of the solid, the solid was filtered, and the cake was rinsed with petroleum ether to obtain 730mg of brown solid compound 3a.
Synthesis of compound 4 a:
a25 mL reaction flask was charged with the crude compound 3 of the previous step, followed by 1.5mL phosphorus oxychloride and 117mg trioctylmethyl ammonium chloride, and stirred at room temperature under nitrogen for 48h. After the reaction was completed, the reaction solution was poured into 20mL of crushed ice, dichloromethane (3×50 mL) was added for extraction, the organic phases were combined, dried and concentrated to obtain a crude product, and the crude product was subjected to column chromatography to obtain 275mg of compound 4a, with a yield of 35%.
Synthesis of compound 5 a:
compound 4a of the previous step was purified using 5mL of dichloromethane/methanol 1:1, dissolving the mixed solvent, adding 190mg of sodium borohydride in an ice bath, and stirring at room temperature for reaction for 1h. After completion of the reaction, 2mL of water was added to quench sodium borohydride, dichloromethane and methanol were removed under reduced pressure at 40 ℃, ethyl acetate (3 x 50 mL) was extracted, the ethyl acetate phases were combined, filtered and concentrated to remove ethyl acetate to give 224mg of crude compound 5 a.
Synthesis of compound 6 a:
The compound 5a of the previous step was added to dry 1, 4-dioxane (11.2 mL) and stirred, 1.1g of potassium hydrogen sulfate was added, and the mixture was heated to 110℃and stirred under reflux for 3 hours. After the reaction was completed, dioxane was removed under reduced pressure, diluted with 30mL of ethyl acetate, washed to neutrality with saturated aqueous sodium carbonate (10 mL), the aqueous phase was back-extracted with ethyl acetate (3 x 50 mL), the ethyl acetate phases were combined and concentrated by drying to yield 196mg of crude compound 6 a.
Synthesis of the vanillin:
in the last step, compound 6a was added with methanol (5 mL), stirred at room temperature, 1mL of sodium methoxide methanol solution (5.4M) was added, and the mixture was heated at 80℃for 3 days under reflux. After the reaction, the methanol is removed under reduced pressure, 50mL of ethyl acetate and 20mL of water are added for extraction, the organic phase is dried and concentrated to obtain a crude product, and the crude product is separated by a silica gel forward chromatographic column (petroleum ether: ethyl acetate 3:1 to petroleum ether: ethyl acetate 1:1) to obtain 113mg of the vanillin.
Results: synthesizing to obtain the vanillin.
Example 3
Furano [2,3-b ] quinoline derivatives for inhibiting proliferation of NRK-49F cells
The method comprises the following steps: NRK-49F cells can be activated after stimulation, and a large amount of extracellular matrix is generated after further proliferation, so that the abnormal deposition of the extracellular matrix in the renal interstitial tissue causes the renal interstitial fibrosis. NRK-49F cells were incubated in complete DMEM medium (10% fetal calf serum, 100. Mu.g/mL penicillin, 100. Mu.g/mL streptomycin) at 37℃with 5% CO 2 Culturing under saturated humidity. The liquid was changed every 24 hours. Log grown cells were taken, digested with 0.25% trypsin, diluted to cell concentration in complete medium and plated into 96 well cell culture plates. After overnight incubation, LYY12, 38, 40, 66, 68, 103, HL-14, 17, 18 (0. Mu.M-320. Mu.M) was added at various concentrations to each well and incubation was continued for 24 hours. The inhibition ability to proliferation of NRK-49F cells was detected using CCK8 detection kit, and the experiment was repeated three times.
Results: as shown in FIG. 29, LYY-12 concentration > 20 mu M, LYY-38>320 mu M, LYY-40>160 mu M, LYY-68 concentration of 20 mu M and 320 mu M, LYY-103>10 mu M, HL-14, 17, 18 >10 mu M has remarkable inhibition effect on proliferation of NRK-49F cells, and preliminary verification that LYY-12, 38, 40, 68, 103 and HL-14, 17, 18 have potential of treating chronic kidney disease activity in cells, and LYY-66 has weak potential of treating chronic kidney disease activity in cells.
Example 4
Furano [2,3-b ] quinoline derivatives for inhibiting HK-2 cell proliferation
The method comprises the following steps: HK-2 cells belong to the normal adult tubular epithelial cell line and are a layer of cells outside the tubular. HK-2 cells were cultured in complete DMEM medium (containing 10% fetal calf serum, 100. Mu.g/mL penicillin, 1) 00 μg/mL streptomycin) at 37 degrees celsius, 5% co 2 Culturing under saturated humidity. The liquid was changed every 24 hours. Log grown cells were taken, digested with 0.25% trypsin, diluted to cell concentration in complete medium and plated into 96 well cell culture plates. After overnight incubation, LYY12, 38, 40, 66, 68, 103, HL-14, 17, 18 (0. Mu.M-640. Mu.M) was added at various concentrations to each well and incubation was continued for 24 hours. The ability to inhibit HK-2 cell proliferation was tested using the CCK8 assay kit, and the experiment was repeated three times.
Results: as shown in FIG. 30, LYY-12, 66, 103 > 160. Mu.M showed significant inhibition of HK-2 cell proliferation; LYY-40> 40. Mu.M, HL-14, 18 > 20. Mu. M, HL-17 > 10. Mu.M has remarkable inhibiting effect on the proliferation of HK-2 cells, LYY38, 68 has no remarkable inhibiting effect on the proliferation of HK-2 cells in the concentration range of 0 μm-320. Mu.M, and does not influence 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 20. Mu.M to 80. Mu.M, but has no inhibitory effect on the proliferation of normal HK-2 cells, so LYY-12 is preferred for the subsequent experiments.
Example 5
0.2% adenine feed-induced chronic kidney disease mice model
The method comprises the following steps: chronic kidney disease models were established by daily intake of 0.2% adenine feed induction for 21 consecutive days in C57BL/6 mice. Experiments were performed in two groups of 6. Normal feed is taken into normal mice, 0.2% adenine feed is taken into experimental mice, the mice are sacrificed on the 22 th day, eyeballs are taken out of the blood of the mice, and after standing for 30min at 37 ℃, the blood is centrifuged at 13000rpm for 10min, and then the serum is obtained for renal function detection; the right kidney of the mouse was taken, 4% paraformaldehyde was fixed, and kidney histopathological changes were detected by HE staining and Masson staining.
Results: as shown in FIG. 31, the creatinine value and urea nitrogen value of the normal group are within the normal range, and the kidney tissue pathology shows no pathological damage and collagen deposition is not obvious. Compared with the normal group, the creatinine value and the urea nitrogen value of the mice in the model group are obviously increased, and the pathological results show that the renal tubular expansion and atrophy condition of the renal tissue cortex of the mice is serious, the tubular type and the protein tubular type in the cortex are increased, the infiltration of interstitial inflammation is increased, and the collagen deposition condition is serious. The method is proved to be successful in modeling the mice with chronic kidney disease.
Example 6
Furano [2,3-b ] quinoline derivatives improving 0.2% adenine feed induced kidney function in chronic kidney disease mice
The method comprises the following steps: a chronic kidney disease model is built by continuously taking 0.2% adenine feed daily for 21 days of C57BL/6 mice, feeding is started while adenine feed is taken, LYY1210mg/kg, 20mg/kg, 40mg/kg and allopurinol 20mg/kg are respectively administrated by stomach infusion for 21 days, and the mice are sacrificed on 22 days, and blood of the normal group, the model group and the three doses LYY12 and allopurinol groups are respectively administrated after 0.2% adenine feed induction, and serum is obtained after centrifugation for 10 minutes at 13000rpm after standing for 30 minutes at 37 ℃ for renal function detection.
Results: as shown in FIG. 32, 10mg/kg, 20mg/kg and 40mg/kg LYY12 were able to decrease the serum creatinine and urea nitrogen values to different extents, improving renal function, and improving effect was better with increasing concentration. LYY-12 40mg/kg showed an effect of improving creatinine elevation equivalent to that of allopurinol and an effect of improving urea nitrogen elevation superior to that of allopurinol (< 0.05, <0.01, < 0.001).
Example 7
Furo [2,3-b ] quinoline derivatives for improving 0.2% adenine feed-induced pathological damage of kidney tissue in mice with chronic kidney disease
The method comprises the following steps: a chronic kidney disease model was established by daily ad hoc intake of 0.2% adenine feed induced by C57BL/6 mice for 21 consecutive days, administration was started while the adenine feed was taken, LYY1210mg/kg, 20mg/kg, 40mg/kg and allopurinol 20mg/kg were administered to the mice by gavage, respectively, for 21 days, and the mice were sacrificed on day 22, and right kidneys of the normal group, model group, and three doses LYY12 and allopurinol groups, respectively, were gavaged after 0.2% adenine feed induction, were fixed with 4% paraformaldehyde, and renal histopathological changes were detected by HE staining and Masson staining.
Results: as shown in FIG. 33, the HE staining score results showed that the renal tissue of the mice was severely distended and atrophic by cortical tubular cells and protein tubular cells in the cortex after 0.2% adenine feed induction, the infiltration of interstitial inflammation was increased, and the renal tissue was improved to different degrees after the administration of LYY12 mg/kg, 20mg/kg, 40mg/kg and allopurinol by stomach irrigation. High doses of LYY12 can improve pathological lesions of the kidney of mice more significantly than allopurinol. The Masson staining scoring result shows that the collagen deposition condition of the renal tubule of the mice after 0.2% adenine feed induction is serious, and the collagen deposition condition is improved to different degrees after the LYY12 mg/kg, 40mg/kg and allopurinol are administrated by stomach irrigation. High doses of LYY12 improved collagen deposition in mice more significantly than allopurinol (< P <0.05, < P <0.01, < P < 0.001).
The research result shows that compared with allopurinol, the high-dose LYY12 can improve the kidney function and the kidney structure injury of the mice more remarkably, and the LYY12 has better treatment effect.

Claims (14)

1. The application of a furo [2,3-b ] quinoline derivative, a tautomer, a medicinal salt, a prodrug or a solvate thereof in preparing a medicament for treating chronic kidney disease is characterized in that the structure of the furo [2,3-b ] quinoline derivative is shown as a formula (I):
Figure FDA0004151307360000011
wherein R is 1 Is methoxy; r is R 2 Is hydrogen, hydroxy, methoxy; r is R 3 Is hydrogen, hydroxy, methoxy; r is R 4 Is hydrogen or methoxy.
2. The use according to claim 1, wherein the structure and substituents of the furo [2,3-b ] quinoline derivative are as shown in the following table:
Figure FDA0004151307360000012
Figure FDA0004151307360000021
3. a method for extracting a furo [2,3-b ] quinoline derivative, which is characterized by comprising the following steps:
s1, crushing branches and leaves of rutin, adding water for ultrasonic extraction, concentrating an extracting solution, filtering, standing residues for later use, adsorbing the filtrate by a macroporous resin column, eluting with water, 50% ethanol and 90% ethanol in sequence, collecting an eluent of 90% ethanol, and concentrating to obtain a fluid extract; loading the fluid extract on a normal phase silica gel column, gradient eluting with dichloromethane-methanol solution, mixing similar fractions, and drying to obtain fractions YXC-1-YXC-14;
S2, loading the YXC-5 on a gel column, eluting with petroleum ether-dichloromethane-methanol solution, merging similar fractions, and drying to obtain fractions YXC 5-1-YXC 5-6;
s3, subjecting YXC5-2 to reverse phase semi-preparative HPLC to obtain a compound furo [2,3-b ] quinoline derivative;
the structure of the furo [2,3-b ] quinoline derivative is shown as follows:
Figure FDA0004151307360000022
4. the method for extracting furo [2,3-b ] quinoline derivatives according to claim 3, wherein the branches and leaves of rutin are extracted by ultrasound twice for 1h each time, and the extracts are combined; preferably, the macroporous resin column is a D101 macroporous resin column.
5. The method for extracting furo [2,3-b ] quinoline derivatives according to claim 3, wherein the gradient elution is carried out by using methylene dichloride-methanol solution, and the volume ratio of methylene dichloride to methanol is from 100:0 to 0:1; preferably, the identification fractions are also included before combining the similar fractions, and are identified by thin layer chromatography tracking spots.
6. A method of extracting a furo [2,3-b ] quinoline derivative according to claim 3, wherein the volume ratio of petroleum ether to dichloromethane to methanol in the petroleum ether to dichloromethane to methanol solution is (5-20): 5:1, preferably 5:5:1.
7. The method for extracting furo [2,3-b ] quinoline derivatives according to claim 3, wherein the column for reversed-phase semi-preparative HPLC is a phenyl column and the mobile phase is acetonitrile-water at a volume ratio of 29:71.
8. The method for extracting furo [2,3-b ] quinoline derivatives according to claim 3, wherein the residue is extracted with 80% ethanol solution by ultrasonic extraction, the extract is concentrated, filtered, the filtrate is extracted three times with petroleum ether, and the petroleum ether layer portions are combined and extracted with ethyl acetate to obtain ethyl acetate extract; loading the ethyl acetate extract on a normal phase silica gel column, gradient eluting with dichloromethane-methanol solution, mixing similar fractions, and drying to obtain fractions YXY-1-YXY-18;
loading the YXY-4 on a normal phase silica gel column, performing gradient elution by using a petroleum ether-ethyl acetate system, merging similar fractions, and drying to obtain fractions YXY 4-1-YXY 4-10;
and (3) separating out crystals of YXY4-6, namely the furo [2,3-b ] quinoline derivative.
9. The method for extracting furo [2,3-b ] quinoline derivative according to claim 8, wherein the dissolved fraction left after YXY4-6 precipitation and crystallization is subjected to a gel column, eluted with a methylene chloride-methanol system, and the similar fractions are combined and dried to obtain fractions YXY4-6-1 to YXY4-6-6;
and (3) separating out crystals from the YXY-4-6-3 and YXY-4-6-5 parts to obtain the furo [2,3-b ] quinoline derivative.
10. The method for extracting furo [2,3-b ] quinoline derivatives according to claim 8, wherein the residue is extracted by ultrasound twice for 1 hour each time, and the extracts are combined.
11. The method for extracting furo [2,3-b ] quinoline derivative according to claim 8, wherein the volume ratio of petroleum ether to ethyl acetate is from 4:1 to 1:2 in the gradient elution process of petroleum ether-ethyl acetate solution; preferably, the identification fractions are also included before combining the similar fractions, and are identified by thin layer chromatography tracking spots.
12. A process for the preparation of a furo [2,3-b ] quinoline derivative comprising the steps of:
(1) Sequentially dripping malonic acid diester, chloroacetyl chloride and a catalyst into strong alkali for reaction to obtain a compound 1;
the malonic diester has the structure that:
Figure FDA0004151307360000031
wherein R is selected from C1-C4 branched or straight chain alkyl, phenyl or substituted phenyl; the substituent of the substituted phenyl group comprises halogen, C1-C4 branched or straight-chain alkyl and C1-C4 alkoxy;
(2) Dripping arylamine into the compound 1 prepared in the step (1), adding an aqueous solution of ethyl acetate for precipitation after reaction, and obtaining a solid-phase compound 2 and a solution;
the structure of the arylamine is as follows:
Figure FDA0004151307360000041
R 2 、R 3 and R is 4 Independently selected from hydrogen, hydroxy, halogen, C1-C4 linear or branched alkyl, C1-C4 alkoxy, benzyloxy;
(3) Adding the compound 2 into an inert solvent, heating to 250-260 ℃, refluxing and stirring for 0.5-1 h, adding petroleum ether, filtering and drying to obtain a compound 3;
(4) Adding a chloro reagent into the compound 3 for reaction, and then extracting and concentrating the mixture at a low temperature by using dichloromethane to obtain a compound 4;
(5) Adding a reducing agent into the compound 4 for reaction in ice bath, concentrating, extracting with dichloromethane, washing with water, and drying to obtain a compound 5;
(6) Adding potassium bisulfate or hydrochloric acid into the compound 5, carrying out reflux reaction for 3-5 h, extracting with ethyl acetate, neutralizing with alkali, and drying to obtain a compound 6;
(7) Adding a nucleophilic reagent into the compound 6 under the action of alkali, carrying out reflux reaction for 2-3d, extracting with ethyl acetate, washing with water, and drying to obtain a crude product, and separating and purifying the crude product by normal phase column chromatography to obtain the furo [2,3-b ] quinoline derivative;
the structure of the furo [2,3-b ] quinoline derivative is as follows:
Figure FDA0004151307360000042
wherein R is 1 Is methoxy; r is R 2 Is hydrogen, hydroxy, methoxy; r is R 3 Is hydrogen, hydroxy, methoxy; r is R 4 Is hydrogen or methoxy.
13. The process for preparing furo [2,3-b ] quinoline derivatives according to claim 12, wherein the mobile phase of the normal phase column chromatography in the step (7) is a mixed solution of petroleum ether/ethyl acetate in a volume ratio of 2/1.
14. The process for the preparation of a furo [2,3-b ] quinoline derivative according to claim 12 or 13, wherein the synthesis route of the furo [2,3-b ] quinoline derivative is as follows:
Figure FDA0004151307360000051
CN202310319881.2A 2023-03-29 2023-03-29 Preparation method and application of furo [2,3-b ] quinoline derivative Pending CN116284018A (en)

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