CN108947859B

CN108947859B - Derivatives of kutkin dimer analogue JJA-D0 or pharmaceutically acceptable salts thereof, preparation method and application

Info

Publication number: CN108947859B
Application number: CN201710347578.8A
Authority: CN
Inventors: 蒋杰; 李沙; 藕洋; 高瑞涛; 张志坚; 王高芳; 祁健斌
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2017-05-17
Filing date: 2017-05-17
Publication date: 2021-06-11
Anticipated expiration: 2037-05-17
Also published as: CN108947859A

Abstract

The invention relates to a derivative of a kutkin dimer analogue JJA-D0 or a pharmaceutically acceptable salt thereof, a preparation method and application. The compound has a general formula (I). The present invention introduces hydrocarbon radical, aryl radical, heteroaryl radical, carbalkoxy alkyl radical, acyl radical, sulfonate radical and antioxidant radical, such as liponic acid radical, H, to JJA-D0₂S donor groups such as cysteinyl, NO donor groups such as nitrate and the like, and a series of compounds with brand-new structures are synthesized and disclosed. The compounds can inhibit NADPH oxidase, have better antioxidant and anti-inflammatory dual pharmacological action mechanism than kutkin, and can provide NO and H₂The donor group of S can further improve the pharmacological activity, and is a novel multifunctional compound. The JJA-D0 derivative disclosed can be used for preventing or treating NADPH oxidase-related diseases, free radical-related diseases, inflammation-related diseases, NO-related diseases, and H₂Preparation of health products or medicaments for S-related diseases.

Description

Derivatives of kutkin dimer analogue JJA-D0 or pharmaceutically acceptable salts thereof, preparation method and application

Technical Field

The invention belongs to the field of medicines, and particularly relates to a derivative of a kutkin dimer analogue JJA-D0 or a pharmaceutically acceptable salt thereof, a preparation method and application.

Background

Picrorhizin is a main active ingredient of a commonly used Chinese medicine rhizoma picrorhizae, is originally separated from Canada hemp (Apocynum Cannabinum L.) of Carciniaceae, and has a structure shown in the following. Picrorhiza rhizome is rhizome of Tibetan picrorhiza rhizome and picrorhiza rhizome of Scrophulariaceae, is a common Chinese medicinal material, and has effects of clearing heat and cooling blood, treating malnutrition disease, etc. Picrorhiza rhizome is mainly distributed in Himalayan mountain areas, and picrorhiza rhizome in Tibet is mainly distributed in Sichuan, Yunnan and Tibet.

Molecular structure of picrorhizin

Picrorhiza rhizome is used in India folk for benefiting gallbladder, resisting malaria, invigorating spleen, treating urinary tract infection and hepatitis. With the intensive research on the chemical components and the pharmacological action of the picrorhiza rhizome, the picrorhiza rhizome is found to be one of the main pharmacological components of the picrorhiza rhizome, and has the effects of oxidation resistance and inflammation resistance. In vivo NADPH oxidase is one of the main ways for regulating the ROS level, and under pathological conditions, the NADPH oxidase is activated, the ROS expression is increased, the structure and the function of tissue protein are damaged, the tissue damage of a disease part is caused, inflammation is caused, and the like. It has been reported that picrorhizin can specifically inhibit the activity of NADPH oxidase, can selectively inhibit polymorphonuclear neutrophils from generating ROS, and has antioxidant effect. It has also been reported that picrorhizin can improve some inflammatory injuries and has a certain anti-inflammatory effect. However, the reported antioxidant and anti-inflammatory effects of the picrorhizine are not ideal, and some researchers modify the structure of the picrorhizine so as to obtain a candidate compound with better effect.

Apocynin-lipid Acid Conjugates and Uses therof (publication No. US2014/0315989A1) discloses that the thioctic Acid derivatives of picrorhizin improve the cerebral tissue infarction injury and have the effects on resisting oxidative stress, apoptosis and the like in the cerebral stroke ischemia-reperfusion model of SD ratsThe application is as follows. Kanegae MP et al performed structure-activity relationship studies on the picrorhizine, and replaced acetyl groups on the molecular structure with different electron-withdrawing groups and electron-donating groups, respectively, to obtain a series of derivatives, and found that the activity of the electron-withdrawing substituent derivatives in inhibiting NADPH oxidase is stronger than that of the electron-donating substituent derivatives (Kanegae MP, da Fonseca LM, Brunetti IL, Silva SO, Ximes VF. the reactivity of the organic-methoxy-substituted catalytic activities with sub-hydroxyl groups: control distribution for the reaction of the mechanism of inhibition of the inhibition of NADPH oxidase. biological chemistry 2007,74 (457) (464)). The phenolic hydroxyl groups of the picrorhizin were modified with carboxylic acids of different carbon chain lengths by Mactias-Perez ME et al, which showed that the synthesized picrorhizin derivatives were associated with P47 of NADPH oxidase^phoxHas good affinity, and thus inhibits NOX activity (Mac i as-Perrez ME, Mart i nez-Ramos F, Padella-Martinez II, Correa-Basuroto J, Kisport L, Mendelia-Weje JE, Rosales-Hern end MC. ethers and esters derivative from adipokine available the interaction between beta and P47^phox and p22^phox subunits of NADPH oxidase:evaluation in vitro and in silico.Bioscience Reports.2013,33(4):605-616.)。

The prior patents of the derivatives of picrorhizin and the preparation method and application (ZL200610037302.1) thereof and the patents of the derivatives of picrorhizin and the preparation and application (ZL201010185981.3) thereof are disclosed aiming at structural modification of the picrorhizin by researchers in the field. The two patents mainly disclose the preparation methods of the derivatives of the picrorhizine by nitration, amination and amidation, the derivatives of the picrorhizine combined with lipoic acid, nitrone and other antioxidant groups, and the dimer analogues of the picrorhizine JJA-D0, and the application of the compounds in the preparation of medicines for treating immune system diseases and anti-oxidant medicines.

With the intensive research on the pharmacology of picrorhizin, it has been reported that picrorhizin is a prodrug and that picrorhizin dimer, which is an in vivo metabolite, actually acts. Picrorhizin is converted into a dimer in vivo by oxidation of Myeloperoxidase (MPO), and the dimer acts on NADPH oxidase to inhibit its activation. The structural modification of the active metabolite of the kutkin dimer is expected to obtain a compound with better pharmacological activity, but the current patents and documents reporting the structural modification of the kutkin dimer mainly refer to the modification of the kutkin monomer, and the reports on the modification of the kutkin dimer are very few.

In earlier work, the picrorhizine dimer has been synthesized and prepared for structural modification in the prior art, but the site of modification is relatively unique for this parent compound. For this purpose, two molecules of picrorhizin are substituted with-CH₂-NH-linked to synthesize picrorhiza dimer analog JJA-D0. JJA-D0 has more modifiable structural sites than the picrorhizine dimer. The structures of the kutkin dimer and the analogues JJA-D0 are shown as follows:

molecular structural formula of kutkin dimer (a) and kutkin dimer analogue JJA-D0(b)

The pharmacological activity of JJA-D0 was preliminarily evaluated by researchers in the prior art, and it was found that the protective effect on LPS-induced RAW264.7 cell damage was significantly better than that of the parent compound picrorhizin (Lu X, Wan S, Jiang J, Jiang X, Yang W, Yu P, Xu L, Zhang Z, Zhang G, Shan L, Wang Y. Synthesis and biological evaluation of novel apolipoprotein analogues. European Journal of Medicinal chemistry.2011,46(7): 2691-2698.). In addition, LPS is used for treating male SD rats to cause an acute lung injury model, and picrorhizin and derivatives thereof are used for treatment, and the results show that JJA-D0 reduces the level of inflammatory factor TNF-alpha, improves the pathological change of the lung and reduces the p47 of NADPH oxidase^phoxWith gp91^phoxThe protein expression and the like are better than the picrorhizin (Xu L, Li Y, Wan S, Wang Y, Yu P.protective effects of adocynin nitride on acid solution in resource induced by lipopolypharmaceutical in rates, 20(2): 377-382.). As can be seen, JJA-D0 is a potential model compound.

In the mid-80 th of the twentieth century, Nitric Oxide (NO) was discovered as the first gas signaling molecule in the body, and its in vivo synthesis was mainly catalyzed by nitric oxide synthase. Nitric oxide synthase is widely present in various tissues and organs of the body, and the produced NO also has various biological activities in the body, and it functions as a signal molecule. NO plays an important role in the maintenance of the function of the nervous system, immune system and cardiovascular system and in the development of diseases. NO, as a messenger substance of central and peripheral nervous systems, may be involved in the processes of development, learning and memory of brain cells, secretion of posterior pituitary hormones, regulation of blood supply to the brain and protection of brain tissue in the case of cerebral ischemia, etc., and also in the airway nerves and intestinal muscle reflex of the gastrointestinal tract system, and participate in the relaxation regulation of airway and gastrointestinal smooth muscle. In the process of pathogen invasion and cancer occurrence, the produced inflammation and tumor-related cytokines such as gamma-interferon, interleukin-1 beta and tumor necrosis factor can induce a large amount of iNOS expression, so that a large amount of NO is produced, and the produced inflammation and tumor-related cytokines can be used as immune functional molecules to play roles in preventing virus replication, eliminating pathogens and killing tumor cells. NO is widely involved in the processes of vascular smooth muscle regulation, blood pressure regulation, platelet aggregation, thrombosis, neointima of blood vessels, vascular remodeling and the like, and thus plays an important role in the occurrence and development of various cardiovascular diseases such as atherosclerosis, hypertension, heart failure, restenosis after angioplasty and the like. NO is widely involved in the regulation of various physiological functions and has close relation with the occurrence and development of various diseases, so that the research on the medicines related to the regulation of NO synthesis and release and exogenous supply is deeply valued.

High concentration of H₂S has great harm to human body, is easy to cause toxicity in various aspects of central nervous system, cardiovascular system and the like, and is considered as toxic waste gas for 300 years. After the important regulatory role played by NO and CO gases in complex life activities was gradually discovered, in the middle of the 90 s of the twentieth century, Kimura discovered the metabolite H of cysteine₂S gas has an effect on the nervous system, in recent years, H₂S is known as a third gas signal molecule, and various physiological functions of S are widely researched. Endogenous H₂S is contained in mammalian bodyThe sulfur amino acid is generated in a metabolic pathway and is mainly generated under the action of cystathionine-beta synthetase, cystathionine-gamma lyase and beta-mercaptopyruvate transferase. H₂S plays an important role in cell proliferation and apoptosis, inflammatory reaction, angiogenesis, ischemia-reperfusion injury, neurotransmission and the like. H₂S can respectively show two-way effects of promotion and inhibition on inflammation under different conditions. In the cardiovascular system, H₂S can mediate vascular smooth muscle relaxation; inhibiting the formation of pulmonary hypertension under hypoxia, and regulating the reconstruction of pulmonary vascular structure; can reduce the blood pressure of the primary hypertension rat; inhibiting myocardial ischemia reperfusion injury. In the respiratory system, H₂S has the relaxation effect on airway smooth muscle, can relieve inflammatory reaction of asthma, and has protection effect. In the nervous system, H₂S can relieve the damage of oxidative stress on neurons through mechanisms such as antioxidant stress, anti-inflammatory action, anti-metabolic inhibition and the like, and antagonize neurodegenerative diseases induced by neurotoxin; brain tissue H₂Decreased S levels are associated with decreased cognitive ability in alzheimer' S patients. Furthermore, H₂S also has an effect on diseases of various systems such as the digestive system, endocrine system, reproductive system, and the like. In view of H₂The important significance of S signal molecules in physiology and pathology makes it one of the concerns of drug development.

The pathogenesis or the course development of a plurality of serious diseases such as cardiovascular and cerebrovascular diseases, metabolic diseases, immunological diseases, nervous system diseases and the like is closely related to oxidative stress injury and inflammatory injury caused by free radicals. With NO, H₂Many diseases related to S and NADPH oxidase have mutual correlation in the aspects of oxidative stress injury and inflammation generation and development, so that the combination of the three targets for drug design is an important method for searching new drugs for treating the diseases.

Disclosure of Invention

To overcome the defects and shortcomings of the prior art, the invention provides a derivative of a kutkin dimer analogue JJA-D0 or a pharmaceutically acceptable salt thereof. Wherein, the invention converts NO and H₂Dimerization by introducing S donor group into picrorhizineIn the structural modification of a body analogue JJA-D0, a series of JJA-D0 derivatives are disclosed in design, so that NO and H can be exerted while the activity of the kutkin dimer for inhibiting NADPH oxidase is exerted₂S novel compound structure type of pharmacological action.

Another object of the present invention is to provide a method for preparing the above derivatives of the kutkin dimer analog JJA-D0 or pharmaceutically acceptable salts thereof.

Still another object of the present invention is to provide the use of the above described derivatives of the kutkin dimer analog JJA-D0, or pharmaceutically acceptable salts thereof. The invention discloses a series of derivatives which are designed and synthesized by taking a kutkin dimer analogue JJA-D0 as a parent compound, most of the derivatives have stronger anti-inflammatory and antioxidant effects, and can be used for diseases related to NADPH oxidase, diseases related to free radicals, diseases related to inflammation, diseases related to NO and H₂Preparation of health products or medicaments for S-related diseases.

The purpose of the invention is realized by the following technical scheme:

a derivative of a kutkin dimer analog JJA-D0, or a pharmaceutically acceptable salt thereof, having a structure of formula (I):

wherein R is₁、R₂、R₃The same or different, are respectively selected from: hydrogen, substituted or unsubstituted, heteroatom-containing or heteroatom-free, straight, branched or cyclic hydrocarbyl carbon chains of up to 10 carbon atoms (preferably 1 to 8, more preferably 1 to 4 carbon atoms), substituted or unsubstituted monocyclic aryl, heteroaryl, alkoxycarbonylalkyl, lipoyl, substituted or unsubstituted cysteinyl, nitrate, acyl, sulfonate.

Preferably, R₁Is a substituted or unsubstituted cysteinyl group, i.e., the compound has the structure of formula (II):

wherein R is₂，R₃The same or different, respectively, hydrogen, substituted or unsubstituted, straight, branched or cyclic hydrocarbon carbon chain of up to 10 carbon atoms with or without heteroatoms (preferably 1 to 8, more preferably 1 to 4 carbon atoms), substituted or unsubstituted monocyclic aryl, heteroaryl; r₄Is hydrogen, substituted or unsubstituted, straight-chain, branched or cyclic alkyl carbon chain with or without heteroatoms and up to 6 carbon atoms, substituted or unsubstituted monocyclic aryl, heteroaryl.

Preferably, R₂And R₃At least one of which is a nitrate group of the NO donor group, i.e. the compound has one of the general formulae (III), (IV), (V):

wherein in the structure of the general formula (III), R₁、R₃Identical or different, hydrogen, substituted or unsubstituted, linear, branched or cyclic, hydrocarbon-based carbon chains of up to 10 carbon atoms, with or without heteroatoms (preferably of 1 to 8, more preferably of 1 to 4 carbon atoms), substituted or unsubstituted monocyclic aryl, heteroaryl; z₁Is a straight chain or branched carbon chain to which a nitrate group is attached, wherein Z₁May be substituted with hetero atom, alkyl group, aryl group or heteroaryl group, and Z₁Contains 1-6 carbon atoms;

wherein in the structure of the general formula (IV), R₁、R₂Identical or different, hydrogen, substituted or unsubstituted, linear, branched or cyclic, hydrocarbon-based carbon chains of up to 10 carbon atoms, with or without heteroatoms (preferably of 1 to 8, more preferably of 1 to 4 carbon atoms), substituted or unsubstituted monocyclic aryl, heteroaryl; z₂Is a straight chain or branched carbon chain to which a nitrate group is attached, wherein Z₂May be substituted with hetero atom, alkyl group, aryl group or heteroaryl group, and Z₂Contains 1-6 carbon atoms;

wherein in the structure of the general formula (V), R₁Is hydrogen, substituted or unsubstituted, straight, branched or cyclic hydrocarbon carbon chain of up to 10 carbon atoms with or without heteroatoms (preferably 1 to 8, more preferably 1 to 4 carbon atoms), substituted or unsubstituted monocyclic aryl, heteroaryl; z₃And Z₄Identical or different, respectively, are straight-chain or branched carbon chains to which nitrate groups are attached, wherein Z is₃And Z₄Each of which may be substituted with a heteroatom, a hydrocarbyl group, an aryl group or a heteroaryl group, and Z₃And Z₄Each containing 1 to 6 carbon atoms.

Preferably, R₁Is a substituted or unsubstituted acyl group, i.e., the compound has the structure of formula (VI):

wherein R is₂，R₃The same or different, respectively, hydrogen, substituted or unsubstituted, straight, branched or cyclic hydrocarbon carbon chain of up to 10 carbon atoms with or without heteroatoms (preferably 1 to 8, more preferably 1 to 4 carbon atoms), substituted or unsubstituted monocyclic aryl, heteroaryl; r₅Is hydrogen, substituted or unsubstituted, straight-chain, branched or cyclic alkyl carbon chain (preferably with 1-5 carbon atoms) with or without heteroatoms, and substituted or unsubstituted monocyclic aryl or heteroaryl.

Preferably, said R is₂And R₃At least one of which is a substituted or unsubstituted sulfonate group, i.e. the compound has one of the general formulae (VII), (VIII), (IX):

wherein in the structure of the general formula (VII), R₁，R₃Identical or different, are each hydrogen, substituted or unsubstituted, straight-chain, branched or cyclic with or without heteroatomsA hydrocarbyl carbon chain of up to 10 carbon atoms (preferably 1 to 8, more preferably 1 to 4 carbon atoms), substituted or unsubstituted monocyclic aryl, heteroaryl; r₆Is hydrogen, substituted or unsubstituted, straight, branched or cyclic, hydrocarbon carbon chain of up to 10 carbon atoms with or without heteroatoms (preferably 1-4 carbon atoms), substituted or unsubstituted monocyclic aryl, heteroaryl;

wherein in the structure of the general formula (VIII), R₁，R₂The same or different, respectively, hydrogen, substituted or unsubstituted, straight, branched or cyclic hydrocarbon carbon chain of up to 10 carbon atoms with or without heteroatoms (preferably 1 to 8, more preferably 1 to 4 carbon atoms), substituted or unsubstituted monocyclic aryl, heteroaryl; r₇Is hydrogen, substituted or unsubstituted, straight, branched or cyclic, hydrocarbon carbon chain of up to 10 carbon atoms with or without heteroatoms (preferably 1-4 carbon atoms), substituted or unsubstituted monocyclic aryl, heteroaryl;

wherein in the structure of the general formula (IX), R₁Selected from: hydrogen, substituted or unsubstituted, heteroatom or heteroatom-free, straight, branched or cyclic hydrocarbyl carbon chains of up to 10 carbon atoms (preferably 1 to 8 carbon atoms, more preferably 1 to 4 carbon atoms), and substituted or unsubstituted monocyclic aryl, heteroaryl; r₈And R₉The same or different, are respectively selected from: hydrogen, substituted or unsubstituted, straight, branched or cyclic hydrocarbyl carbon chains of up to 10 carbon atoms (preferably 1 to 4 carbon atoms) with or without heteroatoms, and substituted or unsubstituted monocyclic aryl, heteroaryl groups.

Preferably, the derivative of the kutkin dimer analogue JJA-D0 or the pharmaceutically acceptable salt thereof is selected from but not limited to one of the following compounds:

the preparation method of the derivatives of the kutkin dimer analogue JJA-D0 or the pharmaceutically acceptable salts thereof comprises the following steps:

using kutkin as raw material, obtaining intermediate Apo-CHO by Reimer-Tiemann reaction, obtaining intermediate Apo-NH by nitration and reduction reaction₂The two parts of the structure are condensed by a Schiff's base forming reaction and then are hydrogenated and reduced to obtain JJA-D0;

wherein JJA-D0 is composed of alkyl, aryl, heteroaryl, alkoxycarbonylalkylation, nitroesterification and sulfonatoesterification derivatives, which are bonded to Apo-CHO and Apo-NH via ether bond and ester bond₂To the phenolic hydroxyl group of (A) to give substituted Apo-CHO and Apo-NH₂Intermediate, substituted or unsubstituted Apo-CHO and substituted or unsubstituted Apo-NH₂The catalyst is prepared by condensing two parts of structures and then hydrogenating and reducing the two parts of structures by a Schiff's alkali forming reaction; wherein the alkylated, arylated, heteroarylated, alkoxycarbonylalkylated Apo-CHO and/or Apo-NH₂The intermediate is prepared by respectively reacting halogenated hydrocarbon, halogenated aromatic hydrocarbon, halogenated heteroarene, halogenated carboxylic ester with Apo-CHO and/or Apo-NH₂Etherified phenolic hydroxyl groups of (a); nitrated Apo-CHO and/or Apo-NH₂The intermediate is prepared by reacting a bihalogenated hydrocarbon with Apo-CHO and/or Apo-NH₂Etherified phenolic hydroxyl, and esterified with silver nitrate; sulfoesterified Apo-CHO and/or Apo-NH₂The intermediate is prepared by reacting sulfonyl chloride with Apo-CHO and/or Apo-NH₂Is esterified to form the phenolic hydroxyl group;

wherein the thioctic acid-base derivative of JJA-D0 is prepared by reacting JJA-D0 (JJA-D0-TBDMS) with both phenolic hydroxyl groups protected, or unprotected JJA-D0 with lipoic acid; JJA-D0 amidated derivative is prepared through the reaction of JJA-D0-TBDMS with both phenolic hydroxyl groups protected and acyl chloride; JJA-D0 cysteine amidated derivatives were prepared byApo-NH protected by phenolic hydroxyl₂I.e. Apo-NH₂-TBDMS，Apo-NH₂Reaction of-TBDMS with Apo-CHO to produce JJA-D0 (Apo-NH) protected by a single phenolic hydroxyl group₂TBDMS), and the intermediate JJA-D0 (Apo-NH)₂TBDMS) with a substituted or unsubstituted cysteine to produce a cysteine amidated derivative.

The preparation method of the derivatives of the kutkin dimer analogue JJA-D0 or the pharmaceutically acceptable salts thereof preferably comprises the following steps:

(1) adding triethylamine and absolute ethyl alcohol into the picrorhizine, dropwise adding a sodium hydroxide aqueous solution, dropwise adding chloroform into the reaction solution, and reacting for 1-4 hours at 70-80 ℃; cooling to room temperature, acidifying with hydrochloric acid, washing with water, extracting, and separating with silica gel column to obtain Apo-CHO; (2) adding glacial acetic acid into picrorhizine to dissolve a substrate, adding concentrated nitric acid under ice bath, reacting for 15-60 min, removing ice water, reacting at normal temperature for 1.5-5 h, adding ice water, performing suction filtration, washing with ice water, and recrystallizing to obtain Apo-NO₂(ii) a Taking Apo-NO₂Dissolving in ethanol, adding 10% Pd/C (mass ratio of Pd to C is Pb: C-1: 9), and introducing H₂Reacting for 7-14 h, washing and extracting, and separating by a silica gel column to obtain Apo-NH₂(ii) a (3) Dissolving a compound Apo-CHO in DMF, adding one of halogenated hydrocarbon, halogenated aromatic hydrocarbon, halogenated heteroarene, halogenated carboxylic ester or sulfonyl chloride, reacting with potassium carbonate or triethylamine at the temperature of 30-60 ℃ or in an ice bath for 1.5-5 h, cooling to room temperature, washing with water for extraction, and separating by using a silica gel column to obtain phenolic hydroxyl substituted Apo-CHO; (4) taking compound Apo-NH₂Dissolved in anhydrous dichloromethane, di-tert-butyl dicarbonate ((Boc) was added₂O) and triethylamine react for 7-14 h, the reaction liquid is washed and extracted, and silica gel column separation is carried out to obtain a compound Apo-NH-Boc; (5) dissolving a compound Apo-NH-Boc, acetone or DMF (dimethyl formamide), adding one of halogenated hydrocarbon, halogenated aromatic hydrocarbon, halogenated heteroarene or sulfonyl chloride, reacting with triethylamine for 1-4 h at room temperature or in an ice bath, washing and extracting, and separating by using a silica gel column to obtain a compound of the phenolic hydroxyl substituted Apo-NH-Boc; (6) taking phenolic hydroxyl substituted Apo-NH-Boc, dissolving in dichloromethane, dropwise adding trifluoroacetic acid, reacting for 0.5-4 h, and directly performing water washing extraction or NaHCO (NaHCO) after the reaction is finished₃Saturated aqueous solutionNeutralizing the reaction liquid, extracting, and separating by a silica gel column to obtain the phenolic hydroxyl substituted Apo-NH₂(ii) a (7) Taking compound of Apo-CHO substituted by phenolic hydroxyl, dissolving with methanol, adding compound of Apo-NH₂Reacting for 0.5-3 h, adding sodium cyanoborohydride and glacial acetic acid, reacting for 1-4 h at normal temperature, washing and extracting, and separating by using a silica gel column to obtain compounds JJA-D14, JJA-D15, JJA-D16, JJA-D17, JJA-D23, JJA-D25, JJA-D26, JJA-D33 and JJA-D34; (8) dissolving Apo-CHO with methanol, adding phenol hydroxyl to substitute Apo-NH₂Reacting for 0.5-3 h, adding a proper amount of sodium cyanoborohydride, dropwise adding glacial acetic acid, reacting for 1-4 h at normal temperature, washing and extracting, and separating by using a silica gel column to obtain solid compounds JJA-D18, JJA-D19, JJA-D20, JJA-D21, JJA-D24, JJA-D27 and JJA-D28; (9) dissolving compound of Apo-CHO substituted by phenolic hydroxyl in methanol, and adding compound of Apo-NH substituted by phenolic hydroxyl₂Reacting for 0.5-3 h, adding sodium cyanoborohydride and glacial acetic acid, reacting for 1-4 h at normal temperature, washing and extracting, and separating by using a silica gel column to obtain compounds JJA-D22, JJA-D29, JJA-D30, JJA-D31, JJA-D32, JJA-D38, JJA-D39 and JJA-D40.

(1) taking Apo-NH₂Dissolving with Apo-CHO by using methanol, reacting for 1-5 h at normal temperature, adding sodium cyanoborohydride and glacial acetic acid, reacting for 1-4 h at normal temperature, and washing and extracting to obtain a compound JJA-D0; (2) adding JJA-D0 into anhydrous dichloromethane, tert-butyl dimethyl silicon chloride (TBDMS-Cl) and imidazole, reacting for 3-7 h at normal temperature, washing with water, and separating by a silica gel column to obtain a compound JJA-D0-TBDMS with both phenolic hydroxyl groups protected; (3) dissolving lipoic acid in dichloromethane, adding 1-hydroxybenzotriazole (HOBt) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) in an ice bath, reacting for 15-60 min, adding JJA-D0-TBDMS and anhydrous triethylamine, reacting for 5-10 h at room temperature, washing with water, extracting, adding tetrabutylammonium fluoride (TBAF) into an organic layer, reacting for 0.5-3 h at room temperature, and separating by using a silica gel column to obtain a compound JJA-D5; (4) dissolving a compound JJA-D0-TBDMS in dichloromethane, adding triethylamine and acyl chloride under the protection of nitrogen, reacting for 1-4 h in an ice bath, removing the ice bath, washing and extracting,concentrating the organic layer; dissolving the residue with anhydrous dichloromethane, dropwise adding TBAF (tert-butyl ammonium fluoride), reacting for 1-4 h, washing with water, extracting, and separating with a silica gel column to obtain a compound JJA-D11, JJA-D12, JJA-D13; (5) dissolving lipoic acid in dichloromethane, adding HOBt and EDCI in ice bath, reacting for 15-60 min, removing ice bath, adding JJA-D0 and N, N-Diisopropylethylamine (DIPEA), reacting at room temperature for 5-10 h,

washing with water, extracting, separating with silica gel column to obtain JJA-D6 and JJA-D7.

(1) dissolving L-cysteine in pre-cooled NH₃·H₂Dropwise adding halogenated hydrocarbon into the O solution under an ice bath condition, reacting for 1.5-5 h, dropwise adding dilute hydrochloric acid under the ice bath condition to adjust the pH value, separating out a solid, performing suction filtration, and recrystallizing to obtain L-cysteine (Cys-a) with mercapto hydrogen of the compound substituted by alkyl; (2) cys-a, water, acetone, triethylamine and (Boc)₂O, reacting for 2-6 h under ice bath; removing ice bath, evaporating acetone under reduced pressure, extracting, mixing water layers, adjusting pH of the water layer with dilute hydrochloric acid, extracting, washing the organic layer with water, evaporating to obtain oily compound, and freezing to obtain solid compound Cys-b, i.e. Cys-a with amino protected by Boc; (3) taking Apo-NH₂Dissolving dichloromethane, adding imidazole and TBDMS-Cl, reacting for 7-14 h, washing with water, and separating by using a silica gel column to obtain a phenolic hydroxyl protected compound Apo-NH₂-TBDMS; (4) dissolving Apo-CHO in methanol, adding Apo-NH₂Reacting for 0.5-3 h with TBDMS, adding sodium cyanoborohydride and glacial acetic acid, reacting for 1-4 h at normal temperature, washing with water, extracting, and concentrating an organic layer to obtain a single phenolic hydroxyl protected compound JJA-D0 (Apo-NH)₂-TBDMS); (5) taking compound JJA-D0 (Apo-NH)₂TBDMS), dissolving dichloromethane, adding Cys-b, HOBt and EDCI, and reacting for 7-14 h; washing with water, extracting, separating with silica gel column to obtain JJA-D0 (Apo-NH) amidated with Cys-b₂-TBDMS); (6) JJA-D0 (Apo-NH) amidated with Cys-b₂TBDMS), dissolving in dichloromethane, dropwise adding tetrabutylammonium fluoride (TBAF), reacting for 0.5-4 h, water washing and extracting, concentrating an organic layer, and obtaining a residueDissolving with anhydrous dichloromethane, dropwise adding trifluoroacetic acid, reacting for 1-4 h, washing with water, extracting, and separating with a silica gel column to obtain a compound JJA-D8, JJA-D9 or JJA-D10.

(1) adding one of DMF, dihalogenated hydrocarbon, dihalogenated aromatic hydrocarbon or dihalogenated heteroarene into Apo-CHO, and mixing with K₂CO₃Reacting for 7-14 h at 60-90 ℃, washing and extracting, and separating by a silica gel column to obtain Apo-CHO of halogenated alkyl alkylation, halogenated aryl alkylation or halogenated heteroaryl alkylation respectively; (2) dissolving Apo-CHO subjected to halogenated alkylation, halogenated aryl alkylation or halogenated heteroaryl alkylation by using acetonitrile, adding silver nitrate, reacting for 6-12 h at 60-90 ℃ under the condition of keeping out of the sun, and separating reaction liquid by using a silica gel column to respectively obtain Apo-CHO subjected to nitrate alkylation, nitrate aryl alkylation or nitrate heteroaryl alkylation; (3) dissolving Apo-NH-Boc with DMF, adding one of dihalogenated hydrocarbon, dihalogenated aromatic hydrocarbon or dihalogenated heteroarene, reacting with DIPEA at normal temperature for 6-12 h, washing and extracting, and separating by using a silica gel column to respectively obtain halogenated alkyl alkylated, halogenated aryl alkylated or halogenated heteroarene alkylated Apo-NH-Boc; (4) dissolving Apo-NH-Boc subjected to halogenated alkylation, halogenated aryl alkylation or halogenated heteroaryl alkylation by using acetonitrile, adding silver nitrate, reacting for 6-12 h at 60-90 ℃ in the dark, and separating by using a silica gel column after the reaction is finished to respectively obtain Apo-NH-Boc subjected to nitrate alkylation, nitrate aryl alkylation or nitrate heteroaryl alkylation; (5) dissolving Apo-NH-Boc subjected to alkylation of nitrate ester, aryl alkylation of nitrate ester or heteroaryl alkylation of nitrate ester with dichloromethane, adding trifluoroacetic acid, reacting for 0.5-3 h, and reacting with NaHCO₃Neutralizing the reaction liquid with saturated water solution, extracting, and vacuum concentrating the organic layer to obtain Apo-NH with alkylated nitrate ester, arylalkylated nitrate ester or heteroaromatic alkylated nitrate ester₂(ii) a (6) Alkylating the obtained nitrate ester, arylating the nitrate ester or heteroaromatizing the obtained Apo-CHO and Apo-NH₂Dissolving with methanol, reacting for 0.5-3 h, adding sodium cyanoborohydride and glacial acetic acid, reacting for 1-4 h, and separating the reaction liquid with a silica gel column to obtain a compound JJA-D1 or JJA-D2. (7) Nail for useApo-NH alcohol-solubilized nitrate hydrocarbylated, nitrate arylated or nitrate heteroaromatized₂Adding Apo-CHO, reacting for 0.5-3 h, adding sodium cyanoborohydride and glacial acetic acid, reacting for 1.5-5 h, and separating by a silica gel column to obtain a compound JJA-D3 or JJA-D4; (8) alkylating, arylating or heteroarylalkylating Apo-CHO with, or optionally with, Apo-NH₂Dissolving with methanol, reacting for 0.5-3 h, adding sodium cyanoborohydride and glacial acetic acid, reacting for 1.5-5 h, and separating the reaction liquid by using a silica gel column to obtain a compound JJA-D35, JJA-D36 or JJA-D37.

The compounds provided by the invention have multiple functions, can inhibit NADPH oxidase, scavenge free radicals, resist inflammation, release NO and release H₂S, therefore, the above-mentioned derivatives of the kutkin dimer analog JJA-D0 or pharmaceutically acceptable salts thereof can be used for the preparation of a pharmaceutical composition for preventing or treating NADPH oxidase-related diseases, free radical-related diseases, inflammation-related diseases, NO-related diseases, or H-related diseases₂A health product or a medicament for S-related diseases.

The diseases related to NADPH oxidase are selected from hypertension, atherosclerosis, cardiac hypertrophy and heart failure, coronary heart disease, arrhythmia, myocardial infarction, ischemia-reperfusion injury, pneumonic injury, acute lung injury, chronic obstructive pulmonary disease, emphysema, chronic bronchitis, acute respiratory distress syndrome, pulmonary hypertension, asthma, idiopathic pulmonary fibrosis, cerebral apoplexy, Alzheimer's disease, renal calculus, chronic kidney disease, arthritis, diabetes and complications thereof, tumor, etc. The diseases related to free radicals are selected from hypertension, keshan disease, heart failure scorpion, atherosclerosis, myocardial infarction, coronary heart disease, acute lung injury, pneumonia, chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, ischemia reperfusion injury, brain trauma, cerebral apoplexy, Alzheimer disease, anaphylaxis, Hashimoto's disease, lupus erythematosus, dermatomyositis, autoimmune vasculitis, amyotrophic lateral sclerosis, arthritis, cataract, glaucoma, fibrosis after crystal, retinal oxidation injuryOptic neuritis, radiation sickness, dermatitis herpetiformis, water immersion dermatitis, solar dermatitis, glomerulonephritis, hepatitis, liver cirrhosis, obesity, diabetes and its complications, and tumor. The disease associated with inflammation is selected from acute lung injury, pneumonia, chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, arthritis, hypertension, atherosclerosis, viral myocarditis, nonspecific ulcerative colitis, Crohn's disease, chronic inflammatory bowel disease, necrotizing enterocolitis neonatorum, inflammatory bowel disease in children, congenital megacolon combined enterocolitis, acute liver injury, non-alcoholic fatty liver, pancreatitis, diabetes and its complications, psoriasis, atopic dermatitis, urticaria, bullous pemphigoid, prostatitis, Behcet's disease, Alzheimer's disease, obesity, and tumor. The NO-related disease is selected from chronic obstructive pulmonary disease, emphysema, chronic bronchitis, arthritis, acute lung injury, acute respiratory distress syndrome, asthma, cerebral ischemia, cerebral stroke, cerebral trauma, epilepsy, alzheimer's disease, amyotrophic lateral sclerosis, migraine, hypertension, atherosclerosis, angina pectoris, myocardial infarction, ischemic heart disease, coronary heart disease, vascular embolism, ischemia reperfusion injury, heart failure, periodontal disease, oral mucositis, salivary gland disease, oral cancer and temporomandibular joint disorder, kawasaki disease, esophagitis, pancreatitis, diabetes and its complications, hypercholesterolemia, and tumor. Said and H₂The S-related disease is selected from hypertension, atherosclerosis, hypoxic pulmonary hypertension, high pulmonary blood flow pulmonary hypertension, myocardial ischemic injury, adriamycin cardiomyopathy, myocardial hypertrophy, heart failure, atherosclerosis, angina pectoris, myocardial infarction, coronary heart disease, traumatic brain injury, febrile convulsion, Alzheimer 'S disease, Huntington' S chorea, amyotrophic lateral sclerosis, cerebral apoplexy, cerebral ischemia reperfusion, acute lung injury, pulmonary fibrosis, pulmonary ischemia reperfusion injury, asthma, chronic obstructive pulmonary disease, pneumonia, liver cirrhosis, stress ulcer, inflammatory bowel disease, acute gastric mucosa injury, colon cancer, congenital megacolon, ulcerative colitis, periodontitis, oral cancer, psoriasis, kidney transplantation, uremia, obesity, diabetes, chronic obstructive pulmonary disease, acute gastric mucosa injury, chronic obstructive pulmonary disease, acute gastric mucosa injury, chronic inflammatory bowel disease, chronic gastric mucosa injury, chronic inflammatory bowel disease, chronic inflammatory bowelDisease and its complications, and tumor.

The novel compounds of the present invention include the structures of the aforementioned general formulae (I) to (IX). The new compound has JJA-D0 structure and contains at least 1 substituent of alkyl, aryl, heteroaryl, alkoxycarbonylalkyl, lipoyl, cysteinyl, nitrate, acyl and sulfonate. The following definitions are set forth to illustrate and define the meaning and scope of various terms used in the present disclosure.

The term "hydrocarbyl" as used herein refers to an unsubstituted or substituted straight, branched or cyclic hydrocarbyl carbon chain of up to 10 carbon atoms, or a hydrocarbyl group containing at least one heteroatom (e.g., nitrogen, oxygen or sulfur) in the chain. Non-limiting examples of the linear hydrocarbon group include saturated hydrocarbon groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl groups, unsaturated hydrocarbon groups having substituents such as ethylenic bonds, acetylenic bonds, carbonyl groups and cyano groups, and heteroatom-containing hydrocarbon groups such as-CH₂CH₂OCH₃、-CH₂CH₂N(CH₃)₂and-CH₂CH₂SCH₃And the like. Non-limiting examples of branched hydrocarbyl groups, free or containing heteroatoms, include, e.g., isopropyl, sec-butyl, isobutyl, tert-butyl, neopentyl, -CH₂CH(OCH₃)CH₃、-CH₂CH(N(CH₃)₂)CH₃and-CH₂CH(SCH₃)CH₃. Non-limiting examples of the cyclic hydrocarbon group containing no or hetero atoms ("cycloalkyl group") include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, and O, N, S-containing six-membered rings such as-CH (CH)₂CH₂)₂O、-CH(CH₂CH₂)₂NCH₃and-CH (CH)₂CH₂)₂S, etc. and the corresponding five-membered heterocyclic ring, etc. The hydrocarbyl group may be substituted with one or more substituents, non-limiting examples of which include N (CH)₃)₂、F、Cl、Br、I、-OCH₃、-CO₂CH₃CN, -OH, aryl and heteroaryl.

The term "aryl" as used herein means unsubstituted or substitutedAromatic compounds, carbocyclic groups and heteroaryl groups. Aryl is either a monocyclic or polycyclic fused compound. The aryl group may be substituted with one or more substituents, non-limiting examples of which include-N (CH)₃)₂、F、Cl、Br、I、-OCH₃、-CO₂CH₃CN, -OH, aryl and heteroaryl.

Heteroaryl refers to a substituted or unsubstituted mono-or polycyclic group containing at least one heteroatom, such as nitrogen, oxygen and sulfur, within the ring. Exemplary heterocyclic groups include, by way of example, one or more nitrogen atoms such as tetrazolyl, pyrrolyl, pyridyl (e.g., 4-pyridyl, 3-pyridyl, 2-pyridyl, etc.), pyridazinyl, indolyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl, etc.), imidazolyl, isoquinolinyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridonyl; typical heterocyclic groups containing one oxygen atom include 2-furyl, 3-furyl or benzofuryl; typical sulfur heteroatom groups include thienyl, benzothienyl; typical mixed heteroatom groups include furazanyl, oxazolyl, isoxazolyl, thiazolyl, and phenothiazinyl. The heterocyclic group can be substituted with one or more substituents including-O-alkyl, -NH-alkyl, -N- (alkyl)₂-NHC (O) -alkyl, F, Cl, Br, I, -OH, -OCF₃、-CO₂-alkyl, -CN and aryl and polyaryl.

The term "pharmaceutically acceptable" as used herein means having no unacceptable toxicity in a compound such as a salt or excipient. Pharmaceutically acceptable salts include inorganic anions such as chloride, bromide, iodide, sulfate, sulfite, nitrate, nitrite, phosphate, hydrogen phosphate and the like. Organic anions include acetate, propionate, cinnamate, benzensulfonate, citrate, lactate, gluconate, fumarate, tartrate, succinate, and the like. The present invention relates to hydrocarbyl, aryl, heteroaryl, alkoxycarbonylalkyl, thioctic, cysteinyl, nitrate, acyl, sulfonate derivatives of the huxadol dimer analog JJA-D0, which may be administered to a patient in the form of a pharmaceutically acceptable salt or pharmaceutical complex. Certain complexes may be mixed with suitable carriers or excipients to form pharmaceutical compositions to ensure that an effective therapeutic agent is achieved. By "therapeutically effective amount" is meant the amount of the compound of the class and derivatives thereof required to achieve a therapeutic effect.

The normal temperature and the room temperature are both 15-25 ℃.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the invention takes a parent compound of the kutkin dimer analogue JJA-D0 with better activity and more structural modification sites than the kutkin dimer as a basic structure, carries out systematic structural modification on the basic structure, and designs and provides a compound with novel structure. And NO and H are reacted₂The S donor group is introduced into JJA-D0 for structural modification, so that NO and H are exerted while the kutkin dimer is exerted to inhibit the activity of NADPH oxidase₂S novel compound structure type of pharmacological action. The compound has dual pharmacological action mechanisms of anti-inflammation and antioxidation, can inhibit NADPH oxidase and provide NO and H₂S donor, and the like. The compounds are useful for preventing or treating NADPH oxidase-related diseases, free radical-related diseases, inflammation-related diseases, NO-related diseases, H-related diseases₂Preparation of health products or medicaments for S-related diseases.

(2) JJA-D0 has simple preparation process, easily obtained raw materials, low cost, economy and effectiveness. JJA-D0 has better pharmacological activity than kutkin like kutkin dimer, and JJA-D0 has more structural modification sites than kutkin dimer, which is beneficial to obtain more abundant brand new compounds with better drugability.

Drawings

FIG. 1 depicts the compounds Apo-CHO and Apo-NH₂And (4) synthesizing. Wherein: (a) 50% sodium hydroxide aqueous solution, ethanol, chloroform, refluxing, and dilute hydrochloric acid; (b) 67% nitric acid and glacial acetic acid are carried out on ice to room temperature; (c) hydrogen, 10% palladium on carbon, ethanol.

FIG. 2 depicts the synthesis of compound JJA-D1. Wherein: (a)1, 2-dibromoethane/dimethylformamide/potassium carbonate, 80 ℃; (b) silver nitrate/acetonitrile, 80 ℃; (c) sodium cyanoborohydride/methanol, glacial acetic acid, room temperature.

FIG. 3 depicts the synthesis of compound JJA-D2. Wherein: (a)1, 4-dibromobutane/dimethylformamide/potassium carbonate, 80 ℃; (b) silver nitrate/acetonitrile, 80 ℃; (c) sodium cyanoborohydride/methanol, glacial acetic acid, room temperature.

FIG. 4 depicts the synthesis of compound JJA-D3. Wherein: (a) di-tert-butyl dicarbonate/triethylamine at room temperature; (b)1, 2-dibromoethane/N, N-diisopropylethylamine at room temperature; (c) silver nitrate/acetonitrile, 80 ℃; (d) dichloromethane/trifluoroacetic acid; (e) sodium cyanoborohydride/methanol, glacial acetic acid, room temperature.

FIG. 5 depicts the synthesis of compound JJA-D4. Wherein: (a) di-tert-butyl dicarbonate/triethylamine at room temperature; (b)1, 4-dibromobutane/dimethylformamide, room temperature; (c) silver nitrate/acetonitrile, 80 ℃; (d) dichloromethane/trifluoroacetic acid; (e) sodium cyanoborohydride/methanol, glacial acetic acid, room temperature.

FIG. 6 depicts the synthesis of compound JJA-D5. Wherein: (a) sodium cyanoborohydride/methanol, glacial acetic acid, room temperature; (b) dichloromethane/tert-butyldimethylsilyl chloride/imidazole, room temperature; (c) lipoic acid, dichloromethane/1-hydroxybenzotriazole/carbodiimide/triethylamine, room temperature; (d) tetrabutylammonium fluoride/dichloromethane, room temperature.

FIG. 7 depicts the synthesis of compounds JJA-D6 and JJA-D7. Wherein: (a) sodium cyanoborohydride/methanol, glacial acetic acid, room temperature; (b) lipoic acid, dichloromethane/1-hydroxybenzotriazole/carbodiimide/N, N-diisopropylethylamine, room temperature.

FIG. 8 depicts the synthesis of compounds JJA-D8, JJA-D9 and JJA-D10. Wherein: (a) ethyl bromide/allyl bromide/propargyl bromide, aqueous ammonia, 0 ℃; (b) acetone/water/di-tert-butyl dicarbonate/triethylamine; (c) dichloromethane, tert-butyldimethylsilyl chloride/imidazole, room temperature; (d) sodium cyanoborohydride/methanol, glacial acetic acid, room temperature; (e) SEC-Boc/SAC-Boc/SPRC-Boc, 1-hydroxybenzotriazole/carbodiimide, dichloromethane, 0 ℃; (f) tetrabutylammonium fluoride/dichloromethane; trifluoroacetic acid/dichloromethane, room temperature.

FIG. 9 depicts the synthesis of compounds JJA-D11, JJA-D12, and JJA-D13. Wherein: (a) dichloromethane/tert-butyldimethylsilyl chloride/imidazole, room temperature; (b) dichloromethane/different acid chlorides/triethylamine, 0 ℃; (c) tetrabutylammonium fluoride/dichloromethane, room temperature.

FIG. 10 depicts the synthesis of compounds JJA-D14, JJA-D15, JJA-D16, JJA-D17, JJA-D23, JJA-D25 and JJA-D26. Wherein: (a) different halogenated hydrocarbons or sulfonyl chlorides, potassium carbonate or triethylamine, dimethylformamide, at 40 ℃ or in an ice bath; (b) sodium cyanoborohydride/methanol, glacial acetic acid, room temperature.

FIG. 11 depicts the synthesis of compounds JJA-D18, JJA-D19, JJA-D20, JJA-D21, JJA-D24, JJA-D27 and JJA-D28. Wherein: (a) di-tert-butyl dicarbonate, triethylamine and dichloromethane at room temperature; (b) different halogenated hydrocarbons or sulfonyl chlorides, triethylamine, acetone or dimethylformamide; (c) trifluoroacetic acid, dichloromethane, room temperature; (d) sodium cyanoborohydride/methanol, glacial acetic acid, room temperature.

FIG. 12 depicts the synthesis of compound JJA-D22. Wherein: (a) benzyl chloride, potassium carbonate and dimethylformamide at 40 ℃ for 3 h; (b) di-tert-butyl dicarbonate, triethylamine and dichloromethane at room temperature; (c) benzyl chloride, triethylamine, acetone; (d) trifluoroacetic acid, dichloromethane, room temperature; (e) sodium cyanoborohydride/methanol, glacial acetic acid, room temperature.

FIG. 13 depicts the results of the safety evaluation of JJA-D0 in example 54. The graph indicates significant differences from the control group (, P < 0.01;, P < 0.001).

Figure 14 depicts the protective effect of the picrorhizin, JJA-D0 and 14 JJA-D0 derivatives of example 55 on LPS-induced RAW264.7 cell damage (#, significant difference compared to the blank control, # # # P < 0.001; # significant difference compared to the model group, # P <0.05, # P < 0.01).

Figure 15 depicts the effect of the picrorhizin, JJA-D0, and 14 JJA-D0 derivatives of example 56 on LPS-induced ROS levels in RAW264.7 cells (#, significant difference compared to the blank control, # # # P < 0.001; # P < 0.05;. P <0.01) compared to the model group.

Figure 16 depicts the down-regulation of TNF- α expression in LPS-induced RAW264.7 cells by picrorhizine, JJA-D0, JJA-D26 in example 57 (#, significant difference compared to the blank control, # # P < 0.01;. a significant difference compared to the model group, # P < 0.05).

FIG. 17 depicts the administration of Picrorrhiza kutkin, JJA-D0, JJA-D26 in example 58 to NADPH oxidase subunit p47 in LPS-induced RAW264.7 cells^phox、gp91^phoxDownregulation of expression (#, significant difference compared to blank control, ### P<0.001; significant differences compared to model groups<0.05,**P<0.01)。

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.

Example 1: compound Apo-NO₂Synthesis of (2)

Taking 1.66g of picrorhizin (10 mmol), adding 10ml of glacial acetic acid for dissolving, slowly adding 1.5ml of 67 wt% concentrated nitric acid under the stirring of ice bath, reacting for 30min, removing ice water, reacting at normal temperature for 3h, adding the ice water into a system after the reaction is finished, performing suction filtration to obtain yellow solid, washing with ice water, and recrystallizing in 95% (v/v) ethanol to obtain yellow needle crystals (1.65g, yield 78%). The compound is a known compound and is consistent with a map recorded in a literature through mass spectrum and nuclear magnetic spectrum display.

Example 2: compound Apo-NH₂Synthesis of (2)

Taking Apo-NO₂(2.11g,10mmol) was dissolved in 30ml of ethanol, 10% by mass Pd/C (950mg) was added and H was passed through the reaction mixture₂Reacting overnight, washing with water after reaction, concentrating organic layer under reduced pressure, separating with silica gel column (ethyl acetate: petroleum ether: 1:2) to obtain light white solid Apo-NH₂(1.32g, 73% yield). The compound is a known compound and is consistent with a map recorded in a literature through mass spectrum and nuclear magnetic spectrum display.

Example 3: synthesis of Compound Apo-CHO

Picrorhizin (1.66g,10mmol) was taken out and placed in a flask, and after a mixed solution of 3ml of triethylamine and 20ml of absolute ethanol was added, 30ml of a 50 wt% aqueous solution of sodium hydroxide was dropped into the system, and stirred and mixed uniformly. Chloroform (5.95g,50mmol) was added dropwise to the reaction solution, and the reaction was carried out at 70-80 ℃ for 2 hours. The reaction was cooled to room temperature and acidified with hydrochloric acid to pH 1. The organic layers were collected, the aqueous layer was extracted with dichloromethane, the combined organic layers were washed with water, dried over anhydrous sodium sulfate and concentrated, and separated on silica gel column (ethyl acetate: petroleum ether ═ 2:3) to give Apo-CHO as a pale yellow powder (0.69g, 36% yield). The compound is a known compound and is consistent with a map recorded in a literature through mass spectrum and nuclear magnetic spectrum display.

Example 4: synthesis of Compound JJA-D0

Taking Apo-NH₂(1.81g,10mmol) and Apo-CHO (1.94g,10mmol) are placed in a flask, a proper amount of methanol is added for dissolution, the reaction is carried out for 3h at normal temperature, a proper amount of sodium cyanoborohydride is added after no precipitation is generated, a catalytic amount of glacial acetic acid is dropwise added, the reaction is carried out for 2h at normal temperature, then water is added for extraction, an organic layer is taken and concentrated under reduced pressure, and compound JJA-D0(2.08g, 58%) is obtained as a brown yellow solid, is a known compound, and is shown to be consistent with the graph recorded in the literature through mass spectrum and nuclear magnetic spectrum.

Example 5: synthesis of Compound JJA-D1

The compound Apo-CHO (1.94g,10mmol) was taken and placed in a flask, DMF10ml, 1, 2-dibromoethane (2.23g,12mmol), K were added₂CO₃(2.76g,20mmol), reaction at 80 ℃ overnight, after completion of the reaction, 60ml of dichloromethane was added, washing with water, concentration under reduced pressure, and silica gel column separation (ethyl acetate: petroleum ether ═ 1:6) to give Apo-CHO- (CH) as a compound₂)₂Br is added. Dissolving Apo-CHO- (CH) with appropriate amount of acetonitrile₂)₂Br (3.0g,10mmol), silver nitrate (3.38g,20mmol) was added and the reaction was carried out at 80 ℃ for 8h in the absence of light. After the reaction, the reaction mixture was separated by silica gel column (ethyl acetate: petroleum ether: 1:5) to obtain Apo-CHO- (CH) as a compound₂)₂NO₃(1.5g, 53% yield). Adding Apo-CHO- (CH)₂)₂O-NO₂(2.83g,10mmol) with Apo-NH₂(1.81g,10mmol) was placed in a flask, and dissolved by adding an appropriate amount of methanol, and after no precipitation occurred, an appropriate amount of sodium cyanoborohydride and a little glacial acetic acid were added, and reacted for 2 hours, and the reaction solution was separated by silica gel column (ethyl acetate: petroleum ether ═ 1:4) to obtain JJA-D1(2.55g, yield 57%) as a white solid. And m.p. 161-163 ℃. ESI-MS of M/z [ M + H ]]⁺449,[M-H]⁺447.¹H-NMR(300MHz,CDCl₃)δ:9.46(s,1H),7.66(d,1H),7.50(d,1H),7.12(d,1H),7.07(d,1H),5.52(s,1H),4.79～4.82(m,2H),4.51(s,2H),4.45～4.48(m,2H),3.93(s,3H),3.91(s,3H),2.56(s,3H),2.52(s,3H).¹³C-NMR(75MHz,CDCl₃)δ:198.69,198.40,153.59,150.79,147.08,138.43,137.09,134.79,133.99,131.11,124.00,112.26,107.57,102.96,73.40,70.25,57.68,57.36,44.50,27.82,27.64.

Example 6: synthesis of Compound JJA-D2

Taking compound Apo-CHO (1.94g,10mmol), placing in a flask, adding DMF10ml, 1, 4-dibromobutane (2.57g,12mmol), K₂CO₃(2.76g,20mmol), reaction at 80 ℃ overnight, after completion of the reaction, 60ml of dichloromethane was added, washed with water, evaporated to dryness under reduced pressure, and separated by silica gel column (ethyl acetate: petroleum ether ═ 1:8) to give Apo-CHO- (CH) as a compound₂)₄Br。Apo-CHO-(CH₂)₄Br (3.28g,10mmol) was dissolved in an appropriate amount of acetonitrile, and silver nitrate (3.38g,20mmol) was added to the solution to react at 80 ℃ for 8 hours in the absence of light. After the reaction was completed, the reaction mixture was separated by silica gel column (ethyl acetate: petroleum ether: 1:6) to obtain Apo-CHO- (CH) as a compound₂)₄O-NO₂(2.66g, yield 86%). Adding Apo-CHO- (CH)₂)₄NO₃(3.11g,10mmol) with Apo-NH₂(1.81g,10mmol) was placed in a flask, an appropriate amount of methanol was added, after no precipitation occurred, an appropriate amount of sodium cyanoborohydride and a little glacial acetic acid were added, reaction was carried out for 2h, and the reaction solution was separated by silica gel column (ethyl acetate: petroleum ether ═ 1:5) to obtain compound JJA-D2(2.81g, yield 59%) as a pale yellow solid. m.p. 151-152 deg.C ESI-MS M/z [ M + H ]]⁺477.4,[M-H]⁺475.4.¹H-NMR(300MHz,CDCl₃)δ:9.46(s,1H),7.61(d,1H),7.47(d,1H),7.05(d,1H),7.00(d,1H),5.52(s,1H),4.48(t,2H),4.43(s,2H),4.14(t,2H),3.88(s,3H),3.85(s,3H),2.52(s,3H),2.50(s,3H),1.87～1.94(m,4H).¹³C-NMR(75MHz,CDCl₃)δ:197.37,197.22,152.49,150.42,145.73,137.22,135.45,132.85,132.11,129.48,122.84,110.84,106.18,101.94,73.00,72.24,56.20,55.85,43.47,26.52,26.42,26.25,23.53.

Example 7: synthesis of Compound Apo-NH-Boc

Taking and combiningSubstance Apo-NH₂(1.81g,10mmol) was dissolved in an appropriate amount of anhydrous dichloromethane and di-tert-butyl dicarbonate ((Boc) was added successively₂O, 4.36g,20mmol), triethylamine (1.01g,10mmol), and reacted overnight. After completion of the reaction, the organic layers were washed with water, combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then separated by a silica gel column (ethyl acetate: petroleum ether ═ 1:8) to obtain the compound Apo-NH-Boc (2.24g, yield 80%). ESI-MS of M/z [ M + H ]]⁺282,[M-H]⁺280.¹H-NMR(300MHz,DMSO-d₆)δ:10.02(s,1H),8.06(d,1H),7.89(s,1H),7.26(d,1H),3.87(s,3H),2.50(s,3H),1.47(s,9H).

Example 8: synthesis of Compound JJA-D3

The compound Apo-NH-Boc was prepared as in example 7 by dissolving Apo-NH-Boc (2.82g,10mmol) in DMF, adding 1, 2-dibromoethane (2.23g,12mmol) and N, N-diisopropylethylamine (DIPEA, 2.58g, 20mmol), reacting at room temperature for 8h, adding dichloromethane 60ml after the reaction was over, washing with water, evaporating to dryness under reduced pressure, separating with silica gel column (ethyl acetate: petroleum ether ═ 1:8) to give the compound Apo-NH-Boc- (CH: -N-methyl-N-propyl-N-methyl-ethyl acetate)₂)₂Br, dissolving Apo-NH-Boc- (CH) with appropriate amount of acetonitrile₂)₂Br (3.87g,10mmol), silver nitrate (3.38g,20mmol) was added and the reaction was carried out for 8h in the absence of light. After the reaction was completed, the reaction mixture was separated by a silica gel column (ethyl acetate: petroleum ether ═ 1:6) to obtain Apo-NH-Boc- (CH) as a compound₂)₂O-NO₂(1.10g, yield 30%). Dissolving all the obtained Apo-NH-Boc- (CH) with dichloromethane₂)₂O-NO₂Trifluoroacetic acid is added, after 1h of reaction, NaHCO is used₃Neutralizing the reaction solution with saturated water solution, extracting with dichloromethane, and concentrating the organic layer under reduced pressure to obtain compound Apo-NH₂-(CH₂)₂O-NO₂. Dissolving Apo-NH in methanol₂-(CH₂)₂O-NO₂(2.70g,10mmol), Apo-CHO (1.94g,10mmol) was added, after no precipitation occurred, an appropriate amount of sodium cyanoborohydride and a little glacial acetic acid were added, the reaction was carried out for 3h, and the reaction solution was separated by silica gel column (ethyl acetate: petroleum ether: 1:2) to obtain JJA-D3(2.15g, 48% yield) as a brown yellow solid m.p. at 138-139 ℃. ESI-MS of M/z [ M + H ]]⁺449.4,[M-H]⁺447.5.¹H-NMR(300MHz,CDCl₃)δ:9.87(s,1H),7.59(d,1H),7.44(d,1H),7.05(d,1H),6.96(d,1H),5.95(s,1H),4.72～4.75(m,2H),4.46(s,2H),4.33～4.36(m,2H),3.92(s,3H),3.87(s,3H),2.53(s,3H),2.51(s,3H).¹³C-NMR(75MHz,CDCl₃)δ:197.61,196.91,151.89,148.62,146.68,141.35,137.51,133.67,129.49,123.72,123.55,109.01,106.02,101.88,71.96,68.20,56.23,55.88,43.00,26.48,26.18.

Example 9: synthesis of Compound JJA-D4

The compound Apo-NH-Boc was prepared as in example 7 by dissolving Apo-NH-Boc (2.81g,10mmol) in the appropriate amount of DMF and adding 1, 4-dibromobutane (2.57g,12mmol) and K₂CO₃(2.76g,20mmol), reaction at room temperature for 8h, after completion of the reaction, addition of dichloromethane 60ml, washing with water, evaporation to dryness under reduced pressure, silica gel column separation (ethyl acetate: petroleum ether ═ 1:10) to give Apo-NH-Boc- (CH) as the compound₄)₄Br, dissolving Apo-NH-Boc- (CH) with appropriate amount of acetonitrile₄)₄Br (4.15g,10mmol), silver nitrate (3.38g,20mmol) was added and the reaction was carried out for 8h in the absence of light. After the reaction was completed, the product was separated by silica gel column (ethyl acetate: petroleum ether ═ 1:8) to obtain Apo-NH-Boc- (CH) as a pure compound₂)₄O-NO₂(1.52g, 38% yield). Dissolving all the obtained Apo-NH-Boc- (CH) with dichloromethane₂)₄O-NO₂Trifluoroacetic acid is added, after 1h of reaction, NaHCO is used₃Neutralizing the reaction solution with saturated water solution, extracting with dichloromethane, and concentrating the organic layer under reduced pressure to obtain compound Apo-NH₂-(CH₄)₄-ONO₂. Dissolving Apo-NH in methanol₂-(CH₄)₄-ONO₂(2.98g,10mmol), Apo-CHO (1.94g,10mmol) was added, an appropriate amount of sodium cyanoborohydride was added after precipitation occurred, the reaction was reacted for 3h, and the reaction solution was separated on a silica gel column (ethyl acetate: petroleum ether ═ 1:3) to obtain compounds JJA-D4 as a white solid (2.09g, 44% yield). m.p. 160-162 ℃. ESI-MS of M/z [ M + H ]]⁺477.6,[M-H]⁺475.6.¹H-NMR(300MHz,CDCl₃)δ:9.87(s,1H),7.60(d,1H),7.45(d,1H),7.08(d,1H),7.00(d,1H),5.95(s,1H),4.53(t,2H),4.48(s,2H),4.07(t,2H),3.93(s,3H₃),3.87(s,3H),2.53(s,3H),2.52(s,3H),1.85～1.97(m,4H).¹³C-NMR(75MHz,CDCl₃)δ:197.58,196.78,151.96,148.60,146.66,141.05,138.97,133.13,129.48,123.63,123.59,109.10,106.25,102.58,72.96,71.49,56.22,55.86,43.53,26.46,26.20,23.61；

Example 10: synthesis of Compound JJA-D0-TBDMS

Taking compound Apo-NH₂(1.81g,10mmol), Apo-CHO (1.94g,10mmol) JJA-D0 was prepared as in example 4. 50ml of anhydrous dichloromethane, tert-butyldimethylsilylchloride (TBDMS-Cl, 6g,40mmol) and imidazole (4.1g,60mmol) were added to JJA-D0, and the mixture was reacted at room temperature for 5 hours, followed by washing with water after completion of the reaction, concentration of the reaction mixture under reduced pressure and silica gel column separation (ethyl acetate: petroleum ether: 1:5) to obtain JJA-D0-TBDMS as a white solid (4.1g, yield 70%). ESI-MS of M/z [ M + H ]]⁺588,[M-H]⁺586.¹H-NMR(300MHz,DMSO-d₆)δ:7.60(s,1H),7.44(s,1H),6.96(s,1H),6.82(s,1H),4.90(s,1H),4.39(d,2H),3.87(d,3H),3.80(s,3H),2.46(s,3H),2.45(s,3H),0.95(s,9H),0.92(s,9H),0.23(d,6H),0.16(s,6H).

Example 11: synthesis of Compound JJA-D5

Taking compound Apo-NH₂(1.81g,10mmol), Apo-CHO (1.94g,10mmol) were reacted according to the method of example 10 to prepare JJA-D0-TBDMS. Lipoic acid (2.08g,10mmol) was dissolved in anhydrous dichloromethane, 1-hydroxybenzotriazole (HOBt, 2.02g,15mmol), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI, 2.87g,15mmol) were added to the solution in an ice bath, after 30min of reaction, the ice bath was removed, JJA-D0-TBDMS (5.87g,10mmol) and anhydrous triethylamine (3.03g,30mmol) were added to the solution, the reaction was carried out at room temperature for 8h, after completion of the reaction, the solution was washed with water, tetrabutylammonium fluoride (TBAF) was added to the organic layer, and after 1h of reaction at room temperature, the solution was separated by silica gel column (ethyl acetate: petroleum ether: 1) to obtain a pale solid compound JJA-D5(3.06g, 5 (yield: 56%). m.p. of 112-114 ℃. ESI-MS of M/z [ M + H ]]⁺548,[M-H]⁺546.¹H-NMR(300MHz,DMSO-d₆)δ:10.11(s,2H),7.50(d,1H),7.43(d,1H),7.35(d,1H),7.27(d,1H),4.43～5.12(dd,2H),3.88(s,3H),3.82(s,3H),3.47～3.58(m,1H),3.03～3.18(m,2H),2.46(s,3H),2.39(s,3H),2.00～2.17(m,2H),1.74～1.85(m,1H),1.40～1.58(m,4H),1.17～1.32(m,3H).¹³C-NMR(75MHz,CDCl₃)δ:197.92,197.53,174.85,151.16,150.15,149.57,149.10,130.72,129.80,129.66,125.48,125.43,125.27,112.01,111.56,57.99,57.89,57.79,47.88,39.94,35.96,34.62,34.56,30.05,28.07,27.96,26.38.

Example 12: synthesis of Compounds JJA-D6 and JJA-D7

Lipoic acid (6.24g,30mmol) was dissolved in anhydrous dichloromethane, HOBt (6.06g,45mmol) and EDCI (8.61g,45mmol) were added to the solution under ice bath, reaction was carried out for 30min, the ice bath was removed, JJA-D0(3.59g,10mmol) and DIPEA (11.6g,90mmol) were added, the mixture was reacted at room temperature for 8h, water washing was carried out, the organic layer was concentrated under reduced pressure, and silica gel column separation was carried out to obtain JJA-D6(3.16g, yield 43%) as a white solid and JJA-D7(3.69g, yield 40%) as a white solid.

Compound JJA-D6: m.p. of 150-152 ℃. ESI-MS of M/z [ M + H ]]⁺736,[M-H]⁺734.¹H-NMR(300MHz,DMSO-d₆)δ:7.58(d,1H),7.45(d,1H),7.38(d,1H),7.37(d,1H),5.95(s,1H),4.41～5.07(dd,2H),3.86(s,3H),3.83(s,3H),3.52～3.68(m,2H),3.07～3.25(m,4H),2.50(s,3H),2.46(s,3H),2.08～2.43(m,4H),1.14～1.96(m,16H).¹³C-NMR(75MHz,CDCl₃)δ:197.67,196.98,176.76,171.15,154.15,151.63,149.90,141.95,137.01,136.47,130.25,127.00,123.27,123.05,113.15,112.25,57.91,57.68,57.64,57.53,50.30,41.62,41.53,39.89,39.82,35.86,34.78,34.74,34.71,29.98,29.85,27.78,27.48,26.02,25.88.

Compound JJA-D7: m.p. 200-203 ℃. ESI-MS of M/z [ M + H ]]⁺924,[M-H]⁺922.¹H-NMR(DMSO-d₆,300MHz)δ:7.58(d,1H),7.50(d,1H),7.32(d,1H),7.27(d,1H),4.30～5.18(dd,2H),3.86(s,3H),3.82(s,3H),3.48～3.68(m,3H),3.04～3.25(m,6H),2.50～2.56(m,2H),2.48(s,3H),2.46(s,3H),2.02～2.43(m,4H),1.15～1.95(m,24H).¹³C-NMR(CDCl₃,300MHz)δ:198.06,197.51,173.56,172.40,171.84,153.90,153.07,144.41,142.67,136.47,136.40,135.91,131.96,126.22,125.45,112.08,112.01,57.90,57.81,57.78,57.69,46.53,41.68,41.61,39.92,39.85,36.09,36.02,35.97,35.12,34.99,34.90,30.17,30.01,29.97,27.82,27.72,26.25,26.07,25.84.

Example 13: synthesis of Compound S-ethylcysteine (SEC)

L-cysteine (1.21g,10mmol) was taken in a flask and dissolved in pre-cooled 20ml NH₃·H₂To the solution O, bromoethane (2.17g,20mmol) was added dropwise under ice-bath conditions. After reacting for 3 hours, slowly dropwise adding dilute hydrochloric acid under an ice bath condition, adjusting the pH to 2-3, precipitating a large amount of white solid, performing suction filtration, and recrystallizing with water/absolute ethyl alcohol to obtain white needle-shaped crystals SEC (1.3g, yield 87%). ESI-MS of M/z [ M + H ]]⁺150,[M-H]⁺148.¹H-NMR(300MHz,DMSO-d₆)δ:11.00(s,1H),4.14(s,1H),3.01(d,2H),2.72(t,2H),2.27(s,2H),1.22～1.01(m,3H).

Example 14: synthesis of Compound SEC-Boc

The flask was charged with SEC (1.5g,10mmol), water (10 ml), acetone (20 ml) in this order, and triethylamine (4g,40mmol), (Boc) was added thereto with stirring₂O (4.36g,20mmol), stirring was continued for 4h under ice bath. Removing the ice bath after the reaction is finished, evaporating acetone under reduced pressure, extracting the water layer by using petroleum ether and ethyl acetate, combining the water layers, adjusting the pH of the water layer to 2-3 by using dilute hydrochloric acid, extracting by using ethyl acetate, combining the organic layers, washing by using a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, filtering, evaporating under reduced pressure to obtain an oily compound, and freezing to obtain a white solid compound SEC-Boc (2.3g, the yield is 92%). ESI-MS of M/z [ M + H ]]⁺250,[M-H]⁺248.¹H-NMR(300MHz,DMSO-d₆)δ:11.00(s,1H),7.10(d,1H),4.04(d,1H),3.20～3.00(m,1H),2.86(dd,J＝4.7Hz,1H),2.70(dd,J＝9.2Hz,1H),2.55(d,1H),1.37(d,9H),1.16(dd,3H).

Example 15: synthesis of Compound S-allylcysteine (SAC)

L-cysteine (1.21g,10mmol) and allyl bromide (2.38g,20mmol) were reacted by the method of example 13 to obtain SAC (1.5g, 93% yield) as a white solid. ESI-MS of M/z [ M + H ]]+162,[M-H]⁺160.¹H-NMR(300MHz,D₂O)δ:11.00(s,1H),5.83～5.64(m,1H),5.17～5.06(m,2H),3.80(dd,1H),3.11(d,2H),2.97(dd,2H),2.86(dd,2H).

Example 16: synthesis of SAC-Boc Compound

The SEC-Boc preparation in example 14 was carried out by substituting the compound SAC (1.61g,10mmol) for the compound SEC with (Boc)₂O (4.36g,20mmol) was reacted to obtain SAC-Boc (2.5g, 96% yield) as a white solid. ESI-MS of M/z [ M + H ]]⁺262,[M-H]⁺260.¹H-NMR(300MHz,Acetone-d₆)δ:11.00(s,1H),6.16(d,1H),5.81(d,1H),5.14(ddd,J＝1.2Hz,2H),4.36(s,1H),3.22(d,2H),2.98(d,1H),2.84(dd,1H),1.43(s,9H).

Example 17: synthesis of compound S- (prop-2-yn-1-yl) cysteine (SPRC)

L-cysteine (1.21g,10mmol) and bromopropyne (2.36g,20mmol) were reacted by the method of example 13 to obtain SPRC (1.5g, 94% yield) as a white solid. ESI-MS of M/z [ M + H ]]⁺160,[M-H]⁺158.¹H-NMR(300MHz,D₂O)δ:11.00(s,1H),3.91(dd,2H),3.30(dd,1H),3.22(dd,2H),3.08(dd,2H),2.60(t,3H).

Example 18: synthesis of Compound SPRC-Boc

The SEC-Boc preparation method in example 14 was followed, taking SPRC (1.59g,10mmol) instead of compound SEC, with (Boc)₂O (4.36g,20mmol) gave SPRC-Boc (2.5g, 97% yield) as a white solid. ESI-MS of M/z [ M + H ]]⁺260,[M-H]⁺258.¹H-NMR(300MHz,DMSO-d₆)δ:11.00(s,1H),7.07(dd,1H),5.09(t,1H),4.01(d,1H),3.24～2.93(m,2H),2.92～2.69(m,1H),2.65(d,1H),1.37(s,9H).

Example 19: synthesis of Compound 8a

Weighing compound Apo-NH₂(1.81g,10mmol) was dissolved in a flask with 40ml of anhydrous dichloromethane, and imidazole (1.33g,20mmol) and TBDMS-Cl (3g,20mmol) were added successively with stirring and reacted overnight. After completion of the reaction, the reaction mixture was washed with water, the combined organic layers were evaporated to dryness under reduced pressure, and the residue was separated with silica gel (ethyl acetate: petroleum ether: 1:8) to obtain compound 8a (2.50g, 85%) as a white solid. ESI-MS of M/z [ M + H ]]⁺296,[M-H]⁺294.¹H-NMR(300MHz,DMSO-d₆)δ:7.03(t,1H),6.86(d,1H),4.72(s,2H),3.79(d,3H),2.47(s,3H),0.97(s,9H),0.15(s,6H).

Example 20: synthesis of Compound 8b

Dissolving a compound Apo-CHO (1.94g,10mmol) in a flask by using methanol, adding a compound 8a (3.24g,11mmol) under stirring, reacting for 1h to generate yellow precipitate, adding a proper amount of sodium cyanoborohydride, dropwise adding a catalytic amount of glacial acetic acid, reacting for 2h at normal temperature, adding water for extraction, taking an organic layer, and evaporating to dryness under reduced pressure to obtain a white solid compound 8 b. ESI-MS of M/z [ M + H ]]⁺474,[M-H]⁺472.¹H-NMR(300MHz,CDCl₃)δ:9.87(s,1H),7.61(d,1H),7.48(d,1H),7.08(d,1H),7.02(d,1H),5.95(s,1H),4.50(s,2H),3.95(s,3H),3.84(s,3H),2.53(s,3H),2.51(s,3H),0.99(s,9H),0.23(s,6H).

Example 21: synthesis of Compound 8c

The compound 8b (4.73g,10mmol) was dissolved in an appropriate amount of anhydrous dichloromethane, and SEC-Boc (4.98g,20mmol), HOBt (2.7g,20mmol) and EDCI (3.8g,20mmol) were added successively under stirring to react overnight. After completion of the reaction, the organic layers were washed with water, combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column (ethyl acetate: petroleum ether: 1:8) to obtain compound 8c as a white solid (4.22g, yield 60%). ESI-MS of M/z [ M + H ]]⁺705,[M-H]⁺703.¹H-NMR(300MHz,DMSO-d₆)δ:9.49(s,1H),7.67(d,1H),7.60(s,1H),7.52(s,1H),6.92(s,1H),6.75(s,2H),5.15(s,1H),4.37(s,1H),3.85(s,3H),3.79(s,3H),3.07(d,2H),3.02～2.82(m,2H),2.60(d,3H),2.41(s,3H),1.37(d,9H),1.17(d,3H),0.97(s,9H),0.20(s,6H).

Example 22: synthesis of Compound JJA-D8

Taking the compound 8c (3.52g,5mmol), dissolving in 20ml dichloromethane, dropwise adding 4ml TBAF, and reacting for 2-3 h. And after the reaction is finished, washing with water, extracting with dichloromethane, combining organic layers, drying with anhydrous sodium sulfate, concentrating under reduced pressure, dissolving the concentrated solution with 20ml of anhydrous dichloromethane, dropwise adding 5ml of trifluoroacetic acid under the stirring condition, and reacting for 2-3 h. After completion of the reaction, the reaction mixture was washed with water, extracted with dichloromethane, the combined organic layers were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated with a silica gel column (dichloromethane: methanol: 100:1) to obtain JJA-D8 as a yellow solid (1.85g, 76% yield). m.p. 80.2-82.1 ℃. ESI-MS of M/z [ M + H ]]⁺491,[M-H]⁺489.¹H-NMR(300MHz,DMSO-d₆)δ:8.51(s,2H),7.77(s,1H),7.67(s,1H),7.19(s,1H),7.11(s,1H),7.01(s,1H),6.92(d,1H),4.71(s,2H),4.22(s,1H),3.80(s,3H),3.74(s,3H),3.17(s,1H),2.71(d,1H),2.52(s,3H),2.49(s,3H),2.45(s,2H),1.24(dd,3H).¹³C-NMR(75MHz,DMSO-d₆):196.69,195.23,164.72,150.80,150.11,149.58,147.68,128.99,128.35,124.73,124.19,123.64,123.10,110.26,109.45,56.19,51.31,48.00,27.10,26.27,26.19,25.63,25.14,14.96.

Example 23: synthesis of Compound 9c

Compound 8b (4.73g,10mmol) and SAC-Boc (5.22g,20mmol) were reacted by the method of example 21 to give compound 9c as a white solid (4.43g, 62% yield). ESI-MS of M/z [ M + H ]]⁺717,[M-H]⁺715.¹H-NMR(300MHz,DMSO-d₆)δ:9.55～9.12(m,1H),7.70(d,1H),7.59(s,1H),7.52(s,1H),6.92(s,1H),6.73(s,1H),5.85～5.69(m,1H),5.18(s,1H),5.12(s,1H),5.09(s,1H),4.36(s,2H),3.85(d,3H),3.79(s,3H),3.24(d,2H),3.01(d,2H),2.91～2.74(m,3H),2.40(d,3H),1.35(s,9H),0.94(d,9H),0.20(s,6H).

Example 24: synthesis of Compound JJA-D9

The phenolic hydroxyl group and amino protecting groups of compound 9c (3.58g,5mmol) were removed in this order and treated accordingly as in example 22 to give JJA-D9 as a yellow solid (1.95g, 78% yield). m.p. 81.6-82.9 deg.C ESI-MS M/z [ M + H ]]⁺503,[M-H]⁺501.¹H-NMR(300MHz,d₆-DMSO)δ:9.85(s,1H),9.48(s,1H),7.60(d,J＝1.8Hz,1H),7.38(d,J＝1.8Hz,1H),6.92(d,J＝1.7Hz,1H),6.86(d,J＝1.8Hz,1H),5.75(dd,J＝17.0,9.9Hz,1H),5.27～4.92(m,2H),4.35(s,2H),4.23(s,1H),3.87(s,3H),3.82(s,3H),3.17(d,J＝2.9Hz,1H),3.15(d,J＝2.8Hz,1H),2.98(dd,J＝13.9,3.5Hz,2H),2.74(dd,J＝13.9,3.9Hz,2H),2.44(s,3H),2.42(s,3H).¹³C-NMR(75MHz,d₆-DMSO)δ:197.06,197.65,167.01,149.51,147.41,146.65,137.99,137.66,134.97,128.76,128.40,126.48,122.93,117.83,110.10,105.05,102.18,56.40,56.38,55.07,41.89,34.83,26.71,26.66.

Example 25: synthesis of Compound 10c

Compound 8b (4.73g,10mmol) and SPRC-Boc (5.18g,20mmol) were reacted by the method of example 21 to give compound 10c as a white solid (4.28g, 60% yield). ESI-MS of M/z [ M + H ]]⁺715,[M-H]⁺713.¹H-NMR(300MHz,DMSO-d₆)δ:9.72(d,1H),7.73(d,1H),7.58(s,1H),7.52(s,1H),6.92(d,1H),6.71(d,1H),4.58～4.44(m,1H),4.37(s,2H),3.86(d,3H),3.78(s,3H),3.48(t,2H),3.32(s,2H),3.04(dd,J＝9.8Hz,1H),2.5～2.43(m,3H),2.40(d,3H),1.34(s,9H),0.97(s,9H),0.20(s,6H).

Example 26: synthesis of Compound JJA-D10

The phenolic hydroxyl group and amino protecting groups of compound 10c (3.57g,5mmol) were removed in this order and treated accordingly as in example 22 to give JJA-D10 as a yellow solid (1.95g, 78% yield). m.p. 82.0-83.5 ℃. ESI-MS of M/z [ M + H ]]⁺501,[M-H]⁺499.¹H-NMR(300MHz,DMSO-d₆)δ:9.86(s,1H),9.46(s,1H),7.60(d,1H),7.37(d,1H),6.92(d,1H),6.86(d,1H),5.49(s,1H),4.35(s,2H),3.87(s,3H),3.82(d,3H),2.44(s,3H),2.42(s,3H),2.40(s,2H),1.63(dd,J＝6.6Hz,2H),1.37(dd,1H),1.23(s,2H).¹³C-NMR(75MHz,DMSO-d₆)δ:196.70,195.75,165.0,149.90,149.34,148.92,148.06,136.35,128.98,128.27,127.48,125.23,123.21,121.71,110.45,79.49,71.60,56.25,56.30,50.93,49.20,44.10,36.75,26.20,26.10.

Example 27: synthesis of Compound JJA-D11

Taking Apo-NH₂(1.81g,10mmol), Apo-CHO (1.94g,10mmol), reacted according to the procedure described in example 10 to produce compound JJA-D0-TBDMS. Dissolving a compound JJA-D0-TBDMS (5.87g,10mmol) with a proper amount of anhydrous dichloromethane, vacuumizing under the protection of nitrogen, adding triethylamine (2.02g,20mmol) and acetyl chloride (1.57g,20mmol), reacting for 2 hours in an ice bath, removing the ice bath after the reaction is finished, washing the reaction solution with water, extracting with dichloromethane, combining organic layers, drying with anhydrous sodium sulfate, and evaporating to dryness under reduced pressure. Dissolving the residue with anhydrous dichloromethane, adding TBAF dropwise under stirring, reacting for 2h, washing with water, extracting with dichloromethane, mixing organic layers, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and separating with silica gel column (Dichloromethane methanol 100: 1). Compound JJA-D11 was obtained as a yellow solid (2.48g, 62% yield). m.p. 187.2-188.9 ℃. ESI-MS of M/z [ M + H ]]⁺402,[M-H]⁺400.¹H-NMR(300MHz,DMSO-d₆)δ:10.36(s,1H),9.75(s,1H),7.50(d,1H),7.41(d,1H),7.34(d,1H),7.31(d,1H),4.49(d,2H),3.88(s,3H),3.81(s,3H),2.46(s,3H),2.40(s,3H),1.81(s,3H).¹³C-NMR(75MHz,DMSO-d₆)δ:196.67,195.81,173.82,150.55,148.66,147.87,146.49,129.46,128.67,127.51,125.24,123.19,122.20,110.90,109.91,56.52,56.14,48.81,26.11,26.15,21.49.

Example 28: synthesis of Compound JJA-D12

Dissolving a compound JJA-D0-TBDMS (5.87g,10mmol) with a proper amount of anhydrous dichloromethane, vacuumizing under the protection of nitrogen, adding triethylamine (2.02g,20mmol) and propionyl chloride (1.85g,20mmol), reacting for 2 hours in an ice bath, removing the ice bath after the reaction is finished, washing the reaction solution with water, extracting with dichloromethane, combining organic layers, drying with anhydrous sodium sulfate, and evaporating to dryness under reduced pressure. The residue was dissolved in anhydrous dichloromethane, TBAF was added dropwise with stirring, reacted for 2 hours, washed with water, extracted with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column (dichloromethane: methanol ═ 100: 1). Compound JJA-D12 was obtained as a white solid (2.37g, 57% yield). m.p. 184.2-185.5 ℃. ESI-MS of M/z [ M + H ]]⁺416,[M-H]⁺414.¹H-NMR(300MHz,DMSO-d₆)δ:10.29(s,1H),9.74(s,1H),7.48(d,J＝1.8Hz,1H),7.41(d,J＝1.8Hz,1H),7.34(d,J＝1.8Hz,1H),7.28(d,J＝1.8Hz,1H),5.08(d,J＝15.2Hz,1H),4.45(d,J＝15.3Hz,1H),3.88(s,2H),3.81(s,1H),2.45(s,2H),2.39(s,1H),2.03(dt,J＝15.9,7.0Hz,1H),1.18(t,J＝7.1Hz,1H),0.95(dd,J＝9.6,5.2Hz,1H).¹³C NMR(75MHz,CDCl₃)δ:196.55,195.70,176.93,150.65,148.72,147.70,146.35,129.62,128.66,127.03,125.23,123.47,122.32,110.99,109.81,56.55,56.16,49.03,26.96,26.09,9.21,1.03.

Example 29: synthesis of Compound JJA-D13

Dissolving compound JJA-D0-TBDMS (5.87g,10mmol) in appropriate amount of anhydrous dichloromethane, vacuumizing under nitrogen protection, adding triethylamine (2.02g,20mmol) and isobutyrylChlorine (2.12g,20mmol) reacts for 2h in an ice bath, after the reaction is finished, the ice bath is removed, the reaction solution is washed by water, dichloromethane is extracted, organic layers are combined, anhydrous sodium sulfate is dried, and reduced pressure evaporation is carried out. The residue was dissolved in anhydrous dichloromethane, TBAF was added dropwise with stirring, reacted for 2 hours, washed with water, extracted with dichloromethane, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column (dichloromethane: methanol ═ 100: 1). Compound JJA-D13 was obtained as a white solid (1.97g, 46% yield). 187.3-188.9 ℃ in m.p. ESI-MS of M/z [ M + H ]]⁺430,[M-H]⁺428.¹H NMR(300MHz,DMSO-d₆)δ:10.34(s,1H),9.79(s,1H),7.47(d,J＝1.8Hz,1H),7.43(d,J＝1.8Hz,1H),7.35(d,J＝1.9Hz,1H),7.26(d,J＝1.9Hz,1H),5.07(d,J＝15.4Hz,1H),4.40(d,J＝15.3Hz,1H),3.88(d,J＝3.4Hz,3H),3.82(s,3H),2.73(s,1H),2.45(s,3H),2.40(s,3H),1.15～0.90(m,6H).¹³C NMR(75MHz,CDCl₃)δ:196.56,195.71,180.60,150.77,148.72,147.65,146.36,129.51,128.57,127.05,125.26,123.39,122.21,110.98,109.85,56.54,56.15,49.15,31.69,26.10,19.85,19.3.

Example 30: synthesis of Compound 14a

The compound Apo-CHO (1.94g,10mmol) was dissolved in DMF and added with methyl iodide (2.84g,20mmol) and potassium carbonate (2.76g,20mmol) and heated at 40 ℃ for reaction for 3 h. After completion of the reaction, the reaction mixture was cooled, washed with water, extracted with dichloromethane, the combined organic layers were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column (ethyl acetate: petroleum ether: 1:8) to obtain compound 14a as a white solid (1.64g, yield 79%). ESI-MS of M/z [ M + H ]]⁺209,[M-H]⁺207.¹H-NMR(300MHz,DMSO-d₆)δ:10.33(s,1H),7.88(d,1H),7.79(d,1H),4.00(s,3H),3.96(s,3H),2.62(s,3H).

Example 31: synthesis of Compound JJA-D14

Taking compound 14a (2.08g,10mmol), dissolving with methanol, adding compound Apo-NH under stirring₂(1.99g,11mmol), reacting for 1h until no yellow precipitate is generated, adding a proper amount of sodium cyanoborohydride while stirring, dropwise adding a catalytic amount of glacial acetic acid, reacting for 2h at normal temperature, washing with water, extracting with ethyl acetate, combining organic layers, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and separating with silica gel column (1.99g,11mmol)Ethyl acetate-petroleum ether-1: 4) to give compound JJA-D14(2.42g, 65% yield) as a white solid. m.p. 182.2-183.9 ℃. ESI-MS of M/z [ M + H ]]⁺374,[M-H]⁺372.¹H-NMR(300MHz,DMSO-d₆)δ:9.46(s,1H),7.61(d,J＝1.9Hz,1H),7.44(d,J＝1.9Hz,1H),6.93(d,J＝1.8Hz,1H),6.83(d,J＝1.8Hz,1H),5.52(s,1H),4.39(s,1H),3.89(s,3H),3.82(s,2H),2.49(s,1H),2.41(s,2H).¹³C-NMR(75MHz,CDCl₃)δ:197.42,197.25,152.60,151.51,145.73,137.28,135.57,132.84,132.26,129.49,122.69,110.88,105.70,101.64,61.00,56.25,55.93,43.21,26.44,26.31.

Example 32: synthesis of Compound JJA-D15

Compound Apo-CHO (1.94g,10mmol) was weighed out and dissolved in DMF, bromoethane (2.18g,20mmol) and potassium carbonate (2.76g,20mmol) were added and heated to 40 ℃ for reaction for 3 h. After completion of the reaction, the reaction mixture was cooled, washed with water, extracted with dichloromethane, the combined organic layers were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column (ethyl acetate: petroleum ether: 1:8) to obtain compound 15a (1.52g, 68%). Taking compound 15a (2.22g,10mmol), dissolving with methanol, adding compound Apo-NH under stirring₂(1.99g,11mmol) and reacted for 1h until no yellow precipitate is formed, an appropriate amount of sodium cyanoborohydride is added with stirring, a catalytic amount of glacial acetic acid is added dropwise, the reaction is carried out at normal temperature for 2h, the reaction product is washed with water, extracted with ethyl acetate, combined with organic layers, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by a silica gel column (ethyl acetate: petroleum ether ═ 1:4) to obtain JJA-D15 as a brown yellow solid (2.39g, 62% yield). m.p. 184.2-185.4 deg.C ESI-MS M/z [ M + H ]]⁺388,[M-H]⁺386.¹H-NMR(300MHz,DMSO-d₆)δ:9.45(s,1H),7.62(d,J＝2.0Hz,1H),7.43(d,J＝1.9Hz,1H),6.93(d,J＝1.8Hz,1H),6.84(d,J＝1.8Hz,1H),5.53(s,1H),4.40(s,2H),4.15(d,J＝7.0Hz,2H),3.87(s,3H),3.82(s,3H),2.49(s,3H),2.41(s,3H),1.34(t,J＝7.0Hz,3H).¹³C-NMR(75MHz,DMSO-d₆)δ:197.24,197.00,152.51,150.20,146.70,138.03,137.53,134.03,132.68,128.79,121.79,111.34,104.91,102.34,68.90,56.38,56.30,41.95,40.49,40.21,39.93,39.66,39.38,26.91,26.66,16.10.

Example 33: synthesis of Compound JJA-D16

Referring to the procedure of example 32, ethyl bromide was converted to isopropyl bromide (2.44g,20mmol) to provide JJA-D16 as a tan solid (2.12g, 53% yield). m.p. 185.2-187.9 ℃. ESI-MS of M/z [ M + H ]]⁺402,[M-H]⁺400.¹H NMR(300MHz,DMSO-d6)δ:9.46(s,1H),7.61(d,J＝2.0Hz,1H),7.42(d,J＝2.0Hz,1H),6.99(d,J＝2.0Hz,2H),6.79(d,J＝1.8Hz,1H),5.60(s,1H),4.70(dt,J＝12.2,6.1Hz,2H),4.38(s,3H),3.83(m,3H),2.48(m,6H),1.28(d,J＝6.1Hz,6H).¹³C NMR(75MHz,CDCl₃)δ:197.47,197.28,152.58,149.35,145.69,137.09,132.89,132.43,129.57,122.47,110.47,106.35,101.27,75.11,56.21,55.85,43.31,26.32,22.71.

Example 34: synthesis of Compound JJA-D17

Referring to the procedure of example 32, ethyl bromide was converted to propyl bromide (2.44g,20mmol) to provide JJA-D17 as a tan solid (2.72g, 68% yield). 185.5-187.3 ℃ in m.p. ESI-MS of M/z [ M + H ]]⁺402,[M-H]⁺400.¹H NMR(300MHz,DMSO-d₆)δ:9.46(s,1H),7.61(d,J＝1.7Hz,1H),7.43(d,J＝1.8Hz,1H),6.93(d,J＝1.6Hz,1H),6.83(s,1H),5.54(s,1H),4.40(s,2H),4.04(t,J＝6.6Hz,2H),3.87(s,3H),3.82(s,3H),2.49(s,3H),2.41(s,3H),1.76(d,J＝7.2Hz,2H),1.01(s,3H).¹³C NMR(75MHz,DMSO-d₆)δ:197.23,196.97,152.46,150.40,146.67,138.05,137.52,133.87,132.64,128.77,121.78,111.40,104.85,102.37,74.76,56.36,56.29,41.89,26.90,26.63,23.67,10.90.

Example 35: synthesis of Compound 18b

Dissolving Apo-NH-Boc (2.81g,10mmol) with appropriate amount of acetone, sequentially adding methyl iodide (2.84g,20mmol) and triethylamine (2.02g,20mmol) under sealed condition, and reacting for 2 h. After completion of the reaction, acetone was evaporated under reduced pressure, the reaction mixture was washed with water, the combined organic layers were extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column (ethyl acetate: petroleum ether: 1:10) to obtain compound 18b (2.41g, yield 82%) as a white solid. ESI-MS of M/z [ M + H ]]⁺296,[M-H]⁺294.¹H-NMR(300MHz,CDCl₃)δ:8.36(s,1H),7.21(d,1H),7.18(s,1H),3.88(s,3H),3.85(s,3H,),2.55(s,3H),1.49(s,9H).

Example 36: synthesis of Compound 18c

Taking the compound 18b (2.95g,10mmol), dissolving in 20ml dichloromethane, dropwise adding 5ml trifluoroacetic acid under stirring, and reacting for 2-3 h. After completion of the reaction, the reaction mixture was washed with water, and the combined organic layers were extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column (dichloromethane: methanol: 100:1) to obtain compound 18c as a white solid (1.52g, yield 78%). ESI-MS of M/z [ M + H ]]⁺196,[M-H]⁺194.¹H-NMR(300MHz,DMSO-d₆)δ:6.99(d,1H,ArH),6.80(d,1H,ArH),5.13(s,2H),3.80(s,3H),3.69(s,3H),2.47(s,3H).

Example 37: JJA-D18 Synthesis

Taking compound Apo-CHO (1.94g,10mmol), dissolving with methanol, adding compound 18c (2.15g,11mmol) under stirring, reacting for 1h until no yellow precipitate is generated, adding a proper amount of sodium cyanoborohydride under stirring, dropwise adding a catalytic amount of glacial acetic acid, reacting for 2h at normal temperature, washing with water, extracting with ethyl acetate, combining organic layers, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and separating with silica gel column (using ethyl acetate: petroleum ether ═ 1:4) to obtain compound JJA-D18 as a brown yellow solid (2.05g, yield 55%). 181.2-182.8 ℃ in m.p. ESI-MS of M/z [ M + H ]]⁺374,[M-H]⁺372.¹H NMR(300MHz,DMSO-d₆)δ:9.87(s,1H),7.60(d,J＝2.0Hz,1H),7.37(d,J＝1.9Hz,1H),6.85(s,2H),5.95(s,1H),4.36(d,J＝6.7Hz,2H),3.87(s,3H),3.81(s,3H),3.75(s,3H),2.44(d,J＝2.9Hz,6H).¹³C NMR(75MHz,CDCl₃)δ:197.75,196.89,152.05,148.64,146.72,141.59,140.14,134.08,129.44,124.01,123.54,108.98,105.70,102.26,60.14,56.22,55.92,43.00,26.44,26.17.

Example 38: synthesis of Compound JJA-D19

Compounds

19b and 19c were synthesized by reaction treatment according to the methods of examples 35, 36 and 37, respectively, by replacing methyl iodide with ethyl bromide (2.16g,20mmol), and finally yielded compounds JJA-D19 as brown yellow solids (1.70g, 44% yield). m.p. 182.4-184.5 ℃. ESI-MS of M/z [ M + H ]]⁺388,[M-H]⁺386.¹H NMR(300MHz,DMSO-d₆)δ:9.89(s,1H),7.59(d,J＝1.8Hz,1H),7.37(d,J＝1.9Hz,1H),6.85(d,J＝2.4Hz,2H),5.85(s,1H),4.37(d,J＝4.0Hz,2H),4.00(d,J＝7.0Hz,2H),3.87(s,3H),3.80(s,3H),2.45(s,3H),2.43(s,3H),1.31(s,3H).¹³C NMR(75MHz,DMSO-d₆)δ:197.70,196.55,152.32,149.55,147.46,142.54,138.16,133.07,128.45,126.31,122.80,110.10,104.89,101.60,67.99,56.40,56.12,41.52,26.97,26.56,15.95.

Example 39: synthesis of Compound JJA-D20

Compounds

20b and 20c were synthesized by reaction treatment according to the methods of examples 35, 36 and 37 by replacing methyl iodide with bromoisopropane (2.46g,20mmol), respectively, to give compounds JJA-D20 as a brown-yellow solid (1.76g, 44% yield). m.p. 186.2-187.5 ℃. ESI-MS of M/z [ M + H ]]⁺402,[M-H]⁺400.¹H NMR(300MHz,DMSO-d₆)δ:9.88(s,1H),7.59(d,J＝1.7Hz,1H),7.37(d,J＝1.8Hz,1H),6.93～6.79(m,2H),5.73(t,J＝6.6Hz,1H),4.55(dt,J＝12.2,6.0Hz,1H),4.36(d,J＝6.4Hz,2H),3.87(s,3H),3.79(s,3H),2.45(s,3H),2.42(s,3H),1.25(s,3H),1.23(s,3H).¹³C NMR(75MHz,CDCl₃)δ:197.71,196.85,152.08,148.62,146.74,142.36,137.82,132.76,129.39,124.07,123.27,108.85,105.71,102.32,75.00,56.18,55.84,43.10,26.39,26.14,22.69.

Example 40: JJA-D21 Synthesis

Compounds

21b and 21c were synthesized by reaction treatment according to the methods of examples 35, 36 and 37 by substituting methyl iodide with bromopropane (2.46g,20mmol), respectively, to give compounds JJA-D21 as a tan solid (2.08g, 52% yield). m.p. 185.2-187.4 ℃. ESI-MS of M/z [ M + H ]]⁺402,[M-H]⁺400.¹H NMR(300MHz,DMSO-d₆)δ:9.88(s,1H),7.60(d,J＝1.8Hz,1H),7.38(d,J＝1.9Hz,1H),6.87(s,2H),5.71(s,1H),4.38(d,J＝6.4Hz,2H),3.87(s,3H),3.90(t,J＝5.2Hz,2H),3.80(s,3H),2.45(s,3H),2.44(s,3H),1.73(dd,J＝14.4,7.1Hz,2H),0.96(t,J＝7.4Hz,3H).¹³C NMR(75MHz,DMSO-d₆)δ:197.72,196.57,152.20,149.57,147.47,142.32,138.47,132.99,128.42,126.24,122.87,110.11,104.97,101.75,74.07,56.40,56.15,41.62,26.99,26.56,23.44,10.79.

Example 41: synthesis of Compound 22a

The compound Apo-CHO (1.94g,10mmol) was dissolved in DMF and added with benzyl chloride (2.52g,20mmol) and potassium carbonate (2.76g,20mmol) and reacted at 40 ℃ for 3 h. After completion of the reaction, the reaction mixture was cooled, washed with water, extracted with dichloromethane, the combined organic layers were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column (ethyl acetate: petroleum ether: 1:8) to obtain compound 22a as a white solid (2.24g, yield 79%). ESI-MS of M/z [ M + H ]]⁺285,[M-H]⁺283.¹H NMR(300MHz,DMSO-d₆)δ:7.68(s,1H),7.52～7.27(m,7H),5.16(s,2H),3.83(s,3H),2.50(s,3H).

Example 42: synthesis of Compound 22b

The compound Apo-NH-Boc (2.81g,10mmol) was dissolved in an appropriate amount of acetone, and benzyl chloride (2.52g,20mmol) and triethylamine (2.02g,20mmol) were added sequentially under a sealed condition to react for 2 h. After completion of the reaction, acetone was evaporated under reduced pressure, the reaction mixture was washed with water, the combined organic layers were extracted with dichloromethane, dried over anhydrous sodium sulfate, and the organic layers were concentrated under reduced pressure and separated by silica gel column (ethyl acetate: petroleum ether: 1:10) to obtain compound 22b as a white solid (3.04g, yield 82%). ESI-MS of M/z [ M + H ]]⁺372,[M-H]⁺370.¹H NMR(300MHz,DMSO-d₆)δ:8.05(s,1H),7.96(d,J＝1.3Hz,1H),7.55～7.26(m,6H),5.09(s,2H),3.92(s,3H),2.53(d,J＝5.8Hz,3H),1.44(s,9H).

Example 43: synthesis of Compound 22c

The compound 22b (3.71g,10mmol) was dissolved in 20ml dichloromethane, and 5ml trifluoroacetic acid was added dropwise with stirring to react for 2-3 h. After completion of the reaction, the reaction mixture was washed with water, extracted with dichloromethane, the combined organic layers were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column (dichloromethane: methanol: 100:1) to obtain compound 22c as a white solid (2.17g, yield 80%). ESI-MS of M/z [ M + H ]]⁺272,[M-H]⁺270.¹H NMR(300MHz,DMSO-d₆)δ:7.50(d,J＝6.7Hz,1H),7.42～7.28(m,4H),7.01(d,J＝1.7Hz,1H),6.84(d,J＝1.6Hz,1H),5.05(s,2H),4.96(s,2H),3.83(s,3H),2.49(d,J＝7.5Hz,3H).

Example 44: synthesis of Compound JJA-D22

Take compound 22a (2.83g,10mmol)Dissolving the compound with methanol, adding compound 22c (2.98g,11mmol) under stirring, reacting for 1h until no yellow precipitate is generated, adding an appropriate amount of sodium cyanoborohydride under stirring, dropwise adding a catalytic amount of glacial acetic acid, reacting for 2h at normal temperature, washing with water, extracting with ethyl acetate, combining organic layers, drying over anhydrous sodium sulfate, concentrating under reduced pressure, and separating with silica gel column (ethyl acetate: petroleum ether ═ 1:4) to obtain compound JJA-D22 as a light yellow solid (2.42g, yield 45%). ESI-MS of M/z [ M + H ]]⁺540,[M-H]⁺538.¹H NMR(300MHz,DMSO-d₆)δ:7.53～7.27(m,12H),6.82(s,1H),6.78(s,1H),5.16(s,4H),5.01(s,1H),4.45(s,2H),3.83(s,6H),2.50(s,6H).¹³C NMR(300MHz,DMSO-d₆)δ:198.44,152.79,152.41,150.94,144.89,137.56,136.96,135.94,134.37,130.50,129.01,128.19,128.16,122.92,114.41,109.75,108.74,74.02,56.83,42.84,27.81.

Example 45: synthesis of Compound JJA-D23

Taking compound 22a (2.83g,10mmol), dissolving with methanol, adding compound Apo-NH under stirring₂(1.99g,11mmol) and reacted for 1h until no yellow precipitate is formed, an appropriate amount of sodium cyanoborohydride is added with stirring, a catalytic amount of glacial acetic acid is added dropwise, the reaction is carried out at normal temperature for 2h, the reaction product is washed with water, extracted with ethyl acetate, combined with organic layers, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by a silica gel column (ethyl acetate: petroleum ether ═ 1:4) to obtain the compound JJA-D23 as a light yellow solid (2.51g, 56% yield). m.p. 135.5-136.4 ℃. ESI-MS of M/z [ M + H ]]⁺450,[M-H]⁺448.¹H NMR(300MHz,DMSO-d₆)δ:9.44(s,1H),7.60(d,J＝1.9Hz,1H),7.49(dd,J＝7.9,1.6Hz,3H),7.43～7.29(m,3H),6.93(d,J＝1.8Hz,1H),6.80(d,J＝1.8Hz,1H),5.50(s,1H),5.15(s,2H),4.30(s,2H),3.94(s,3H),3.82(s,3H),2.50(s,3H),2.37(s,3H).¹³C NMR(75MHz,,CDCl₃)δ:197.45,197.30,152.68,150.65,145.62,137.10,136.95,135.60,133.04,132.90,129.58,128.53,128.47,128.34,122.51,110.65,106.36,101.23,75.07,56.25,55.98,43.14,26.49,26.35。

Example 46: synthesis of Compound JJA-D24

Compound Apo-CHO (1.94g,10mmol) was taken, dissolved in methanol and compound 22c (2) was added with stirring.98g,11mmol) to no longer generate yellow precipitate, adding a proper amount of sodium cyanoborohydride while stirring, dropwise adding a catalytic amount of glacial acetic acid, reacting at normal temperature for 2h, then adding water for washing, extracting with ethyl acetate, combining organic layers, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and separating with silica gel column (ethyl acetate: petroleum ether ═ 1:4) to obtain JJA-D24 as a yellow solid (2.47g, yield 55%). m.p. 119.4-120.4 ℃. ESI-MS of M/z [ M + H ]]⁺450,[M-H]⁺448.¹H NMR(300MHz,DMSO-d₆)δ:9.91(s,1H),7.68～7.55(m,1H),7.50(dd,J＝6.2,1.8Hz,2H),7.43～7.27(m,4H),6.87(d,J＝3.4Hz,2H),5.73(s,1H),4.93(t,J＝27.8Hz,2H),4.28(d,J＝41.2Hz,2H),3.92～3.71(m,6H),2.67～2.14(m,6H).¹³C NMR(75MHz,DMSO-d₆)δ:197.73,196.61,152.29,149.53,147.47,142.40,137.99,137.80,133.27,128.83,128.67,128.45,126.21,122.95,110.17,105.05,101.58,73.84,56.44,56.23,41.70,27.03,26.65。

Example 47: synthesis of Compound 25a

Compound Apo-CHO (1.94g,10mmol) was dissolved in DMF and added with methanesulfonyl chloride (2.28g,20mmol) and triethylamine (2.02g,20mmol) and reacted in ice bath for 3 h. After completion of the reaction, the reaction mixture was washed with water, extracted with dichloromethane, the combined organic layers were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by silica gel column (ethyl acetate: petroleum ether: 1:8) to obtain compound 25a as a white solid (1.45g, yield 53%). ESI-MS of M/z [ M + H ]]⁺273,[M-H]⁺271.¹H NMR(300MHz,DMSO-d₆)δ:10.18(d,J＝5.0Hz,1H),8.04～7.84(m,2H),4.01(s,3H),2.75～2.60(m,3H),1.59～1.36(m,3H).

Example 48: synthesis of Compound JJA-D25

Taking compound 25a (2.72g,10mmol), dissolving with methanol, adding compound Apo-NH under stirring₂(2.17g,12mmol) and reacted for 1h until no yellow precipitate is formed, an appropriate amount of sodium cyanoborohydride is added with stirring, a catalytic amount of glacial acetic acid is added dropwise, the reaction is carried out at normal temperature for 2h, the reaction product is washed with water, extracted with ethyl acetate, combined with organic layers, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by a silica gel column (ethyl acetate: petroleum ether ═ 1:4) to obtain JJA-D25 as a brown yellow solid (2.79g, 64% yield). m.p. 172.4-173.6 deg.C。ESI-MS:m/z[M+H]⁺438,[M-H]⁺436.¹H NMR(300MHz,DMSO-d₆)δ:9.54(s,1H),7.62(s,1H),7.55(s,1H),6.94(d,J＝1.0Hz,1H),6.70(s,1H),5.82(s,1H),4.49(d,J＝4.8Hz,2H),3.96(s,3H),3.83(s,3H),3.59(s,3H),2.53(s,3H),2.37(s,3H).¹³C NMR(75MHz,DMSO-d₆)δ:197.44,196.91,152.10,146.77,140.30,138.08,137.12,136.41,136.12,128.77,120.51,111.66,104.74,102.35,56.76,56.37,41.98,27.19,26.64.

Example 49: synthesis of Compound JJA-D26

Compound Apo-CHO (1.94g,10mmol) was dissolved in DMF and reacted with ethylsulfonyl chloride (2.56g,20mmol) and triethylamine (2.76g,20mmol) according to the methods of examples 47 and 48 to give compound 26a and compound JJA-D26 as a tan solid (3.06g, 68% yield). m.p. 174.2-176.5 ℃. ESI-MS of M/z [ M + H ]]⁺452,[M-H]⁺450.¹H-NMR(300MHz,DMSO-d₆)δ:9.53(s,1H),7.62(d,J＝1.9Hz,1H),7.53(d,J＝1.9Hz,1H),6.93(d,J＝1.8Hz,1H),6.69(d,J＝1.7Hz,1H),5.83(s,1H),4.48(d,J＝5.8Hz,2H),3.94(s,3H),3.83(d,J＝4.7Hz,3H),3.71(q,J＝7.3Hz,2H),2.53(s,3H),2.37(s,3H),1.48(t,J＝7.3Hz,3H).¹³C NMR(75MHz,CDCl₃)δ:197.72,197.03,152.05,145.74,140.45,136.93,136.17,135.75,135.13,129.69,121.62,110.42,106.66,101.25,56.25,47.76,43.06,26.60,26.36,8.39.

Example 50: synthesis of Compound 27c

Taking Apo-NH₂(1.81g,10mmol), Apo-NH-Boc was prepared as in example 7, and Apo-NH-Boc (2.81g,10mmol) was dissolved in an appropriate amount of DMF, followed by addition of methanesulfonyl chloride (1.36g,12mmol) and triethylamine (2.02g,20mmol), reaction in ice bath for 2h, and after completion of the reaction, 60ml of dichloromethane was added, washing with water, evaporation to dryness under reduced pressure, and separation on silica gel column (ethyl acetate: petroleum ether ═ 1:10) to give compound 27b as a white solid. ESI-MS of M/z [ M + H ]]⁺360.5.¹H NMR(300MHz,DMSO-d₆) δ 8.67(s,1H),7.80(d, J ═ 1.7Hz,1H),7.41(d, J ═ 1.9Hz,1H),3.91(s,3H),3.59(s,1H),2.59(s,3H),1.78 to 1.15(m, 9H). Dissolve compound 27b (3.59g,10mmol) in dichloromethane, add trifluoroacetic acid, react for 1h with NaHCO₃The reaction mixture was neutralized with a saturated aqueous solution, extracted with dichloromethane, and the organic layer was concentrated under reduced pressure to give compound 27c (1.76g, 68%) as a white solid. ESI-MS of M/z [ M + H ]]⁺260.4,[M-H]⁺258.5.¹H NMR(300MHz,DMSO-d₆)δ:7.07(d,J＝2.0Hz,1H),6.84(d,J＝2.0Hz,1H),5.39(s,2H),3.84(s,3H),3.41(d,J＝26.4Hz,3H),2.61–2.29(m,3H).

Example 51: synthesis of Compound JJA-D27

Compound 27c (2.8g,11mmol) was taken, dissolved in methanol, Apo-CHO (1.94g,10mmol) was added, the reaction was carried out for 1h, after no precipitation occurred, an appropriate amount of sodium cyanoborohydride was added with stirring, a catalytic amount of glacial acetic acid was added dropwise, the reaction was carried out at room temperature for 2h, water washing and ethyl acetate extraction were carried out, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure and separated by a silica gel column (ethyl acetate: petroleum ether ═ 1:3), to obtain compound JJA-D27(2.57g, 59% yield) as a tan solid. m.p. 179.2-181.4 ℃. ESI-MS of M/z [ M + H ]]⁺438,[M-H]⁺436.¹H NMR(300MHz,DMSO-d₆)δ:9.95(s,1H),7.64(s,1H),7.38(s,1H),6.90(s,2H),5.95(s,1H),4.41(t,J＝10.0Hz,2H),3.87(d,J＝6.8Hz,6H),3.52(s,3H),2.50(s,3H),2.44(s,3H).¹³C NMR(75MHz,DMSO-d₆)δ:197.86,196.64,152.79,149.48,147.54,142.63,136.31,129.05,128.57,125.53,122.95,109.78,105.21,100.72,56.48,56.38,41.13,27.19,26.59.

Example 52: synthesis of Compound 28c

Taking Apo-NH₂(1.81g,10mmol), Apo-NH-Boc was prepared as in example 7, and Apo-NH-Boc (2.81g,10mmol) was dissolved in an appropriate amount of DMF, ethylsulfonyl chloride (1.53g,12mmol) and triethylamine (2.02g,20mmol) were added, and the reaction was performed in ice bath for 2h, followed by addition of 60ml of dichloromethane, washing with water, evaporation to dryness under reduced pressure, and silica gel column separation (ethyl acetate: petroleum ether ═ 1:10) to obtain compound 28b as a white solid (3.02g, yield 81%). ESI-MS: [ M + H ]]⁺374.3,[M-H]⁺372.3.¹H-NMR(300MHz,DMSO-d₆) δ 8.67(s,1H),7.80(d, J ═ 1.7Hz,1H),7.41(d, J ═ 1.9Hz,1H),3.91(s,3H),3.58(q, J ═ 7.3Hz,2H),2.59(s,3H),1.54 to 1.35(m,12H), compound 28b (3.73g,10mmol) was dissolved in dichloromethane, trifluoroacetic acid was added, reaction was carried out for 1H, and NaHC was usedO₃The reaction mixture was neutralized with a saturated aqueous solution, extracted with dichloromethane, and the organic layer was concentrated under reduced pressure to give compound 28c (1.77g, 65%). ESI-MS of M/z [ M + H ]]⁺274.3,[M+NH₄]⁺291.2.¹H NMR(300MHz,DMSO-d₆)δ:7.07(d,J＝1.9Hz,1H),6.84(d,J＝1.8Hz,1H),5.34(s,2H),3.83(s,3H),3.61(q,J＝7.3Hz,2H),2.52(s,3H),1.41(t,J＝7.3Hz,3H).

Example 53: synthesis of Compound JJA-D28

Compound 28c (2.99g,11mmol) was taken, dissolved in methanol, Apo-CHO (1.94g,10mmol) was added, after no more precipitation occurred, an appropriate amount of sodium cyanoborohydride was added, a catalytic amount of glacial acetic acid was added dropwise, reaction was carried out for 3h, and the reaction solution was separated on a silica gel column (ethyl acetate: petroleum ether ═ 1:3) to give compound JJA-D28(2.84g, 63% yield) as a tan solid. 177.2-180.1 ℃ in m.p. ESI-MS of M/z [ M + H ]]⁺452,[M-H]⁺450.¹H NMR(300MHz,DMSO-d₆)δ:9.96(s,1H),7.66(s,1H),7.38(d,J＝1.3Hz,1H),6.91(s,2H),5.85(t,J＝6.0Hz,1H),4.42(d,J＝5.9Hz,2H),3.88(s,3H),3.85(s,3H),3.66(q,J＝7.3Hz,2H),2.50(s,3H),2.44(s,3H),1.26(dd,J＝15.3,5.5Hz,3H).¹³C NMR(75MHz,DMSO-d₆)δ:197.84,196.63,152.66,149.49,147.55,142.79,136.26,129.01,128.56,125.48,123.02,109.77,105.26,100.75,56.48,56.38,47.66,41.23,27.20,26.58,8.65.

Example 54: JJA-D0 evaluation of safety

This example used the picrorhizine derivative JJA-D0, and logarithmic growth of RAW264.7 cells was performed at 1.0X 10⁴The culture medium was inoculated into a 96-well plate at a concentration of 12.5. mu.M, 25. mu.M, 50. mu.M, 100. mu.M, 200. mu.M and 400. mu.M, treated with JJA-D0 at a concentration of 12.5. mu.M, 25. mu.M, 50. mu.M, 100. mu.M, 200. mu.M and 400. mu.M, and after 24 hours, 36 hours, 48 hours and 72 hours, the supernatant was discarded, the culture medium containing 0.5mg/₂Culturing at 37 deg.C for 4h in incubator, removing culture solution, adding 100 μ l DMSO, shaking for 10min, detecting light absorption value at wavelength of 570nm, and calculating cell survival rate. The purpose was to test the cytotoxicity of compound JJA-D0 on RAW264.7 cells. The experimental data show (fig. 13) that the JJA-D0 compound did not exhibit any cytotoxicity at 24 h; at 36h, only at 400. mu.M, the composition is shown to be betterSmall toxicity, corresponding to a cell survival rate of 88.66% in Control (Ctrl); at 48h, the cell survival rate at 200. mu.M was equivalent to 91.73% of that of the blank control group, and at 400. mu.M was equivalent to 75.96% of that of the blank control group; at 72h, the cell viability at 200. mu.M corresponded to 83.91% of that of the blank control, and at 400. mu.M, the cell viability at 400. mu.M corresponded to 50.73% of that of the blank control. As can be seen from the results, JJA-D0 exhibited cytotoxicity and good safety after treating cells for 48h and 72h for a long time and at a concentration of more than 200. mu.M.

Example 55: MTT method for testing protection effect of Apo, JJA-D0 and 14 JJA-D0 derivatives on LPS-induced RAW264.7 cell injury

Taking logarithmic growth RAW264.7 cells, at 1.0 × 10⁴The culture medium was inoculated in a 96-well plate at a concentration of one ml, Apo (50,100, 200. mu.M), JJA-D0(50,100, 200. mu.M) and 14 JJA-D0 derivatives (50,100, 200. mu.M) in the above examples were added at different concentrations after 24 hours of growth, and the blank medium and Model medium were added in equal amounts, and after 1 hour of pretreatment, the supernatant was aspirated. Except for the blank control group, the same amount of blank culture medium was added, and the culture medium containing 100. mu.g/mL LPS was added to each of the other groups. Is placed in CO₂Culturing at 37 deg.C for 24 hr, discarding supernatant, adding culture solution containing 0.5mg/ml MTT, and placing in CO₂Culturing at 37 deg.C for 4h in incubator, removing culture solution, adding 100 μ l DMSO, shaking for 10min, detecting light absorption value at wavelength of 570nm, and calculating cell survival rate. The experimental results (FIG. 14) show that the tested JJA-D0 derivatives all have a certain protective effect on LPS-induced RAW264.7 cell damage, and the cell survival rate is highest at 200 μ M under the condition of JJA-D26.

Example 56: effect of Apo, JJA-D0, 14 JJA-D0 derivatives on LPS-induced intracellular ROS levels in RAW264.7 cells

This example examined the effect of the derivatives of picrorhizin, JJA-D0 and 14 of JJA-D0 in the above examples on LPS-induced ROS levels in RAW264.7 cells. RAW264.7 cells in logarithmic growth phase were seeded in a black bottom-penetrating 96-well plate at a density of 1X 10⁴One cell/100. mu.l, after 24h of growth, equal amount of air was added except for the blank control group and the model groupOutside the white culture solution, the experimental groups were pretreated with different compounds of different concentrations for 1h, and then the supernatants were aspirated. Except for the blank control group, the culture medium containing 100. mu.g/mL LPS was added to each group for 24 hours. The supernatant was discarded, washed 2 times with PBS, the cells were labeled with a fluorescent probe DCFH-DA sensitive to active oxygen (final concentration 20. mu.M), incubated at 37 ℃ for 30min, the supernatant discarded, and washed 2 times with PBS. And immediately detecting the fluorescence intensity of ROS in RAW264.7 cells induced by LPS by using an enzyme-labeling instrument, wherein the excitation wavelength is 488nm, and the emission wavelength is 525 nm. The results (fig. 15) show that the tested JJA-D0 derivatives all had some reduction in LPS-induced ROS levels in RAW264.7 cells, and that most of the derivatives were more effective than the monomeric compound picrorhizin, with significant differences in the reduction in intracellular ROS levels at the tested concentrations compared to the model group.

Example 57: JJA-D26 downregulation of the expression of the inflammatory factor TNF-alpha

In this example, a western blot experiment was used to examine the effect of kutkin, JJA-D0, JJA-D26, on TNF- α expression in LPS-induced RAW264.7 cells. In the same manner as in example 55, at 5X 10⁵Each of RAW264.7 cells was seeded on a 6-well plate and cultured for 24 hours, and after the cells were pretreated with kutkin (200. mu.M), JJA-D0 (100. mu.M) and JJA-D26 (200. mu.M) for 1 hour, LPS (100. mu.g/mL) was added for 24 hours of stimulation. Cytoplasmic extracts were prepared, subjected to SDS-acrylamide gel electrophoresis, and then transferred to PVDF membranes. The membrane was blocked in TBST and incubated with TNF-alpha antibody (Cell Signaling Technology,1:1000) and beta-actin antibody (Cell Signaling Technology,1:1000) overnight at 4 ℃. Then the membrane is incubated with goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Santa Cruz,1:2000) for 1h at room temperature, the membrane is washed with TBST, and working solution (0.1ml working solution/cm) prepared from ECL solution A and B (1:1) is used²) And (5) exposing and developing. The results (FIG. 16) show that the intracellular TNF-. alpha.protein expression level was up-regulated 2.58-fold in the model cells compared to the blank control group (P)<0.001) in the cells treated with the picrorhizin group (200. mu.M), 1.99-fold in the cells treated with the JJA-D0 group (100. mu.M), 1.65-fold in the cells treated with the JJA-D26 group (200. mu.M), and 1.48-fold in the cells treated with the JJA-D26 group (200. mu.M). Form a model setIn comparison, JJA-D0 and JJA-D26 groups have significant difference on the down-regulation effect of the expression level of the TNF-alpha protein in cells, and P<0.05。

Example 58: JJA-D26 on NADPH oxidase subunit p47^phox、gp91^phoxDown-regulation of expression

In this example, a western blot experiment was used to detect the NADPH oxidase subunit p47 in LPS-induced RAW264.7 cells from kutkin, JJA-D0, JJA-D26^phox、gp91^phoxThe effect of expression. In the same manner as in example 55, at 5X 10⁵Each of RAW264.7 cells was seeded on a 6-well plate and cultured for 24 hours, and after the cells were pretreated with kutkin (200. mu.M), JJA-D0 (100. mu.M) and JJA-D26 (200. mu.M) for 1 hour, LPS (100. mu.g/mL) was added for 24 hours of stimulation. Cytoplasmic extracts were prepared, subjected to SDS-acrylamide gel electrophoresis, and then transferred to PVDF membranes. The membrane was blocked in TBST and reacted with p47^phoxAntibodies (Cell Signaling Technology,1:1000), gp91^phoxAntibodies (Cell Signaling Technology,1:1000) and β -actin antibodies (Cell Signaling Technology,1:1000) were incubated overnight at 4 ℃. Then the membrane is incubated with goat anti-mouse IgG horseradish peroxidase-conjugated antibody (Santa Cruz,1:2000) for 1h at room temperature, the membrane is washed with TBST, and working solution (0.1ml working solution/cm) prepared from ECL solution A and B (1:1) is used²) And (5) exposing and developing. The results of the experiment (FIG. 17) showed that the picrorhizin, JJA-D0, JJA-D26, induced the NADPH oxidase subunit p47 in the RAW264.7 cells induced by LPS^phox、gp91^phoxThe expression has obvious down-regulation effect, and the effect of the kutkin is the weakest, and the effect of JJA-D26 is the strongest.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A derivative of a kutkin dimer analog JJA-D0, or a pharmaceutically acceptable salt thereof, wherein the derivative of kutkin dimer analog JJA-D0, or a pharmaceutically acceptable salt thereof, has a structure of formula (VI):

in the structure of the general formula (VI), R₂，R₃The same or different, are respectively selected from: hydrogen, unsubstituted, straight or branched hydrocarbon carbon chain with 1-8 carbon atoms and no hetero atom; r5 is an unsubstituted, heteroatom-free, straight or branched, hydrocarbyl carbon chain having from 1 to 5 carbon atoms.

2. A derivative of a kutkin dimer analog JJA-D0, or a pharmaceutically acceptable salt thereof, wherein the derivative of kutkin dimer analog JJA-D0, or a pharmaceutically acceptable salt thereof, has a structure of one of general formulas (VII), (VIII), (IX):

in the structure of the general formula (VII), R1 is hydrogen, R3 is selected from hydrogen, unsubstituted and heteroatom-free straight-chain hydrocarbyl carbon chain with 1-8 carbon atoms; r6 is an unsubstituted, heteroatom-free, straight-chain hydrocarbyl carbon chain having from 1 to 4 carbon atoms;

wherein in the structure of the general formula (VIII), R1 is hydrogen, R2 is selected from hydrogen, unsubstituted and heteroatom-free straight-chain hydrocarbyl carbon chain with 1-8 carbon atoms; r7 is an unsubstituted, heteroatom-free, straight-chain hydrocarbyl carbon chain having from 1 to 4 carbon atoms;

wherein in the structure of the general formula (IX), R1 is hydrogen; r8 and R9 are the same or different and are unsubstituted, heteroatom-free, straight-chain hydrocarbyl carbon chains having 1 to 4 carbon atoms.

3. A derivative of a kutkin dimer analog JJA-D0 or a pharmaceutically acceptable salt thereof, wherein the derivative of the kutkin dimer analog JJA-D0 or the pharmaceutically acceptable salt thereof is selected from one of the following compounds:

4. a process for the preparation of a derivative of the picrorhizin dimer analogue JJA-D0 or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 3, comprising the steps of:

wherein the alkylated and sulfonate-esterified derivatives of JJA-D0 are bonded to Apo-CHO and Apo-NH via ether bond and ester bond respectively₂To the phenolic hydroxyl group of the (A) to obtain correspondingly substituted Apo-CHO and Apo-NH₂Intermediate, substituted or unsubstituted Apo-CHO and substituted or unsubstituted Apo-NH₂The catalyst is prepared by condensing two parts of structures and then hydrogenating and reducing the two parts of structures by a Schiff's alkali forming reaction; wherein the Apo-CHO and/or Apo-NH are hydrocarbylated₂The intermediate is prepared by respectively reacting halogenated hydrocarbon with Apo-CHO and/or Apo-NH₂Etherified phenolic hydroxyl groups of (a); sulfoesterified Apo-CHO and/or Apo-NH₂The intermediate is prepared by reacting sulfonyl chloride with Apo-CHO and/or Apo-NH₂Is esterified to form the phenolic hydroxyl group;

wherein the amidated derivative of JJA-D0 is prepared by reacting JJA-D0-TBDMS with both phenolic hydroxyl groups protected with acyl chloride;

the structural formula of Apo-CHO is shown in the specification

Said Apo-NH₂Has the structural formula

The structural formula of the JJA-D0 is shown in the specification

The structural formula of the JJA-D0-TBDMS is shown as

5. Use of the derivative of the kutkin dimer analog JJA-D0 or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 for the preparation of a medicament for the prophylaxis or treatment of NADPH oxidase-related diseases, free radical-related diseases, or inflammation-related diseases.