Berberine derivative, pharmaceutical composition and application of berberine derivative

Abstract

The invention provides a berberine derivative, a pharmaceutical composition and application thereof, and relates to the technical field of pharmaceutical chemistry, a main skeleton of the berberine derivative is berberine with ureido, different sites of the berberine skeleton are connected with different substituents through the ureido, and the oxidation state of a C ring is changed, so that the berberine derivative is obtained. A series of berberine derivatives with high activity on the soluble epoxide hydrolase are obtained, and the IC50 value of the berberine derivatives on inhibition of the soluble epoxide hydrolase reaches a nanomole level, which indicates that the berberine derivatives have efficient inhibition effect on the soluble epoxide hydrolase; therefore, the inhibition effect on inflammation can be achieved by inhibiting soluble epoxide hydrolase, and the method has a good application scene.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a berberine derivative, a pharmaceutical composition and application thereof. Background Art

[0002] Inflammation is an evolutionarily conservative defense response of the immune system to protect the host from harmful stimuli. Common clinical diseases such as sepsis, cardiovascular and cerebrovascular diseases, neurodegenerative diseases, gastrointestinal diseases, and diabetes are often induced by factors such as infection and local tissue damage, and are accompanied by local inflammation and increased levels of pro-inflammatory cytokines. This complex and diverse pattern of inflammatory development and its role in various diseases have attracted widespread attention from researchers. Therefore, it has become possible to assist in the treatment of many inflammatory diseases by targeting inflammation-related pathways.

[0003] Existing anti-inflammatory drugs can be divided into steroidal anti-inflammatory drugs (hormones) that act on phospholipase A2 and non-steroidal anti-inflammatory drugs that act on cyclooxygenase (COX-2) and lipoxygenase (LOX-2). They have been widely used in clinical practice due to their significant therapeutic effects. Steroidal drugs have stronger anti-inflammatory effects, but they also bring greater potential side effects to patients, such as respiratory diseases such as asthma, osteoporosis, etc. Non-steroidal anti-inflammatory drugs have fewer side effects overall, but their widespread use has also led to an increasing number of adverse reactions such as gastrointestinal bleeding.

[0004] Studies have found that soluble epoxide hydrolase (sEH) plays an important role in the process of inflammation. Compared with the existing drug action pathways, it has a new anti-inflammatory pathway, which may be a new direction for the treatment of inflammatory diseases. Arachidonic acid (AA) is the direct precursor for the synthesis of active mediators such as prostaglandins and leukotrienes. These bioactive substances can regulate a series of different inflammatory processes. Epoxyeicosatrienoic acid (EETs) produced by AA through cytochrome P450 enzymes is a beneficial arachidonic acid metabolite with anti-inflammatory and anti-apoptotic effects. The main metabolic pathway of EETs is hydrolysis by sEH to generate proinflammatory factors such as dihydroxyeicosatrienoic acid (DHETs). The decrease in the concentration of EETs in the body and the increase in the concentration of DHETs can induce the production of inflammation. Therefore, inhibiting the activity of sEH and thus increasing the concentration of EETs in the body can effectively treat inflammation and related diseases. Current studies have also shown that sEH is also closely related to cardiovascular, central nervous system and metabolic diseases. Several research groups at home and abroad have designed and synthesized compounds with different skeletons for sEH, and some have entered clinical trials, such as EC5026.

[0005]

[0006] Natural products are important resources for new drug development. Berberine (BBR), also known as berberine, is a typical natural product with rich pharmacological activities in current research. It has a long history of medicinal use in China and has multiple pharmacological activities such as anti-inflammatory, antibacterial, and antiviral. More and more in vitro and in vivo experimental models have shown that BBR is a multi-effect molecule that can simultaneously target multiple inflammation-related signaling molecules and has multiple anti-inflammatory mechanisms. However, its development is severely restricted due to its unclear target and low bioavailability.

[0007] Therefore, there is an urgent need to design and develop a compound with a novel berberine skeleton and an IC50 value for sEH at the nanomolar level. Summary of the invention

[0008] The present invention provides a berberine derivative, a pharmaceutical composition and application thereof, so as to solve the problem that the IC50 value of the existing inflammation-inhibiting compounds is high and the drugability is poor.

[0009] In a first aspect, the present invention provides a berberine derivative, wherein the berberine derivative has the following general structural formula I or II:

[0010]

[0011] In the formula I or II, R 1 and R 2 All are methoxy, or R 1 and R 2 They are cyclically bonded to form methylenedioxy groups;

[0012] R 3 Selected from C 1-4 Alkyl, five-membered to six-membered cycloalkane methyl, five-membered to six-membered oxygen-containing heterocycloalkane methyl, N-(C 1-4 alkylformyl)piperidinyl, N-(C 1-4 alkylformyl)piperidinylmethyl, N-(halogenated C 1-4 alkylformyl)piperidinyl, N-(halogenated C 1-4 Alkylformyl)piperidinylmethyl, halo C 1-3 Alkoxyphenyl, halogenated C 1-3 Any one of alkoxybenzyl, methylsulfonylbenzyl and aminosulfonylbenzyl;

[0013] The C ring is selected from any of the following structures:

[0014]

[0015] Preferably, the R 1 , R 2 They form a ring to form methylenedioxy.

[0016] Preferably, the R 3 Any one selected from isopropyl, isobutyl, cyclopentyl, cyclohexyl, oxetanemethyl, tetrahydrofuranmethyl, tetrahydropyranylmethyl, N-acetylpiperidinyl, N-propionylpiperidinyl, N-acetylpiperidinylmethyl, N-propionylpiperidinylmethyl, difluoromethoxyphenyl, trifluoromethoxyphenyl, difluoromethoxybenzyl, trifluoromethoxybenzyl, methylsulfonylbenzyl and aminosulfonylbenzyl.

[0017] Preferably, the R 3 Any one group selected from trifluoromethoxybenzyl and difluoromethoxybenzyl.

[0018] Preferably, the C ring is selected from any one of the following structures:

[0019]

[0020] Preferably, the C ring is

[0021] Preferably, the R 1 and R 2 The ring is formed between methylenedioxy or the R 1 , R 2 are methoxy, the R 3 Any one of tetrahydropyranyl, N-acetylpiperidinylmethyl, N-propionylpiperidinylmethyl, trifluoromethoxybenzyl, difluoromethoxybenzyl, methylsulfonylbenzyl or aminosulfonylbenzyl, wherein the C ring is

[0022] Preferably, the structural formula of the berberine derivative is as follows:

[0023]

[0024]

[0025] In a second aspect, the present application provides a berberine derivative pharmaceutical composition, which comprises at least one of the compounds described in any one of the first aspect above, their isotope-labeled substances, solvates, polymorphs, pharmaceutically acceptable salts or prodrug compounds thereof.

[0026] In a third aspect, the present application provides a berberine derivative as described in any one of the first aspects, or a berberine derivative pharmaceutical composition as described in the second aspect for use in the preparation of an anti-inflammatory drug, wherein the anti-inflammatory drug is used to inhibit inflammation caused by soluble epoxide hydrolase.

[0027] The present invention includes the following advantages:

[0028] (1) The embodiments of the present invention obtain a novel soluble epoxide hydrolase inhibitory compound by introducing the berberine skeleton described in general formula I and II. Most of the compounds have an IC50 value of less than 20 nM for soluble epoxide hydrolase, and the lowest IC50 value is about 0.67 nM;

[0029] (2) The main skeleton of the berberine derivative for inhibiting soluble epoxide hydrolases prepared in the embodiment of the present invention is berberine with urea groups. Different substituents are connected to different sites of the berberine skeleton through urea groups, and the oxidation state of the C ring is changed to obtain a series of berberine derivatives with high activity against soluble epoxide hydrolases. The IC50 value of the berberine derivatives for inhibiting soluble epoxide hydrolases reaches the nanomolar level, indicating that the berberine derivatives have a high inhibitory effect on soluble epoxide hydrolases, thereby achieving an inhibitory effect on inflammation by inhibiting soluble epoxide hydrolases, and have good application prospects. DETAILED DESCRIPTION

[0030] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0031] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the prior art can be used. The various chemicals used in the present invention are commercially available, and the nuclear magnetic resonance and mass spectrometers used to determine the structures of the following compounds are provided by the Analysis and Testing Center of Beijing Institute of Technology.

[0032] The first aspect of the present invention provides a berberine derivative, a pharmaceutical composition and its application, wherein the berberine derivative has the following general structural formula I or II:

[0033]

[0034] In the formula I or II, R 1 , R 2 Combined into methylenedioxy or selected as methoxy;

[0035] R 3 Selected from C 1-4 Alkyl, five-membered to six-membered cycloalkane methyl, five-membered to six-membered oxygen-containing heterocycloalkane methyl, N-(C 1-4 alkylformyl)piperidinyl, N-(C 1-4 alkylformyl)piperidinylmethyl, N-(halogenated C 1-4alkylformyl)piperidinyl, N-(halogenated C 1-4 Alkylformyl)piperidinylmethyl, halo C 1-3 Alkoxyphenyl, halogenated C 1-3 Any one of alkoxybenzyl, methylsulfonylbenzyl and aminosulfonylbenzyl;

[0036] The C ring is selected from any of the following structures:

[0037]

[0038] C 1-4 Alkyl refers to a straight or branched saturated monovalent hydrocarbon group having 1 to 4 carbon atoms. 1-4 Alkyl represents straight-chain and branched alkyl groups having 1, 2, 3 or 4 carbon atoms, such as methyl, ethyl, propyl, butyl, isopropyl, isobutyl, sec-butyl or tert-butyl.

[0039] C 1-4 Alkylformyl is represented by RC(=O)-, where R is C 1-4 The alkylformyl group is, for example, acetyl, propionyl, butyryl, valeryl, isobutyryl.

[0040] The above five-membered to six-membered cycloalkane methyl is represented by RCH2-, where R is C 5-6 Cycloalkyl, such as cyclopentyl and cyclohexyl.

[0041] The above five-membered to six-membered oxygen-containing heterocycloalkane methyl group is represented by a 5-membered or 6-membered monocyclic ring and contains at least one, for example 1, 2, 3 or more O heteroatoms, a five-membered ring such as tetrahydrofuranyl, dioxolyl; or a 6-membered ring such as tetrahydropyranyl, morpholinyl.

[0042] C 1-3 Alkoxyphenyl is represented by ROPh-, where RO is C 1-3 Alkoxy. The alkoxy group may be connected to the 2, 3, or 4 position of the phenyl group, and the alkoxy group is, for example, methoxy, difluoromethoxy, trifluoromethoxy, ethoxy, or propoxy.

[0043] C 1-3 Alkoxybenzyl is represented by ROBn-, where RO is C 1-3 Alkoxy. The alkoxy group may be attached to the 2, 3, or 4 position of the benzyl group, and the alkoxy group is, for example, methoxy, difluoromethoxy, trifluoromethoxy, ethoxy, or propoxy.

[0044] In the present invention, the halogen refers to fluorine, chlorine, bromine and / or iodine. Accordingly, the halogenated refers to fluorinated, chloro, brominated and / or iodinated. Within the scope of the present invention, when an atom, residue, group or part is halogenated, the atom at the halogenated position may be monosubstituted, disubstituted or polysubstituted up to full substitution by halogen atoms, for example, halogenated C 1-4 The alkylformyl group may be a trifluoroacetyl group, and the halogenated methoxy group may be a difluoromethoxy group or a trifluoromethoxy group.

[0045] An embodiment of the present invention further provides a berberine derivative pharmaceutical composition, which comprises at least one of the berberine derivatives described in any one of the first aspect above, its isotope-labeled substances, solvates, polymorphs, pharmaceutically acceptable salts or prodrug compounds thereof.

[0046] An embodiment of the present invention also provides a use of the berberine derivative described in any one of the first aspect above, or the berberine derivative pharmaceutical composition described in the second aspect above, in the preparation of an anti-inflammatory drug, wherein the anti-inflammatory drug is used to inhibit inflammation caused by soluble epoxide hydrolase.

[0047] An embodiment of the present invention also provides a method for preventing and / or treating inflammation-related diseases, comprising administering to a patient a preventive or therapeutically effective amount of a compound represented by Formula I or Formula II, its racemate, stereoisomer, tautomer, isotope-labeled substance, solvate, polymorph, pharmaceutically acceptable salt, prodrug compound thereof, or at least one of the pharmaceutical compositions.

[0048] In some alternative embodiments, the patient is a mammal, preferably a human.

[0049] The pharmaceutical composition of the present invention also includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents for pharmaceutically active substances, which are agents and materials known in the art. In addition to any conventional media or agents that are incompatible with the compound, any conventional media or agents can be considered for use in the pharmaceutical composition, and supplementary compounds can also be added to the composition.

[0050] In the embodiments of the present invention, various pharmaceutically acceptable acids can form pharmaceutically acceptable salts on the basic nitrogen of the compound. The acid can be an inorganic acid or an organic acid; the inorganic acid is hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid or phosphoric acid; the organic acid is acetic acid, malonic acid, methanesulfonic acid, succinic acid, p-toluenesulfonic acid, citric acid, maleic acid, fumaric acid, malic acid or citric acid.

[0051] The prodrug compounds in this specification are administered with a certain structure that is not disclosed, but are metabolized or converted into the compounds disclosed in this article in the human body and exert pharmacological effects as active ingredients. The conventional method for preparing prodrugs can be found in "Design of Prodrugs" (H.Bundgaad, Elsevier, 1985).

[0052] The patient refers to any animal including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses or primates in the present embodiment, and most preferably humans.

[0053] The therapeutically effective amount refers to the amount of active compound or drug that causes the biological or medical response that the researcher, veterinarian, physician or other clinician is seeking in a tissue, system, animal, individual or human, and includes one or more of the following: (1) Preventing disease: for example, preventing a disease, disorder or condition in an individual who is susceptible to the disease, disorder or condition but has not yet experienced or developed the pathology or symptoms of the disease. (2) Inhibiting disease: for example, inhibiting a disease, disorder or condition in an individual who is experiencing or developing the pathology or symptoms of the disease, disorder or condition (i.e., preventing further development of the pathology and / or symptoms). (3) Alleviating disease: for example, alleviating a disease, disorder or condition in an individual who is experiencing or developing the pathology or symptoms of the disease, disorder or condition (i.e., reversing the pathology and / or symptoms).

[0054] In order to enable those skilled in the art to further understand the technical solution of the present invention, the berberine derivative, pharmaceutical composition and application thereof provided by the present invention are further described below by way of specific embodiments.

[0055] The following is a brief description of the synthesis method of the compounds of the present invention. In the synthesis examples listed below, the synthesis of the intermediates mainly involves Friedel-Crafts acylation reaction, coupling reaction, ring closure reaction and reduction reaction.

[0056] Specifically, the synthesis methods of representative specific compounds of the present invention are shown below.

[0057] Synthesis Example 1: When the urea group is connected to the C10 position of berberine, the synthesis route of the berberine derivative precursor shown in Formula I is as follows:

[0058]

[0059] The reaction reagents and conditions involved in the above-mentioned synthesis route are as follows: a: borane-tetrahydrofuran complex, tetrahydrofuran (THF), 4h; b: pivaloyl chloride, pyridine, dichloromethane (DCM), 5h; c: zinc chloride, acetic anhydride, 24h; d: ethylene glycol, p-toluenesulfonic acid, toluene, 12h; e: palladium carbon, hydrogen, methanol, 3h; f: N-bromosuccinimide (NBS), N,N-dimethylformamide (DMF), 12h; g: acetic anhydride, pyridine, 4-dimethylaminopyridine (DMAP), DCM, 10min; h: palladium acetate, Xantphos, potassium phosphate, THF, 3d; i: ammonium chloride, ethanol / water, 2d; j: sodium borohydride, methanol, 10min.

[0060] The synthetic route of compound 4 is as follows:

[0061]

[0062] 3,4-dimethoxyphenylacetic acid 1 (10.19mmol, 2.00g) was dissolved in 30mL tetrahydrofuran, and borane tetrahydrofuran complex (1mol / L, 20.39mmol, 20.39mL) was slowly added dropwise at 0℃ under argon protection, and the temperature was slowly raised to room temperature for reaction after the addition was completed. After the reaction was completed, the mixture was quenched with sodium hydroxide solution, extracted with dichloromethane, and concentrated by rotary evaporation to obtain 3,4-dimethoxyphenylethanol 2. 2 was dissolved in dry dichloromethane, pyridine (20.39mmol, 1.61mL) was added, and pivaloyl chloride (20.39mmol, 2.54mL) was added dropwise at 0℃. After the reaction was completed, saturated ammonium chloride solution was added to terminate the reaction, and the organic phase was washed with saturated ammonium chloride solution, distilled water, saturated sodium bicarbonate solution, and saturated sodium chloride solution, respectively, and concentrated by rotary evaporation to obtain colorless transparent liquid 3. The obtained liquid was redissolved in 40 mL of acetic anhydride, and zinc chloride (51.04 mmol, 6.95 g) was added. The reaction was started at room temperature under an argon atmosphere. After the reaction was completed, distilled water was slowly added dropwise in an ice bath, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated sodium bicarbonate solution, and concentrated by rotary evaporation to obtain a brown viscous liquid. Compound 4 (1.93 g, total yield 61.5%) was obtained by column chromatography (silica gel 200-300 mesh, V (petroleum ether): V (ethyl acetate) = 15:1).

[0063] HRMS(ESI)(M+Na) + m / z 331.1514,calcd for C 17 H 24 NaO5 + 331.1516.

[0064] 1H NMR (400MHz, Chloroform-d) δ7.27(s,1H),6.77(s,1H),4.29(t,J=6.7Hz,2H),3.93(s,6H),3.22(t,J=6.7Hz,2H),2.58(s,3H),1.16(s,9H).

[0065] 13 C NMR (100MHz, Chloroform-d) δ199.30,178.52,151.53,146.87,133.51,129.39,114.97,113.54,64.98,56.18,55.94,38.70,33.74,29.33,27.24.

[0066] The synthetic route of compound 10 is as follows:

[0067]

[0068] Dissolve m-nitrobenzaldehyde 6 (39.70mmol, 6.00g) in 100mL toluene, add ethylene glycol (79.41mmol, 4.34mL) and p-toluenesulfonic acid (1.19mmol, 0.23g), start the reaction at 120℃, wash with saturated sodium bicarbonate solution and saturated sodium chloride solution respectively after the reaction, dry and concentrate to obtain a yellow solid. The yellow solid is redissolved in 100mL methanol, palladium carbon is added to react under hydrogen atmosphere, after the reaction is completed, filter and evaporate methanol, after drying, redissolve the compound in DMF, add NBS (43.67mmol, 7.77g) under ice bath, and react at room temperature under argon atmosphere, after the reaction is completed, add water to terminate the reaction, extract with ethyl acetate several times, combine the organic phases and wash with half-saturated sodium chloride solution, dry and concentrate to obtain a black oily compound, and obtain a brown-red oily compound by column chromatography (silica gel 200-300 mesh, V (petroleum ether): V (ethyl acetate) = 2:1). The above obtained compound was redissolved in dry dichloromethane, and pyridine (75.12 mmol, 6.05 mL), DMAP (2.57 mmol, 0.32 g) and acetic anhydride (37.51 mmol, 3.55 mL) were added to the reaction system for reaction. After the reaction was completed, distilled water was added to terminate the reaction. The organic phase was washed with saturated ammonium chloride solution, saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively, concentrated by rotary evaporation, and dried to obtain white solid compound 10 (5.33 g, total yield 46.9%).

[0069] HRMS(ESI)(M+Na) + m / z 307.9893,calcd for C 11 H12 BrNNaO3 + 307.9893.

[0070] 1 H NMR (400MHz, Chloroform-d) δ7.63–7.52(m,2H),7.49(d,J=8.4Hz,1H),7.35–7.30(m,1H),6.05(s,1H),4.22–3.98(m,4H),2.15(s,3H).

[0071] 13 C NMR (100MHz, Chloroform-d) δ168.35,137.42,137.10,133.49,122.01,118.89,117.05,102.34,65.49,24.59.

[0072] The synthetic route of compound 13 is as follows:

[0073]

[0074] Compounds 4 (2.80 mmol, 0.86 g) and 10 (1.40 mmol, 0.40 g) were dissolved in 10 mL of tetrahydrofuran, and palladium acetate (0.07 mmol, 0.016 g), Xantphos (0.14 mmol, 0.081 g) and potassium phosphate (2.80 mmol, 0.61 g) were added. The reaction system was replaced with argon and reacted at 85°C for 48 h. After the reaction was completed, the mixture was filtered, concentrated by rotary evaporation and dried to obtain a brown oily compound. The compound was purified by column chromatography (silica gel 200-300 mesh, V (petroleum ether): V (ethyl acetate) = 1:1) to obtain a white solid compound 11 (0.58 g, 82.8%).

[0075] HRMS(ESI)(M+Na) + m / z 536.2251,calcd for C 28 H 35 NNaO8 + 536.2255.

[0076] 1H NMR (400MHz, DMSO-d6) δ9.96 (s, 1H), 7.65 (t, J = 1.9 Hz, 1H), 7.60 (dt, J = 8.3, 2.0 Hz ,1H),7.50(d,J=1.6Hz,1H),7.12(dd,J=8.2,1.6Hz,1H),6.91(d,J=1.5Hz,1H),5.7 4(d,J=1.6Hz,1H),4.38(s,2H),4.19–4.12(m,2H),3.95–3.83(m,4H),3.82(dd,J=4 .3,1.6Hz,6H),3.02(t,J=6.7Hz,2H),2.03(d,J=1.6Hz,3H),1.09(d,J=1.6Hz,9H).

[0077] 13 C NMR(100MHz,DMSO-d6)δ199.95,177.76,168.67,151.26,147.04,138.26,136.52,132.44,132.39,129.78,129 .30,119.70,117.83,115.63,113.62,101.98,65.13,64.98,56.27,56.00,44.60,38.59,33.03,27.36,24.41.

[0078] Compound 11 (1.16 mmol, 0.58 g) was dissolved in 15 mL of 1 M ethanol / water (3:1) ammonium chloride solution, reacted at 90°C for 24 h, then heated to 110°C and continued to react for 48 h. After the reaction was completed, the mixture was cooled to room temperature and filtered to obtain a yellow solid 12 (0.21 g, 62.4%).

[0079] HRMS(ESI)(M-Cl) + m / z 307.1428,calcd for C 19 H 19 N2O2 + 307.1441.

[0080] 1 H NMR (400MHz, DMSO-d6) δ9.55(s,1H),8.82(s,1H),7.98(d,J=8.9Hz,1H),7.66(s,1H),7.57(d,J=9.0Hz,1H),7 .12(s,1H),7.07(s,1H),6.56(s,2H),4.78(t,J=6.4Hz,2H),3.92(s,3H),3.86(s,3H),3.19(t,J=7.3Hz,2H).

[0081] Compound 12 (0.61 mmol, 0.21 g) was dissolved in methanol, and sodium borohydride was added to the system at 45°C. After the reaction, the methanol was evaporated and re-dissolved in dichloromethane, washed with water and saturated sodium chloride solution in turn, and concentrated by rotary evaporation to obtain a yellow solid. After column chromatography (silica gel 200-300 mesh, V (dichloromethane): V (ethanol): V (ammonia water) = 10:0.1:0.05), a light yellow solid 13 (compound 13) (0.14 g, 74.6%) was obtained.

[0082] HRMS(ESI)(M+H) + m / z 311.1754,calcd for C 19 H 23 N2O2 + 311.1754.

[0083] 1 H NMR (400MHz, Methanol-d4) δ6.99 (d, J = 8.1Hz, 1H), 6.89 (s, 1H), 6.72 (s, 1H), 6.63(dd,J=8.1,2.4Hz,1H),6.53(d,J=2.3Hz,1H),3.93(d,J=15.0Hz,1H),3. 84(d,J=8.8Hz,6H),3.70–3.57(m,2H),3.40(dd,J=16.0,4.2Hz,1H),3.23–3. 14(m,1H),3.13–3.04(m,1H),2.79–2.68(m,2H),2.62(td,J=11.3,3.8Hz,1H).

[0084] 13 C NMR(175MHz,Methanol-d4)δ147.82,147.71,145.24,134.02,129.54,129.01,126.33,12 3.66,114.59,112.36,111.52,108.99,60.11,58.22,55.27,54.99,51.18,34.96,27.87.

[0085] The synthetic route of compound 5 is as follows:

[0086]

[0087] According to the preparation method of compound 4, compound 5 (2.19 g, total yield 67.5%) was obtained from piperoacetic acid (11.10 mmol, 2.00 g) via borane reduction, acyl chloride protection and acetylation.

[0088] HRMS(ESI)(M+Na) + m / z 315.1198,calcd for C 16 H 20 NaO5 + 315.1203.

[0089] 1 H NMR (400MHz, Chloroform-d) δ7.23(s,1H),6.75(s,1H),6.02(s,2H),4.26(t,J=6.6Hz,2H),3.17(t,J=6.6Hz,2H),2.53(s,3H),1.16(s,9H).

[0090] The synthetic route of compound 14 is as follows:

[0091]

[0092] According to the preparation method of compound 13, compound 14 (0.15 g, total yield 36.2%) was obtained from compound 5 (2.80 mmol, 0.82 g) and 10 (1.40 mmol, 0.40 g) through coupling, ring closure and reduction reactions.

[0093] HRMS(ESI)(M+H) + m / z 295.1438,calcd for C 18 H 19 N2O2 + 295.1441.

[0094] 1 H NMR(400MHz, Methanol-d4)δ6.93(d,J=8.1Hz,1H),6.79(s,1H),6.63–6.55(m,2H) ,6.49(d,J=2.3Hz,1H),5.87(s,2H),3.90(d,J=15.0Hz,1H),3.63(d,J=15.0Hz,1H) ,3.55(dd,J=11.6,4.1Hz,1H),3.28(dd,J=16.1,3.7Hz,2H),3.18–3.10(m,1H),3. 05(ddd,J=16.8,11.6,5.3Hz,1H),2.74–2.62(m,2H),2.58(td,J=11.4,3.8Hz,1H).

[0095] 13C NMR(100MHz,Methanol-d4)δ146.49,146.32,145.32,133.75,130.18,129.05,126.96 ,123.39,114.62,112.28,107.76,105.06,100.76,60.35,58.05,50.99,35.01,28.26.

[0096] Synthesis Example 2-Synthesis of Compound C1:

[0097]

[0098] The synthetic route of compound C1 is as follows:

[0099]

[0100] Compound 14 (0.34mmol, 0.10g) was dissolved in 10mL of dichloromethane, 4-dimethylaminopyridine (DMAP, 0.68mmol, 0.083g) was added, and finally carbonyldiimidazole (CDI, 0.68mmol, 0.11g) was added, and the mixture was reacted at room temperature for 6h under argon atmosphere. After the reaction was completed, the mixture was concentrated by rotary evaporation and redissolved in dry DMF, and isobutylamine (0.85mmol, 0.062g) was added, and stirred at room temperature for 5h. After the reaction was completed, distilled water was added to the reaction solution to terminate the reaction, and the mixture was extracted twice with ethyl acetate. The organic phases were combined and washed with water, saturated ammonium chloride solution, and saturated sodium chloride solution in turn, concentrated by rotary evaporation, and subjected to column chromatography (silica gel 200-300 mesh, V (dichloromethane): V (ethanol) = 10: 0.15) to obtain a light yellow solid compound 15 (compound C1) (0.075g, yield 56.2%).

[0101] mp126.7-127.5℃.HRMS(ESI)(M+H) + m / z 394.2120,calcd for C 23 H 28 N3O3 + 394.2125.

[0102] 1H NMR(400MHz,Methanol-d4)δ7.19(d,J=2.1Hz,1H),7.15–7.04(m,2H),6.81(s,1 H),6.59(s,1H),5.89(s,2H),3.97(d,J=15.1Hz,1H),3.69(d,J=15.1Hz,1H),3.6 0(dd,J=11.5,4.0Hz,1H),3.41–3.32(m,1H),3.17(ddd,J=11.1,5.4,1.9Hz,1H) ,3.12–2.95(m,3H),2.81–2.56(m,3H),1.84–1.69(m,1H),0.94(d,J=6.7Hz,6H).

[0103] 13 C NMR(100MHz,Methanol-d4)δ157.04,146.52,146.38,137.74,133.70,130.00,128.76,127.66,126.9 9,117.58,116.03,107.78,105.06,100.78,60.09,57.98,50.94,46.91,35.12,28.76,28.27,18.99.

[0104] Synthesis Example 3-Synthesis of Compound C2:

[0105]

[0106] The synthetic route of compound C2 is as follows:

[0107]

[0108] According to the preparation method of compound C1, compound 13 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-aminomethyltetrahydropyran (0.81 mmol, 0.092 g). After purification and drying, a light yellow solid 16 (compound C2) (0.085 g, 58.4%) was obtained.

[0109] mp125.4-126.6℃.HRMS(ESI)(M+H) + m / z 452.2539,calcd for C 26 H 34 N3O4 + 452.2544.

[0110] 1H NMR(400MHz, Methanol-d4)δ7.19(d,J=2.0Hz,1H),7.15–7.06(m,2H),6.87(s,1H),6.70(s,1H),4.04– 3.91(m,3H),3.81(d,J=9.9Hz,6H),3.69(d,J=15.1Hz,1H),3.62(dd,J=11.5,4.1Hz,1H),3.48–3.35(m ,3H),3.18(ddd,J=11.0,5.4,1.9Hz,1H),3.09(d,J=6.7Hz,3H),2.82–2.67(m,2H),2.62(td,J=11.4,3 .8Hz,1H),1.82–1.69(m,1H),1.65(ddd,J=13.3,4.0,2.0Hz,2H),1.30(dtd,J=13.4,11.8,4.6Hz,2H).

[0111] 13 C NMR(100MHz,Methanol-d4)δ157.03,147.95,147.80,137.63,133.86,129.27,128.77,127.92,126.32,117 .66,116.18,111.63,109.11,67.36,59.82,58.07,55.34,55.04,51.09,45.07,35.67,35.02,30.34,27.84.

[0112] Synthesis Example 4-Synthesis of Compound C3:

[0113]

[0114] The synthetic route of compound C3 is as follows:

[0115]

[0116] According to the preparation method of compound C1, compound 14 (0.34 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-aminomethyltetrahydropyran (0.85 mmol, 0.098 g). After purification and drying, a light yellow solid 17 (compound C3) (0.08 g, 53.9%) was obtained.

[0117] mp117.5-118.4℃.HRMS(ESI)(M+H) + m / z 436.2224,calcd for C 25 H 30 N3O4+ 436.2231.

[0118] 1 H NMR(400MHz, Methanol-d4)δ7.18(d,J=2.1Hz,1H),7.14–7.04(m,2H),6.81(s,1H),6.58(s,1H),5.88(s,2H),4.02–3.91(m,3H),3.66(d,J=15.0Hz, 1H),3.57(dd,J=11.8,4.0Hz,1H),3.45–3.32(m,3H),3.19–2.99(m,4H),2 .79–2.53(m,3H),1.82–1.68(m,1H),1.68–1.59(m,2H),1.37–1.22(m,2H).

[0119] 13 C NMR(100MHz,Methanol-d4)δ157.02,146.48,146.33,137.62,133.91,130.16,128.77,127.87,127.08,1 17.62,116.14,107.77,105.06,100.75,67.36,60.08,58.05,50.98,45.07,35.67,35.21,30.34,28.36.

[0120] Synthesis Example 5-Synthesis of Compound C4:

[0121]

[0122] The synthetic route of compound C4 is as follows:

[0123]

[0124] According to the preparation method of compound C1, compound 13 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with (S)-tetrahydrofuran-2-methylamine (0.81 mmol, 0.081 g). After purification and drying, a light yellow solid 18 (compound C4) (0.062 g, 43.9%) was obtained.

[0125] mp105.7-106.3℃.HRMS(ESI)(M+H) + m / z 438.2385,calcd for C 25 H 32 N3O4 + 438.2387.

[0126] 1 H NMR(400MHz, Methanol-d4)δ7.18(d,J=2.1Hz,1H),7.16–7.06(m,2H),6.87(s,1H),6.70(s,1H),4.03–3.92( m,2H),3.88(dt,J=8.3,6.7Hz,1H),3.81(d,J=9.7Hz,6H),3.78–3.70(m,1H),3.70–3.62(m,1H),3.58(dd,J=1 1.5,4.1Hz,1H),3.42(dd,J=16.3,4.1Hz,1H),3.35(dd,J=13.8,4.1Hz,1H),3.26–3.12(m,2H),3.08(ddd,J=1 6.6,11.8,3.4Hz,1H),2.81–2.65(m,2H),2.60(td,J=11.3,3.8Hz,1H),2.08–1.84(m,3H),1.69–1.56(m,1H).

[0127] 13 C NMR(100MHz,Methanol-d4)δ156.99,147.87,147.74,137.59,134.02,129.37,128.77,128.00,126.36,117.53 ,116.09,111.56,109.03,78.22,67.70,59.81,58.14,55.30,55.00,51.12,43.22,35.09,28.10,27.90,25.37.

[0128] Synthesis Example 6-Synthesis of Compound C5:

[0129]

[0130] The synthetic route of compound C5 is as follows:

[0131]

[0132] According to the preparation method of compound C1, compound 13 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with cyclohexylmethylamine (0.81 mmol, 0.091 g). After purification and drying, a light yellow solid 20 (compound C5) (0.086 g, 59.4%) was obtained.

[0133] mp108.4-109.0℃.HRMS(ESI)(M+H) +m / z 450.2751,calcd for C 27 H 36 N3O3 + 450.2751.

[0134] 1 H NMR (400MHz, Methanol-d4) δ7.21(d,J=2.1Hz,1H),7.18–7.08(m,2H),6.89(s,1H),6.73(s,1H),4.00(d,J=15.1Hz,1H),3.8 4(d,J=9.6Hz,6H),3.72(d,J=15.1Hz,1H),3.65(dd,J=11.8,3.8Hz,1H),3.46(dd,J=16.3,4.1Hz,1H),3.21(ddd,J=11.1,5.4 ,1.9Hz,1H),3.12(td,J=11.4,5.8Hz,1H),3.05(d,J=6.8Hz,2H),2.84–2.70(m,2H),2.65(td,J=11.4,3.8Hz,1H),1.83–1.74 (m,4H),1.76–1.66(m,1H),1.48(dddd,J=14.8,11.4,5.9,3.5Hz,1H),1.35–1.19(m,3H),0.98(qd,J=13.6,12.7,3.6Hz,2H).

[0135] 13 C NMR(100MHz,Methanol-d4)δ157.05,147.92,147.77,137.74,133.75,129.17,128.77,127.74,126.24,117.58 ,116.05,111.55,109.00,59.82,58.04,55.30,55.01,51.08,45.74,38.41,34.98,30.53,27.80,26.23,25.67.

[0136] Synthesis Example 7-Synthesis of Compound C6:

[0137]

[0138] The synthetic route of compound C6 is as follows:

[0139]

[0140] According to the preparation method of compound C1, compound 13 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-aminopropionylpiperidine (0.81 mmol, 0.13 g). After purification and drying, a light yellow solid 21 (compound C6) (0.095 g, 60.1%) was obtained.

[0141] mp122.3-122.9℃.HRMS(ESI)(M+H) + m / z 493.2811,calcd for C 28 H 37 N4O4 + 493.2809.

[0142] 1 H NMR(400MHz, Methanol-d4)δ7.18(d,J=3.5Hz,1H),7.15–7.07(m,2H),6.88(s,1H),6.71(s,1H),4.37(dt,J=16.2,4.4Hz,1H),3 .98(d,J=15.1Hz,1H),3.94–3.86(m,1H),3.82(d,J=9.9Hz,7H),3.69(d,J=15.1Hz,1H),3.62(dd,J=11.4,4.0Hz,1H),3.44(dd,J =16.3,4.1Hz,1H),3.27–3.14(m,2H),3.09(ddd,J=16.7,11.9,5.5Hz,1H),2.89(ddd,J=13.9,11.4,3.0Hz,1H),2.82–2.67(m,2 H),2.63(td,J=11.3,3.8Hz,1H),2.42(q,J=7.5Hz,2H),1.98(dd,J=30.4,12.9Hz,2H),1.46–1.30(m,2H),1.12(t,J=7.5Hz,3H).

[0143] 13 C NMR(175MHz,Methanol-d4)δ173.32,156.10,147.89,147.74,137.48,134.00,129.32,128.78,128.04,126.34,117.55,1 16.10,111.55,109.02,59.81,58.12,55.30,55.00,51.12,46.70,44.12,40.40,35.07,32.67,31.78,27.89,25.90,8.61.

[0144] Synthesis Example 8-Synthesis of Compound C7:

[0145]

[0146] The synthetic route of compound C7 is as follows:

[0147]

[0148] According to the preparation method of compound C1, compound 14 (0.34 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-aminopropionylpiperidine (0.85 mmol, 0.133 g). After purification and drying, a light yellow solid 22 (compound C7) (0.083 g, 51.4%) was obtained.

[0149] mp185.7-187.1℃.HRMS(ESI)(M+H) + m / z 477.2485,calcd for C 27 H 33 N4O4 + 477.2496.

[0150] 1 H NMR(400MHz, Methanol-d4)δ7.17(d,J=2.7Hz,1H),7.14–7.03(m,2H),6.81(s,1H),6.58(s,1H),5.88(s,2H),4.40–4.32(m,1H),3.99– 3.85(m,2H),3.81(tt,J=10.4,4.1Hz,1H),3.65(d,J=15.0Hz,1H),3.56(dd,J=11.5,4.0Hz,1H),3.34(dd,J=16.7,4.4Hz,1H),3.22(dd d,J=14.1,11.4,2.9Hz,1H),3.14(ddd,J=11.1,5.4,1.9Hz,1H),3.05(ddd,J=16.8,11.5,5.4Hz,1H),2.89(ddd,J=13.9,11.3,3.0Hz,1 H),2.79–2.63(m,2H),2.58(td,J=11.3,3.7Hz,1H),2.42(q,J=7.5Hz,2H),2.05–1.89(m,2H),1.47–1.26(m,2H),1.12(t,J=7.5Hz,3H).

[0151] 13C NMR(100MHz,Methanol-d4)δ173.33,156.11,146.48,146.32,137.48,133.97,130.20,128.77,127.96,127.10,117.55 ,116.09,107.77,105.07,100.75,60.07,58.06,50.98,46.70,44.11,40.39,35.23,32.67,31.77,28.38,25.90,8.61.

[0152] Synthesis Example 9-Synthesis of Compound C8:

[0153]

[0154] The synthetic route of compound C8 is as follows:

[0155]

[0156] According to the preparation method of compound C1, compound 13 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-aminomethylpropionylpiperidine (0.81 mmol, 0.14 g). After purification and drying, a light yellow solid 23 (compound C8) (0.080 g, 49.1%) was obtained.

[0157] mp121.3-122.5℃.HRMS(ESI)(M+H) + m / z 507.2971,calcd for C 29 H 39 N4O4 + 507.2966.

[0158] 1H NMR(400MHz, Methanol-d4)δ7.19(d,J=1.9Hz,1H),7.16–7.05(m,2H),6.88(s,1H),6.71(s,1 H),4.54(ddt,J=13.0,4.5,2.4Hz,1H),4.02–3.94(m,2H),3.82(d,J=9.8Hz,6H),3.70(d,J=15 .1Hz,1H),3.63(dd,J=11.6,4.1Hz,1H),3.45(dd,J=16.3,4.1Hz,1H),3.23–3.01(m,5H),2.82 –2.67(m,2H),2.67–2.56(m,2H),2.40(q,J=7.5Hz,2H),1.85–1.73(m,3H),1.24–1.02(m,5H).

[0159] 13 C NMR (100MHz, Methanol-d4) δ173.25,157.00,147.91,147.76,137.61,133.90,129.26,128.78,127.96,126.31,117.64,116. 16,111.56,109.02,59.83,58.09,55.30,55.01,51.11,45.40,44.55,41.54,36.81,35.04,30.08,29.27,27.85,25.99,8.65.

[0160] Synthesis Example 10-Synthesis of Compound C9:

[0161]

[0162] The synthetic route of compound C9 is as follows:

[0163]

[0164] According to the preparation method of compound C1, compound 14 (0.34 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-aminomethylpropionylpiperidine (0.85 mmol, 0.15 g). After purification and drying, a light yellow solid 24 (compound C9) (0.075 g, 44.9%) was obtained.

[0165] mp120.7-122.1℃.HRMS(ESI)(M+H) + m / z 491.2650,calcd for C 28 H 35 N4O4+ 491.2653.

[0166] 1 H NMR (400MHz, Methanol-d4) δ7.18 (d, J = 2.1Hz, 1H), 7.15–7.05 (m, 2H), 6.81 (s, 1H), 6. 59(s,1H),5.89(s,2H),4.54(ddt,J=13.3,4.5,2.3Hz,1H),4.00–3.91(m,2H),3.66(d, J=15.1Hz,1H),3.57(dd,J=11.2,4.0Hz,1H),3.36(dd,J=16.3,4.2Hz,1H),3.19–2.99( m,5H),2.79–2.55(m,4H),2.40(q,J=7.5Hz,2H),1.84–1.73(m,3H),1.24–1.03(m,5H).

[0167] 13 C NMR(175MHz,Methanol-d4)δ173.24,156.99,146.49,146.33,137.60,133.92,130.15,128.77,127.88,127.08,117.59,11 6.11,107.77,105.06,100.76,60.09,58.06,50.99,45.39,44.55,41.54,36.80,35.21,30.08,29.27,28.36,25.99,8.65.

[0168] Synthesis Example 11-Synthesis of Compound C10:

[0169]

[0170] The synthetic route of compound C10 is as follows:

[0171]

[0172] According to the preparation method of compound C1, compound 13 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-trifluoromethoxybenzylamine (0.81 mmol, 0.15 g). After purification and drying, a light yellow solid 25 (compound C10) (0.073 g, 42.9%) was obtained.

[0173] mp112.2-113.1℃.HRMS(ESI)(M+H) +m / z 528.2106,calcd for C 28 H 29 F3N3O4 + 528.2105.

[0174] 1 H NMR(400MHz,Methanol-d4)δ7.45–7.37(m,2H),7.26–7.17(m,3H),7.17–7.06(m,2 H),6.86(s,1H),6.69(s,1H),4.39(s,2H),3.95(d,J=15.2Hz,1H),3.81(d,J=9.8H z, 6H), 3.70–3.62 (d, J = 15.2Hz, 1H), 3.58 (dd, J = 11.5, 4.0Hz, 1H), 3.42 (dd, J = 16. 3,4.1Hz,1H),3.19–3.03(m,2H),2.81–2.65(m,2H),2.59(td,J=11.3,3.7Hz,1H).

[0175] 13 C NMR (100MHz, Methanol-d4) δ156.83,148.08,147.88,147.74,139.18,137.46,134.04,129.34,128.80,128.48,128.18,126.35,120.72,120.55 117.76,116.36,111.56,109.02,59.78,58.11,55.29,55.00,51.11,42.38,35.08,27.89.

[0176] Synthesis Example 12-Synthesis of Compound C11:

[0177]

[0178] The synthetic route of compound C11 is as follows:

[0179]

[0180] According to the preparation method of compound C1, compound 14 (0.34 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-trifluoromethoxybenzylamine (0.85 mmol, 0.16 g). After purification and drying, a light yellow solid 26 (compound C11) (0.089 g, 51.1%) was obtained.

[0181] mp168.4-169.8℃.HRMS(ESI)(M+H)+ m / z 512.1808,calcd for C 27 H 25 F3N3O4 + 512.1792.

[0182] 1 H NMR(400MHz, Methanol-d4)δ7.44(d,J=8.3Hz,2H),7.31–7.20(m,3H),7.19–7.08(m, 2H),6.84(s,1H),6.61(s,1H),5.91(s,2H),4.42(s,2H),3.99(d,J=15.1Hz,1H),3.69 (d,J=15.1Hz,1H),3.60(dd,J=11.1,4.0Hz,1H),3.39(dd,J=16.3,4.2Hz,1H),3.18( ddd,J=11.2,5.4,1.9Hz,1H),3.08(ddd,J=16.8,11.6,5.4Hz,1H),2.86–2.56(m,3H).

[0183] 13 C NMR(100MHz,Methanol-d4)δ156.84,146.50,146.35,139.19,137.50,133.88,130.09,128.79,128.48,12 8.02,127.05,120.72,117.77,116.30,107.77,105.05,100.77,60.09,58.01,50.97,42.37,35.19,28.32.

[0184] Synthesis Example 13-Synthesis of Compound C12:

[0185]

[0186] The synthetic route of compound C12 is as follows:

[0187]

[0188] According to the preparation method of compound C1, compound 13 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-trifluoromethoxyaniline (0.81 mmol, 0.14 g). After purification and drying, a light yellow solid 27 (compound C12) (0.081 g, 49.0%) was obtained.

[0189] mp110.5-111.3℃.HRMS(ESI)(M+H) + m / z 514.1945,calcd for C 27 H 27 F3N3O4 + 514.1948.

[0190] 1 H NMR(400MHz, Methanol-d4)δ7.55–7.46(m,2H),7.27(d,J=2.2Hz,1H),7.23–7.15(m,3H),7.14(d, J=8.3Hz,1H),6.88(m,1H),6.70(s,1H),3.99(d,J=15.1Hz,1H),3.82(d,J=10.8Hz,6H),3.70(d,J =15.1Hz,1H),3.61(dd,J=11.5,4.0Hz,1H),3.45(dd,J=16.3,4.0Hz,1H),3.18(ddd,J=11.0,5.5, 1.9Hz,1H),3.10(ddd,J=16.7,12.2,3.5Hz,1H),2.83–2.67(m,2H),2.62(td,J=11.3,3.7Hz,1H).

[0191] 13 C NMR(100MHz,Methanol-d4)δ153.81,147.90,147.75,143.94,138.42,136.94,134.14,129.32,128.86,128.66,12 6.36,121.34,120.62,119.87,117.86,116.49,111.56,109.03,59.78,58.10,55.30,55.00,51.12,35.11,27.90.

[0192] Synthesis Example 14-Synthesis of Compound C13:

[0193]

[0194] The synthetic route of compound C13 is as follows:

[0195]

[0196] According to the preparation method of compound C1, compound 13 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 3-trifluoromethoxybenzylamine (0.81 mmol, 0.15 g). After purification and drying, a light yellow solid 28 (compound C13) (0.097 g, 57.4%) was obtained.

[0197] mp85.2-86.1℃.HRMS(ESI)(M+H) + m / z 528.2106,calcd for C 28 H 29 F3N3O4 + 528.2105.

[0198] 1 H NMR (400MHz, Methanol-d4) δ7.42(t,J=7.9Hz,1H),7.33(dt,J=8.0,1.3Hz,1H),7.24(s,1H),7.20(d,J= 2.0Hz,1H),7.18–7.08(m,3H),6.87(s,1H),6.70(s,1H),4.42(s,2H),3.97(d,J=15.1Hz,1H),3.81(d,J =9.9Hz,6H),3.68(d,J=15.0Hz,1H),3.64–3.56(m,1H),3.44(dd,J=16.3,4.1Hz,1H),3.17(ddd,J=11.0 ,5.5,1.9Hz,1H),3.09(ddd,J=16.8,12.0,3.5Hz,1H),2.82–2.66(m,2H),2.61(td,J=11.4,3.8Hz,1H).

[0199] 13 C NMR (175MHz, Methanol-d4) δ156.85,149.34,147.88,147.74,142.80,137.42,134.04,129.77,129.35,128.79,128.23,126.3 5,125.56,120.54,119.30,119.06,117.83,116.42,111.55,109.00,59.82,58.12,55.28,55.00,51.12,42.51,35.09,27.89.

[0200] Synthesis Example 15-Synthesis of Compound C14:

[0201]

[0202] The synthetic route of compound C14 is as follows:

[0203]

[0204] According to the preparation method of compound C1, compound 14 (0.34 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 3-trifluoromethoxybenzylamine (0.85 mmol, 0.16 g). After purification and drying, a light yellow solid 29 (compound C14) (0.073 g, 42.2%) was obtained.

[0205] mp149.7-151.5℃.HRMS(ESI)(M+H) + m / z 512.1790,calcd for C 27 H 25 F3N3O4 + 512.1792.

[0206] 1 H NMR(400MHz, Methanol-d4)δ7.42(t,J=7.9Hz,1H),7.33(d,J=7.7Hz,1H),7.24(s,1H),7.20 (s,1H),7.18–7.06(m,3H),6.82(s,1H),6.59(s,1H),5.89(s,2H),4.42(s,2H),3.96(d,J=15 .1Hz,1H),3.67(d,J=15.1Hz,1H),3.57(dd,J=11.3,4.0Hz,1H),3.36(dd,J=16.3,4.2Hz,1H) ,3.15(ddd,J=11.2,5.6,2.0Hz,1H),3.06(ddd,J=16.8,11.9,5.4Hz,1H),2.80–2.54(m,3H).

[0207] 13 C NMR(100MHz,Methanol-d4)δ156.84,149.35,146.48,146.32,142.79,137.43,133.98,130.17,129.77,128.80,128.12,12 7.08,125.57,121.81,119.30,119.06,117.82,116.38,107.77,105.05,100.75,60.08,58.05,50.99,42.52,35.23,28.36.

[0208] Synthesis Example 16-Synthesis of Compound C15:

[0209]

[0210] The synthetic route of compound C15 is as follows:

[0211]

[0212] According to the preparation method of compound C1, compound 13 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-difluoromethoxybenzylamine (0.81 mmol, 0.14 g). After purification and drying, a light yellow solid 30 (compound C15) (0.077 g, 46.9%) was obtained.

[0213] mp113.2-114.5℃.HRMS(ESI)(M+H) + m / z 510.2222,calcd for C 28 H 30 F2N3O4 + 510.2199.

[0214] 1 H NMR (400MHz, DMSO-d6) δ8.46 (s, 1H), 7.38-7.33 (d, J = 8.6Hz, 2H), 7.20 (s, 1H), 7.19 (t, J=74.4Hz,1H),7.18–7.12(m,3H),7.03(d,J=8.3Hz,1H),6.89(s,1H),6.69(s,1H),6.6 1(t,J=6.0Hz,1H),4.28(d,J=5.8Hz,2H),3.92(dd,J=14.4,4.1Hz,1H),3.74(d,J=9.0H z,6H),3.61–3.37(m,3H),3.08(d,J=10.4Hz,1H),3.01–2.88(m,1H),2.69–2.52(m,3H).

[0215] 13 C NMR(100MHz,DMSO-d6)δ155.74,150.14,147.70,138.55,138.08,129.23,129.11,119.27,118.06, 116.88,115.36,114.33,112.18,109.84,59.80,56.21,55.89,55.37,51.23,42.56,35.94,29.49.

[0216] Synthesis Example 17-Synthesis of Compound C16:

[0217]

[0218] The synthetic route of compound C16 is as follows:

[0219]

[0220] According to the preparation method of compound C1, compound 14 (0.34 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-difluoromethoxybenzylamine (0.85 mmol, 0.15 g). After purification and drying, a light yellow solid 31 (compound C16) (0.087 g, 52.0%) was obtained.

[0221] mp176.9-177.4℃.HRMS(ESI)(M+H) + m / z 494.1905,calcd for C 27 H 26 F2N3O4 + 494.1886.

[0222] 1 H NMR(400MHz, Methanol-d4)δ7.41–7.34(m,2H),7.22(d,J=2.2Hz,1H),7.18–7.09(m,4H),6.84(s, 1H),6.79(t,J=73.2Hz,1H),6.61(s,1H),5.91(s,2H),4.39(s,2H),3.98(d,J=15.1Hz,1H),3.69(d ,J=15.1Hz,1H),3.59(dd,J=11.4,4.0Hz,1H),3.38(dd,J=16.3,4.2Hz,1H),3.17(ddd,J=11.2,5.5 ,2.0Hz,1H),3.08(ddd,J=16.8,11.6,5.3Hz,1H),2.82–2.65(m,2H),2.61(td,J=11.4,3.8Hz,1H).

[0223] 13C NMR(100MHz,Methanol-d4)δ146.49,146.32,137.50,136.62,133.99,130.01,128.78,128.44,128.06 ,127.11,118.89,117.73,116.29,107.77,105.05,100.75,60.10,58.07,50.99,42.45,35.24,28.37.

[0224] Synthesis Example 18-Synthesis of Compound C17:

[0225]

[0226] The synthetic route of compound C17 is as follows:

[0227]

[0228] According to the preparation method of compound C1, compound 13 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-methylsulfonylbenzylamine (0.81 mmol, 0.15 g). After purification and drying, a light yellow solid 32 (compound C17) (0.084 g, 50.2%) was obtained.

[0229] mp161.7-162.4℃.HRMS(ESI)(M+H) + m / z 522.2073,calcd for C 28 H 32 N3O5S + 522.2057.

[0230] 1 H NMR(400MHz, Methanol-d4)δ7.93(d,J=8.3Hz,2H),7.61(d,J=8.3Hz,2H),7.26–7.20(m, 1H),7.20–7.10(m,2H),6.91(s,1H),6.73(s,1H),4.51(s,2H),4.06–3.95(m,1H),3.85( d,6H),3.71(d,J=15.0Hz,1H),3.64(dd,J=11.3,4.1Hz,1H),3.47(dd,J=16.3,4.2Hz,1H ),3.25–3.14(m,1H),3.17–3.05(m,4H),2.85–2.69(m,2H),2.64(td,J=11.3,3.8Hz,1H).

[0231] 13 C NMR (100MHz, Methanol-d4) δ156.83,147.89,147.75,146.70,139.24,137.42,134.01,129.30,128.81,128.23,127. 61,127.25,126.33,117.80,116.39,111.56,109.02,59.81,58.09,55.30,55.01,51.11,43.02,42.66,35.07,27.87.

[0232] Synthesis Example 19-Synthesis of Compound C18:

[0233]

[0234] The synthetic route of compound C18 is as follows:

[0235]

[0236] According to the preparation method of compound C1, compound 13 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with sulfamethoxazole hydrochloride (0.81 mmol, 0.18 g). After purification and drying, a light yellow solid 33 (compound C18) (0.069 g, 40.9%) was obtained.

[0237] mp184.5-185.1℃.HRMS(ESI)(M+H) + m / z 523.2026,calcd for C 27 H 31 N4O5S + 523.2010.

[0238] 1 H NMR(400MHz, Methanol-d4)δ7.86(d,J=8.0Hz,2H),7.49(d,J=8.0Hz,2H),7.23(s,1H),7.20–7.09(m,2H),6.88(s,1H),6.72(s,1H),4.46(s,2H),4.0 2(d,J=15.2Hz,1H),3.82(d,J=9.8Hz,6H),3.79–3.66(m,2H),3.47(dd,J= 16.4,4.2Hz,1H),3.27–3.18(m,1H),3.17–3.04(m,1H),2.84–2.63(m,3H).

[0239] 13C NMR(100MHz,Methanol-d4)δ156.84,147.98,147.81,144.62,142.24,137.56,133.51,128.89,128.83,127.90,1 27.20,126.10,125.98,117.88,116.31,111.54,108.97,59.83,57.88,55.31,55.02,51.02,42.64,34.87,27.66.

[0240] Synthesis Example 20-Synthesis of Compound C19:

[0241]

[0242] The synthetic route of compound C19 is as follows:

[0243]

[0244] Compound C10 (0.15 mmol, 0.080 g) and potassium acetate (0.17 mmol, 0.016 g) were dissolved in 10 mL of ethanol and poured into an ethanol solution of iodine (0.15 mmol, 0.038 g). The mixture was stirred at room temperature for 1 h, and then saturated sodium thiosulfate solution was added to quench the mixture. Then, sodium hydroxide solution and ethyl acetate were added to separate the liquids. The organic phase was washed with 2 M hydrochloric acid solution and saturated sodium chloride solution, respectively. The mixture was concentrated by rotary evaporation and recrystallized to obtain a yellow solid 34 (compound C19) (0.071 g, yield 84.6%).

[0245] mp greater than 280℃.HRMS(ESI)(M-Cl) + m / z 524.1785,calcd for C 28 H 25 F3N3O4 + 524.1792.

[0246] 1 H NMR (400MHz, Methanol-d4) δ9.59(s,1H),8.81(s,1H),8.63(d,J=2.2Hz,1H),8.20(d,J=9.0Hz,1H),7.98(dd,J=9.0,2.2Hz,1H),7.69(s,1H), 7.49(d,J=8.7Hz,2H)7.28(d,J=8.0Hz,2H),7.08(s,1H),4.90(t,J=6.4Hz,2H),4.50(s,2H),4.02(s,3H),3.96(s,3H),3.31(t,J=6.4Hz,2H).

[0247] 13 C NMR(175MHz,Methanol-d4)δ155.91,152.47,149.53,148.18,147.93,141.85,138.78,135.12,130.00,128.61,128 .58,127.93,127.37,120.78,120.54,119.95,119.13,112.92,110.84,108.56,55.78,55.59,55.27,42.43,26.34.

[0248] Synthesis Example 21-Synthesis of Compound C20:

[0249]

[0250] The synthetic route of compound C20 is as follows:

[0251]

[0252] Potassium ferrocyanide (0.90 mmol, 0.29 g) was dissolved in 20 mL of 5 M sodium hydroxide solution, and compound C19 (0.18 mmol, 0.10 g) was slowly added. After reflux for 5 h, the mixture was cooled to room temperature and filtered. The filter residue was purified by column chromatography (silica gel 100-200 mesh, V (dichloromethane): V (ethanol) = 10:0.1) to give a light yellow solid compound 35 (compound C20) (0.060 g, yield 61.7%).

[0253] mp248.1-248.7℃.HRMS(ESI)(M+H) + m / z 540.1741,calcd for C 28 H 25 F3N3O5 + 540.1741. 1 H NMR(400MHz, DMSO-d6)δ9.05(s,1H),8.29(d,J=2.3Hz,1H),7.78(dd,J=8.7,2.4Hz,1H),7.60(d,J=8.6Hz,1H),7.49–7.41(m,3H),7.34(d,J=8.2Hz, 2H),7.21(s,1H),6.95(s,1H),6.84(t,J=6.1Hz,1H),4.36(d,J=5.9Hz,2H ),4.21(t,J=6.1Hz,2H),3.88(s,3H),3.82(s,3H),2.91(t,J=6.2Hz,2H).13 C NMR(175MHz,DMSO-d6)δ161.18,155.63,150.20,148.58,147.57,140.51,139.30,135.46,131.27,129.42,128.50,1 27.38,125.01,124.12,122.34,121.44,120.58,114.48,111.49,108.66,101.33,56.34,56.08,42.56,40.48,27.72.

[0254] Synthesis Example 22: When the urea group is connected to the C3 position of berberine, the synthesis route of the berberine derivative precursor shown in general formula II is as follows:

[0255]

[0256] The reaction reagents and conditions involved in the above-mentioned synthesis route are as follows: a: borane-tetrahydrofuran complex, THF, 4h; b: pivaloyl chloride, pyridine, DCM, 5h; c: stannous chloride, ethanol / water, 5h; d: benzyl bromide, potassium carbonate, ethanol / water, 0.5h; e: zinc chloride, acetic anhydride, 24h; f: ethylene glycol, p-toluenesulfonic acid, toluene, 12h; g: palladium acetate, Xantphos, potassium phosphate, THF, 3d; h: ammonium chloride, ethanol / water, 2d; i: sodium borohydride, methanol, 10min; j: palladium carbon, hydrogen, ethanol, 3h.

[0257] The synthetic route of compound 41 is as follows:

[0258]

[0259] m-Nitrophenylacetic acid 36 (11.04mmol, 2.00g) was dissolved in 30mL tetrahydrofuran, and borane tetrahydrofuran complex (1mol / L, 22.08mmol, 22.08mL) was slowly added dropwise at 0℃ under argon protection, and the temperature was slowly raised to room temperature for reaction. After the reaction was completed, sodium hydroxide solution was used for quenching, and dichloromethane was extracted and concentrated by rotary evaporation to obtain m-nitrophenylethanol 37. 37 was dissolved in dry dichloromethane, pyridine (22.08mmol, 1.74mL) was added, and pivaloyl chloride (22.08mmol, 2.75mL) was added dropwise at 0℃. After the reaction was completed, saturated ammonium chloride solution was added to terminate the reaction, and the organic phase was washed with saturated ammonium chloride solution, distilled water, saturated sodium bicarbonate solution, and saturated sodium chloride solution, respectively, and concentrated by rotary evaporation to obtain a light yellow liquid 38 (compound 38) (2.32g, yield 83.49%).

[0260] Compound 38 (7.96mmol, 2.00g) was redissolved in 50mL of ethanol / water 3:1 solution, stannous chloride (31.84mmol, 7.18g) was added and refluxed for 5h. After the reaction, 1M sodium hydroxide solution was added to adjust the pH to 7, excess ethyl acetate was added to extract the organic phase, and the organic phase was washed with saturated sodium chloride solution, concentrated and dried to obtain a light yellow liquid 39 (1.41g, yield 79.81%). 39 (8.94mmol, 1.98g), potassium carbonate (26.83mmol, 3.71g) and benzyl bromide (35.77mmol, 6.12g) were added to 50mL of ethanol / water 3:1 solution and reacted at 80℃ for 0.5h. After the reaction was completed, it was cooled to room temperature, saturated sodium chloride solution and ethyl acetate were added for separation, the organic phase was washed with saturated sodium bicarbonate solution and sodium chloride solution in sequence, dried and concentrated to obtain a yellow oily liquid. Column chromatography (silica gel 200-300 mesh, petroleum ether) gave colorless oily liquid 40 (Compound 40) (3.21 g, 89.31%).

[0261] HRMS(ESI)(M+H) + m / z 402.2427,calcd for C 27 H 32 NO2 + 402.2428.

[0262] 1 H NMR(400MHz,Chloroform-d)δ7.45–7.36(m,4H),7.33(tt,J=6.6,1.4Hz,6H),7.18(td,J=7.6,0.8 Hz,1H),6.74–6.64(m,3H),4.71(s,4H),4.29(t,J=7.1Hz,2H),2.90(t,J=7.1Hz,2H),1.24(s,9H).

[0263] Compound 40 (2.49 mmol, 1.00 g) was dissolved in 30 mL of acetic anhydride, and zinc chloride (12.45 mmol, 1.70 g) was added. The mixture was reacted at 105 °C under an argon atmosphere for 0.5 h. After the reaction, distilled water was slowly added dropwise in an ice bath, and the mixture was extracted three times with ethyl acetate. The organic phases were combined and washed with saturated sodium bicarbonate solution, and concentrated by rotary evaporation to obtain a brown viscous liquid. The mixture was purified by column chromatography (silica gel 200-300 mesh, V (petroleum ether): V (ethyl acetate) = 15:1) to obtain a light yellow oily liquid compound 41 (0.47 g, yield 42.1%).

[0264] HRMS(ESI)(M+H) + m / z 444.2528,calcd for C29 H 34 NO3 + 444.2533.

[0265] 1 H NMR(400MHz,Chloroform-d)δ7.63(d,J=8.8Hz,1H),7.29–7.18(m,6H),7.15–7.10(m,4H),6.55(d,J=2.8Hz,1H), 6.51(dd,J=8.8,2.8Hz,1H),4.62(s,4H),4.16(t,J=6.8Hz,2H),3.15(t,J=6.8Hz,2H),2.42(s,3H),1.05(s,9H).

[0266] 13 C NMR(100MHz,Chloroform-d)δ198.13,178.50,151.60,142.10,137.25,133.84,128.8 8,127.32,126.48,125.08,115.37,109.38,64.98,53.64,38.65,35.05,28.62,27.23.

[0267] The synthetic route of compound 43 is as follows:

[0268]

[0269] 6-Bromoveratraldehyde 42 (4.08 mmol, 1.00 g) was dissolved in 30 mL of toluene, and ethylene glycol (8.16 mmol, 0.45 mL) and p-toluenesulfonic acid (0.21 mmol, 0.035 g) were added. The reaction was started at 120°C. After the reaction was completed, the mixture was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, respectively, and dried and concentrated to obtain a white solid 43 (compound 43) (1.14 g, yield 96.3%).

[0270] HRMS(ESI)(M+H) + m / z 289.0050,calcd for C 11 H 14 BrO4 + 289.0070.

[0271] 1 H NMR (400MHz, Chloroform-d) δ7.04 (s, 1H), 6.94 (s, 1H), 5.92 (s, 1H), 4.15–3.95 (m, 4H), 3.81 (d, J = 5.7Hz, 6H).

[0272] The synthetic route of compound 47 is as follows:

[0273]

[0274] Compound 41 (2.25 mmol, 1.00 g) and 43 (3.16 mmol, 0.91 g) were dissolved in 10 mL of tetrahydrofuran, and palladium acetate (0.11 mmol, 0.026 g), Xantphos (0.22 mmol, 0.13 g) and potassium phosphate (5.63 mmol, 1.23 g) were added. The reaction system was replaced with argon and reacted at 95°C for 48 h. After the reaction was completed, the mixture was filtered and concentrated by rotary evaporation to obtain a dry brown oily compound. After column chromatography (silica gel 200-300 mesh, V (petroleum ether): V (ethyl acetate) = 4:1), a yellow oily compound 44 (0.68 g, 46.3%) was obtained.

[0275] 1 H NMR (400MHz, DMSO-d6) δ7.87(d,J=8.7Hz,1H),7.30(m,10H),7.00(s,1H),6.75(s,1H),6.68–6.59(m,2H),5.65(s,1H),4.7 9(s,4H),4.24(s,2H),4.05(t,J=6.4Hz,2H),3.93–3.77(m,4H),3.71(d,J=17.1Hz,6H),3.02(t,J=6.8Hz,2H),1.03(s,9H).

[0276] Compound 44 (1.04 mmol, 0.68 g) was dissolved in 15 mL of 1 M ethanol / water (3:1) ammonium chloride solution, reacted at 90°C for 24 h, then heated to 110°C and continued to react for 48 h. After the reaction was completed, the mixture was cooled to room temperature and filtered to obtain yellow solid compound 45 (0.21 g, 38.5%).

[0277] 1 H NMR(400MHz, Methanol-d4)δ9.09(s,1H),8.29(s,1H),7.77(d,J=9.0Hz,1H),7.42(s,1H),7.38(s,1H),7.28–7.11(m,10H),6.77( dd,J=8.9,2.7Hz,1H),6.67(d,J=2.6Hz,1H),4.73(s,4H),4.60(t,J=6.4Hz,2H),3.98(s,3H),3.92(s,3H),3.05(t,J=6.3Hz,2H).

[0278] Compound 45 (0.40 mmol, 0.21 g) was dissolved in methanol, and sodium borohydride was added to the system at 45°C. After the reaction was completed, the methanol was evaporated and re-dissolved in dichloromethane, and the mixture was washed with water and saturated sodium chloride solution in turn. The mixture was concentrated by rotary evaporation to obtain a yellow solid, which was purified by column chromatography (silica gel 200-300 mesh, V (dichloromethane): V (ethanol) = 10:0.1) to obtain a light yellow solid 46 (0.11 g, 57.6%).

[0279] 1 H NMR(400MHz,Chloroform-d)δ7.49–7.26(m,10H),7.10(d,J=8.6Hz,1H),6.69-6.6 5(m,2H),6.59(s,1H),6.54(d,J=2.7Hz,1H),4.66(s,4H),3.96(d,J=14.5Hz,1H), 3.88(d,J=2.9Hz,6H),3.67(d,J=14.6Hz,1H),3.63–3.56(m,1H),3.26(dd,J=15.9 ,3.9Hz,1H),3.20–3.09(m,2H),2.86(dd,J=15.8,11.3Hz,1H),2.70–2.59(m,2H).

[0280] Compound 46 (0.39 mmol, 0.19 g) was dissolved in ethanol, and 0.5 mL of 2M hydrochloric acid solution and palladium carbon (0.019 g) were added to the system and reacted under a hydrogen atmosphere. After reacting for 24 h, the mixture was filtered and the ethanol was evaporated. Column chromatography (silica gel 200-300 mesh, V (dichloromethane): V (ethanol) = 10:0.2) gave a light yellow solid 47 (0.09 g, 75.6%).

[0281] HRMS(ESI)(M+H) + m / z 311.1751,calcd for C 19 H 23 N2O2 + 311.1754.

[0282] 1H NMR(400MHz,Chloroform-d)δ6.99(d,J=8.3Hz,1H),6.58(s,1H),6.53-6.48(m,2H),6.40(d,J=2.4Hz,1H),3.89(d,J=14.5Hz,1H),3 .78(d,J=2.7Hz,6H),3.68–3.49(m,2H),3.18(dd,J=16.1,4.1Hz,1H),3.12-3.03(m,2H),2.76(t,J=13.7Hz,1H),2.65–2.52(m,2H).

[0283] Synthesis Example 23-Synthesis of Compound C21:

[0284]

[0285] The synthetic route of compound C21 is as follows:

[0286]

[0287] According to the preparation method of compound C1, compound 47 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-aminomethyltetrahydropyran (0.81 mmol, 0.092 g). After purification and drying, a light yellow solid 48 (compound C21) (0.081 g, 55.9%) was obtained.

[0288] mp131.7-132.4℃.HRMS(ESI)(M+H) + m / z 452.2541,calcd for C 26 H 34 N3O4 + 452.2544.

[0289] 1 H NMR (400MHz, Methanol-d4) δ7.16–7.08 (m, 2H), 7.05 (d, J = 2.0Hz, 1H), 6.67 (s, 1H) ,6.60(s,1H),3.90–3.81(m,3H),3.70(d,J=2.8Hz,6H),3.59-3.48(m,2H),3.36–3 .25(m,3H),3.12–2.97(m,4H),2.72–2.61(m,2H),2.54(td,J=10.9,10.3,3.7Hz,1 H),1.66(dtt,J=11.4,8.0,3.6Hz,1H),1.56(d,J=13.1Hz,2H),1.27–1.13(m,2H).

[0290] 13 C NMR(175MHz,Methanol-d4)δ156.99,148.00,147.70,137.94,134.22,131.10,126.06,125.64,125.60,118 .43,117.19,111.69,109.25,67.36,59.70,57.52,55.10,55.07,51.01,45.05,35.66,35.13,30.33,28.44.

[0291] Synthesis Example 24-Synthesis of Compound C22:

[0292]

[0293] The synthetic route of compound C22 is as follows:

[0294]

[0295] According to the preparation method of compound C1, compound 47 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-aminomethylpropionylpiperidine (0.81 mmol, 0.14 g). After purification and drying, a light yellow solid 49 (compound C22) (0.076 g, 46.8%) was obtained.

[0296] mp126.7-127.1℃.HRMS(ESI)(M+H) + m / z 507.2969,calcd for C 29 H 39 N4O4 + 507.2966.

[0297] 1H NMR (400MHz, Methanol-d4) δ7.30–7.21(m,2H),7.20(d,J=2.1Hz,1H),6.81(s,1H),6.74(s,1H) ,4.56(d,J=13.7Hz,1H),4.07-3.95(m,2H),3.83(d,J=3.0Hz,6H),3.78–3.70(m,2H),3.47(dd, J=16.5,4.3Hz,1H),3.31–3.22(m,1H),3.22–3.03(m,4H),2.87–2.68(m,3H),2.70–2.59(m,1H) ,2.43(q,J=7.5Hz,2H),1.88–1.75(m,3H),1.21(dd,J=12.5,4.1Hz,1H),1.13(t,J=7.5Hz,3H).

[0298] 13 C NMR (175MHz, Methanol-d4) δ173.25,156.95,148.10,147.77,138.07,134.02,130.71,125.85,125.66,125.14,118.42,117. 25,111.65,109.21,59.74,57.35,55.11,55.06,50.94,45.40,44.54,41.54,36.80,34.96,30.08,29.27,28.26,25.99,8.65.

[0299] Synthesis Example 25-Synthesis of Compound C23:

[0300]

[0301] The synthetic route of compound C23 is as follows:

[0302]

[0303] According to the preparation method of compound C1, compound 47 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-trifluoromethoxybenzylamine (0.81 mmol, 0.15 g). After purification and drying, a light yellow solid 50 (compound C23) (0.10 g, 60.3%) was obtained.

[0304] mp136.7-137.2℃.HRMS(ESI)(M+H) + m / z 528.2103,calcd for C 28 H29 F3N3O4 + 528.2105.

[0305] 1 H NMR (400MHz, Methanol-d4) δ7.32(d,J=8.3Hz,2H),7.16–7.08(m,5H),6.68(s,1H),6.61(s,1H),4.30(s,2H),3.93(d,J=15.3Hz,1H ),3.70(d,J=3.0Hz,6H),3.69–3.60(m,2H),3.35(dd,J=16.5,4.1Hz,1H),3.20–3.12(m,1H),3.10-2.99(m,1H),2.75–2.60(m,3H).

[0306] 13 C NMR(175MHz,Methanol-d4)δ156.75,148.22,148.10,147.85,139.15,138.12,133.81,130.38,128.49,125.70,125.60,1 24.61,120.73,120.54,119.82,118.55,117.44,111.64,109.19,59.74,57.12,55.11,55.08,50.84,42.37,34.73,28.03.

[0307] Synthesis Example 26-Synthesis of Compound C24:

[0308]

[0309] The synthetic route of compound C24 is as follows:

[0310]

[0311] According to the preparation method of compound C1, compound 47 (0.32 mmol, 0.10 g) was reacted with CDI and DMAP to insert the carbonyl imidazole group and then reacted with 4-difluoromethoxybenzylamine (0.81 mmol, 0.14 g). After purification and drying, a light yellow solid 51 (compound C24) (0.091 g, 55.2%) was obtained.

[0312] mp188.2-188.9℃.HRMS(ESI)(M+H) + m / z 510.2198,calcd for C 28 H 30 F2N3O4 +510.2199.

[0313] 1 H NMR (400MHz, DMSO-d6) δ8.51 (s, 1H), 7.35 (d, J = 8.4Hz, 2H), 7.20 (t, J = 74.2Hz, 1H), 7.23–7.11(m,5H),6.73(s,1H),6.70–6.61(m,2H),4.28(d,J=5.9Hz,2H),3.87(d,J= 14.8Hz,1H),3.72(s,6H),3.56–3.39(m,2H),3.29(dd,J=16.0,3.9Hz,1H),3.10–3.0 2(m,1H),2.97(ddd,J=16.5,11.4,5.4Hz,1H),2.67–2.52(m,2H),2.50–2.40(m,1H).

[0314] 13 C NMR(175MHz,DMSO-d6)δ155.74,150.13,147.63,147.49,138.70,138.09,134.99,131.44,129.20,126.85,126.61, 126.18,119.26,117.79,116.89,116.46,112.34,109.91,59.56,58.00,55.98,55.93,51.24,42.54,36.27,29.63.

[0315] Synthesis Example 27-Synthesis of Compound C25:

[0316]

[0317] The synthetic route of compound C25 is as follows:

[0318]

[0319] According to the preparation method of compound C19, compound C23 (0.15 mmol, 0.080 g), an ethanol solution of potassium acetate and an ethanol solution of iodine were mixed and reacted, and after purification and drying, a yellow solid 52 (compound C25) (0.068 g, yield 81.1%) was obtained.

[0320] mp greater than 240℃.HRMS(ESI)(M-Cl) + m / z 524.1794,calcd for C 28 H 25 F3N3O4 + 524.1792.

[0321] 1 H NMR (400MHz, Methanol-d4) δ9.23(s,1H),8.49(s,1H),7.94(d,J=8.7Hz,1H),7.55–7.47(m,3H),7.44(dd,J=8.6,2.2H z,1H),7.35(d,2H),7.15(d,2H),4.70(t,J=6.4Hz,2H),4.34(s,2H),4.03(s,3H),3.96(s,3H),3.17(t,J=6.4Hz,2H).

[0322] 13 C NMR(175MHz,Methanol-d4)δ158.57,156.08,153.20,148.13,144.70,143.29,139.20,138.92,137.59,135.85,128 .55,126.52,123.25,120.75,120.54,120.36,117.76,116.89,105.87,104.91,56.06,55.63,54.82,42.36,27.21.

[0323] The technical effects and advantages of the present invention are verified below by applying the berberine derivatives of the present invention in anti-inflammatory drugs, which are used to inhibit inflammation caused by soluble epoxide hydrolases.

[0324] Specifically, the verification method of the anti-inflammatory drug in the embodiment of the present invention is to incubate the berberine derivative with a sample containing a soluble epoxide hydrolase and the endogenous hydrolysis substrate 14,15-EET of the hydrolase, detect the content of the hydrolysis product 14,15-DHET, and use its relative production amount to reflect the inhibitory effect of the compound on the soluble epoxide hydrolase. The specific steps are as follows:

[0325] 1:10 (mg / μL) pre-cooled PBS (phosphate buffer saline) (pH=7.4) was added to the collected brain tissue samples of adult (weight 25-30g) male C57BL / 6J mice, and the samples were disrupted with a homogenizer (Bertin Precellys 24-Dual, Bertin Technologies) for 30 seconds (4°C, 4500rpm), and then centrifuged at 9000g for 15 minutes at 4°C in a small low-temperature centrifuge, and the supernatant was diluted 20 times. In an EP (eppendorf) tube, 160 μL PBS, 20 μL tissue fluid diluted 20 times, and 2 μL of a compound with a concentration gradient of 10000, 5000, 1000, 500, 100, 50, 10, 1, 0.1, and 0.01 μg / mL (final concentration 100, 50, 10, 5, 1, 0.5, 0.1, 0.01, 0.001, and 0.0001 μg / mL) were added and incubated at room temperature for 15 minutes. The negative control was PBS without tissue diluent but with solvent added (180 μL PBS plus 2 μL DMSO), and the positive control was the same concentration sample diluent plus an equal volume of solvent (160 μL PBS plus 20 μL tissue fluid diluted 20 times plus 2 μL DMSO). After the room temperature incubation, all samples were placed on ice, and the enzyme reaction substrate (10 μL 14,15-EET (10 μg / mL)) was quickly added and mixed, and then incubated in a shaker at 37°C. After the incubation, the samples were placed on ice and 10 μL 800 nM t-TUCB (No. 6757, Tocris Bioscience, UK, Bristol) was quickly added to terminate the reaction, and the final volume of the reaction system was 200 μL. In order to detect the enzyme activity, the concentration of the reaction product 14,15-DHET was detected using high performance liquid chromatography-mass spectrometry. The pretreatment is as follows: 1. Add an equal volume (200 μL) of a mixture of methanol and acetonitrile (50 / 50, v / v) to the sample, the mixture contains 0.4% acetic acid and internal standards 11,12-EET-d11 and 11,12-DHET-d11 (20 ng / mL); 2. Mix with a homogenizer at 4500 rpm for 10 minutes and place at -20°C for 2 hours to precipitate the protein; 3. After the sample is centrifuged for 10 minutes (14000 rpm), the supernatant is collected for UPLC-MS / MS analysis. The relative enzyme activity of sEH is calculated by comparing the amount of 14,15-DHET produced at a certain concentration of the test compound with that without an inhibitor, and the inhibition rate is 100% minus the relative enzyme activity.

[0326] Here, inhibition rate % (5 μg / mL) is the inhibition rate of the compound on sEH at 5 μg / mL.

[0327] Among them, the control compounds are TPPU, EC5026 and BBR.

[0328] The specific verification results of the berberine derivatives prepared in the examples of the present invention are shown in Table 1.

[0329] Table 1 Inhibition rate and IC50 of berberine derivatives prepared in the examples of the present invention on sEH

[0330]

[0331]

[0332]

[0333]

[0334] The specific validation results of the control compounds TPPU, EC5026 and BBR on soluble epoxide hydrolases are shown in Table 2.

[0335] Table 2. Inhibition rate and IC50 of control compounds on sEH under the same conditions

[0336]

[0337] As can be seen from Tables 1 and 2, compared with the control compound berberine BBR, the berberine derivatives prepared by the present invention have excellent soluble epoxide hydrolase (sEH) inhibitory activity, and the IC50 values ​​of some berberine derivatives are better than 43.77nM of the positive control compound TPPU and 19.11nM of EC5026, and berberine has excellent sEH inhibitory activity through structural modification. The unique tertiary amine structure of the berberine skeleton greatly increases its drugability, which has a huge advantage over other sEH inhibitor skeletons. Overall, the berberine skeleton is best active when connected to benzyl groups containing different substituents through urea groups, and the activity is equivalent when connected to alkyl chains and heterocycles; the different oxidation states of the C ring of the berberine skeleton lead to huge spatial conformational differences, and the tetrahydrogen structure is better than the quaternary ammonium salt structure and the lactam structure; the activity of connecting the urea group at the C-10 position of the berberine skeleton is better than that at the C-3 position. Therefore, the present invention obtains a novel soluble epoxide hydrolase inhibitory compound by introducing the berberine skeleton described in general formula I and II. Most of the compounds have an IC50 value of less than 20nM for soluble epoxide hydrolase, and the lowest IC50 value is about 0.67nM.

[0338] In summary, the main skeleton of the berberine derivatives for inhibiting soluble epoxide hydrolases prepared by the present invention is berberine with a urea group. Different substituents are connected to different sites of the berberine skeleton through the urea group, and the oxidation state of the C ring is changed, thereby obtaining a series of berberine derivatives with high activity against soluble epoxide hydrolases, and the IC50 value of the berberine derivatives for inhibiting soluble epoxide hydrolases reaches the nanomolar level, indicating that the berberine derivatives have a high inhibitory effect on soluble epoxide hydrolases, thereby achieving the inhibitory effect on inflammation by inhibiting soluble epoxide hydrolases, and have good application prospects.

Claims

1. A berberine derivative, characterized in that The berberine derivative has the following general structural formula I or II: In the formula I or II, R 1 and R 2 All are methoxy, or R 1 and R 2 They are cyclically bonded to form methylenedioxy groups; R 3 Selected from C 1-4 Alkyl, five-membered to six-membered cycloalkane methyl, five-membered to six-membered oxygen-containing heterocycloalkane methyl, N-(C 1-4 alkylformyl)piperidinyl, N-(C 1-4 alkylformyl)piperidinylmethyl, N-(halogenated C 1-4 alkylformyl)piperidinyl, N-(halogenated C 1-4 Alkylformyl)piperidinylmethyl, halo C 1-3 Alkoxyphenyl, halogenated C 1-3 Any one of alkoxybenzyl, methylsulfonylbenzyl and aminosulfonylbenzyl; The C ring is selected from any of the following structures:

2. The berberine derivative according to claim 1, characterized in that The R 1 , R 2 They form a ring to form methylenedioxy.

3. The berberine derivative according to claim 1, characterized in that The R 3 Any one selected from isopropyl, isobutyl, cyclopentyl, cyclohexyl, tetrahydrofuranyl, tetrahydropyranyl, N-acetylpiperidinyl, N-propionylpiperidinyl, N-acetylpiperidinylmethyl, N-propionylpiperidinylmethyl, difluoromethoxyphenyl, trifluoromethoxyphenyl, difluoromethoxybenzyl, trifluoromethoxybenzyl, methylsulfonylbenzyl and aminosulfonylbenzyl.

4. The berberine derivative according to claim 3, characterized in that The R 3 Any one group selected from trifluoromethoxybenzyl and difluoromethoxybenzyl.

5. The berberine derivative according to claim 1, characterized in that The C ring is selected from any one of the following structures:

6. The berberine derivative according to claim 5, characterized in that The C ring is selected from 7. The berberine derivative according to claim 1, characterized in that The R 1 and R 2 Ring-forming methylenedioxy or R 1 , R 2 are selected as methoxy, the R 3 Any one of tetrahydropyranyl, N-acetylpiperidinylmethyl, N-propionylpiperidinylmethyl, trifluoromethoxybenzyl, difluoromethoxybenzyl, methylsulfonylbenzyl or aminosulfonylbenzyl, wherein the C ring is 8. The berberine derivative according to claim 1, characterized in that The structural formula of the berberine derivative is shown below:

9. A berberine derivative pharmaceutical composition, characterized in that: The pharmaceutical composition comprises at least one of the berberine derivatives according to any one of claims 1 to 8, its isotope-labeled substance, solvate, polymorph, pharmaceutically acceptable salt or prodrug compound thereof.

10. Use of the berberine derivative according to any one of claims 1 to 8, or the berberine derivative pharmaceutical composition according to claim 9, in the preparation of an anti-inflammatory drug, wherein: The anti-inflammatory drug is used for inhibiting inflammation caused by soluble epoxide hydrolase.

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