6-(N-substituted) carbamoyl methyl dihydrobenzophenanthridine alkaloid as well as preparation method and application thereof

Abstract

The invention discloses 6-(N-substituted) carbamoyl methyl dihydrobenzophenanthridine alkaloid as well as a preparation method and application thereof, and relates to the technical field of pharmaceutical compound synthesis. The compound is prepared by the following steps: by taking chelerythrine or sanguinarine as a raw material, firstly carrying out nucleophilic substitution reaction on the chelerythrine or sanguinarine and ethyl acetate to obtain a product A, namely 6-ethoxycarbonyl methyl dihydrochelerythrine or 6-ethoxycarbonyl methyl dihydrosanguinarine; hydrolyzing the ester group of the product A under an alkaline condition, and acidifying to obtain a corresponding product B, namely alpha-(dihydrochelerythrine 6-yl) substituted acetic acid or alpha-(dihydrosanguinarin-6-yl) substituted acetic acid; and dissolving the product B in a solvent, and carrying out condensation with different substituted primary amines under the action of a condensing agent to prepare 6-(N-substituted) carbamoyl methyl dihydrochelerythrine or 6-(N-substituted) carbamoyl methyl dihydrosanguinarine, thereby obtaining the 6-(N-substituted) carbamoyl methyl dihydrochelerythrine or 6-(N-substituted) carbamoyl methyl dihydrosanguinarine. According to the invention, chelerythrine or sanguinarine is used as a raw material, (N-substituted) carbamyl methyl is introduced to the No.6 site of chelerythrine or sanguinarine for structural modification, the yield is high, and the preparation is easy. The prepared series of derivatives can significantly inhibit the release of an inflammatory medium NO in RAW264.7 cells induced by LPS, and the expression level of inflammatory cytokines TNF-alpha and IL-6 proteins.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical compound synthesis, and in particular to a 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid, a preparation method and application thereof, and in particular to a derivative obtained by introducing a (N-substituted)carbamoylmethyl group into the 6th position of dihydrochelerythrine or dihydrosanguinarine for structural modification, a preparation method thereof, and application thereof in the preparation of anti-inflammatory drugs. Background Art

[0002] The inflammatory response is the body's self-defense mechanism against tissue damage, infection, and antigen attack. Moderate inflammation can eliminate pathogens and promote recovery. However, persistent inflammation can produce excessive inflammatory factors and reactants, causing tissue damage and a variety of diseases, such as arthritis, atherosclerosis, obesity, diabetes, inflammatory bowel disease, and even cancer. It is precisely because of the diverse patterns of inflammation and its association with a variety of diseases that it is attracting more and more attention from researchers.

[0003] Lipopolysaccharide (LPS) can stimulate macrophages to produce proinflammatory mediators and proinflammatory cytokines, among which proinflammatory substrates include nitric oxide (NO) and prostaglandin E2 (PGe2), and proinflammatory cytokines include tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Studies have confirmed that inhibiting these inflammatory factors can weaken the inflammatory response in the disease. At present, nonsteroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs (SAIDs) are the main drugs for the treatment of chronic inflammation. However, long-term use of these drugs can cause serious side effects, including gastrointestinal damage and cardiovascular toxicity, so the development of new drugs with fewer side effects is urgent.

[0004] Chelerythrine and sanguinarine belong to quaternary ammonium benzophenanthridine alkaloids (QBAs), which are widely distributed in plants of the Papaveraceae, Violaceae, and Ranunculaceae. QBAs have a wide range of important biological activities, including anti-inflammatory, anti-tumor, antibacterial, antiviral, anti-HIV, and anti-acetylcholinesterase. Due to their wide range of biological activities, some plant extracts containing chelerythrine and sanguinarine have been used as folk medicine for centuries.

[0005]

[0006] Natural plant ingredients can be modified by reasonable structures to improve their biological activity, reduce toxicity, change solubility and thus change bioavailability. In the past few decades, there have been hundreds of cases of structural modification of chelerythrine, such as: CN103374007A discloses a salt of a dihydrochelerythrine derivative, specifically discloses a fumarate of a dihydrochelerythrine derivative or a pharmaceutically acceptable solvate thereof, these derivative compounds have significant stability, water solubility and antiviral activity, and can be used to prepare hepatitis B virus. However, most of the derivative compounds of chelerythrine and sanguinarine after these structural modifications are concentrated in the research fields of antiviral, anticancer, antimicrobial, and anti-insect activities, and there is no structural modification for their anti-inflammatory activity, which limits the development and utilization of chelerythrine or sanguinarine.

[0007] The present invention intends to provide a dihydrobenzophenanthridine alkaloid derivative with high anti-inflammatory activity and a preparation method and application thereof. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid and its preparation method and application in view of the defects of the prior art. This series of derivatives of dihydrobenzophenanthridine alkaloids can significantly inhibit the release of inflammatory mediator NO in LPS-induced RAW264.7 cells and the expression levels of inflammatory cytokines TNF-α and IL-6 proteins; have high in vitro anti-inflammatory activity and low cytotoxicity, and can be used to prepare anti-inflammatory drugs.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid, specifically 6-(N-substituted)carbamoylmethyldihydrochelerythrine as shown in formula (I) or 6-(N-substituted)carbamoylmethyldihydrosanguinarine as shown in formula (II):

[0011]

[0012] In the formula, R is any one of a phenyl group, an alkyl group, and a substituted phenyl group.

[0013] Preferably, the substituted phenyl group includes, but is not limited to, any one of methylphenyl, methoxyphenyl, bromophenyl, iodophenyl and nitrophenyl.

[0014] Preferably, the alkyl group includes any one of n-butyl, isobutyl or n-propyl.

[0015] In a second aspect, the present invention provides a method for preparing the above-mentioned 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid, which specifically comprises the following steps:

[0016] S1, using chelerythrine or sanguinarine as a raw material, first undergoing a nucleophilic substitution reaction with ethyl acetate, and after the reaction is complete, obtaining product A, i.e. 6-ethoxycarbonylmethyldihydrochelerythrine or 6-ethoxycarbonylmethyldihydrosanguinarine;

[0017] S2, hydrolyzing the ester group of product A under alkaline conditions and acidifying, and after the reaction is complete, obtaining the corresponding product B, i.e., α-(dihydrochelerythrine 6-yl) substituted acetic acid or α-(dihydrosanguinarine-6-yl) substituted acetic acid;

[0018] S3, dissolving the product B in a solvent, and condensing it with a primary amine under the action of a condensing agent to obtain 6-(N-substituted)carbamoylmethyl-dihydrochelerythrine or 6-(N-substituted)carbamoylmethyl-dihydrosanguinarine.

[0019] Furthermore, the preparation process of step S1 is as follows:

[0020] (1) dissolving 1 equivalent of ethyl acetate in tetrahydrofuran, then adding dropwise 2 to 3 equivalents of 1.0 mol / L tetrahydrofuran solution of lithium bis(trimethylsilyl)amide to obtain a mixture, stirring the mixture at -40 to -20°C for 20 to 40 minutes, then slowly adding 0.5 to 1.0 equivalents of chelerythrine / sanguinarine and stirring continuously for 3 to 5 hours;

[0021] (2) After the reaction is complete, wait for the temperature of the reaction solution to rise to room temperature, add water, extract with ethyl acetate 3 to 5 times, recover the organic layer, dry and concentrate, and separate and purify using a silica gel column to obtain the corresponding 6-ethoxycarbonylmethyldihydrochelerythrine or 6-ethoxycarbonylmethyldihydrosanguinarine.

[0022] Furthermore, the preparation process of step S2 is as follows:

[0023] (1) adding 6-ethoxycarbonylmethyldihydrochelerythrine or 6-ethoxycarbonylmethyldihydrosanguinarine prepared in S1 to a 5% to 20% NaOH aqueous solution, and reacting at 100 to 120° C. for 24 to 48 hours;

[0024] (2) then adding dilute hydrochloric acid dropwise to adjust the pH value to 4-7; then extracting with ethyl acetate 3-5 times, retaining the organic layer, drying and concentrating the organic layer to obtain the corresponding α-(dihydrochelerythrine 6-yl) substituted acetic acid or α-(dihydrosanguinarine-6-yl) substituted acetic acid.

[0025] Furthermore, the preparation process of step S3 is as follows:

[0026] (1) dissolving 1 equivalent of α-(dihydrochelerythrine-6-yl) substituted acetic acid or α-(dihydrosanguinarine-6-yl) substituted acetic acid prepared in S2 in a solvent, adding 3 to 4 equivalents of a condensing agent and 2 to 4 equivalents of a primary amine, and reacting with stirring at room temperature for 12 to 24 hours;

[0027] (2) After the reaction is complete, the reaction solution is washed with water for 3 to 5 times, the organic layer is retained, dried and concentrated, and separated by column chromatography to obtain 6-(N-substituted)carbamoylmethyldihydrochelerythrine or 6-(N-substituted)carbamoylmethyldihydrosanguinarine.

[0028] Preferably, the solvent in step S3 (1) is dichloromethane.

[0029] Preferably, the condensing agent in step S3 (1) is EDCI.

[0030] Furthermore, the primary amine is any one of aniline, alkylammonium or substituted phenylamine.

[0031] Preferably, the alkylammonium is any one of n-butylammonium, isobutylammonium or n-propylammonium.

[0032] Preferably, the substituted phenylamine is any one of methylphenylammonium, methoxyphenylammonium, brominated phenylammonium, iodinated phenylammonium, and nitrophenylammonium.

[0033] In a third aspect, the present invention also provides the use of the above 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid or the 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid prepared by the above preparation method in the preparation of anti-inflammatory drugs.

[0034] The present invention has the following beneficial effects:

[0035] 1. The preparation method provided by the present invention uses chelerythrine / sanguinarine as raw materials, and introduces a carbamoyl methyl group at position 6 for the first time, including N-phenylcarbamoyl methyl, N-n-butylcarbamoyl methyl, N-isobutylcarbamoyl methyl, N-substituted phenylcarbamoyl methyl, etc., which is easy to prepare.

[0036] 2. The derivatives obtained by introducing a (N-substituted) carbamoylmethyl group at position 6 of chelerythrine / sanguinarine for structural modification include 6-(N-substituted) carbamoylmethyl dihydrochelerythrine or 6-(N-substituted) carbamoylmethyl sanguinarine, etc., which can significantly inhibit the expression of NO and the protein expression levels of TNF-α and IL-6, and the cytotoxicity is greatly reduced compared with chelerythrine / sanguinarine. Therefore, the derivatives can be used to prepare anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1The present invention is a schematic diagram of the process of synthesizing 6-(N-substituted)carbamoylmethyldihydrochelerythrine or 6-(N-substituted)carbamoylmethylsanguinarine.

[0038] Figure 2 This is the 1H spectrum of the NMR nuclear magnetic results of 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine synthesized in Example 1 of the present invention.

[0039] Figure 3 This is the 13C spectrum of the NMR nuclear magnetic resonance results of 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine synthesized in Example 1 of the present invention.

[0040] Figure 4 This is the 1H spectrum of the NMR nuclear magnetic resonance results of 6-(N-n-butyl)-carbamoylmethyl-dihydrochelerythrine synthesized in Example 2 of the present invention.

[0041] Figure 5 This is the 13C spectrum of the NMR nuclear magnetic resonance results of 6-(N-n-butyl)-carbamoylmethyl-dihydrochelerythrine synthesized in Example 2 of the present invention.

[0042] Figure 6 This is the 1H spectrum of the NMR nuclear magnetic results of 6-(N-phenyl)-carbamoylmethyl-dihydrosanguinarine synthesized in Example 3 of the present invention.

[0043] Figure 7 This is the 13C spectrum of the NMR nuclear magnetic results of 6-(N-phenyl)-carbamoylmethyl-dihydrosanguinarine synthesized in Example 3 of the present invention.

[0044] Figure 8 This is the 1H spectrum of the NMR nuclear magnetic results of 6-(N-n-butyl)-carbamoylmethyl-dihydrosanguinarine synthesized in Example 4 of the present invention.

[0045] Fig. 9 This is the 13C spectrum of the NMR nuclear magnetic resonance result of 6-(N-n-butyl)-carbamoylmethyl-dihydrosanguinarine synthesized in Example 4 of the present invention.

[0046] Fig.10 The figures are the effects of 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine on the expression levels of inflammatory factors in LPS-stimulated RAW264.7 cells, wherein: 10(A) is the effect of 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine on the level of IL-6, and 10(B) is the effect of 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine on the level of TNF-α. DETAILED DESCRIPTION

[0047] As used herein:

[0048] "Prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0049] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing the specific embodiments and are not intended to limit the present invention. In the embodiments, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0051] The present invention provides a dihydrobenzophenanthridine alkaloid derivative, specifically a derivative obtained by introducing a (N-substituted) carbamoylmethyl group at position 6 of a dihydrobenzophenanthridine alkaloid chelerythrine or sanguinarine for structural modification, and in the scheme of the present invention, specifically a 6-(N-substituted) carbamoylmethyl dihydrochelerythrine as shown in formula (I) or a 6-(N-substituted) carbamoylmethyl dihydrosanguinarine as shown in formula (II):

[0052]

[0053] In the formula, R is any one of phenyl, alkyl, and substituted phenyl. In a preferred embodiment, the substituted phenyl includes any one of methylphenyl, methoxyphenyl, bromophenyl, iodophenyl, and nitrophenyl. In a preferred embodiment, the alkyl includes any one of n-butyl, isobutyl, or n-propyl.

[0054] like Figure 1 As shown, the present invention provides a method for preparing the above-mentioned 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid, which specifically comprises the following steps:

[0055] S1. Using chelerythrine or sanguinarine as a raw material, a nucleophilic substitution reaction is carried out with ethyl acetate in an organic lithium reagent lithium bis(trimethylsilyl)amide solution. After the reaction is completed, a product A, i.e., 6-ethoxycarbonylmethyldihydrochelerythrine or 6-ethoxycarbonylmethyldihydrosanguinarine, is obtained.

[0056] S2. The ester group of product A is hydrolyzed under alkaline conditions and acidified. After the reaction is complete, the corresponding product B, i.e., α-(dihydrochelerythrine-6-yl)-substituted acetic acid or α-(dihydrosanguinarine-6-yl)-substituted acetic acid, is obtained.

[0057] S3, dissolving product B in a solvent, condensing with a primary amine under the action of a condensing agent, and obtaining 6-(N-substituted)carbamoylmethyl-dihydrochelerythrine or 6-(N-substituted)carbamoylmethyl-dihydrosanguinarine, that is, obtaining. In a preferred embodiment, the solvent is dichloromethane; the condensing agent is EDCI (carbodiimide); the primary amine is aniline, alkylammonium, or substituted phenylamine. In a preferred embodiment, the alkylammonium is any one of n-butylammonium, isobutylammonium, or n-propylammonium; the substituted phenylamine is any one of methylphenylammonium, methoxyphenylammonium, bromophenylammonium, iodophenylammonium, or nitrophenylammonium.

[0058] In order to further illustrate the scheme of the present invention, the following preferred embodiments of the present invention are given as examples for illustration. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0059] 1. Preparation of 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloids

[0060] Embodiment 1:

[0061] This embodiment provides a method for preparing 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine, comprising the following steps:

[0062] S1, dissolve 1 equivalent of ethyl acetate in tetrahydrofuran, in this embodiment, the volume percentage concentration of ethyl acetate and tetrahydrofuran is configured to be 10%; then add 2 equivalents of ethyl acetate and 1.0 mol / L tetrahydrofuran solution of lithium bis(trimethylsilyl)amide to obtain a mixture, stir the mixture for 20 min at -40°C, then slowly add 0.5 equivalent of chelerythrine and stir continuously for 4 h. After the reaction is complete, the temperature of the reaction solution is raised to room temperature, water is added, extracted with ethyl acetate 3 times, the organic layer is recovered, dried and concentrated, and separated and purified by silica gel column to obtain product A, i.e. 6-ethoxycarbonylmethyldihydrochelerythrine.

[0063] The synthetic reaction equation of this step is as follows:

[0064]

[0065] S2. Add 6-ethoxycarbonylmethyldihydrochelerythrine prepared in S1 to a 5% NaOH aqueous solution and react at 100° C. for 24 hours. Then add dilute hydrochloric acid dropwise and adjust the pH value to 6; then extract with ethyl acetate three times, retain the organic layer, dry and concentrate the organic layer to obtain product B, i.e., α-(dihydrochelerythrine 6-yl) substituted acetic acid.

[0066] The synthetic reaction equation of this step is as follows:

[0067]

[0068] S3, dissolving 1 equivalent of α-(dihydrochelerythrine-6-yl) substituted acetic acid obtained in S2 in dichloromethane, adding 3 equivalents of condensing agent EDCI (carbodiimide) and 2 equivalents of aniline, stirring at room temperature for 14 hours. After the reaction is complete, the reaction solution is washed with water 3 times, the organic layer is retained, dried and concentrated, and separated by column chromatography to obtain 6-(N-substituted)carbamoylmethyldihydrochelerythrine.

[0069] The synthetic reaction equation of this step is as follows:

[0070]

[0071] Embodiment 2:

[0072] This embodiment provides a preparation method of 6-(N-n-butyl)-carbamoylmethyl-dihydrochelerythrine, comprising the following steps:

[0073] S1, dissolve 1 equivalent of ethyl acetate in tetrahydrofuran, and the volume percentage concentration of ethyl acetate and tetrahydrofuran in this embodiment is configured to be 10%; then add 3 equivalents of ethyl acetate and 1.0 mol / L tetrahydrofuran solution of lithium bis(trimethylsilyl)amide to obtain a mixture; after the mixture is stirred at -30°C for 20 minutes, 0.7 equivalents of chelerythrine are slowly added and stirred continuously for 4 hours. After the reaction is complete, the temperature of the reaction liquid is raised to room temperature, water is added, and the mixture is extracted with ethyl acetate 3 times, the organic layer is recovered, dried and concentrated, and separated and purified by silica gel column to obtain product A, i.e. 6-ethoxycarbonylmethyldihydrochelerythrine.

[0074] S2. Add 6-ethoxycarbonylmethyldihydrochelerythrine prepared in S1 to a 10% NaOH aqueous solution and react at 120° C. for 24 hours. Then add dilute hydrochloric acid dropwise to adjust the pH value to 6; then extract with ethyl acetate 5 times, retain the organic layer, dry and concentrate the organic layer to obtain product B, i.e., α-(dihydrochelerythrine 6-yl) substituted acetic acid.

[0075] S3, dissolving 1 equivalent of α-(dihydrochelerythrine-6-yl) substituted acetic acid obtained in S2 in dichloromethane, adding 3 equivalents of condensing agent EDCI (carbodiimide) and 2 equivalents of n-butylamine, stirring at room temperature for 14 hours. After the reaction is complete, the reaction solution is washed with water 3 times, the organic layer is retained, dried and concentrated, and separated by column chromatography to obtain 6-(N-n-butyl)-carbamoylmethyl-dihydrochelerythrine.

[0076] The synthetic reaction equation of this step is as follows:

[0077]

[0078] Example 3

[0079] This embodiment provides a method for preparing 6-(N-phenyl)-carbamoylmethyl-dihydrosanguinarine, which specifically comprises the following steps:

[0080] S1. Dissolve 1 equivalent of ethyl acetate in tetrahydrofuran. In this embodiment, the volume percentage concentration of ethyl acetate and tetrahydrofuran is 10%; then add 2.5 equivalents of ethyl acetate and 1.0 mol / L tetrahydrofuran solution of lithium bis(trimethylsilyl)amide to obtain a mixture; after stirring the mixture at -40°C for 20 minutes, slowly add 1 equivalent of sanguinarine and stir continuously for 3 hours. After the reaction is complete, wait for the temperature of the reaction liquid to rise to room temperature, add water, extract with ethyl acetate 3 times, recover the organic layer, dry and concentrate, and separate and purify by silica gel column to obtain product A, i.e. 6-ethoxycarbonylmethyldihydrosanguinarine.

[0081] The synthetic reaction equation of this step is as follows:

[0082]

[0083] S2, add 6-ethoxycarbonylmethyl sanguinarine prepared in S1 to a 10% NaOH aqueous solution, and react at 110°C for 24 hours. Then add dilute hydrochloric acid dropwise, and adjust the pH value to 5; then extract with ethyl acetate 4 times, retain the organic layer, dry and concentrate the organic layer, and obtain product B, i.e., α-(sanguinarine-6-yl) substituted acetic acid.

[0084] The synthetic reaction equation of this step is as follows:

[0085]

[0086] S3, dissolve 1 equivalent of α-(dihydrosanguinarine-6-yl) substituted acetic acid obtained in S2 in dichloromethane, add 3 equivalents of condensing agent EDCI (carbodiimide) and 2 equivalents of aniline, and react at room temperature for 24 hours with stirring. After the reaction is complete, wash the reaction solution with water 5 times, retain the organic layer, dry and concentrate, and separate the corresponding 6-(N-substituted)carbamoylmethylsanguinarine by column chromatography.

[0087] The synthetic reaction equation of this step is as follows:

[0088]

[0089] Example 4

[0090] This embodiment provides a preparation method of 6-(N-n-butyl)-carbamoylmethyl-dihydrosanguinarine, comprising the following steps:

[0091] S1. Dissolve 1 equivalent of ethyl acetate in tetrahydrofuran. In this embodiment, the volume percentage concentration of ethyl acetate and tetrahydrofuran is 10%; then add 2.5 equivalents of ethyl acetate and 1.0 mol / L tetrahydrofuran solution of lithium bis(trimethylsilyl)amide to obtain a mixture; after stirring the mixture at -30°C for 30 minutes, slowly add 0.7 equivalents of sanguinarine and stir continuously for 4 hours. After the reaction is complete, wait for the temperature of the reaction liquid to rise to room temperature, add water, extract with ethyl acetate 4 times, recover the organic layer, dry and concentrate, and separate and purify by silica gel column to obtain product A, i.e. 6-ethoxycarbonylmethyldihydrosanguinarine.

[0092] S2, add 6-ethoxycarbonylmethyl sanguinarine prepared in S1 to a 5% NaOH aqueous solution, and react at 110°C for 48 hours. Then add dilute hydrochloric acid dropwise, and adjust the pH value to 5; then extract with ethyl acetate three times, retain the organic layer, dry and concentrate the organic layer, and obtain product B, i.e., α-(sanguinarine 6-yl) substituted acetic acid.

[0093] S3, dissolve 1 equivalent of α-(dihydrosanguinarine-6-yl) substituted acetic acid obtained in S2 in dichloromethane, add 4 equivalents of condensing agent EDCI (carbodiimide) and 4 equivalents of n-butylamine, and stir at room temperature for 14 hours. After the reaction is complete, wash the reaction solution with water 3 times, retain the organic layer, dry and concentrate, and separate the corresponding 6-(N-n-butyl)-carbamoylmethyl-dihydrosanguinarine by column chromatography.

[0094] The synthetic reaction equation of this step is as follows:

[0095]

[0096] 2. Structural Characterization of Products

[0097] The NMR results of the products obtained in Examples 1-4 are as follows: Figure 2-9 shown.

[0098] The NMR result data of 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine prepared in Example 1 are as follows:

[0099] 1 H NMR (400MHz, CDCl3): δ8.88(s,1H),7.76(d,J=8.7Hz,1H),7.58–7.54(m,5H),7.36(d,J=8.1Hz,2H),7.16–7.11(m,2H),7.01(dd, J=8.6Hz,1H),6.04(dd,J=1.0,16.1Hz,2H),5.04(dd,J=1.0,16.1Hz,2H),3.98(d,J=15.8Hz,6H),2.76(s,3H),2.44–2.34(m,2H). 13 C NMR(100MHz, CDCl3):169.9,152.5,148.6,147.7,145.7,138.2,137.7,131.1,128.9(×2),127.9,126.9,124 .7,124.2,124.0,123.9,120.0,119.8(×2),119.1,111.9,104.7,101.3,99.8,61.1,55.9,55.4,42.3,40.9.

[0100] The NMR result data of 6-(N-n-butyl)-carbamoylmethyl-dihydrochelerythrine prepared in Example 2 are as follows:

[0101] 1 H NMR (400MHz, CDCl3): δ7.73 (d, J=8.6Hz, 1H), 7.55–7.52 (m, 3H), 7.15 (s, 1H), 6.9 8(d,J=8.6Hz,1H),6.67(s,1H),6.07(d,J=3.2Hz,2H),4.95(dd,J=3.6,10.9Hz,1 H),3.96(d,J=14.1Hz,6H)=3.2Hz,2H),3.28(dd,J=6.6,13.3Hz,2H),2.69(s,3H) ,2.27–2.14(m,2H),1.54–1.44(m,2H),1.41–1.31(m,2H),0.96(d,J=14.4Hz,3H).13 C NMR (100MHz, CDCl3):171.4,152.4,148.4,148.0,145.7,138.2,131.0,128.3,127.0,124.4,124.3,1 23.8,120.0,119.0,111.6,104.7,101.2,100.0,61.1,55.8,55.2,42.4,40.0,39.1,31.7,20.3,13.8.

[0102] The NMR result data of 6-(N-phenyl)-carbamoylmethyl-dihydrosanguinarine obtained in Example 3 are as follows:

[0103] 1 H NMR (400MHz, CDCl3): δ8.88(s,1H),7.76(d,J=8.7Hz,1H),7.58–7.54(m,5H),7.36(d,J=8.1Hz,2H),7.16–7.11(m,2H),7.01(dd, J=8.6Hz,1H),6.04(dd,J=1.0,16.1Hz,2H),5.04(dd,J=1.0,16.1Hz,2H),3.98(d,J=15.8Hz,6H),2.76(s,3H),2.44–2.34(m,2H). 13 C NMR(100MHz, CDCl3):169.9,152.5,148.6,147.7,145.7,138.2,137.7,131.1,128.9(×2),127.9,126.9,124 .7,124.2,124.0,123.9,120.0,119.8(×2),119.1,111.9,104.7,101.3,99.8,61.1,55.9,55.4,42.3,40.9.

[0104] The NMR result data of 6-(N-n-butyl)-carbamoylmethyl-dihydrosanguinarine obtained in Example 4 are as follows:

[0105] 1H NMR (400MHz, CDCl3): δ7.72(d,J=8.6Hz,1H),7.53(d,J=7.7Hz,2H),7.34(d,J=8.2Hz,1H),7.15(s,1H),6.88(d,J=8.2Hz,1H),6.41(s,1H),6.08–6.0 6(m,4H),4.76(dd,J=4.7,9.9Hz,1H),3.35–3.22(m,2H),2.71(s,3H),2.29 –2.19(m,2H),1.54–1.45(m,2H),1.39–1.32(m,2H),0.95(t,J=7.3Hz,3H). 13 C NMR(100MHz, CDCl3):171.0,148.4,147.6,147.4,144.4,131.0(×2),127.2,125.3,124.5,124.0, 120.2,116.5,116.1,107.7,104.6,101.7,101.2,100.1,55.2,42.8,39.9,39.2,31.7,20.2,13.8.

[0106] 3. Performance Test

[0107] 1. Cell culture

[0108] RAW264.7 cells were maintained in DMEM medium containing 10% heat-inactivated fetal bovine serum and 1% double antibody and incubated at 37°C in a humidified environment with 5% CO2.

[0109] 2. Cell Viability Assay

[0110] The effects of each compound on the viability of RAW264.7 cells were examined by MTT assay. 4 The cells were inoculated at a density of 100 μM in a 96-well culture plate and incubated for 24 h. The cells were treated with 20 μM of the compound for 24 h. 20 μL of MTT solution (5 mg / mL) was added and incubated for 4 h. The absorbance was monitored at a wavelength of 490 nm using an ELISA reader to calculate the cell viability.

[0111] The cell survival rate was calculated by the formula: cell survival rate = (sample absorbance - plate bottom absorbance) / (blank absorbance - plate bottom absorbance) × 100%. The results are shown in Table 1 below.

[0112] Table 1 Cell viability of each group at 20 μM

[0113] Compound / Group Name Cell viability at 20 μM (%) Blank group 100 Model Group / 6-(N-n-butyl)-carbamoylmethyl-dihydrosanguinarine (Example 4) 100.62±0.83 6-(N-n-butyl)-carbamoylmethyl-dihydrochelerythrine (Example 2) 92.09±1.37 6-(N-phenyl)-carbamoylmethyl-dihydrosanguinarine (Example 3) 125.38±5.4 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine (Example 1) 135.63±0.82

[0114] From the data in Table 1, it can be seen that 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid derivatives at a concentration of 20 μM have no significant cytotoxicity to LPS-stimulated RAW264.7 cells, especially 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine prepared in Example 1 has the least toxicity.

[0115] 3. Effects on NO production in LPS-stimulated RAW264.7 cells

[0116] RAW264.7 cells (2×10 4 Cells / well) were inoculated into 96-well plates and cultured overnight. Blank group, control group and sample group were set up. The blank group only cultured cells normally, the control group contained cells and LPS (400ng / mL), and the cells in the sample group were pretreated with 20μM compound for 1h and incubated with LPS for 24h. The supernatant was collected and the NO release was determined using Griess reagent. The results are shown in Table 2 below.

[0117] Table 2 NO release and NO inhibition rate at 20 μM in each group

[0118]

[0119] From the data in Table 2, it can be seen that the NO level in the LPS-induced group was significantly higher than that in the blank control group (P<0.01); the NO production level of 20μM 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid derivatives was significantly different from that in the induced group (P<0.01), among which 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine reduced the NO production to 8.16±0.82μM (P<0.001). The above results indicate that 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine can inhibit the production of NO by LPS-stimulated RAW264.7 mouse macrophages. 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine was selected for further study.

[0120] 4. Effects on IL-6 and TNF-α production in LPS-stimulated RAW264.7 cells

[0121] RAW264.7 cells (2×10 5 Cells were inoculated into 6-well plates (Cell-nest, China) and cultured overnight. Cells were pretreated with different concentrations of compounds for 1 h and incubated with LPS (400 ng / mL) for 24 h. The supernatant was quickly collected and centrifuged at 4 °C (2500 rpm, 20 min). Cytokine levels were determined according to the instructions of the ELISA kit (TNF-α and IL-6, Hangzhou Zhenyoupin Biotechnology Co., Ltd.).

[0122] The results are as follows Fig.10 As shown in (A) and 10 (B), the levels of IL-6 and TNF-α in the LPS-induced group were significantly higher than those in the blank control group (P<0.001). The levels of IL-6 and TNF-α in the 40, 80 and 160 μM 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine groups were significantly different from those in the induced group (P<0.01). The 160 μM level of 6-(N-aryl)-carbamoylmethyl-dihydrochelerythrine group reduced the production of IL-6 and TNF-α to a level close to that of the blank group. The experimental results showed that 6-(N-substituted)carbamoylmethyl-dihydrochelerythrine significantly inhibited the production of IL-6 and TNF-α in LPS-stimulated RAW264.7 mouse macrophages in a dose-dependent manner.

[0123] The above experimental results show that 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloids can be synthesized and structurally identified through the above synthetic route. This series of derivatives can significantly inhibit the release of NO in RAW264.7 cells stimulated by LPS, as well as the protein expression levels of TNF-α and IL-6. These results show that 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloids are non-cytotoxic at the experimental setting concentration and have good anti-inflammatory pharmacodynamic activity, so they can be used to prepare anti-inflammatory drugs.

[0124] Those skilled in the art will appreciate that the method of the present invention can be extended to the synthesis of structurally similar quaternary ammonium benzophenanthridine alkaloids and radioactive quaternary ammonium benzophenanthridine alkaloids, which should also be included in the protection scope of the present invention.

[0125] The above descriptions are only some preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid, characterized in that Specifically, it is 6-(N-substituted)carbamoylmethyldihydrochelerythrine as shown in formula (I) or 6-(N-substituted)carbamoylmethyldihydrosanguinarine as shown in formula (II): In the formula, R is any one of a phenyl group, an alkyl group, and a substituted phenyl group.

2. The 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid according to claim 1, characterized in that The substituted phenyl group includes any one of a methylphenyl group, a methoxyphenyl group, a bromophenyl group, an iodophenyl group, and a nitrophenyl group.

3. The 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid according to claim 1, characterized in that The alkyl group includes any of n-butyl, isobutyl or n-propyl.

4. A method for preparing a 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid as claimed in claim 1, characterized in that: The specific steps include: S1, using chelerythrine or sanguinarine as a raw material, first undergoing a nucleophilic substitution reaction with ethyl acetate, and after the reaction is complete, obtaining product A, i.e. 6-ethoxycarbonylmethyldihydrochelerythrine or 6-ethoxycarbonylmethyldihydrosanguinarine; S2, hydrolyzing the ester group of product A under alkaline conditions and acidifying, and after the reaction is complete, obtaining the corresponding product B, i.e., α-(dihydrochelerythrine 6-yl) substituted acetic acid or α-(dihydrosanguinarine-6-yl) substituted acetic acid; S3, dissolving the product B in a solvent, and condensing it with a primary amine under the action of a condensing agent to obtain 6-(N-substituted)carbamoylmethyl-dihydrochelerythrine or 6-(N-substituted)carbamoylmethyl-dihydrosanguinarine.

5. The preparation method according to claim 4, characterized in that: The preparation process of step S1 is as follows: (1) dissolving 1 equivalent of ethyl acetate in tetrahydrofuran, then adding dropwise 2 to 3 equivalents of 1.0 mol / L tetrahydrofuran solution of lithium bis(trimethylsilyl)amide to obtain a mixture, stirring the mixture at -40 to -20°C for 20 to 40 minutes, then slowly adding 0.5 to 1.0 equivalent of chelerythrine or sanguinarine and stirring continuously for 3 to 5 hours; (2) After the reaction is complete, wait for the temperature of the reaction solution to rise to room temperature, add water, extract with ethyl acetate 3 to 5 times, recover the organic layer, dry and concentrate, and separate and purify using a silica gel column to obtain the corresponding 6-ethoxycarbonylmethyldihydrochelerythrine or 6-ethoxycarbonylmethyldihydrosanguinarine.

6. The preparation method according to claim 4, characterized in that: The preparation process of step S2 is as follows: (1) adding 6-ethoxycarbonylmethyldihydrochelerythrine or 6-ethoxycarbonylmethyldihydrosanguinarine prepared in S1 to a 5% to 20% NaOH aqueous solution, and reacting at 100 to 120° C. for 24 to 48 hours; (2) then adding dilute hydrochloric acid dropwise to adjust the pH value to 4-7; then extracting with ethyl acetate 3-5 times, retaining the organic layer, drying and concentrating the organic layer to obtain the corresponding α-(dihydrochelerythrine 6-yl) substituted acetic acid or α-(dihydrosanguinarine-6-yl) substituted acetic acid.

7. The preparation method according to claim 4, characterized in that: The preparation process of step S3 is as follows: (1) dissolving 1 equivalent of α-(dihydrochelerythrine-6-yl) substituted acetic acid or α-(dihydrosanguinarine-6-yl) substituted acetic acid prepared in S2 in a solvent, adding 3 to 4 equivalents of a condensing agent and 2 to 4 equivalents of a primary amine, and reacting with stirring at room temperature for 12 to 24 hours; (2) After the reaction is complete, the reaction solution is washed with water for 3 to 5 times, the organic layer is retained, dried and concentrated, and separated by column chromatography to obtain 6-(N-substituted)carbamoylmethyldihydrochelerythrine or 6-(N-substituted)carbamoylmethyldihydrosanguinarine.

8. The preparation method according to claim 7, characterized in that: The solvent in step S3 (1) is dichloromethane; the condensation agent is EDCI.

9. The preparation method according to claim 7, characterized in that: The primary amine in step S3 (1) is any one of aniline, alkylammonium or substituted phenylamine.

10. Use of the 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid according to any one of claims 1 to 3 or the 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid prepared by the preparation method according to any one of claims 4 to 9 in the preparation of anti-inflammatory drugs.

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