A 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid and its preparation method and application

Abstract

The present invention discloses a 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid and its preparation method and application, relating to the technical field of pharmaceutical compound synthesis. Such compounds are prepared by the following method: using chelerythrine or sanguinarine as raw materials, first undergoing a nucleophilic substitution reaction with ethyl acetate to obtain product A, i.e., 6-ethoxycarbonylmethyldihydrochelerythrine or 6-ethoxycarbonylmethyldihydrosanguinarine; hydrolyzing the ester group of product A under alkaline conditions and acidifying to obtain the corresponding product B, i.e., α-(dihydrochelerythrine 6-base) substituted acetic acid or α-(dihydrosanguinarine 6-base) substituted acetic acid; dissolving product B in a solvent, and condensing with different substituted primary amines under the action of a condensing agent to obtain 6-(N-substituted)carbamoylmethyldihydrochelerythrine or 6-(N-substituted)carbamoylmethyldihydrosanguinarine, that is, This invention uses chelerythrine or sanguinarine as raw materials and introduces a (N-substituted) carbamoylmethyl group at position 6 for structural modification. This series of derivatives, with high yield and ease of preparation, significantly inhibits LPS-induced release of the inflammatory mediator NO and expression of the inflammatory cytokines TNF-α and IL-6 in RAW264.7 cells.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical compound synthesis, and specifically to a 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid, a preparation method and application thereof, and in particular to a derivative obtained by structurally modifying dihydrochelerythrine or dihydrosanguinarine by introducing a (N-substituted)carbamoylmethyl group at position 6 thereof, 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 antigenic 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. Precisely because of its diverse patterns and association with a wide range of diseases, inflammation is attracting increasing attention from researchers.

[0003] Lipopolysaccharide (LPS) can stimulate macrophages to produce proinflammatory mediators and proinflammatory cytokines, among which proinflammatory matrices 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. Currently, 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 the quaternary ammonium benzophenanthridine alkaloids (QBAs), widely distributed in plants of the Papaveraceae, Violaceae, and Ranunculaceae families. QBAs exhibit a wide range of important biological activities, including anti-inflammatory, anti-tumor, antibacterial, antiviral, anti-HIV, and anti-acetylcholinesterase activities. Due to their broad range of biological activities, plant extracts containing chelerythrine and sanguinarine have been used in folk medicine for centuries.

[0005]

[0006] Natural plant ingredients can be modified structurally to enhance their biological activity, reduce toxicity, and alter solubility, thereby improving bioavailability. Over the past few decades, hundreds of cases involving the structural modification of chelerythrine have been reported. For example, CN103374007A discloses a salt of a dihydrochelerythrine derivative, specifically a fumarate salt or a pharmaceutically acceptable solvate thereof. These derivatives exhibit significant stability, water solubility, and antiviral activity, and can be used to prepare hepatitis B virus. However, most of these structurally modified chelerythrine and sanguinarine derivatives have focused on research areas such as antiviral, anticancer, antimicrobial, and anti-insect activities. No structural modifications have yet been made to target their anti-inflammatory activity, limiting their development and utilization.

[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 present invention addresses the technical problem of providing a 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid, its preparation method, and its application, addressing the deficiencies of the prior art. This series of dihydrobenzophenanthridine alkaloid derivatives can significantly inhibit the release of the inflammatory mediator NO and the expression levels of the inflammatory cytokines TNF-α and IL-6 in LPS-induced RAW264.7 cells. They exhibit 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 the product. After the reaction is complete, the corresponding product B is obtained, i.e., α-(dihydrochelerythrine-6-yl)-substituted acetic acid or α-(dihydrosanguinarine-6-yl)-substituted acetic acid;

[0018] S3. Dissolve the product B in a solvent, and condense 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) Dissolve 1 equivalent of ethyl acetate in tetrahydrofuran, then dropwise add 2 to 3 equivalents of a 1.0 mol / L tetrahydrofuran solution of lithium bis(trimethylsilyl)amide to obtain a mixture. Stir the mixture at -40 to -20°C for 20 to 40 minutes, then slowly add 0.5 to 1.0 equivalents of chelerythrine / sanguinarine and continue stirring for 3 to 5 hours.

[0021] (2) After the reaction is complete, the reaction solution is heated to room temperature, water is added, and the mixture is extracted with ethyl acetate 3 to 5 times. The organic layer is recovered, dried, and concentrated, and separated and purified 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) Dilute hydrochloric acid is then added dropwise to adjust the pH value to 4-7; the mixture is then extracted with ethyl acetate 3-5 times, the organic layer is retained, and the organic layer is dried and concentrated 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) Dissolve 1 equivalent of α-(dihydrochelerythrine-6-yl) substituted acetic acid or α-(dihydrosanguinarine-6-yl) substituted acetic acid prepared in S2 in a solvent, add 3 to 4 equivalents of a condensing agent and 2 to 4 equivalents of a primary amine, and stir at room temperature for 12 to 24 hours;

[0027] (2) After the reaction is complete, the reaction solution is washed with water 3 to 5 times, and the organic layer is retained, dried and concentrated, and separated by column chromatography to obtain the corresponding 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 further provides the use of the above-mentioned 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid or the 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid prepared by the above-mentioned 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 carbamoylmethyl group at position 6 for the first time, including N-phenylformylmethyl, N-n-butylformylmethyl, N-isobutylformylmethyl, N-substituted phenylformylmethyl, etc., which is easy to prepare.

[0036] 2. The derivatives obtained by structurally modifying the 6th position of chelerythrine / sanguinarine by introducing an (N-substituted) carbamoylmethyl group, including 6-(N-substituted) carbamoylmethyldihydrochelerythrine or 6-(N-substituted) carbamoylmethylsanguinarine, can significantly inhibit the expression of NO and the protein expression levels of TNF-α and IL-6. The cytotoxicity is also greatly reduced compared to 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 for synthesizing 6-(N-substituted)carbamoylmethyldihydrochelerythrine or 6-(N-substituted)carbamoylmethylsanguinarine.

[0038] Figure 2 This is the 1H spectrum of the NMR 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 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 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 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 NMR result 13C spectrum 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] Figure 9 This is the NMR result 13C spectrum of 6-(N-n-butyl)-carbamoylmethyl-dihydrosanguinarine synthesized in Example 4 of the present invention.

[0046] Figure 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 IL-6 levels, and 10(B) is the effect of 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine on TNF-α levels. 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 variations 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 alone. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including the range "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 herein, 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 to which the present invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. Where specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are 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 the dihydrobenzophenanthridine alkaloid chelerythrine or sanguinarine for structural modification. In the scheme of the present invention, the derivative is specifically 6-(N-substituted)carbamoylmethyldihydrochelerythrine as shown in formula (I) or 6-(N-substituted)carbamoylmethyldihydrosanguinarine as shown in formula (II):

[0052]

[0053] Wherein, 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 a solution of lithium bis(trimethylsilyl)amide, an organic lithium reagent. After the reaction is completed, 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. Dissolve product B in a solvent and condense 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. In a preferred embodiment, the solvent is dichloromethane; the condensing agent is EDCI (carbodiimide); and 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; and the substituted phenylamine is any one of methylphenylammonium, methoxyphenylammonium, bromophenylammonium, iodophenylammonium, or nitrophenylammonium.

[0058] To further illustrate the present invention, preferred embodiments of the present invention are provided below as examples. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

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

[0060] Example 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 percent concentration of ethyl acetate and tetrahydrofuran is 10%. Then, add dropwise 2 equivalents of ethyl acetate and a 1.0 mol / L solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran to obtain a mixture. After stirring the mixture at -40°C for 20 minutes, slowly add 0.5 equivalents of chelerythrine and stir continuously for 4 hours. After the reaction is complete, the reaction solution is heated to room temperature, water is added, and the mixture is extracted three times with ethyl acetate. The organic layer is recovered, dried, and concentrated. The product A, i.e., 6-ethoxycarbonylmethyldihydrochelerythrine, is obtained by separation and purification on a silica gel column.

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

[0064]

[0065] S2. 6-Ethoxycarbonylmethyldihydrochelerythrine prepared in S1 was added to a 5% aqueous NaOH solution and reacted at 100°C for 24 hours. Dilute hydrochloric acid was then added dropwise to adjust the pH to 6. The mixture was then extracted three times with ethyl acetate. The organic layer was retained, dried, and concentrated 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. Dissolve 1 equivalent of α-(dihydrochelerythrine-6-yl)-substituted acetic acid prepared in S2 in dichloromethane, add 3 equivalents of condensing agent EDCI (carbodiimide) and 2 equivalents of aniline, and stir at room temperature for 14 hours. After the reaction is complete, wash the reaction solution with water three times, retain the organic layer, dry and concentrate, and separate by column chromatography to obtain the corresponding 6-(N-substituted)carbamoylmethyldihydrochelerythrine.

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

[0070]

[0071] Example 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. In this embodiment, the volume percent concentration of ethyl acetate and tetrahydrofuran is 10%. Then, add dropwise 3 equivalents of ethyl acetate and a 1.0 mol / L solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran to obtain a mixture. After stirring the mixture at -30°C for 20 minutes, slowly add 0.7 equivalents of chelerythrine and continue stirring for 4 hours. After the reaction is complete, the reaction solution is heated to room temperature, water is added, and the mixture is extracted three times with ethyl acetate. The organic layer is recovered, dried, and concentrated. The product A, i.e., 6-ethoxycarbonylmethyldihydrochelerythrine, is obtained by separation and purification on a silica gel column.

[0074] S2. 6-Ethoxycarbonylmethyldihydrochelerythrine prepared in S1 was added to a 10% aqueous NaOH solution and reacted at 120°C for 24 hours. Dilute hydrochloric acid was then added dropwise to adjust the pH to 6. The mixture was then extracted five times with ethyl acetate. The organic layer was retained, dried, and concentrated to obtain product B, i.e., α-(dihydrochelerythrine 6-yl)-substituted acetic acid.

[0075] S3. Dissolve 1 equivalent of α-(dihydrochelerythrine-6-yl)-substituted acetic acid prepared in S2 in dichloromethane, add 3 equivalents of condensing agent EDCI (carbodiimide) and 2 equivalents of n-butylamine, and stir at room temperature for 14 hours. After the reaction is complete, wash the reaction solution with water three times, retain the organic layer, dry and concentrate, and separate by column chromatography to obtain the corresponding 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 percent concentration of ethyl acetate and tetrahydrofuran is 10%. Then, add dropwise 2.5 equivalents of ethyl acetate to a 1.0 mol / L solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran to obtain a mixture. Stir the mixture at -40°C for 20 minutes, then slowly add 1 equivalent of sanguinarine and continue stirring for 3 hours. After the reaction is complete, warm the reaction mixture to room temperature, add water, and extract with ethyl acetate three times. The organic layer is recovered, dried, and concentrated. The product A, i.e., 6-ethoxycarbonylmethyldihydrosanguinarine, is obtained by separation and purification on a silica gel column.

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

[0082]

[0083] S2. Add 6-ethoxycarbonylmethylsanguinarine prepared in S1 to a 10% aqueous NaOH solution and react at 110°C for 24 hours. Then, add dilute hydrochloric acid dropwise to adjust the pH to 5. Extract the mixture four times with ethyl acetate, retain the organic layer, dry it, and concentrate it to 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 prepared in S2 in dichloromethane, add 3 equivalents of condensing agent EDCI (carbodiimide) and 2 equivalents of aniline, and stir at room temperature for 24 hours. After the reaction is complete, wash the reaction solution with water five times, retain the organic layer, dry and concentrate, and separate by column chromatography to obtain the corresponding 6-(N-substituted)carbamoylmethylsanguinarine.

[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 percent concentration of ethyl acetate and tetrahydrofuran is 10%. Then, add dropwise 2.5 equivalents of ethyl acetate to a 1.0 mol / L solution of lithium bis(trimethylsilyl)amide in tetrahydrofuran to obtain a mixture. After stirring the mixture at -30°C for 30 minutes, slowly add 0.7 equivalents of sanguinarine and continue stirring for 4 hours. After the reaction is complete, the reaction mixture is heated to room temperature, water is added, and the mixture is extracted four times with ethyl acetate. The organic layer is recovered, dried, and concentrated. The product A, i.e., 6-ethoxycarbonylmethyldihydrosanguinarine, is obtained by separation and purification using a silica gel column.

[0092] S2. Add 6-ethoxycarbonylmethylsanguinarine prepared in S1 to a 5% aqueous NaOH solution and react at 110°C for 48 hours. Then, add dilute hydrochloric acid dropwise to adjust the pH to 5. Extract the mixture three times with ethyl acetate, retain the organic layer, dry it, and concentrate it to 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 prepared 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 three times, retain the organic layer, dry and concentrate, and separate by column chromatography to obtain the corresponding 6-(N-n-butyl)-carbamoylmethyl-dihydrosanguinarine.

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

[0095]

[0096] 2. Structural Characterization of Products

[0097] The products obtained in Examples 1-4 were identified by nuclear magnetic resonance. The NMR results were as follows: Figure 2-9 shown.

[0098] The NMR results of 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine obtained 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 results of 6-(N-n-butyl)-carbamoylmethyl-dihydrochelerythrine obtained 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 results 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 results 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 Testing

[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 atmosphere of 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 Cells were seeded at a density of 100 μM in a 96-well culture plate and incubated for 24 hours. The cells were treated with 20 μM of the compound for 24 hours. 20 μL of MTT solution (5 mg / mL) was added and incubated for 4 hours. The absorbance at 490 nm was monitored using a microplate reader to calculate cell viability.

[0111] The cell viability was calculated using the formula: Cell viability = (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] The data in Table 1 show 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 were seeded into 96-well plates (100 cells / well) and cultured overnight. Blank, control, and sample groups were set up. The blank group contained only normal cell culture; the control group contained cells and LPS (400 ng / mL); and the sample group was pretreated with 20 μM of the compound for 1 hour and incubated with LPS for 24 hours. The supernatant was collected and NO release was measured 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] As shown in Table 2, the NO level in the LPS-induced group was significantly higher than that in the blank control group (P < 0.01). The NO production levels of 20 μM 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid derivatives were significantly different from those in the induced group (P < 0.01). 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine reduced NO production to 8.16±0.82 μM (P < 0.001). These results indicate that 6-(N-phenyl)-carbamoylmethyl-dihydrochelerythrine can inhibit NO production in 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 seeded into 6-well plates (Cell-nest, China) and cultured overnight. Cells were pretreated with various concentrations of the compounds for 1 hour and incubated with LPS (400 ng / mL) for 24 hours. The supernatant was quickly collected and centrifuged at 4°C (2500 rpm, 20 minutes). Cytokine levels were measured according to the ELISA kit instructions (TNF-α and IL-6, Hangzhou Zhenyoupin Biotechnology Co., Ltd.).

[0122] The results are as follows Figure 10 As shown in Figures (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 IL-6 and TNF-α production levels 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 6-(N-aryl)-carbamoylmethyl-dihydrochelerythrine group reduced the production of IL-6 and TNF-α to levels close to those in the blank control group. The 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 experimental results above demonstrate that 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloids can be synthesized and structurally characterized using the aforementioned synthetic route. This series of derivatives significantly inhibits the release of NO and the protein expression of TNF-α and IL-6 in LPS-stimulated RAW264.7 cells. These results demonstrate that 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloids are non-cytotoxic at the experimental concentrations and exhibit excellent anti-inflammatory activity, thus potentially being useful in the preparation of 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 scope of protection 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 scope of protection 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 phenyl, alkyl, and substituted phenyl; the substituted phenyl is any one of methylphenyl, methoxyphenyl, bromophenyl, iodophenyl, and nitrophenyl; and the alkyl is any one of n-butyl, isobutyl, and n-propyl.

2. A method for preparing a 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid according to 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 the product. After the reaction is complete, the corresponding product B is obtained, i.e., α-(dihydrochelerythrine-6-yl)-substituted acetic acid or α-(dihydrosanguinarine-6-yl)-substituted acetic acid; S3. Dissolve the product B in a solvent, and condense 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.

3. The preparation method according to claim 2, characterized in that The preparation process of step S1 is as follows: (1) Dissolve 1 equivalent of ethyl acetate in tetrahydrofuran, then add dropwise 2 to 3 equivalents of 1.0 mol / L lithium bis(trimethylsilyl)amide in tetrahydrofuran to obtain a mixture. Stir the mixture at -40 to -20°C for 20 to 40 minutes, then slowly add 0.5 to 1.0 equivalents of chelerythrine or sanguinarine and continue stirring for 3 to 5 hours. (2) After the reaction is complete, the reaction solution is heated to room temperature, water is added, and the mixture is extracted with ethyl acetate 3 to 5 times. The organic layer is recovered, dried, and concentrated, and separated and purified using a silica gel column to obtain the corresponding 6-ethoxycarbonylmethyldihydrochelerythrine or 6-ethoxycarbonylmethyldihydrosanguinarine.

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

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

6. The preparation method according to claim 5, characterized in that The solvent in step S3 (1) is dichloromethane; the condensation agent is EDCI.

7. Use of the 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid according to claim 1 or the 6-(N-substituted)carbamoylmethyldihydrobenzophenanthridine alkaloid prepared by the preparation method according to any one of claims 2 to 6 in the preparation of anti-inflammatory drugs.

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